Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
PAPERS
IN
GENETieS
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c LASSIC TAPERS IN
G ENETICS
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Edited by
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James A. Peters
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(0- Associate Professor
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San Fernando Valley State College
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Preface
Each paper in this collection has each author was as new to biology at
been selected for one or more reasons. the time of writing as to any reader
It may have served to focus attention meeting it for the first time today.
on a particular facet of genetics. It may This collection of papers served as
well the impact the study of
illustrate the basis for a course in introductory
genetics has on biology or on social biology taught for two years at Brown
and racial relationships. It may have University. Many of these students had
embodied a particular idea unique at had no previous training in biologv%
the time of publication that has led to but they demonstrated most satisfac-
extensive research by other geneticists, torily that a neophyte in science can
in many cases still continuing today. read, understand, and profit from a
It may provide a brilliant example of direct experience with the original
the use of the scientific method. It may literature of a particular field. Some
furnish a clear-cut, concise illustration guidance was necessary, and much was
of incisive reasoning. One or two have given in class. It is presented here in the
the added virtue of having been writ- form of an introduction to each paper.
ten in an entertaining style. Little or no interpretation of the paper
In each case, they are evidence of will be found in the introduction, how-
work considered to constitute "classic" ever, for this interferes with the rela-
contributions to the science of biology. tionship between the author and the
Taken as a unit, they have done much reader. All authors attempt to express
to give form and direction to genetic their ideas clearly to the reader, and it
research. Their vitality is unimpaired is only fair to let them do so if they
by age, and their repeated citation in can. At the same time, the reader who
bibliographies of current literature or follows an author's logic can feel that
on seminar reading lists testifies that he has received his information from
they are still important sources of in- the primary source, and he is no longer
formation. dependent upon second hand interpre-
You should not expect, and will not tation of research.
find, any attempt by an author to It is my pleasure to acknowledge the
"write down" to the level of his permission granted by the publishers
readers, for the primary concern is and authors to reproduce the papers in
neither popularization nor condensa- this volume. Citations to the original
tion, but rather, adequate presentation. source are included with each paper.
There is an assumption by the authors All of the journals are still being pub-
that the reader has some biological lished except the Report of the Evolu-
background. Lack of this background tion Committee of the Royal Society,
should not handicap anyone in follow- and they contain a continuing record
ing the development of the basic ideas. of recent activities and researches. It
Most of the major steps in the develop- would repay the reader to look over
ment of the gene theory are here, and them occasionally to see what solutions
the nature of the material discussed by have been oflFered to the many ques-
iii
IV PREFACE
tions left unsolved by the authors in- discussing, and understanding these
cluded in this book. papers. A student who generates an
It is also mv pleasure to thank my interest in the subject he is studying is
students at Brown University for their a joy forever, and I found myself
work and study expended in reading, blessed with a bountiful crop of them.
Note: All page references co72tai?ied hi the individual papers have been carried
over into this volimie froju the original publications, for any value th^y ?nigbt have
for the researcher. Page mnnbers cited iji the text do Jiot refer to pages i?i this volume
unless specifically so stated.
Table of Contents
Preface \^ ^ ^^ iii
1903 • W. JOHANNSEN
Heredity in Populations and Pure Lines 20
1908 • G. H. Hardy
Mendelian Proportions in a Mixed Population 60
1910 • T. H. Morgan
Sex Limited Inheritance in Drosophila 63
1913 • A. H. Sturtevant
The Linear iVrrangement of Six Sex-linked Factors
in Drosophila, as Shown by their Mode of Association 67
1921 • L. C. Dunn
Unit Character Variation in Rodents 92
1922 • H. J. MuLLER
Variation Due to Change in the Individual Gene 104
1925 • A. H. Sturtevant
The Effects of Unequal Crossing Over at the Bar Locus
in Drosophila 124
1927 • H. J. MuLLER
Artificial Transmutation of the Gene 149
V
VI TABLE OF CONTENTS
1931 • Harriet S. Creighton and Barbara McClintock
A Correlation of Cytological and Genetical Crossing-
over in Zea mays 155
1933 • T. S. Painter
A New Method for the Study of Chromosome Re-
arrangements and Plotting of Chromosome Maps 161
1946 •
J. Lederberg and E. L. Tatum
Gene Recombination in Escherichia coli 192
1953 •
J. D. Watson and F. H. C. Crick
Molecular Structure of Nucleic Acids 241
1954 • L. J. Stadler
The Gene 244
1954 • A. H. Sturtevant
Social Implications of the Genetics of Man 259
GREGOR MENDEL
arranged his data so as to shed the best possible light on his con-
clusions, I believe that the only way he might have ma7iipzdated his
data is through oviissioyi of certain results that woidd have led to un-
necessary complications. When Mendel specified that his experime?its
were to deal with ^^consta?itly differe?itiating characters'^ that oc-
curred in pairs, he relieved himself of the necessity of considering
some of the interrelatio7iships that exist in genetic phenomena, and
which will be discussed in the Bateson and Punnett papers (see pp.
44 and 54). Mendel probably knew of these interrelationships, be-
cause he tested many characters before selecting the seven pairs he
used. The fact that he chose to utilize only those characteristics that
fitted hisconcepts ca?mot be interpreted as an act of dishonesty on
his part. As
I see it, he recognized several of the basic cojicepts of
heredity, a?id prese?ited as Jimch of his data as was necessary to
validate those co?icepts.
I have not included the last few pages of MendeVs original paper,
Wichura published the results of some cluded in all essentials. WTiether the
profound investigations into the hy- plan upon which the separate experi-
brids of the Willow. That, so far, no ments were conducted and carried out
generally applicable law governing- the was the best suited to attain the de-
formation and development of hybrids sired end is left to the friendly decision
has been successfully formulated can of the reader.
hardly be wondered at by anyone who
is acquainted with the extent of the
SELECTION OF THE EXPERIMENTAL
task, and can appreciate the difficulties
PLANTS
with which experiments of this class The value and utility of any experi-
have to contend. A final decision can ment are determined by the fitness of
only be arrived at when we shall have the material to the purpose for which
before us the results of detailed ex- it isused, and thus in the case before
periments made on plants belonging to us cannot be immaterial what plants
it
sible care if it be desired to avoid from organs are closely packed inside the
the outset every risk of questionable keel and the anther bursts within the
results. bud, so that the stigma becomes cov-
The experimental plants must neces- ered with pollen even before the
sarily— flower opens. This circumstance is of
1. Possess constant differentiating especial importance. As additional ad-
characters. vantages worth mentioning, there may
2. The hybrids of such plants must, be cited the easy culture of these plants
during the flowering period, be pro- in the open ground and in pots, and
tected from the influence of all foreign also their relatively short period of
pollen, or be easily capable of such growth. Artificial fertilisation is cer-
protection. tainly a somewhat elaborate process,
The hybrids and their offspring but nearly always succeeds. For this
should suffer no marked disturbance purpose the bud is opened before it is
in their fertility in the successive gen- perfectly developed, the keel is re-
erations. moved, and each stamen carefully ex-
Accidental impregnation by foreign tracted by means of forceps, after
pollen, if it occurred during the experi- which the stigma can at once be dusted
ments and were not recognized, would over with the foreign pollen.
lead to entirely erroneous conclusions. In all, thirty-four more or less dis-
Reduced fertility or entire sterility of tinct varieties of Peas were obtained
certain forms, such as occurs in the from several seedsmen and subjected
offspring of many hybrids, would to a two years' trial. In the case of one
render the experiments very difficult were noticed, among a
variety there
or entirely frustrate them. In order to number of plants all alike, a few
larger
discover the relations in which the forms which were markedly different.
hybrid forms stand towards each other These, however, did not vary in the
and also towards their progenitors it following year, and agreed entirely
appears to be necessary that all mem- with another variety obtained from the
bers of the series developed in each same seedsman; the seeds were there-
successive generation should be, with- fore doubtless merely accidentally
out exception, subjected to observa- mixed. All the other varieties yielded
tion. perfectly constant and similar off-
At the ver\' outset special attention spring; at any rate, no essential differ-
was devoted to the Legimiinosae on ence was observed during two trial
account of their peculiar floral struc- years. For fertilisation twenty-two of
ture. Experiments which were made these were selected and cultivated
with several members of this family during the whole period of the experi-
led to the result that the genus Pisum ments. They remained constant with-
was found to possess the necessary out any exception.
qualifications. Their systematic classification is dif-
Some thoroughly distinct forms of ficult and uncertain. If we adopt the
this genus possess characters which are strictest definition of a species, accord-
constant, and easily and certainly rec- ing to which only those individuals
ognizable, and when their hybrids are belong to a species which under pre-
mutually crossed they yield perfectly ciselv^the same circumstances display
fertile progeny. Furthermore, a dis- precisely similar characters, no two of
turbance through foreign pollen can- these varieties could be referred to one
not easily occur, since the fertilising species. According to the opinion of
.
MENDEL
experts, however, the majority belong do not permit of sharp and certain
a
to the speciesPisum sativimi; while the separation, since the differenceis of a
rest are regarded and classed, some as "more or less" nature, which is often
sub-species of P. sativimi, and some as difficult to define. Such characters
independent species, such as P. quadra- could not be utilised for the separate
turn, P. saccharatum, and P. umbella- experiments; these could only be ap-
tum. The positions, however, which plied to characters which stand out
may be assigned to them in a classifica- clearly and definitely in the plants.
tory system are quite immaterial for Lastly, the result must show whether
the purposes of the experiments in they, in their entirety, observe a reg-
question. It has so far been found to ular behaviour in their hybrid unions,
be just as impossible to draw a sharp and whether from these facts any con-
line between the hybrids of species and clusion can be come to regarding those
varieties as between species and vari- characters which possess a subordinate
eties themselves. significance in the type.
The characters which were selected
DIVISION AND ARRANGEMENT for experiment relate:
OF THE EXPERIMENTS 1. To the difference in the form of
If two plants which differ constantly the ripe seeds. These are either round
in one or several characters be crossed, or roundish, the depressions, if any,
numerous experiments have demon- occur on the surface, being always
strated that the common characters are only shallow; or they are irregularly
transmitted unchanged to the hybrids angular and deeply wrinkled (P. qua-
and their progeny; but each pair of dratwn)
on the other
differentiating characters, 2. To the difference in the colour of
hand, unite in the hybrid to form a the seedalbumen (endosperm).^ The
new character, which in the progeny albumen of the ripe seeds is either pale
of the hybrid is usually variable. The yellow, bright yellow and orange
object of the experiment was to ob- coloured, or it possesses a more or less
serve these variations in the case of intense green tint. This difference of
each pair of differentiating characters, colour is easily seen in the seeds as
and to deduce the law according to [= if] their coats are transparent.
which they appear in the successive 3. To the difference in the colour of
generations. The experiment resolves the seed-coat. This is either white,
itself therefore into just as many sepa- with which character white flowers
rate experiments as there are con- are constantly correlated; or it is grey,
stantly differentiating characters pre- grey-brown, leather-brown, with or
sented in the experimental plants. without violet spotting, in which case
The various forms of Peas selected the colour of the standards is violet,
for crossing showed differences in the thr.t of the wings purple, and the stem
length and colour of the stem; in in the axils of the leaves is of a reddish
the size and form of the leaves; in tint. The grey seed-coats become dark
the position, colour, and size of the brown in boiling water.
flowers; in the length of the flower 4. To the difference in the for/n of
stalk; in the colour, form, and size of the ripe pods. These are either simply
the pods; in theform and size of the
3 [Mendel uses the terms "albumen" and
seeds;and in the colour of the seed- "endosperm" somewhat loosely to denote the
coats and of the albumen [cotyle- cotyledons, containing food-material, within
dons]. Some of the characters noted the seed.!
.
MENDEL
inflated, not contracted in places; or 1st trial 60 fertilisations on 15 plants.
'
s
[This also happens in Sweet Peas.] ^^ [Note that Mendel, with true penetra-
Alendel throughout speaks of his cross-
^ f tion, avoidsspeaking of the hybrid-character
bred Peas as "hybrids," a term which many as "transmitted"by either parent, thus escap-
restrict to the offspring of two distinct ing the error pervading the older views of
species. He, as he explains, held this to be heredity.]
only a question of degree.] 11 [Gartner, p. 223.]
MENDEL
which were used in the experiments following year in such plants as have
the following are dominant: been raised from the crossed seed.
1. The round or roundish form of
[Fo] THE GENERATION [bRED]
the seed with or without shallow de-
FROM THE HYBRIDS
pressions.
2. The yellow
colouring of the seed In this generation there reappear, to-
albumen [cotyledons]. gether with the dominant characters,
3. The grey, grey-brown, or leather- also the recessive ones with their pecu-
brown colour of the seed-coat, in asso- liarities fully developed, and this
ciation with violet-red blossoms and occurs in the definitely expressed aver-
reddish spots in the leaf axils. age proportion of three to one, so that
4. The simply inflated form of the among each four plants of this genera-
pod. tion three display the dominant char-
5. The green
colouring of the un- acter and one the recessive. This relates
ripe pod in association with the same without exception to all the characters
colour in the stems, the leaf-veins and which were investigated in the experi-
MENDEL
lifted and transferred to a special bed. the same ratio as the hybrid forms,
This precaution was necessary, as while only one-X.\\\vd remains with the
otherwise they would have perished dominant character constant.
through being overgrown by their tall The separate experiments yielded
relatives. Even in their quite young the following results:
state they can be easily picked out by Expt. 1. Among 565 plants which
their compact growth and thick dark- were raised from round seeds of the
green foliage. ^^ first generation, 193 yielded round
If now the results of the whole of seeds only, and remained therefore
the experiments be brought together, constant in this character; 372, how-
there is found, as between the number ever, gave both round and wrinkled
of forms with the dominant and re- seeds, in the proportion of 3 to 1. The
cessive characters, an average ratio of number of the hybrids, therefore, as
2.98 to 1, or 3 to 1. compared with the constants is 1.93
The dominant character can have to 1.
here a dozible signification— vh.. that of Expt. 2. Of 519 plants which were
to the hybrid, and not "transmitted" to it — of 67, on the other hand, some had
true and fundamental conception here ex- axial and some terminal flowers.
pressed probably for the first time.] Expt. 7. The offspring of 28 plants
10 MENDEL
inherited the long axis, and those of characters [respectively^ in equal
72 plants some the long and some the numbers.
short axis.
In each of these experiments a cer- the subsequent generations
tain number of the plants came con- [bredI from the hybrids
stant with the dominant character. For The proportions in which the de-
the determination of the proportion in scendants of the hybrids develop and
which the separation of the forms with split up in the first and second genera-
the constantly persistent character re- tions presumably hold good for all sub-
sults, the two first experiments are of sequent progeny. Experiments 1 and 2
especial importance, since in these a have already been carried through six
larger number of plants can be com- generations, 3 and 7 through five, and
pared. The ratios 1.93 to 1 and 2.13 to 1 4, 5, and 6 through four, these experi-
gave together almost exactly the aver- ments being continued from the third
age ratio of 2 to 1. The sixth experi- generation with a small number of
ment gave a quite concordant result; plants, and no departure from the rule
in the others the ratio varies more or has been perceptible. The offspring of
less, as was only to be expected in view the hybrids separated in each genera-
of the smaller number of 100 trial tion in the ratio of 2:1:1 into hybrids
plants. Experiment 5, which shows the and constant forms.
greatest departure, was repeated, and If A be taken as denoting one of the
then, in lieu of the ratio of 60 and two constant characters, for instance
40, that of 65 and 35 resulted. The the dominant, a, the recessive, and Aa
average ratio of 2 to 1 appears, there- the hybrid form in which both are
fore, as fixed with certainty. It is conjoined, the expression
therefore demonstrated that, of those
forms which possess dominant
the
A + lAa + a
character in the first generation, two- shows the terms in the series for the
thirds have the hybrid-character, while progeny of the hybrids of two differ-
one-third remains constant with the entiating characters.
dominant character. The observation made by Gartner,
The ratio of 3 to 1, in accordance Kolreuter, and others, that hybrids are
with which the distribution of the inclined to revert to the parental forms,
dominant and recessive characters re- is also confirmed by the experiments
sults in the first generation, resolves described. It is seen that the number
itself therefore in experiments into
all of the hybrids which arise from one
the ratio of 2 1 1 if the dominant char-
: : fertilisation, as compared with the
acter be differentiated according to its number of forms which become con-
significance as a hybrid-character or stant, and their progeny from genera-
as a parental one. Since the members of tion to generation, is continually di-
the first generation [F2I spring di- minishing, but that nevertheless they
rectly from the seed of the hybrids could not entirely disappear. If an
[Fi], it is ?20iv clear that the hybrids average equality of fertility in all plants
forvi seeds havifig one or other of the in all generations be assumed, and if,
two differ entiatifig characters, and of furthermore, each hybrid forms seed
these one-half develop again the hy- of which one-half yields h\'brids again,
brid while the other half yield
foriJi, while the other half is constant to both
plants which remain constant and re- characters in equal proportions, the
ceive the dominant or the recessive ratio of numbers for the offspring in
MENDEL
each generation is seen by the follow-
ing summary, in which A
and a denote
again the two parental characters, and
Aa the hybrid forms. For brevity's
sake it may be assumed that each plant
in each generation furnishes only 4
seeds.
Genera-
12
38 plants
MENDEL
A + 2Aa + a
B + 2Bb + b.
Expt. 2.
the series, and 2" the number of unions If we endeavour to collate in a brief
which remain constant. The series form the results arrived at, we find
therefore contains, if the original that those differentiating characters,
stocks differ in four characters, 3^ = 81 which admit of easy and certain rec-
classes, 4"* = 256 individuals, and 2^ = ognition in the experimental plants, all
16 constant forms; or, which is the behave exactly alike in their hybrid
same, among each 256 offspring of the associations. The offspring of the hy-
hybrids there are 8 1 different combina- brids of each pair of differentiating
tions, 16 of which are constant. characters are, one-half, hybrid again,
All constant combinations which in while the other half are constant in
Peas are possible by the combination equal proportions having the char-
of the said seven differentiating char- acters of the seed and pollen parents
acters were actually obtained by re- respectively. If several differentiating
peated crossing. Their number is given characters are combined by cross-fer-
by 2'^ = 128. Thereby is simultane- tilisation in a hybrid, the resulting off-
ously given the practical proof that spring form the terms of a combination
the constant characters which appear series in which the combination series
if? the several varieties of a group of for each pair of differentiating char-
plants may be obtained w all the asso- acters are united.
ciatio?Js which are possible according The uniformity of behaviour shown
to the [mathematical] laws of com- by the whole ojF the characters sub-
As regards the flowering time of the ciple that a similar relation exists in the
hybrids, the experiments are not yet other characters which appear less
concluded. It can, however, already be sharply defined in plants, and there-
stated that the time stands almost ex- fore could not be included in the
actly between those of the seed and separate experiments. An experiment
pollen parents, and that the constitu- with peduncles of different lengths
tion of the hybrids with respect to this gave on the whole a fairly satisfactorv^
character probably follows the rule result, although the differentiation and
ascertained in the case of the other serial arrangement of the forms could
characters. The forms which are se- not be effected with that certainty
lected for experiments of this class which is indispensable for correct ex-
must have a difference of at least periment.
twenty days from the middle flower-
ing period of one to that of the other; THE REPRODUCTIVE CELLS
furthermore, the seeds when sown OF THE HYBRIDS
must all be placed at the same depth in The results of the previously de-
the earth, so that they may germinate scribed experiments led to further ex-
MENDEL 15
periments, the results of which appear form of the seed and the colour
in the
fitted to afford some conclusions as of the albumen were united by ferti-
regards the composition of the egg and lisation.
pollen cells of hybrids. An important If the differentiating characters are
clue afforded in Pisimi by the cir-
is again indicated as A, B, a, b, we have:
cumstance that among the progeny of
the hybrids constant forms appear, and AB, seed parent;
that this occurs, too, in respect of all A, form round;
combinations of the associated char- B, albumen yellow.
acters. So far as experience goes, we ab, pollen parent;
find it in every case confirmed that a, form wrinkled;
constant progeny can only be formed b, albumen green.
when the egg cells and the fertilising
pollen are of like character, so that The artificially fertilised seeds were
both are provided with the material for sown together with several seeds of
creating quite similar individuals, as is
both original stocks, and the most
the case with the normal fertilisation vigorous examples were chosen for the
of pure species. We
must therefore reciprocal crossing. There were ferti-
regard it as certain that exactly similar lised:
factors must be at work also in the
production of the constant forms in
1. The hybrids with the pollen of
the hybrid plants. Since the various
AB
constant forms are produced in ojie
2. The hybrids with the pollen of
plant, oreven in ojie flower of a plant,
ab.
the conclusion appears logical that in
the ovaries of the hybrids there are
3. AB with the pollen of the hy-
brids.
formed as many sorts of egg cells, and
4. ab with the pollen of the hybrids.
in the anthers as many sorts of pollen
cells, as there are possible constant
combination forms, and that these egg For each of these four experiments
and pollen cells agree in their internal the whole of the flowers on three
plants were fertilised. If the above
composition with those of the separate
forms. theory be correct, there must be de-
In point of fact it is possible todem- veloped on the hybrids egg and pollen
cells of the forms AB, Ab, aB, ab, and
onstrate theoretically that this hypo-
thesis would fully suffice to account
there would be combined:
brid union with the recessive char- 1st Exp. 3rd Exp.
acters a and b, for which reason they 20 25 round yellow seeds . AB
must impress their peculiarity upon 23 19 round yellow and
the whole of the seeds. All seeds should
green seeds ABb
IS 22 round and wrinkled
therefore appear round and yellow, if
yellow seeds AaB
18 [To prove, namely, that both were 22 21 round and wrinkled
similarly differentiated, and not one or other green and yellow
only.] seeds AaBb
MENDEL 17
4-+
A
-+
a
^+-^A
A a
+ 2Aa + a.
of these will on the average figure four
times in the fertilisation, since sixteen
individuals are included in the series.
This represents the average result of Therefore the participators in the fer-
the self-fertilisation of the hybrids tilisation are
when two differentiating characters
are united in them. In individual Pollen cells AB + AB + AB + AB
flowers and in individual plants, how- + Ab + Ab + Ab + Ab
ever, the ratios in which the forms of + aB + aB + aB + aB
the series are produced may suffer not + ab + ab + ab + ab.
inconsiderable Apart
fluctuations.^^ Egg cells AB + AB + AB + AB
from the fact that the numbers in + Ab + Ab + Ab + Ab
which both sorts of egg cells occur in + aB + aB + aB + aB
the seed vessels can only be regarded + ab + ab + ab + ab.
as equal on the average, it remains
purely a matter of chance which of the In the process of fertilisation each pol-
two sorts of pollen may fertilise each len form unites on an average equally
separate egg cell. For this reason the often with each egg cell form, so that
separate values must necessarily be each of the four pollen cells AB unites
subject to fluctuations, and there are once with one of the forms of egg cell
even extreme cases possible, as were AB, Ab, aB, ab. In precisely the same
described earlier in connection with way the rest of the pollen cells of the
the experiments on the form of the forms Ab, aB, ab unite with all the
seed and the colour of the albumen. other egg cells. We
obtain therefore
The true ratios of the numbers can
only be ascertained by an average de- AB
duced from the sum of as many single AB
values as possible; the greater the num-
ber, the more are merely chance effects
eliminated.
The developmental series for hy-
brids in which two kinds of differen-
tiating characters are united contains,
among sixteen individuals, nine differ-
ent forms, viz.,
AB + Ab + aB + ab + lABb + laBb
+ lAaB -f 2Aab + '\AaBb.
Betu^een the differentiating characters
of the original stocks, Aa and Bb, four
constant combinations are possible, and
consequently the hybrids produce the
corresponding four forms of egg and
pollen cells AB, Ab, aB, ab, and each
20 JOHANNSEN
characters are conjoined in them. The ment of the hybrids, finds therefore its
hybrids form eight various kinds of foundation and explanation in the prin-
egg and pollen cells— ^BC, ABc, AbC, ciple enunciated, that the hybrids pro-
Abc, aBC, aBc, abC, abc—iLnd each duce egg cells and pollen cells which
pollen form unites itself again on the in equal numbers represent all constant
average once with each form of egg forms which result from the combina-
cell. tions of the characters brought to-
The law of combination of different gether in fertilisation,
characters, which governs the develop-
W. JOHANNSEN
/ have translated here only the final swfrmary and discussion fro?n
Johannsen's lo?]g paper on pure lines, which was written in German.
This thorough and meticidous ifjvestigation of the true significa?7ce of
selection was a bombshell to evolutio?iary thought. The efficacy of
selection in the production of fiew species had beeji one of the main-
stays of Darwin's theory of evolution. Johajmsen's studies deinon-
strated conclusively that selectiofj coidd not extend the limits of
previously This fact became important iii
established variability.
arguments against Darwinism, and led to a period when selection was
discredited as evolutionarily sig?iifica?Jt. The mutatiojj theory became
genotypes— an event that not without dently has its natural peculiarities
reason has aroused the skepticism of analogous to those of Galton, has its
biometricians. It is to be hoped that value as a basis for analysis of the
the studies presented here will shed Galtonian laws applying to popula-
some light on this problem, which per- tions. My statements conflict neither
haps only appears to detroy the wiih Galton's statements nor with
boundary between fluctuating varia- those of de Vries.
tion and mutation. If my investigations are sound, and
(Postscript: In the final part of his their significance is grasped beyond the
Mutationstheorie — which appeared special case here discussed, the general
during the editing of this work— de work would form a not
results of this
Vries (I.e., pp. 503-504) has shown in unimportant support for the modern
a most ingenious way how in most concepts of Bateson and de Vries on
cases mutations are first expressed. the great significance of "discontinu-
Therein lies an outstandingly impor- ous" variations, or "mutations," for
JOHANNSEN 25
the theorv' of evolution. For a selec- possibility of mutation; for thereby
tion in cases such as mine is effective even the strongest correlative relation-
only in so far as it selects out repre- ship could be destroyed. I do not wish
sentatives of an already existant geno- to take up question at this time; in
this
type. These genotypes would not be a later publication I hope to shed more
successively originated through the re- light on it, using the principle of pure
tention of those individuals which vary lines as the basis for the research.
in the desired direction; they would I would be sorry indeed if the reader
merely he found and isolated. of this work would come to feel that
The knowledge that has been gained the value of the significant work of
from studies on pure lines, combined Galton, Pearson, and other biometrical
with a knowledge of hybridization, research workers were to be placed in
must serve as the starting point in the doubt. I would not have the audacity
case of studies on heredity within to criticize the treatment which Pear-
population in which pure lines can not son in particular has given to the ques-
be completely isolated as a conse- tion of the ancestral influence within
quence of the necessity of constant a specific population. I do think, how-
cross-fertilization or hybridization. ever, that the principle of pure lines
This knowledge is, as has been pointed in the hands of a man such as Pearson
out earlier, in complete agreement can carry biometric studies much far-
with the basic ideas in the great work ther along than his studies of popula-
of de \^ries— as has been seen, my con- tions. Obviously the relationships
cepts have been arrived at via a some- studied by Pearson have great scientific
what different path than that followed significance and they have consider-
by de Vries, and it is also important to able practical value as well— but they
note, based on a different kind of in- are not suitable to illuminate com-
formation. pletely the fundamental laws of hered-
In addition the important question it)\
of correlative variation takes on a And what particularly affects Gal-
somewhat changed character depend- ton's research, in my estimation, is that
ing upon whether one works with the results presented here support in a
pure lines or with populations. In the beautiful way the basic ideas of Gal-
latter case a given "ratio of correla- ton's "Stirp" theory,^ which was al-
tion" (Pearson's term) will not neces- ready worked out in 1876. This law
sarily represent a strongly legitimate includes almost all that is of actual
relationship, as I have sought to dem- value in the more recent Weismann
onstrate earlier. An indicated correla- theory on the "Continuitv' of the
tive relationship is much more signifi- Gerniplasm." That the speculation of
cant within a pure line. My
summation Weismann ^ could overshadow the
table speaks very well for this concept,
in that it was not possible to change 3 Galton's theory is known to me from his
original paper in Revue Scientifique, vol. 10,
through selection within pure lines the
1876, p. 198 ("Theorie de I'heredite").
correlation between length and width •*
position of biometricians on "Weis-
The
of the beans, while it was simple to mannism" has been clearly evaluated by
isolate truly different genot\^pes, as for Pearson in his characterization of this move-
example narrow and broad forms, ment in "Socialism and Natural Selection"
{Fortnightly Review, July, 1894. Reprinted
from the original population, which in Pearson's Chafices of Death, vol. 1, 1897,
appeared to be entirely homogeneous. p. 104) . I do not plan to go further into this
Again, we have to, reckon with the question at this time.
26 JOHANNSEN
more simply put but no less ingenious Should the present publication be
and quite original idea of Galton's is successful in bringing the principle of
perhaps due to some extent to Galton pure lines recognition as an absolutely
himself, for he has not seen fit in his necessary principle in truly intensive
more recent publications to adhere to research in the study of heredity, then
his Stirp theory in the light of research its highest purpose would be achieved.
progress. The Stirp theory does not Later publications will attempt to illu-
correlate too well with Galton's Law minate the activity of lines which vary
of Regression, it is true, but it could polymodally. I have investigated only
scarcely be better supported or illus- unimodal variation in this paper,'^ in
trated than by the results which I have order to present my concept in its
described: a usually complete regres- simplest instance.
sion to the genotype of a pure line The train of thought which under-
seems to me the most beautiful evi- lies this investigation is expressed in
dence for a slightly modified Stirp its simplicity most clearly by the often
concept. It is true that Galton's Stirp cited words of Goethe:
concept cannot be maintained un-
"Dich im UnendHchen zu finden
changed. Although Weismann •'
very
Musst unterscheiden und dann
recently regarded Galton as the ^
verbinden."
"voice" of cellular limitation through
"Determinants" — or however one
Vilmorin has emphasized the differ-
might name these theoretical hered-
entiation of the parts, Galton has dem-
itary corpuscles, de Vries deserves the
onstrated the legitimate basis for re-
great credit for having recognized the
combination; I have tried here to
unitary nature of hereditary particles,
combine the points of view for which
which he called "pangenes"— a concept
these two ingenious investigators are
he first published in 1889 ^ and further
honored.
advanced in the "Mutationstheorie." It
seems to me that the Galton-de Vries
theory is the only truly useful theory de Vries has publislied a special case con-
"^
WALTER S. SUTTON
It has often happened that two people have nmde practically iden-
tical discoveries ajid published their findi?jgs ahiost simnltajieoiisly.
This has bee?i particidarly true in getietics, which fiiids its 7?ioder?i
can make no pretensions to complete suspecting that this fact must corre-
familiarity with the results of experi- spond with some symmetrical figure
mental studies on heredity. As will of distribution of the gametes in the
appear hereafter, they completely sat- cell divisions by which they are pro-
isfy the conditions in typical Mende- duced."
lian cases, and it seems that many of Nearly a year ago it became appar-
the known deviations from the Men- ent to the author that the high degree
delian type may be explained by easily of organization in the chromosome-
conceivable variations from the nor- group of the germ-cells as shown in
mal chromosomic processes. Brachystola could scarcely be without
It has long been admitted that we definite significance in inheritance, for,
as shown in the paper ^ already re-
1Sutton, Walter S., "On the Morphology
of the Chromosome Group in Brachystola ferred to, it had appeared that:
magna," Biol. Bull, IV., 1, 1902.
2 Mendel, Gregor Johann, "Versuche iiber 1. The chromosome group of the
Pflanzen-Hybriden," Verb, naturf. Vers, in presynaptic germ-cells is made up of
BrUnn IV., and in Osterwald's Klassiker der two equivalent chromosome-series,
exakten Wissevschajt. English translation in
and that strong ground exists for the
]oiirn. Roy. Hon. Soc, XXVI., 1901. Later
conclusion that one of these is paternal
reprinted with modifications and corrections
in Bateson's "Mendel's Principles of Hered- and the other maternal.
ity," Cambridge, 1902, p. 40.
3 Wilson, E. B., "Mendel's Principles of » Bateson, W., "Mendel's Principles of
Heredity and the Maturation of the Gemi- Heredity," Cambridge, 1902, p. 30.
Cells," Science, XVI., 416. ^ Sutton, W. S., loc. cit.
SUTTON 29
2. The process of synapsis (pseudo- breeding could accomplish more than
reduction) consists in the union in the condition of a first-cross.
pairs of the homologous members 2. If any animal or plant has but two
(i. e., those that correspond in size) of categories of germ-cells, there can be
the two series.^ only four different combinations in the
3. The first post-synaptic or ma- offspring of a single pair.
turation mitosis is equational and hence 3. If either maternal or paternal
results in no chromosomic differentia- chromosomes are entirely excluded
tion. from every ripe germ-cell, an individ-
4. The second post-sv^naptic divi- ual cannot receive chromosomes (qual-
sion is a reducing division, resulting in ities) from more than one ancestor in
the separation of the chromosomes each generation of each of the parental
which have conjugated in synapsis, lines of descent, e. g., could not inherit
and their relegation to different germ- chromosomes (qualities) from both
cells. paternal or both maternal grandpar-
5. The chromosomes retain a mor- ents.
phological individuality throughout
the various cell-divisions. Amoved by these considerations a
It is well known that in the eggs of more careful study was made of the
many forms the maternal and paternal whole division-process, including the
chromosome groups remain distinctly positions of the chromosomes in the
independent of each other for a con- nucleus before division, the origin and
siderable number of cleavage-mitoses, formation of the spindle, the relative
and with this fact in mind the author positions of the chromosomes and the
was at first inclined to conclude that diverging centrosomes, and the point
in the reducing divisions all the ma- of attachment of the spindle fibers to
ternal chromosomes must pass to one the chromosomes. The results gave no
pole and all the paternal ones to the evidence in favor of parental purity
other, and that the germ-cells are thus of the gametic chromatin as a whole.
divided into two categories which On the contrary, many points were
might be described maternal and as discovered which strongly indicate ^
paternal respectively. But this concep- that the position of the bivalent chro-
tion, which is identical with that mosomes in the equatorial plate of the
recently brought forward by Cannon,'^
was soon seen to be at variance with 8 Absolute proof is impossible in a pure-
.many well-known facts of breeding; bred form on account of the impossibility
thus: of distinguishing between maternal and pa-
ternal members of any synaptic pair. If,
however, such hybrids as those obtained by
1. germ-cells of hybrids are
If the
Moenkhaus (Moenkhaus, W. J., "Early De-
of pure descent, no amount of cross- velopment in Certain Hybrid Species," Re-
port of Second Meeting of Naturalists at
® The conclusion that synapsis involves a Chicago, Sciefice, XIII., 323), with fishes can
union of paternal and maternal chromosomes be reared to sexual maturity absolute proof
in pairs was first reached bv Montgomery in of this point may be expected. This observer
1901. was able in the early cells of certain fish hy-
Montgomery, T. H., Jr., "A Study of the brids to distinguish the maternal from the
Chromosomes of the Germ-Cells of Me- paternal chromosomes by differences in
tazoa," Trans. Amer. Phil. Soc, XX. form, and if the same can be done in the
'
Cannon, W. A., "A Cytological Basis for maturation-divisions the question of the dis-
the A-Iendelian Laws," Bull. Torrey Botanical tribution of chromosomes in reduction be-
Club, 29, 1902. comes a very simple matter of observation.
30 SUTTON
reducing division is purely a matter of of the synaptic pairs, but there are
chance— that is, that any chromosome sixteen possible combinations of ma-
pair may lie with maternal or paternal ternal and paternal chromosomes that
chromatid indifferently toward either will form a complete series, to wit: a,
pole irrespective of the positions of B, C, D; A, b, C, D; A, B, c, D; A, B,
other pairs— and hence that a large C, d; a, b, C, D; a, B, c, D; a, B, C, d;
number of different combinations of a, b, c, d; and their conjugates A, b, c,
maternal and paternal chromosomes d; a, B, c, d; a, b, C, d; a, b, c, D; A, B,
are possible in the mature germ-prod- c, d; A, b, C, d; A, b, c, D; A, B, C, D.
ucts of an individual. To illustrate this, Hence instead of two kinds of gametes
we may consider a form having eight an organism with four chromosomes in
chromosomes in the somatic and pre- its reduced series may give rise to 16
Table 1
Chromosomes
SUTTON 31
plain that in the ordinary form having larvae actually lacking in certain chro-
from 24 to 36 chromosomes, the pos- mosomes of the normal series, which
sibilities are immense. Table below1 seem to leave no alternative to the
shows the number of possible com- conclusion that the chromosomes
binations in forms having from 2 to 36 differ qualitatively and as individuals
chromosomes in the presynaptic cells. represent distinct potentialities. Ac-
Thus if Bardeleben's estimate of six- cepting this conclusion we should be
teen chromosomes for man (the lowest able to find an exact correspondence
estimate that has been made) be cor- between the behavior in inheritance of
rect, each individual is capable of pro- any chromosome and that of the char-
ducing 256 different kinds of germ- acters associated with it in the organ-
products with reference to their ism.
chromosome combinations, and the In regard to the characters, Mendel
numbers of combinations possible in found that, if a hybrid produced by
the offspring of a single pair is 256 X crossing two individuals differing in
256 or 65,536; while ToxopJieitstes, a particular character be self-fertilized,
with 36 chromosomes, has a possibility the offspring, in most cases, conform
of 262,144 and 68,719,476,736 differ- to a perfectly definite rule as regards
ent combinations in the gametes of a the differential character. Represent-
single individual and the zygotes of a ing the. character as seen in one of the
pair respectively. It is this possibility original parents by the letter A and
of so great a number of combinations that of the other by a, then all the
of maternal and paternal chromosomes offspring arising by self-fertilization of
in the gametes which serves to bring the hybrid are represented from the
the chromosome-theory into final rela- standpoint of the given character by
tion with the known facts of heredity^; the formula 2 Aa AA
aa.— that is, one
: :
for Mendel himself followed out the fourth receive only the character of
actual combinations of two and three one of the original pure-bred parents,
distinctive characters and found them one fouith only that of the other;
to be inherited independently of one while one half the number receive the
another and to present a great variety characters of both original parents and
of combinations in the second genera- hence present the condition of the hy-
tion. brid from which they sprang.
The constant size-differences ob- We have not heretofore possessed
served in the chromosomes of Brachy- graphic formulae to express the com-
jtola early led me to the suspicion, binations of chromosomes in similar
which, however, a study of spermato- breeding experiments, but it is clear
genesis alone could not confirm, that from the data already given that such
the individual chromosomes of the re- formulae may now be constructed. The
duced series play different roles in reduced chromosome series in Brachy-
development. The confirmation of this stola made up of eleven members, no
is
surmise appeared later in the results two of which are exactly of the same
obtained by Boveri ^^ in a study of size. These I distinguished in my pre-
vious paper by the letters A, B, C, . . .
i**
Boveri, Th., "Ueber Mehrpolige Alitosen K. In the unreduced series there are
als Mittel zur Analyse des Zellkems," Verb,
d. Phys.-Med. Ges. zu Wurzbiirg, N. F., Bd.
XXXV., 1902. It appears from a personal ducible from his experiments and the results
letter that Boveri had noted the correspond- —
on plant hybrids as indicated also in foot-
ence between chromosomic behavior as de- note 1, /. c, p. 81.
32 SUTTON
twenty-two elements which can be ^^ which would yield four combinations,
seen to make up two series like that of
the mature germ-cells, and hence may
As + A9 = AA
be designated as A, B, C K -h A, . . .
As + a 9 = Aa
as + A9 = aA
B, CK. Synapsis results in the
. . .
separation of these pairs so that one same given for any character
as that
member of each passes to each of the in a Mendelian case. Thus the phe-
resulting germ-products. ?iomejia of germ-cell divisio?i and of
There is reason to believe that the heredity are see?i to have the same
division-products of a given chromo- essential features, viz., purity of units
some in Brachystola maintain in their {chromosomes, characters) and the in-
respective series the same size relation depeiident transijussion of the saine;
as did the parent element; and this, while as a corollary, it follows in each
taken together with the evidence that case that each of the two antagonistic
the various chromosomes of the series units (chromosomes, characters) is
represent distinctive potentialities,
contained by exactly half the gametes
make it probable that a given size-
produced.
relation is characteristic of the physical
The observations which deal with
basis of a definite set of characters.
characters have been made chiefly
But each chromosome of any reduced upon hybrids, while the cytological
series in the species has a homologue in
data are the result of study of a pure-
any other series, and from the above bred form; but the correlation of the
consideration it should follow that
two is justified by the observation of
these homologues cover the same field
Cannon ^- that the maturation mitoses
in development. If this be the case
of fertile hybrids are normal. This
chromosome A
from the father and its being the case it is necessary to con-
homologue, chromosome a, from the clude, as Cannon has already pointed
mother in the presynaptic cells of the out, that the course of variations in
offspring may be regarded as the phys-
hybrids either is a result of normal
ical basesof the antagonistic unit-char- maturation processes or is entirely in-
acters A
and a of father and mother dependent of the nature of those divi-
respectively. In synapsis, copulation sions. If we conclude from the evi-
of the homologues gives rise to the dence already given that the double
bivalent chromosome Aa, which as is
basis of hybrid characters is to be
indicated above would, in the reducing found in the pairs of homologous chro-
division, be separated into the com-
mosomes of the presynaptic germ-
ponents A and a. These would in all cells, then we must also conclude that
cases pass to different germ-products in pure-bred forms likewise, the paired
and hence in a monoecious form we arrangement of the chromosomes indi-
should have four sorts of gametes. cates a dual basis for each character.
nant during successive generations if of this class Bateson clearly states the
the environment is not materially possibility that the case may be one
changed. Nevertheless, some experi- entirely "apart from those to which
ments cited by Bateson " go to show Mendel's principles apply," but goes
that dominance may be variable or on to show how it may possibly be
defective. Furthermore, it is not only brought into relation Math true Men-
conceivable, but highly probable that delian cases. He says in part: "It must
in most, if not all cases, there are be recognized that in, for example, the
many different expressions of each stature of a civilized race of man, a
character (/. e., many different allelo- typically continuous character, there
morphs as suggested by Bateson in must certainly be on any hypothesis
regard to human stature), which on more than one pair of possible allelo-
various combinations would neces- morphs. There may be many such
dominance. The
sarily exhibit relative pairs, but we have no certainty that
experiments with peas show an almost the number of such pairs and conse-
constant dominance of certain allelo- quently of the different kinds of
morphs, such as round over wrinkled gametes are altogether iinlhiiited, even
in regard to stature. If there were even
^^ Cf. Bateson and Saunders, loc. cit.
so few as, say, four or five pairs of
1" Ibid. possible allelomorphs, the various
36 SUTTON
homo- and heterozygous combinations morph were alone produced by the
might, on seriation, give so near an ap- male and the other by the female we
proach to a continuous curve that the should have a species consisting only
purity of the elements would be un- of heterozygotes," which would come
suspected, and their detection prac- true as long as bred together, at first
tically impossible." This hypothesis, sight seems logically applicable to
which presents no difficulties from the these cases.For such an idea, however,
point of view of the chromosome we can find no cytological justifica-
theory, is sufficient in the present state tion, since if any reduction occurs
of our knowledge to bring many cases both chromosomes occur in both male
of apparently continuous variation and female germ-cells in equal num-
into definite relation with strictly bers; and further, the evidence is in
Mendelian cases; but, on the other favor of a great variety of combina-
hand, it seems probable, as already tions of maternal and paternal chro-
noted (p. 221), that the individual mosomes in the germ-cells so that the
variation in many characters now exact chromosome group of a hybrid
thought to be strictly Mendelian may parent could hardly be duplicated ex-
prove to be due to the existence in the cept by fusion of the very pair of cells
species of many variations of what separated by the reducing division. A
may be regarded as the type allelo- more plausible explanation from the
morphs, accompanying similar varia- cytological standpoint is that the union
tions of the homologous chromatin of the chromosomes in synapsis is so
entities representing those types. firm that no reduction can take place,
First Crosses that Breed Tnie.—\t
2. /. e., that in each case, a paternal and
characters of the hybrid are frequently and hence it is not surprising that such
shown by the fact that all combina- cross between dissimilar individuals
tions produce similar offspring which showed the character of one parent
in turn are similar to the parent. The only, those of the other parent being
suggestion made by Bateson in an- shown by further experiment to be
other connection, that "if one allelo- lost permanently. The obvious cyto-
logical explanation of such a phenome-
18 Cf. Mendel's experiments on Hieracium. non is hinted at by Bateson in the
SUTTON 37
observed in eggs treated with chloral saic of maternal and paternal chromo-
(Hertwig brothers) or ether (Wil- somes, but very evidently this is not
son) and may be supposed to occur Bateson's meaning.
under certain unusual conditions in From the standpoint of the chromo-
nature. In the experiments mentioned, some theory I would suggest a possible
however, both pronuclei continue to explanation of the conditions as fol-
divide separately, while for a cytologi- lows: Wehave already assumed that
cal explanation of the occurrence of the somatic chromosome group, hav-
"false hybrids" it is necessary to con- ing a similar number of members to
ceive not only the failure of the nu- that of the cleavage nucleus and de-
clei to copulate but the entire disap- rivedfrom it by equation divisions, is
pearance of one of them. Such a case made up in the same way of pairs of
would be comparable to that of chem- homologous chromosomes. Every so-
ically induced parthenogenesis or to matic cell, by this conception, must
the fertilization of enucleate egg-frag- contain a double basis in the field of
ments, according as the nucleus re- each character it is capable of express-
maining was maternal or paternal. Mendelian cases one of
ing. In strictly
Speculation in this connection, how- the homologues is uniformly dominant
19 Bateson and Saunders, loc. cit., p. 154. 20 Cf. Bateson and Saunders, pp. 135, 136.
38 SUTTON
common function of one of these, group, is thrown out by the reduction-
there is nothing to show why as a division in each generation.
result of some disturbing factor one In considering the behavior of the
body of chromatin may not be called two chromosomes forming the basis of
into activity in one group of cells and any given character, it was noted that
its homologue in another. This would in some cases the heterozygote char-
produce just the sort of a mosaic which acter resulting from the combinations
Bateson and Saunders found in Dattira of dissimilar allelomorphs is sometimes
or as Tchermak's pied yellow and totally unlike either of the latter. Thus
green peas obtained by crossing the Mendel found that in crosses between
Telephone pea with yellow varieties. peas respectively 1 and 6 feet in height
Correns describes the condition as the offspring ranged from 6 to VA
poecilodyjimnous and his conception of feet. In discussing similar cases, Bate-
the causes of the phenomenon as I un- son calls attention to which
the light
derstand it is parallel with that which would be thrown on the phenomenon
I have outlined above. The logical if weventured to assume that the
possibility suggested by Bateson ^^ that bases two allelomorphs con-
of the
the recessive islands in such cases as cerned are chemical compounds; and
the mosaic pea may be due to recessive he compares the behavior of the allelo-
allelomorphs in the paired state does morphs to the reaction of sodium and
not accord with the theory of a chro- chlorine in the formation of salt. The
mosomic basis for those allelomorphs, results of chemical analysis show that
since the chromosome groups, both of one of the most characteristic features
cells showing the recessive character of chromatin is a large percentage
and of neighboring cells showing the content of highly complex and variable
dominant one, are derived, so far as we chemical compounds, the nucleo-pro-
know, by longitudinal or equation teids, and therefore if, as assumed in
division from the chromosomes of the the theory here advanced, the chromo-
same original cleavage nucleus and somes are the bases of definite heredi-
hence must be alike. tary characters, the suggestion of Bate-
The application of the theory here son becomes more than a merely in-
suggested may be put to test by an teresting comparison.
experiment in which hybrids of dis- We have seen reason in the case of
similar true-breeding parentage are the true-breeding hybrids to suspect
crossed and a third generation of that the transmission by the hybrid
"quarter-bloods" produced. Mosaics of heterozyo-ote characters may be due
occurring in such an organism, if this to permanent union of the homologous
theory be correct, would show one chromosomes. From this it is but a
character resembling that of one of the short step to the conclusion that even
maternal grandparents and one resem- if, as is normally the case, the chro-
bling that of one of the original pure- mosomes do not fuse permanently, the
breds of the paternal side. If both very fact of their association in the
characters of the mosaic should be same liquid medium may allow a pos-
clearly paternal or maternal the theory sibility of a certain degree of chemical
as outlined is proven inadequate, since interaction. This must normally be
one of each pair of chromosomes, and slight, since its effects do not appear
hence the corresponding character- to be visible in a single generation; but
the slightest of variation as a result of
21 Bateson and Saunders, p. 156. repeated new association, even though
SUTTON 39
it tend in diverse directions, must in that division must be conceived as a
time, guided by natural selection, re- true reduction. A number of observa-
sult in an appreciable difference in a tions supporting this view will be
definite direction bet\veen a chromo- brought forward in my forthcoming
some and its direct descendant and work on Brachystola.
hence between the characters asso- A^ain, if the normal course of in-
ciated with them. In this we have a heritance depends upon the accurate
sus^S^estion of a possible cause of in- chromatin-division accomplished by
dividual variation in homologous chro- mitosis, it would appear that the inter-
mosomes which we have already seen jection, into any part of the germ
reason to suspect (pp. 221 and 226). cycle, of the gross processes of amitosis
Finally, we may briefly consider could result only in a radical deviation
certain observations which seem at first from that normal course. Such an oc-
sight to preclude the general applica- currence has actually been described
bility of the conclusions here brought by Meves, McGregor and others in the
out. If it be admitted that the phe- primary spermatogonia of amphibians.
nomenon of character-reduction dis- In these cases, however, it appears that
covered by Mendel is the expression fission of the cell-body does not neces-
of chromosome-reduction, it follows sarily follow^ amitotic division of the
that forms which vary according to nucleus. I would suggest, therefore,
Mendel's law must present a reducing the possibility that the process may be
division. But the vertebrates and of no significance in inheritance, since
flowering plants— the very forms from by the disappearance of the nuclear
which most of the Mendelian results membranes in preparation for the first
have been obtained— have been re- mitotic division, the original condition
peatedly described as not exhibiting a is restored, and the chromosomes may
reducing division. Here, therefore, is enter the equatorial plate as if no
a discrepancy of which I venture to amitotic process had intervened.^^
indicate a possible explanation in the There is one observation in connec-
suggestion first made by Fick -- and tion with the accessory chromosome
more recently by Montgomery.-^ This which deserves mention in any treat-
is to the eff^ect that in synapsis as it ment of the chromosomes as agents in
occurs in vertebrates and other forms heredity. This element always divides
possessing loop-shaped chromosomes, longitudinally and hence probably
the union is side by side instead of equationally. It fails to divide in the
end-to-end as in Arthropods. In ver- first maturation mitosis, in which the
chromosomes side by side in synapsis, more than the enclosure of the different
chromosomes in partially separated vesicles.
" Fick, R., "Mittheilung ueber Eireifung Cf. Sutton, W. S., "The Spermatogonial
bei Amphibien," Supp. Afiat. Anz., XVI. Divisions in Brachystola Magna," Kans. Univ.
23 Montgomery, T. H., Jr., loc. cit. Quart., IX., 2.
40 SUTTON
tion of the ordinary chromosomes is the reduction division might contain
effected, the accessory divides longi- the series A, b, c, D, in which case its
the organism.
Bateson and Pimnett experienced great difficidty in the analysis of
their experimental residts. For one familiar with the work, the inter-
pretation is obvious, but it must be remejnbered that these men were
pioiieers in virgin territory. In each of their reports it will be easier
to understand just what has taken place by ignoring the conclusions
the authors draw, and investigatiiig the experimeiital residts. The
data themselves show the way to the reader, and atte?npts to follow
the authors'' lead result i?i confusion and misijiterpretation. It is not a
common occurrence to fi^id that an experiment points the way, but
the author does 7Wt follow the arrow. It does soinetimes happen,
however, ajid shoidd always be looked for by a reader. One cannot
challe?ige an authors data except through repetition of his experi-
ments, but his conclusions are always subject to the tests of analysis
a?id logic.
In the analysis of the combs cf poultry, Batesoji and Fimnett use a
termmology entirely theirown: It is not unusual to have several
symbolic systems during the early development of a field, with all
but one eventually dropped. This particular experiment, with con-
ventional syjnbols, has been used in many if not all genetics texts,
but will be ufifamiliar to almost everyone iji Bateson and Puiinetfs
language. 0?ie of the challenges in reading the original literature,
however, lies in the fact that no one but the original author is placing
an interpretatio?! on the inaterial.
The poultry paper is important in that it demonstrates the inde-
42
BATESON AND PUNNETT 43
pende?7ce of particle (gejie) wheritance, although the final expression
of the two indepe7ide7it pairs of gejies depejids iipoji an interde-
pendent action. All we have learned of the mode of gene action since
the tifne of this paper indicates that this is by far the most commoii
situation, and that co777pletely i7idependc7it actio7i of a pair of genes
at a single locus is rare i7jdeed. It is perhaps imfortimate that most
people whose knowledge of ge7ietics comes fro77i high school courses
or a ge7ieral university course are exposed principally to the atypical
results of Me7idel rather tha7i to a7i a7ialysis of the mode of actio?! of
the gene.
In their original paper Bateso72 a7id Punnett included eight pages of
data 071 their experime7its. I have deleted most of these tables, and
have retained only those experiments necessary for the aiialysis
(Table 1). I have removed all notes 07i the extra toe character, which
are 7iot pertijient to our a7ialysis. I have added an extra column to
those of Bateson and Pu7mett so that the reader can test his imder-
stafiding of the experiment by filling in the mati77g as sy7nbolized by
conve7itio7ial 7nethods.
The first part of the poultry paper includes the authors^ i7Jterpre-
tatio7i of their residts. Carefid reading will show that it is almost
co77ipletely erroTieous. This was realized by the authors, and the
second part of the paper co7nes fro77i a later vohm7e of the Reports.
Here the authors correct thetjtselves, and re-evaluate their residts.
Again the sy77ibolis772 is likely to be but the Pimnett
co7ifusi7ig,
square 07i p. 51 becomes Tnore clear if the reader imderstands that
''''no P" or '''no 7?" actually refers to the occurrence of a recessive
is
IT
Fi,—r-,
DD
DR — r— are,
artificial.
The same bird is given as DD in its
,
C bo
>. c
c
.2^
*c
o bo
<u C .-; eri Q
X X X X X
X X X
Q Pi X
Q Q Q
J3
6
U ^
O
_w
E ^
D M
J3
E
1) o
I ^
6
Si
46 BATESON AND PUNNETT
E
BATESON AND PUNNETT 47
DISCUSSION OF RESULTS Another, ditto '
An example of a
Comb-characters S extracted pure
An example of a
$ extracted pure
dominant (rose)
occurs in Experiment 103 and 157
48 BATESON AND PUNNETT
types of comb, viz., s.s. singles, r.s. of 1903 with various s.c. birds (Ex-
roses, p.s. peas,and finally r.p. combs, periments 163-170, 174, 176, 176a-b),
the latter being a type not before seen and gave—
in the course of the work.^ It is char-
comb that it is wide
acteristic of their r.p. r. p. s.
ford, of Woodbridge, who has a long and the s. was very low. In the light
experience of the breed, was kind of later experience, it is perhaps pos-
enough to give much help in this mat- sible that both were wrongly classi-
ter, and further assistance was also fied. Both were dead in the shell, and
received from Mr. Wootten, a breeder at that date the distinctions were not
of White Malays. It appears that familiar to us.
Malays bred inter se sometimes pro- In these results there are still certain
duce true peas as well as the normal points obscure. The "artificial" r.p.
walnut. Occasionally in certain strains birds are giving off the four types of
peas come often, but generally speak- gamete. Therefore there must be five
ing thev are uncommon. Whether types of r.p. comb possible, namely r.
pure Malays ever throw actual roses or X p., r.p. X r-p-, r.p. X r., r.p. X p-,
singles is not quite certain, but if they r.p. X but owing to the fact stated
s.;
do, these occurrences must be exces- above, that s. may be created afresh by
sively rare. at least one of the combinations into
A
pure black-red Malay $ and two which it does not directly enter, the
pulletswere obtained from Mr. Bran- subsequent analysis becomes very
ford. The had been used by him in
$, complex.
1902 with pure hens, and certainly A further difficulty is suggested by
then gave no rose or single, though the behavior of the natural r.p.
possibly occasional peas. Though the $ r.p. with s. 9 's gave
The Malay $ was bred with 5 s.c. the four forms in equal numbers, it is
hens of various origins and gave (Ex- practically certain that in Malay breed-
periments 188-191) — ing, if r.c. and s.c are ever seen, they
are excessive rarities; whereas if there
r.p. r. s. were gametic equality on both sides,
P-
22 21 20 20 3 in 16 should be r.c, and 1 in 16
should be s.c. For some time the con-
clusion seemed irresistible that the ap-
the equality types of
of the four
pearance of these forms on crossing
gamete being evident. The Malay hens
with s. must be due to some imperfec-
are notoriously bad layers and only
tion of fertilisation, leading to a partial
gave small totals. Each was bred with
monolepsis and this account was pro-
a s.c. cock and the result was
visionally adopted. At that time the
diflFerence between reciprocal crosses
• r-P- P- was not fully appreciated; but after a
14 13, no true r.c. or s.c.
critical study of their material it is
s.c, but none was a true s.c. Of the It is 's with wal-
possible that only $
r.p. group, 1 on hatching was regarded nut combs produce all four kinds of
as an irregular rose, but it developed gamete, r.p., r., p., s., other males giv-
into a fairly ordinary type of walnut ing only r.p., or r.p. and p., or r.p. and
comb, though having two rather prom- r.; while r.p. 9 's give either r.p. and p.,
cross were in the following year mated gether, 2 such r.p. birds will produce
with their father (Experiments 342 r.p., r., p., and s. birds in the ratio
and 343). From these matings all the 9:3:3:1. The constitution of such
4 types of comb resulted. In other birds is set forth in the accompanying
words, the gametic output of birds table (p. 13), which shows that the r.p.
produced by the union of r.p. with s. birds may be of 4 sorts, viz. ( 1 ) homo-
cannot be distinguished from that of zygous for both R. and P.; (2) homo-
birds produced by mating r. with p. zygous for R. and heterozygous for
Judged, therefore, by gametic output, P.; (3) homozygous for P. and hetero-
only 4 types of comb are known to z\'gous for R.; and (4) heterozygous
exist. for both R. and P. The relative pro-
Since the publication of the last Re- portions of birds belonging to the 4
port we have been led to alter our classes (1), (2), (3), and (4) will be
views as to the nature of the r.p. comb, 1 2
: 2 :4. As the table shows, one of
:
and have recently pointed out ^ that the 3 roses and one of the peas will be
the case may be looked upon as one of homozygous.
simple dihybridism. On this view the We have already pointed out - that
allelomorphic pairs are ( ) Rose and 1
the.proportion of the different combs
no-rose; (2) Pea and no-pea— the first in the various matings affords no cri-
member of the pair being in either terion for judging between this view
case dominant to the second. The and the one previously suggested. The
rose-comb is, therefore, in constitu- new view, however, explains the iden-
tion Rose +
no-pea {R. no P.) and tity of the combs produced by the
the pea-comb Pea -\- no-rose (P. no crossing of r. with p. and of r.p. with
RP
RP
(r.p.)
52 BATESON AND PUNNETT
to 307, 309, 311 to 313, 315, 317 to 320, 1904) was originally mated with 3
323 to 327, 329, 331 to 332, 334, 342 to single combed 9 s. to test the nature
343, 345 to 348, 359, 361 to 365, 373 to of his comb. Later on a black Andalu-
374, 382, 386.) sian 9 and 2 Wh. L. 9 s. were intro-
Of the 1,251 birds produced in the duced. Theturned out to be giving
$
above experiments 373 were r.p., 425 off all 4 kinds of gametes, giving with
pea, 413 rose, and 440 single, expecta- the 3 original 9 s. and the black An-
tion in each case being 413. There is dalusian 18 s., 26 r., 26 p., and 25 r.p.
a marked deficiency of r.p. and a dis- With the 2 White Leghorn hens, how-
tinct excess of singles. These disturb- ever, he gave an extraordinary defi-
ances in the expected ratio of equality ciency of r. and r.p., the numbers
we are inclined to attribute to the being 17 s., 2 r., 17 p., and 3 r.p.
behaviour of the 4 5 s. used in Experi- In the case of each of these 4 birds
ments 306, 347 to 349, and 373 to 374- the numbers are so aberrant that we
cases which must be considered in can for the present hardly avoid re-
rather more detail. garding them as due to some disturb-
The $ s. Experiments 306 and
in ing circumstances. The number of
349 gave only 10 r.p. birds out of a chickens involved is 309, viz., 107 s.,
total of 93, i.e., only half the number 75 r., 86 p., and 41 r.p. Subtracting
expected, although the total number these numbers from the sum totals
of chicks hatched was fairly large. given above, we are left with 333 s.,
These cases were not followed up, but 338 r., 329 p., and 332 r.p., which is
when the same phenomenon reap- a very close approach to the expected
peared in Experiment 373, the $ bird ratio of equality.
was mated also to 4 pure Br. L. 9 s.
(Experiment 374). Again, there was a Rose Pea-combs Mated Together.
marked deficiency of r.p. chicks, Zygote R. ?w P. P. no R. zygote X
pointing to something unusual hap- R. no P. P. no R. In the last Report (p.
pening among the gametes of this $ . 110) were mentioned four experiments
The bird has been kept in order that of this nature in which the figures ob-
further experiments may be made next tained were not far removed from the
year. expected ratio of 9 3 3 1. : : :
The result discloses a distinct excess singles and 8 peas. In Experiment 228
of singles and peas, together with a an extracted p.c. S was bred with 3
deficiency of r.p.s. Examination of the extracted p.c. 9 s., and gave 20 singles
table shows that whilst the result in to 34 peas, the expectation being 13*5 :
certain cases (e.g., Experiments 201, 40*5. Such an aberration from the ex-
205, 216, 245) fits closely with ex- pected ratio may easily be the result
pectation, it is in other cases far re- of chance, though it is possible that
moved from it. Thus in Experiment it may be due to the prevalence of
Table I
56 BATESON AND PUNNETT
from Blanche Burpee (hood white) X E. Henderson (erect white), giving F^ purple
erect. I. Painted Lady; II. Purple Invincible; III. Duke of Westminster.
The erect type of standard is dominant (some being whites, others P. L., ac-
to the hooded type. cording to the factorial composition of
The original cross with which we the parents), and it is from such plants
started was made 1904 between the
in and their offspring that the data put
two whites, Blanche Burpee ( long pol- together in Table II were obtained.
len,hooded) and Emily Henderson From the four Fi plants 773 plants
(round pollen, erect standard). The were raised in Fo, with the following
majority of the Fi plants were P. I. results:
427 [435J
As was to be expected, the purples and the coloured are to the whites as
are three times as numerous as the reds. 9:7. From the F^ generation families
.
1. Every hooded purple must be homo- In Experiments 48-53, six hooded purples,
zygous for the blue factor. although from families in which reds occur,
bred true to purple (except in so far as
whites might appear)
2. Every red must be homozygous for the In Experiments 54-59, six reds were bred
erect standard. from, and proved to be all homozygous for
the erect standard.
Every erect purple must be hetero-
3. The erect purples, whose offspring are
zygous for both the erect standard and for recorded Experiments 1-47, all gave
in
blueness, and must therefore give hooded hooded purples and erect reds. Of their 3707
purples, erect purples, and reds, in the ratio coloured offspring, 902 were hooded purples,
1:2:1. 939 were erect reds, and 1920 were erect
purples.
4. Every hooded white must be homo- In the only two cases in which the cross
zygous for the blue factor. between a hooded white and a red has suc-
ceeded, the coloured offspring have been
purple. Wehope to obtain further results in
the present year.
5. Since every hooded plant is homozygous So far only two round hooded purples and
for purple, and since long pollen is partially one round hooded white have been met with
coupled with the blue factor, it follows that in families heterozygous for hood and purple
round pollen should be much rarer among (Experiments 1-47). With this point we shall
the hooded than among the erect purples. deal later, in connection with partial coupling
(see p. 13).
G. H. HARDY
This short paper has more of the air of a kindly old professor
gently reprijnanding an irrepressible stude?it inclined to go off half-
cocked tha?i that of a major contribution to genetic thought. Hardy
had noted the tendency of Ji07i-mathematically inclined biologists to
make assumptions a?id draw cojiclusions based upo?i erroneous in-
terpretatiojis of the statistics i?i McfidePs work, a?id wrote this letter
HARDY 6
bases for the co?itribution of genetics to evolutionary thought.
Hardy, as a mathej/iaticiaji, did not differentiate between the individ-
ual and the genes that individual carries, so he based his calculatioris
of frequency on the nmnbers of honiozygotes and heterozygotes in
the popidation. Because of the redistribution of genes between iji-
dividuals, his first generation, which was made up entirely of ''''pure'"
individuals, differs i?j proportio?7S fro?n his second ge?ieratiojj, which
ijic hidesheterozygotes. Geneticists soo?i recogjiized that the cojj-
stancy and stability Hardy observed after his second gejieration ex-
isted equally m
the transitioji from first to seco7id, if one compares
the total number of "^" afid 'V genes in the population, rather than
the innnbers of different kinds of individuals. A direct co7isequence
of this awareness is the ^^gene-pooV^ concept, which is cojicerned
primarily with the total number of genes and their proportions in a
population, and not with the appearance of the individuals carrying
those genes. From the viewpoint that the number of genes in a gene
pool tends to remain stable and unchanging comes the concept of
evolution defined as any situatioii which tends to change the propor-
tional distribution of genes in a gene pool. Hardy perceived several
of the factors that coidd affect the 'proportional distribution, and
pointed them out in his cojicluding paragraph. He jnissed one of the
prijnary forces, however, i?i that the fruit of Darwiji's thought,
fiatural selectioji, is 07mtted.
The concept of stability of gene proportions in a populatio7i has
come to be kfiown as the ^''Hardy-Weifjberg Law,^'' as a conseqitence
of another of those dramatic coincidences that were pointed out
earlier,for Wei?jberg (Uber den Nachweis des Verebung beim
Menschen, 1908) pointed out the same facts at much the same time
as didHardy. This law still forins the core about which the field of
population genetics revolves today.
T. H. MORGAN
spermatozoa WX—
W.
Assume all of the eggs of the
that
red-eyed female carry the red-eyed
"factor" R; and that all of the eggs
(after reduction) carrv one X each,
the svmbol for the red-eved female
will be therefore RRXX and that for
her eggs will be RX— RX.
When the white-eyed male (sport)
is crossed with his red-eyed sisters, the
MORGAN 65
All of the offspring should be white, to a wild, red-eyed male, /. e., to an
and male and female in equal numbers; individual of an unrelated stock. The
this in fact is the case. anticipation was that wild males and
Second Verification. — As stated, females alike carry the factor for red
there should be two classes of females eyes, but the experiments showed that
in the Fo generation, namely, RRXX all wild males are heterozyCTous for red
and RWXX. This can be tested bv eyes, and that all the wild females are
pairing individual females with white homozygous. Thus when the white-
males. In the one instance (RRXX) all eyed female is crossed with a wild red-
the offspring should be red— eyed male, all of the female offspring
are red-eyed, and all of the male off-
RX-RX (female) spring white-eyed. The
results can be
WX-W(male) accounted for on the assumption that
RWXX - RWX the wild male is RWX. Thus:
A. H. STURTEVANT
chromosome.
The work provided the basis for the co7istruction of chromosome
maps i?i many species besides Drosophila, because the ?nethod used is
occurs (in the female only, since the an explanation will account for all the
male has only one sex-chromosome).
many phenomena that I have observed
and will explain equally, I think, the
A point not noticed at this time came other cases so far described. The results
out later in connection with other sex- are a simple mechanical result of the loca-
linked factors in Drosophila (Morgan tion of the materials in the chromosomes,
'11 d). It became evident that some of
and of the method of union of homolo-
the sex-linked factors are associated, gous chromosomes, and the proportions
i.e., that crossing over does not occur that result are not so much the expression
freely between some factors, as shown of a numerical system as of the relative
by the fact that the combinations pres- location of the factors in the chromo-
retical side of the matter with Messrs. the same point because they are com-
H. J. Aluller, E. Altenburg, C. B. pletely linked. Thousands of flies had
Bridges, and others. Mr. Muller's sug- been raised from the cross CO (red)
gestions have been especially helpful by CO (white) before it was known
during the actual preparation of the that there were two factors concerned.
paper. The discovery was finally made be-
cause of a mutation and not through
THE SIX FACTORS CONCERNED any crossing over. It is obvious, then,
In this paperI shall treat of six sex- that unless coupling strength be vari-
linked factors and their inter-relation- able, the same gametic ratio must be
ships. These factors I shall discuss in obtained whether, in connection with
the order in which they seem to be other allelomorphic pairs, one uses CO
arranged. (red) as against co (white), Co
B stands for the black factor. Flies (eosin) against co (white), or CO
recessive with respect to it (b) have (red) against Co (eosin) (the cO com-
yellow body color. The factor was bination is not known).
first described and its inheritance given
70 STURTEVANT
winged, vermilion-eyed female by without complication by the F2 males,
rudimentary winged, red-eyed male. since the male-producing sperm of the
The analysis and results are seen in Fi male bore no sex-linked genes.
Table 1. There are in this case 349 males in the
It is of course obvious from the non-cross-over classes and 109 in the
figures that there is something peculiar cross-overs. The method which has
about the rudimentary winged flies, seemed most satisfactory for express-
since they appear in far too small num- ing the relative position of factors, on
bers. This point need not detain us the theory proposed in the beginning
here, as it always comes up in connec- of this paper, is as follows. The unit
tion with rudimentary crosses, and is of 'distance' taken as a portion of the
is
point of interest at present is the link- bers for C, for O, and for C and O
age. In the F2 generation the original taken together, giving the total re-
combinations, red rudimentary and sults in the lines beginning (C, O) P,
vermilion long, are much more fre- B (C, O), etc., and have used these
quent in the males (allowing for the figures, instead of the individual C, O,
low viability of rudimentary) than or CO results, in my calculations. The
are the two new or cross-over com- fractions in the column marked 'pro-
binations, red long and vermihon rudi- portion of cross-overs' represent the
mentary. obvious from the anal-
It is number of cross-overs (numerator) to
ysis that no evidence of association can total available gametes (denominator).
be found in the females, since the M As will be explained later, one is
out the distances between the various pected being 3.0. (C, 0)R is less than
72
Table 3
STURTEVANT 73
TABLE 4
NO CROSSING OVER
—
74 STURTEVANT
Table 8 Continned.
1464
Proportion of cross-overs, adding 9 9 from BOPR (below),
Proportion or cross-overs,
D •
260
Proportion orf cross-overs, .^,
' 693
Table 8 Continued.
Proportion or cross-overs, -
Morgan ('11 d) and Morgan and Cattell ('12) as are not complicated
247
——-
V,
Proportion orc cross-overs,
836
n r • 183
Proportion of cross-overs, -rr—
J JO
r, r 218
Proportion
^ of cross-overs, ttt-
404
76 STURTEVANT
Table 8 Continued.
Fj^ long red 9 9 from above X miniature red $ $ oi other stock, gave
D •
r 109
Proportion or cross-overs, -rrr
SEWALL WRIGHT
All of the emphasis during the early history of the field of genetics
was placed upon the kijid of researches dealt with in the first papers
of this collectio?i. The authors were concerned primarily with what
was inherited and how. Their informatio?i came principally from the
final stages of the organism's development, whe?j it was fidly adidt,
and all of its perma72ent characteristics were fixed ajid recognizable.
All of this assumes a direct relationship between the gene and its
characteristic expression, but the pathways the ge?ie took in produc-
ing that expression had not been i?]vestigated, primarily because of
WRIGHT 79
the lack of adequate tools and research methods to permit such
studies.There was little questio?i that the gene played an important
part in growth and development, but as little ijiformatio?! as to how
it did it.
of color formatio?!, and ijidicates the mode of actio?j of the gene i?i
this process. This paper lays the foujidatioji for the field of biochem-
ical or physiological ge?ietics, and in fact still remains as the basic
source of informatio?i ajid the startifjg point of ?noder?2 investigatio?i.
Wright conti?Jued his thoughtfid investigations i?i this field for many
and in 1941, he wrote a paper called ""The Physiology of the
years,
Ge?2e," published i?i Physiological Reviews, vol. 21, pp. 487 -S 27.
This paper is too long to reproduce here, but I recommend it most
strongly to the reader.
One of the most m
the history of twe?itieth
sigfiifica?it events
century genetics, in my the shift in emphasis exemplified
opinion, is
by this paper. The focus of attention on the ways the gene goes
about doing what it does, rather tha?j on the ?nechanics of its tra?is-
missio7i plus its idejitification through its terminal expression, has
given ge?ietics a central positio?i iji the u?iderstanding and inter-
pretation of biological phenomena. A thorough knowledge of the
various modes of action of the gene will re-emphasize the imitary
nature of the field of biology. The more we learn about the ge?7e^s
mode of action the more obvious it becomes that the phe?iomena of
embryology, biochemistry physiology, evolutio?i, ajid systematics—
,
in fact, all major fields of biology— are for the most part only facets
of this one coinmon problem, a?id solutiofi of one aspect of gene
actio?! is likely to have repercussions in several other diverse areas.
Heredity as looked upon since the than it did half a century ago, the
time of Weismann is relatively simple problem of development has become
to understand. It consists merely in the more complex. We see clearly that de-
persistence of a certain cell constitu- velopment is no mere unfolding and
tion (in the germ cells) through an growth of elements already present in
unending succession of cell divisions, the fertilized t^^ or even a sorting out
We see something of the mechanism, of germinal rudiments for parts of the
back of this persistence, in mitosis. We organism. The germ cell has a certain
understand complications brought highly complex constitution, the adult
about by the reduction division and organism another such constitution,'
bv^ the union of two germ cells at and between the two is no simple one-,
fertilization. Thus we no longer puz- to-one relationship. An almost infi- ,
notably the melanins, with which we mals are dealt with, as in other classes
are chiefly concerned in the higher on which genetic work has been done
animals. The very fact that it has been masked
the pigment colors are largely
relatively easy to isolate unit factors by structural effects.
in work on color inheritance suggests The mosthighly pigmented condi-
that in this case the chain of processes tion found in the color black. The
is
between germ cell and adult may be pigment granules in this case are not
relatively simple. Observations which really black but a very dark sepia
indicate that melanin pigment is brown. White in mammals seems al-
formed in the cytoplasm of cells by ways to be a structural color found
the secretion of oxidizing enzymes 1 Cuenot, Arch. Zoo/. Exp. et
L., 1903.
from the nucleus suggest that the Gen. (4), vol. et Revue, p. 33.
i. Notes
may be 2 Castle, W. E., H. E. Walter, R. C. Mul-
\ chain very short indeed when
lenix and S. Cobb, 1909. Cam. Inst. Wash.
it is remembered that genetic factors
Pub!., 114.
are probably characters of the chro- Wash.
3 Little, C. C, 1913. Cam. Inst.
mosomes and that these seem to be Piibl, 179.
WRIGHT 81
in the absence of all pigment. There Finally there are intergrades of vari-
are two distinct series by which black ous sorts between the different sepia
may be reduced toward white. There and yellow series. A coarse-grained
is first the type of dilution found in mixture gives the effect of bay, dun,
brown and tow-colored human hair, or sooty yellow depending on the in-
or in dilute black guinea-pigs, which tensity^ of the colors. A finer t\'pe of
reveals the sepia color of the pigment. intergrade seems to be present in the
A very different kind of dilution of chocolate color of brown mice, rab-
black is to be seen in the so-called bits, guinea-pigs and liver-colored
blue or maltese mammals— blue mice dogs. These browns, however, are
and rabbits, maltese cats, etc. The much closer to the sepias than to the
effect is somewhat similar to that in reds and yellows and are not always
blue roans among horses and cattle distinguishable. Genetic evidence
and seems to be due to a similar cause sharply browns which
distinguishes
on a finer scale. Blue roans have an are due to reduction of black toward
intermingling of jet black hairs and white, and browns which are reduc-
white hairs while the maltese mam- tions toward yellow.
mals have dense black pigment masses By combining the different kinds of
alternating with colorless spaces dilution with the different kinds of
within the hair. intergrades between sepia and yellow
The colors which do not enter into an almost infinite variety of colors is
either of these dilution series or their produced, while the complex patterns
combinations are those which have a in which these colors may be dis-
distinct orange-yellow tinge such as is tributed make possible still further
seen conspicuously in red human hair, diversity in color effect.
red and yellow cattle, bay, chestnut Skin color in general corresponds
and dun horses, tan dogs, etc. The roughly with hair color. The pig-
most highly pigmented colors of this ments in the eyes are like those in the
kind are the so-called reds. The pig- skin and fur, but the appearance is
ment granules appear orange-yellow generally much modified by struc-
in such hair, but it does not seem to be tural effects. In dilute human eyes, for
settled whether there is an essential example, the appearance is blue, al-
chemical difference from sepia-brown though the pigment is brown.
sepia
granules or merely some structural In the rodents the reflection from the
difference. The appearance of the in- back of the retina gives a red color
tense reds varies somewhat in differ- when the pigment is reduced, which
ent mammals but there seems little in the complete absence of pigment
reason for doubting their essential becomes pink as in albinos.
similarity. Red undergoes different
modes of dilution comparable to those CHEMISTRY OF MELANIN
described for black. Reduction to yel- A amount of work in the last
large
low or cream is comparable to the twenty years has firmly established
sepia type of dilution of black, while the hypothesis that melanin is an
a more coarsely granular type of dilu- oxidation product of tyrosin or re-
tion comparable to maltese is found in lated products of protein metabolism.
light reds. These light reds are slightly Enzymes have been extracted in a
redder in hue than the yellows of sim- great manyanimals and plants, which
ilar intensity. The two series may, of have the power of oxidizing tyrosin
course, be combined. and related substances to dark brown
82 WRIGHT
pigments closely resembling the nat- of protein nature. He considered it to
ural melanins. Studies of Hooker ^ on be a diffuse coloration of the keratin
cultures in vitro of frog mesenchyme structure, while he identified the in-
pigment granules are
indicate that the soluble type with the granules. In red
formed cytoplasm immediately
in the human hair he found only the acid-
surrounding the nucleus, presumably soluble type. In brown horse hair and
under the influence of oxidizing en- black wool he found both, while in
zymes secreted by the latter. brown and black human hair, black
The nature of the differences be- rabbit hair and black feathers of do-
tween colors is still far from clear. mestic poultry and crows he found
Onslow could find no chemical dif-
^ only the insoluble type. His two types
ferences betAveen pigments extracted evidently correspond more or less
from black and yellow rabbits and closely with yellow and black pig-
considers that they differ merely ment. This difference in solubility does
quantitatively. Most others do find not necessarily indicate that yellow
chemical differences as well as phys- and black are produced from different
ical ones. Black pigment seems always chromogens. Indeed, Gortner has
to be granular, while red may be either shown that the insoluble type may be
granular or diffuse. Lloyd-Jones ® produced from the soluble by treat-
found only granules in both intense ment with strong alkali. In a brief
and dilute black pigeons, and found paper in 1912, however, Gortner ^°
red granules in red pigeons, but merely reports on a more fundamental differ-
a diffuse yellow in the dilute yellows. ence between the insoluble melanin
The Davenports found granules in "^
from black feathers, black rabbit hair
brown and black human hair, but and brown horse hair, and the soluble
merely a diffuse color in auburn hair. melanin from black wool and brown
There is a distinct difference in horse hair. The former yielded some
between the dark colors and
solubility 3% ash consisting largely of iron
yellow. Durham ^ found that
Miss oxide, while the latter yielded little or
yellow granules in yellow mice dis- no ash. This seems to demonstrate a
CYTOPLASM NUCLEUS
II. Supplementary to I.
Has no effect alone, on chro-
mogen or yellow pigment.
CHROMOGEN ENZYMES
Fig. 1.
WRIGHT 85
There are three points in tlie dia- ranee of the real physiology. In order
gram (Fig. 1) at which physiological to make wholly clear the relations sup-
processes may affect color-production posed to hold between the different
independently,viz., by influence on colors on the hypothesis, the accom-
chromogen, on enzyme I, and enzyme panying diagram (Fig. 2.) is given. Full
II. In the first two cases color should quantity represented
is by two sym-
be modified regardless of its quality. bols, reduced quantity by a single
But as there is as yet no experimental s\'mbol, reduced potency by a symbol
evidence that genetic factors deter- of small size, and complete inhibition
mine variations in the chromogen ele- or impotency by absence of symbol.
ment it will be convenient at present X^ariations in enzyme I are given hori-
to consider all such cases as due to zontally, variations of enzyme II verti-
give the chocolates and the pale for the present they are most con-
browns of the pink-eyed rodents, in veniently put with the pattern fac-
which there seems to be little more tors. They can be considered as deter-
tendency for red to win in competi- mining a fine pattern within the
tion than in intense blacks. Similar individual hairs.
relations with respect to enzyme I may
account for the differences between CLASSIFICATION OF COLOR FACTORS
the maltese and sepia types of dilution 1. Factors which affect distribution
of black and the correlated light red and intensity of color, largely irre-
and cream types of dilution of red. Of spective of the kind of color. (Act as
course, any such definite assignment of if on enzyme I.)
<33 <30 n
BLACK SEPIA
=0
MALTESE
o WHITE
i^n
<3®
BROWN LIGHT BROWN MALTESE BROWN WHITE
^ <3
RED
^CREAM LIGHT RED WHITE
Fig. 2. Production of coat colors in mammals. The inherited color factors can be
classified as if acting on one or other of two enzymes. For explanation see text.
WRIGHT 87
sity of only the dark color? with consideration of classes lb and 2b. Eye
effects visible wherever such colors color is generally determined wholly
develop in skin, fur and eyes. by these last two classes, but occa-
This classification differs slightly sionally extreme white patterns of
from one previously advanced by the class la invade the eye.
writer ^^ in that classes 2 and 3 of the
^*
earlier paper are brought more closely
DISCUSSION OF CLASSES OF FACTORS
together as classes 2a and 2b. Class 1^.— White patterns are very
As an illustration of the classes of common in mammals and most of
factors, consider a guinea-pig which them are obviously determined by fac-
is like a solid black except for the fol- tors of this class even where the mode
lowing factors: Sw Cd Cd A Abb. 2w of inheritance has not yet been thor-
represents unanalyzed hereditary fac- oughly analyzed. The factors which
tors of class la which determine a pat- determine the white face of the red
tern of white regardless of anything Hereford cattle are a good example.
else. Factor A of class 2a puts a yellow The same white face appears in the
band in each colored hair. Factor Cd black cattle from the cross of Here-
of class lb makes the yellow a dilute ford with Aberdeen-Angus. Evidently
yellow instead of red, and makes the the factors involved strike at color in
dark parts of the hair lighter than general regardless of its quality. The
otherwise and perhaps slightly dilutes same is true of the different types of
eye color. Finally factor b of class 2b white patterns found in roan and
further modifies the sepia in the coat in white Shorthorns, in the black and
and eye producing light brown but white Holsteins, and in Dutch belted
does not affect the yellow. The animal cattle. The common white patterns of
is a brown-eyed light brown agouti horses, dogs and cats are similarly in-
with yellowing ticking and a white dependent of the ground colors of the
pattern in the coat. animals. This seems to be true of the
It is often difficult for one who is belt in Hampshire hogs, but is not so
not working in the inheritance of coat certain in other white patterns in hogs
color to understand just what color is which may correspond to extreme
supposed to be determined by a given dilution of yellow patterns. One or
array of factors. Probably this can be more recessive Mendelian factors have
done most easily by considering the
factors in the order just given. First are 1^ Most of the statements in regard to
the factors of class la which determine color inheritance are based on well-known
patterns of color and white. No fac- investigations. Avery detailed review of the
literature to 1913 is given by Lang, 1914
tors considered later can change these
(Experimentelle Vererbungslebre, pp. 467-
white areas. Next to be considered is 888). A discussion of our present knowledge
the pattern of dark and yellow colors on the subject with extensive bibliog-
visible in the colored areas. These are raphy is given by Castle, 1916 (Genetics
determined by class 2a. Finally the and Eugenics). The writer may say that all
of the statements in regard to guinea-pigs
kind of yellow in the vellow areas may can be based on his own experience, and
be seen by noting the factors of class he has also had the opportunity of becoming
lb and the kind of dark color in the directly acquainted with the mode of in-
remaining areas by a simultaneous heritance of most of the color varieties
among rats, mice and rabbits as an assistant
in Professor Castle's laboratory for three
15 Wright, S., 1916. Carn. Inst. Wash. years. Only a few references are cited in
Piibl, 241, part II. this paper.
WRIGHT
been demonstrated for the patterns common in mammals. A case which
of piebald mice, hooded rats, and seems to be dominant is found in the
Dutch rabbits, while a dominant fac- factor by which dun, mouse and
tor is responsible for the English pat- cream-colored horses differ from bays,
tern of rabbits and the white blaze in blacks and chestnuts respectively. An
man. imperfectly dominant factor differen-
The mechanism by which such pat- tiates dun and yellow cattle from
terns are determined is interesting to blacks and reds.^^ The sepia and yel-
speculate upon, but very little is vet low guinea-pigs differ from black and
known. In the case of the English red ones by a unit recessive factor.^^
rabbit, Onslow has demonstrated the There are two more allelomorphs of
presence of an inhibitor which pre- this dilution factor. In red-eved dilutes
vents the oxidation of tyrosin bv tyro- vellow disappears entirelv, giving place
sinase. Apparently the power of a cell to white, and eye color becomes dis-
to produce this inhibitor is determined tinctly dilute while in the lowest re-
by a variety of conditions of which cessives of the series— the albinos-
the level of the English factor (absent, sepia as well as yellow nearly disap-
heterozygous or homozygous) is one pears and the eyes become pink.
and differences brought about in re- Rats show a series of three allelo-
gional differentiation another. As a re- morphs which seem to be comparable
sult of the combined effects of these to the guinea-pig albino series. ^^ The
conditions a given cell either has no lowest recessive determines complete
power to produce the inhibitor or can albinism while the second member of
produce sufficient to inhibit anv in- the series is much like red-eyed dilu-
tensitv of color. The extent of white tion in guinea-pigs. Black is diluted,
patterns seems to be the same in gen- vellow is reduced to white and the eve
eral whether the ground color is color to red. Rabbits show a series of
intenseor dilute. In Dutch rabbits three allelomorphs in the complete
Onslow found no enzyme inhibitor albino, Himalavan albino and fullv
but simply an absence of peroxidase. colored varieties. Albinism is found in
Here we must suppose that some es- manv other mammals and alwavs
sential condition in the cells for pro- seems to be recessive in inheritance.
duction of enzyme I is determined bv There are a number of curious fea-
the array of recessive white pattern tures in the albino series in guinea-pigs
factors in conjunction with regional which have had much to do with
differentiation. shaping the hypothesis advanced here.
The maltese type of dilution which The table below shows roughlv the
appears under the microscope in such model grades of intensity^ of vellow
a case as the blue rabbit as due to an and black fur and eve color found
alternation of colorless spaces with in- with each of the ten possible zvgotic
tense pigmentation within each hair formulae. Guinea-pigs of these ten
is put provisionallv in class la. Reces- formulae can easily^ be distinguished
sive factors which determine simul- bv the results of crosses with albinos,
taneouslv maltese dilution of black and the lowest recessives. The full evi-
the homologous kind of dilution of
vellow have been demonstrated in 17 Wilson,
J.,
1909. Sci. Proc. Roy. Dub.
Soc, 12:66.
dogs, cats, mice and rabbits.
isCasde, W. E., and S. Wright, 1916.
Class 1/7.— Correlated dilution of Cam. hist. Wash. Publ., 241.
black and yellow is probably very 19 Whiting, P., 1916. Set., n.s., 43:781.
WRIGHT
dence has been given in a recent pub-
lication.-"
90 WRIGHT
all cases agouti is dominant over the cannot be made of the agouti pattern.
absence of the pattern in blacks, The latter is greatly modified in extent
browns, Onslow was able to ex-
etc. by conditions which have differential
tract a substance which inhibits the effects on the production of black and
oxidation of tyrosin by tyrosinase, yellow. In pink-eyed guinea-pigs with
from the white belly of gray rabbits, a much reduced potency of enzyme II,
where the agouti factor removes all the agouti band is greatly widened. On
black from the hair. Onslow compared the other hand, the agouti band in
the case with the enzyme inhibitor ordinary intense guinea-pigs can be
which he found in English rabbits but greatly reduced by crossing with ex-
on our interpretation, the latter was an ceptionally intense blacks. Punnett's
inhibitor of enzyme I, the former of density factor in rabbits ^^ eliminates
enzyme II. Since only one genetic the agouti pattern altogether. It is also
factor is involved, it seems likely that more effective in the ordinary black
the same cause which modifies a solid agoutis than in brown agoutis. Thus
black rabbit in such a way that yellow the agouti factor seems to determine
ticking appears on the back is respon- a certain quantity of inhibitor which
sible for the white belly. According to is not as a rule sufficient to eliminate
the hypothesis presented here, white all black and the effect depends not
appears on the belly when black is in- merely on the level of the agouti fac-
hibited not because there is a general tor but also on the level of potency or
inhibition of pigment production but quantity of the substance to be in-
because enzyme I is below the thresh- hibited. The dominant agouti factor
old for the appearance of yellow. (A) and the recessive factor of sooty
There is a parallel case in the white yellow rabbits (e) make an interesting
bellied agouti mice. In this case, how- contrast between two factors of class
ever, it is possible to increase the gen- 2a.In the former case, as we have seen,
eral intensity of pigmentation in the Onslow demonstrated that an enzyme
animal so that yellow appears on the inhibitor was produced, in the latter
belly. In agouti guinea-pigs the belly he was simply unable to demonstrate
is normally yellow but paler than the peroxidase, indicating a reduced quan-
back. Such white patterns as those of tity or potency as compared with
the gray rabbit and white-bellied blacks. The following table shows the
agouti mouse illustrate the possibility effects produced by these factors
of confusing white patterns due really when added to those of a solid black
to yellow pattern factors of class 2a rabbit (aaEE):
acting where enzyme I is below the
yellow threshold with white patterns aaEE
of class la. It is likely that many dark-
eyed whites among mammals are of
the former kind. The polar bear is
probably an example.^^
It has been mentioned that the ex-
tent of white patterns seems to be
independent of the intensity of color
due to enzyme I. A parallel statement
WRIGHT 91
but feeble on the belly in all rabbits able to what an extent black is diluted
due to a regional differentiation. We without bringing out any distinctly
will suppose that enzyme I is below reddish tinge although in red regions
the yellow threshold on the belly. Sec- of the fur as in the agouti band of gray
ond, we will suppose that factor A varieties the red appears in full inten-
determines the production of an in- sity.There is evidently a different sort
hibitor with the same subtraction of reduction of enzyme II from that
effect on enzyme II everywhere, while in the sooty yellow rabbit. A
normal
factor e determines a general propor- quantity of enzyme II but reduced
tional reduction in rate of production potency in some other way would
of enzyme II. On this basis it follows seem to be required in class 2b.
that factor A produces a partial inhibi-
tion of black on the back, revealing
SIMULTANEOUS EFFECTS
yellow but a complete inhibition of It has been assumed so far that fac-
black on the belly where, however, tors act only on one or the other of
only white can be revealed. Factor e the hypothetical enzymes I and II. In
reduces black on the back sufficiently the great majority of cases this is satis-
to permit yellow to predominate in factory but it is not impossible that a
competition while on the belly, where factor may influence both enzymes in
there is no competition from yellow, the course of development. In fact the
what little of the black producing en- writer will soon publish evidence on
zyme I-II is produced is fully effective one such case in guinea-pigs. Tri-color
and black, or at least blue, results. male guinea-pigs of many different
Class 2b.— The factors which reduce stocks agree in showing a slightly
the intensity of black areas in skin, fur greater average area of color as op-
and eyes without affecting red areas posed to white than their sisters, and
form a clearly defined class. The they also show relatively more black
brown-eyed chocolate mice, guinea- as opposed to red. Maleness seems to
pigs and rabbits; the pink-eyed pale determine a higher level of both en-
sepia or "Hlac" mice, guinea-pigs and zymes I and II as regards pattern. The
rats, and the red-eyed rats of similar effect on color is perhaps due rather
coat color ^^ differ from blacks by fac- to a general metabolic difference in
tors of this class. Among the larger the cells of males and females early
animals the difference between liver- in ontogeny than to any specific modes
colored and black dogs seems to be of of action on the two enzymes. The
this kind. Probably chestnut and liver- same may be true of certain coat pat-
colored horses differ from bays and terns in which it seems necessary to
blacks by such a factor. All of these suppose that the level of enzymes I
factors are recessive. In the pink-eyed and II is raised or lowered simultane-
mice, guinea-pigs and rats it is remark- ously in some respect by regional dif-
ferentiation. We have cited the cases
23 These red-eyed described by
rats first of the rabbit, mouse, and guinea-pig
Castle (1914, A?ner. Nat.) under the name in which it was found convenient to
of yellows must not be confused with the
red-eyed rats described by Whiting (loc.
suppose that both enzymes I and II are
cit.). In the former yellow pigment is un- strong on the back and weak on the
affected and to a large extent gives the color belly. Similar cases are common. An-
to the fur in agoutis, owing to the great other sort of example seems to be
reduction of black. In the latter, yellow is
present in the tiger pattern of cats. In
reduced to white, while black is only slightly
affected. yellow cats the pattern is shown by
92 WRIGHT
alternate orange and cream stripes and classification.
which on our interpretation must indi- Finally the bringing under one point
cate alternate stripes of high and low of view of biochemical and genetic
potency of enzyme I. In tabby cats, the facts would have great intrinsic inter-
intense stripes are solid black, the pale est. The present paper attempts merely
stripes show yellow ticking which to trace the character— coat color-
seems to require that enzyme II also back one stage in development. Instead
be strong in the former, weak in the of considering factors as acting on this
latter. one character, they have been divided
into two sets acting on two characters,
CONCLUSION production of the hypothetical en-
In the present paper an attempt has zymes I and II. Suggestions have been
been made to relate the findings of the made in certain cases in regard to fur-
biochemist in regard to melanin pig- ther tracing back of the action of the
ment with the great mass of curious factors. A more thorough comparison
relations between colors which have than has yet been made of the effects
come to light in genetic work. A of factors in all combinations should
scheme is given which designed to
is yield much data bearing on the process
show the inter-relations of the differ- of pigmentation and give a very much
ent mammalian coat colors and a clas- more complete understanding of the
sification of color factors is suggested. heredity of color than we have at
It is hoped that these will be of use in present. By constant comparison of
organizing our present very extensive the deductions from such work with
knowledge of color inheritance and in the findings of the biochemist, it
aiding in the discovery of new facts, should be possible in the end to estab-
or at least in leading to a better scheme lish a very pretty correlation of results.
L. C. DUNN
By the time this paper by Dimn was written, it had become ap-
parent that many of the visible variations that occurred in laboratory
animals could be accoimted for by assuming the existence of minute
These germi?ial variations,
variatiojis in their hereditary materials.
called mutations,became an important part of the Darwinian theory
of evolution, after a period during which they were thought to
DUNN 93
demojistrate it to he invalid (see Johajuisev, p. 20). The application
Species'' (1941, p. 60) says, "-Dmin was the first to apply the
. . .
the same phenomena while studying ably constant and individual organiza-
inheritance of certain characters which tions of chromatin known as chromo-
had arisen by mutation in the vinegar somes, which appear at the time of cell
fly (Drosophila melmiogaster). He division and which probably retain
conceived these two exceptions to their individualityeven in the resting
Mendel's principle of independent as- stages of the nucleus. Of the evidence
sortment as two aspects of a single it must be observed that the interpreta-
phenomenon which he termed Liyikage tion and proof of the theory rest en-
or associated inheritance. tirely on the study of linkage, or asso-
The interpretation of these events ciated inheritance. This phenomenon
has constituted one of the great ad- is observed in the tendency which
vances of biological science. Sutton, in characters exhibit of remaining
1902, suggested that the marked paral- through several generations in their
lelism between the discreteness and as- original combinations, resulting in an
sortment of unit characters and the be- alteration of the expected Mendelian
havior of the chromosomes might be ratios basedon independent assort-
due to the residence in the chromo- ment. This tendency may be absolute,
somes of the determinants or genes in which case linkage is said to be com-
representing unit characters. Immedi- plete. More often it is partial, that is,
the evidence on unit variation in color always of a lighter shade than the
in the Rodentia is to describe the ap- dorsum, due to a lesser concentration
pearance and genetic behavior of each of black pigment and a wider area of
of the principal variations with a short pale dusky yellow in the hairs. The
list of the species in which it has been "agouti" coat is seen in a typical form
studied, and of the species in which a in the familiar wild house mouse (Aiiis
variation of similar appearance has musnihis), the common rat of this
been reported.- Where the inheritance country {Rattiis jwrvegims), etc. It
of a variation has not been determined characterizes the wild type forms of
all the species included in the follow-
- This proceeding may be expected to lead ing list.
to some errors since similarity of appearance
is not always evidence of similarity in germi- ALBINO
nal constitution, but in the absence of breed-
ing data we must use the only criterion From wild type distinct graded
this
available. losses of pigment have taken place,
— — —— — —
DUNN 97
the extreme of which is complete al- Ratrus norvegicus —
binism, or entire absence of pigment, Common rat.
leaving the fur clear white and the *Microtus pennsylvanicus
eyes pink. The pinkness of the eye is
— Meadow vole.
*Fiber zibethicus
due to the absence of pigment in the
Muskrat.
iris, which is typically colored by Peromyscus leucopus
black or brown pigment granules, so noveboracensis Deer —
that the blood in the capillaries on mouse.
the retina is directly visible. This Hystricidce- *Erethizon dorsatum
variation is to be sharply distinguished Canada porcupine.
from "partial albinism," a term which Caviidce — Cavia cobaya — Guinea-
has been applied, unwisely it now ap- Pig-
pears, to the occurrence of white
spotting in animals whose eyes retain
their full color.The color of the eyes The inheritance of the albinism has
isan important point of distinction be- been studied in the rabbit, the house
tween complete albinos and spotted mouse, the house rat, the deer mouse
animals. Cases of true albinism have and the guinea-pig. In all of these it
been reported in nearly ali he families is due to a gene which acts as a Men-
of rodents. Data from only h " of the delian recessive to full color. At the
commonest families are given here, same (albino) locus in the germ plasm
the families being listed roughly in have occurred other mutations. In the
the order of their relationship from the rat, a change in this locus has produced
more primitive to the more special- both albino and its dominant allelo-
ized.^ morphs ruby-eyed dilute, in which
the reduction of the melanic pigments
Leporid<£— Oryctolagus cuniculus isvisible in the generally lighter tone
European "rabbit."
of black, coupled with a complete ab-
Scmridce — *Marmota monax
sence of yellow. In the guinea-pig
Woodchuck.
three graded variations have occurred:
*Sciurus hudsonicus
( 1 ) dilution, resulting in a reduction
Northern red squirrel.^
*Sciurus carolinensis leu- of all pigments; (2) ruby, resulting in
cotis —American gray the absence of yellow, and the further
squirrel. reduction of black and brown in fur
*Tamias striatus lysteri and eyes to very light shades (prob-
Chipmunk. ably homologous with the ruby varia-
Muridce — Mus musculus House — tion in rats); and (3) Himalayan al-
mouse. binism, which determines the absence
^ I have followed the older order of clas-
of yellow and the restriction of black
sification which includes the Leporids in the and brown to the extremities, ears,
Rodentia. nose, feet, and rump, while the eyes
* Through the kindness of Professor Bar- are pink. These three conditions are
rows of the Michigan Agricultural College
distinct in appearance, do not blend
and Prof. W. E. Castle of Harvard Univer-
sity the writer has learned of the capture of a in crosses and are all alternative allelo-
pair of albino red squirrels by A.
E. Secord, morphs with fullcolor and with each
of Wheeler, Michigan. Breeding experiments other. No complete albinism is known
to test the inheritance of this variation were
in the guinea-pig. In the rabbit two
to have been attempted but expense and
pressure of other work have prevented the changes have taken place: Himalayan
writer from undertaking the project. albinism (probably homologous with
98 DUNN
the Himnlayan albinism of guinea- both fur and eyes. A certain amount
pigs)ai:J albinism. These are allelo- of pigment is present in the iris but
morphic with full color and with each not enough to obscure the blood color
other; that is, crosses of full colored of the retina. Yellow pigment is not
animals with albinos produce only full affected. Pink-eyed animals with the
colored young and in the second gen- "agouti" coat pattern therefore appear
eration only colored and albinos. The yellow since the black bases of the
same is true of the cross colored X dorsal hairs are a reduced slaty or
Himalayan, while the cross Himalayan bluish tint and are covered by the fully
X produces only Himalayan
albino intense yellow parts of the hair. Black
and in second generation only
the animals with this variation are slaty or
Himalayan and albino. The occur- bluish all over in mice and a dirty
rence of this variation in several near-white in rats and guinea-pigs. Its
species, its similarities in appearance distinctness from albinism becomes
and in inheritance, and finally the pro- evident when pink-eyed colored ani-
duction at the same locus as indicated mals are crossed with albinos. The first
by allelomorphism of other similarly generation offspring in this case are
appearing variations indicate that the allas fully colored as the wild type
other piebald mice and in guinea-pigs in that it is less pigmented than any
especially it appears purely at ran- other type of spotting studied, some
dom, in irregular blotches hardly ap- black-eyed white-spotted mice having
proximating any pattern at all. The pigment only in the eyes, while the
spotting may also vary in amount from rest of the pelt is pure white. It is
a few white hairs to over half the sur- discontinuous with piebald spotting,
face of the animal, although in general and is, like yellow, an unfixable hy-
the pigmented areas exceed the white brid, always throwing, when bred
portions in total size. The belly is like- pure, a ratio of two black-eyed whites
wise more susceptible to spotting than to one piebald. The cause of this pecu-
the dorsum. This variation has been liarity has lately been traced to its
noted many times in wild species and association with another lethal factor
I am certain the present list which has which determines the death in utero
been hastily compiled does not repre- of all pure black-eyed white zy-
sent the true distribution of this varia- gotes.
tion among rodents in general.
BLACK
Leporidie— Oryctolagus cuniculus Oneother color variation is com-
Sciuridte— *Sciurus finlaysoni
mon enough in rodents to make com-
Muridee— Mus musculus
parison profitable. This is the discon-
Rattus norvegicus
tinuous change from the "agouti" coat
*Evotomys gapperi
Caviidie — Cavia cobaya to one which is black all over and it is
probably due in all the species in
In the rabbit, rat, mouse, and which occurs to a gene determining
it
probably not located in the same chro- the change producing non-agouti, or
mosome with albinism and pink-eye, the absence of the "agouti" pattern.
nor with the black-eyed white-spot- Its recessive allelomorph is brown,
ting about to be discussed. In rats and which has occurred in rabbits, mice,
guinea-pigs it is likewise not linked as guinea-pigs and possibly in rats. This
far as is known with any other color gene is probably not linked with any
variation, while in rabbits it may be a of the other known genes in mice, but
property of the same locus at which its relationships in other species have
English or dominant spotting is de- not been studied. The variation from
termined. agouti to black occurs in the follow-
The two other categories of spot- ing species:
ting are peculiar each to a single spe-
cies.The English broken spotting of Leporidce — Oryctolagus cuniculus
domesticated rabbits is a Mendelian *Lepus americanus
dominant to self-color and has no virginianus —
Eastern
varying hare
probable homologue in other species,
while the black-eyed white-spotting
Sciuridce — *Sciurus hudsonicus
*Sciurus niger ludovicianus
of mice, likewise a dominant, is ap- *Sciurus niger niger
parently peculiar to mice although *Sciurus carolinensis leucotis
wild rodents resembling this type have *Tamias striatus lysteri
102 DUNN
Muridte — Mus musculus tion, producing when acting on black
Rattus norvegicus varieties the familiar maltese color of
*Fiber zibethicus blue rabbits and mice. The maltese cat
Caviidce — Cavia cobaya
is the result of a similar variation from
causes are to be sought, and, once pigs are not the same as those in mice
having arisen by a mysterious occur- and rats. It may mean that the chro-
rence called mutation, we have learned mosome which contains both genes in
something of the manner in which the the more primitive Muridae may in
variations are inherited, and by a the more specialized Caviidae be repre-
process of inference have been able to sented by two chromosomes, the sum
localize still more exactly the region of which rather than either one sepa-
of change. The only permissible gen- rately may be homologous with the
erality, then, concerns a general sim- one chromosome of mice and rats. Al-
ilarity in the germ plasm and probably though this will be recognized as
in the individual chromosomes of speculation, there is some slight evi-
these many species of rodents. But in dence that in the evolution of the
one case the similarity between species rodents a fractionation of chromo-
has been found to be more than gen- somes may have occurred, for the
eral. It has been found to be quite a mice and rats have 19 (haploid) while
specific similarit\". the guinea-pigs have 28. In the rabbit
If we examine this case in detail we (Oryctolagus cimiculiis) a member
find that in two distinct, inter-sterile now judged too primitive for the true
species, mice and rats, two similarly rodents and recently placed in the
appearing variations have occurred, al- Lagomorpha with the others of the
binism and pink-eye. In rats the genes old rodent suborder Duplicidentata,
for these variations are linked with a the chromosome number is probably
strength of about 21 per cent, which 12. If this progressive increase in the
is possibly slightly in excess of the number of chromosomes through the
actual. In mice the linkage between order Rodentia is found to be a fact
these genes is something less than 15 and not a chance phenomenon asso-
per cent, which is based on observa- ciated with the smallness of the sample
tions of 6700 animals raised solely for of four species from which our cyto-
the purpose of determining this link- logical evidence is drawn, it may fur-
age and is probably reasonably accu- nish a very important clue to a series
rate. In terms of the chromosome of evolutionary relationships of more
hypothesis these facts mean that these than ordinary interest.
two genes are present in the same In the concluding chapter of his
chromosome in rats and mice, in rats recent book Professor Morgan (1919)
at a distance of 21 units apart, in mice has referred to the possible evolution-
at a distance of about 15 units apart. ary significance of the localization of
The difference in location is so small genes as determined by the study of
that for practical purposes we can say linkage. He has there reviewed some
that they are located at homologous of the work on similar variations in
points in the two species. several species of insects by Metz and
In guinea-pigs where both of these Sturtevant, pointing out the difficulties
variations occur, there is incomplete to be encountered in applying this
evidence, but the data which Doctor method to the analysis of species, chief
Wright has supplied indicate that of which is the necessity of establish-
pink-eye and albinism in guinea-pigs ing the same linear order in each spe-
are probably not linked and may there- cies of the genes for similar variations.
fore be determined in different chro- A species in his point of view, and in
mosomes. This does not prove, how- this he follows De Vries, may ulti-
ever, that these variations in guinea- mately prove to be a "community of
104 MULLER
genes." We may expect evidence of of descent in the terms of current
this community in the variations evolutionary theory or to relationship
which arise from time to time within through some other cause is one of
the species, whether they be at the the questions which genetics, aided by
time of specific value or not. Such the chromosome notation, may be ex-
community is not to be inferred from pected at some time to answer.
mere similarity in appearance but must
BIBLIOGRAPHY
rest on a more real homology of
germinal cause. This kind of similarity
Castle, W. E. 1920 Genetics and Eugenics.
Harvard University Press, Cambridge,
is now apparent between Miis rmiscu-
Mass.
lus and Rattus ?iorvegicJis, which have Morgan, T. H., Sturtevant, A. H., Muller,
varied so far from a common type that H. J., and Bridges, C. B. 1915 The Mecha-
thev are now inter-sterile and have nisjn of Mendelian Heredity. Henry Holt
and Co., New York.
been placed recently in different gen-
era. Yet they have retained a genetic
Morgan, T. H. 1919 The Physical Basis of
Heredity. J. B. Lippincott Co., Philadel-
constitution so similar that it contains phia and London.
genes common to both species. Wright, S. 1917 "Color Inheritance in Mam-
Whether this is due to a community mals." Journal of Heredity, vol. 8, nos. 5-9.
H. J. MULLER
Tny general biology course found this article the easiest of the entire
collection to read and digest. In Tny opinion, it is the paper that
taught them the most.
We have noted two differ eyit types of papers already; first, the
research paper, and secotid, the paper e?nbodying syjithesis of several
areas of study. Muller' s co?itributio7i is a third type. It is a thoughtful
ajialysis of the progress that had been made in the understanding of
MULLER 105
a specific pheno?ne?ion, a summary of our knowledge concenmig it,
and then indicatioji of the pathways that 7night prove fruitful for
future research. This type of contribution is know7i as a '''review
and, whe'n properly done, serves as a primary tool used by
paper,''''
and they play a fundamental role in acid, or any related substance, and it
determining the nature of all cell sub- would be similarly absurd, therefore,
stances, cell structures, and cell activ- to regard cases of the former kind as
ities. Through these cell effects, in giving any evidence that the gene is
turn, the genes afi^ect the entire organ- an enzyme, or an agglutinin-like body.
ism. The reactions whereby the genes pro-
It is not mere guesswork to say that duce their ultimate effects are too
the genes are ultra-microscopic bodies. complex for such inferences. Each of
For the work on Drosophila has not these effects, which we call a "char-
only proved that the genes are in the acter" of the organism, is the product
chromosomes, in definite positions, but of a highly complex, intricate, and
it has shown that there must be hun- delicately balanced system of reac-
dreds of such genes within each of the tions, caused by the interaction of
106 MULLER
countless genes, and every organic gene becomes changed, through some
structure and activity is therefore "chance variation," the catalytic prop-
liable to become
increased, diminished, erty of the gene may ^ become corre-
abolished, or altered in some other spondingly changed, in such a way as
way, when the balance of the reaction to leave it still autocatalytic. In other
system is disturbed by an alteration in words, the change in gene structure-
the nature or the relative quantities of accidental though it was— has some-
any of the component genes of the how resulted in a change of exactly
system. To return now to these genes appropriate nature in the catalytic re-
themselves. actions, so that the new reactions are
now accurately adapted to produce
II. THE PROBLEM OF more material just like that in the new
GENE MUTABILITY changed gene itself. It is this paradoxi-
The most distinctive characteristic cal phenomenon which is implied in
of each of these ultra-microscopic par- the expression "variation due to
ticles—that characteristic whereby we change in the individual gene," or, as
identify it as a gene— is its property it is often called, "mutation."
of self-propagation: the fact that, What sort of structure must the
within the complicated environment gene possess to permit it to mutate
of the cell protoplasm, it reacts in such in this way? Since, through change
a way as to convert some of the com- after change in the gene, this same
mon surrounding material into an end- phenomenon persists, it is evident that
product identical in kind with the it must depend upon some general fea-
original gene itself. This action fulfills ture of gene construction— common to
the chemist's definition of "autocatal- all genes— which gives each one a gen-
ysis"; it is what the physiologist would eral autocatalytic power— a "carte
call "growth"; and when it passes blanche"— to build material of what-
through more than one generation it ever specific sort it itself happens to
becomes "heredity." It may be ob- be composed of. This general principle
served that this reaction is in each of gene structure might, on the one
instance a rather highly localized one, hand, mean nothing more than the
since the new material is laid down by possession b\^ each gene of some very
the side of the original gene. simple character, such as a particular
The fact that the genes have this radicle or "side-chain"— alike in them
autocatalytic power is in itself suffi- all— which enables each gene to enter
ciently striking, for they are un- into combination with certain highly
doubtedly complex substances, and it organized materials in the outer proto-
is difficult to understand by what plasm, in such a way as to result in
strange coincidence of chemistry a the formation, "by" the protoplasm,
gene can happen to have just that very of material like this gene which
more
special series of physico-chemical is combination with it. In that case
in
effects upon its surroundings which the gene itself would only initiate and
produces— of all possible end-products guide the direction of the reaction. On
—just this which is
particular one, the other hand, the extreme alternative
identical with its own complex struc- to such a conception has been gen-
ture. But the most remarkable feature
1 It is of course conceivable, and even
of the situation is not this oft-noted
unavoidable, that so7?ie tj'pes of changes do
autocatalytic action in itself— it is the destroy the gene's autocatalytic power, and
fact that, when the structure of the thus result in its eventual loss.
MULLER 107
erally assumed, perhaps gratuitously, this matter and other matter, which
in nearly all previous theories con- would keep growing wider, with the
cerning hereditar\' units; this postu- increasing complexity, diversit\^ and
lates that the chief feature of the auto- so-called "adaptation" of the selected
catalytic mechanism resides in the mutable material.
structure of the genes themselves, and
that the outer protoplasm does little
III. A POSSIBLE ATTACK THROUGH
more than provide the building ma- CHROMOSOME BF:HAVI0R
ordinary forces of so-called cohesion, between portions of the gene and sim-
adhesion and adsorption known to ilar protoplasmic building blocks (de-
physical science. In this sense, then, pendent on their force-field patterns),
the physicist has no parallel for this evident that the very same forces
it is
force. There seems, however, to be no which cause the genes to grow should
way of escaping the conclusion that in also cause like genes to attract each
the must be of the same
last analysis it other, but much more strongly, since
nature as these other forces which here all the individual attractive forces
cause inorganic substances to have of the different parts of the gene are
specific attractions for each other, ac- summated. If the two phenomena are
cording to their chemical composition. thus really dependent on a common
These inorganic forces, according to principle in the make-up of the gene,
the newer depend upon the
physics,
2 It can hardly be true, as Troland in-
arrangement and mode of motion of similar attract each
timates, that all fields
the electrons constituting the mole- other more than they do dissimilar fields,
cules, which set up electro-magnetic otherwise all substances would be autocata-
fields of force of specific patterns. To lytic, and, in fact, no substances would be
soluble. Moreover, if the parts of a molecule
find the principle peculiar to the con-
are in anv kind of "solid," three dimen-
struction of the force-field pattern of sional formation, it would seem that those in
genes would accordingly be requisite the middle would scarcely have opportunity
for solving the problem of their tre- to exert the moulding effect above men-
tioned. It therefore appears that a special
mendous auto-attraction.
manner of construction must be necessary,
Now, according to Troland (1917), in order that a complicated structure like a
the growth of cr)stals from a solution gene may exert such an effect.
MULLER 109
progress made in the study of one of it is one of our few conceivable modes
them should help in the solution of of approach to an all-important prob-
the other. lem.
Great opportunities are now open It may also be recalled in this con-
for the study of the nature of the nection that besides the genes in the
synaptic attraction, especially through chromosomes there is at least one sim-
the discovery of various races having ilarly autocatalytic material in the
abnormal numbers of chromosomes. chloroplastids, which likewise may be-
Here we have already the finding by come permanently changed, or else
Belling,that where three like chro- lost, as has been shown by various
mosomes are present, the close union studies on chlorophyll inheritance.
of any two tends to exclude their close Whether this plastid substance is sim-
union with the third. This is very ilar to the genes in the chromosomes
suggestive, because the same thing is we can not say, but of course it can
found in the cases of specific attrac- not be seen to show synaptic attrac-
tionsbetween inorganic particles, that tion, and could not be studied by the
are due to their force field patterns. method suggested above.^
And through Bridges' finding of tri-
ploid Drosophila, the attraction phe- IV. THE ATTACK THROUGH STUDIES OF
nomena can now be brought down to MUTATION
by the intro-
a definitely genie basis, There is, however, another method
duction of specific genes— especially of attack, in a sense more direct, and
those known to influence chromosome not open to the above criticisms. That
behavior— into one of the chromo- is the method of investigating the in-
somes of a triad. The amount of influ- dividual gene, and the structure that
ence of this gene on attraction may permits it to change, through a study
then be tested quantitatively, by gen- of the changes themselves that occur
etic determination of the frequencies in it, as observed by the test of breed-
of the various possible types of segre- ing and development. It was through
gation. By extending such studies to the investigation of the chajiges in the
include the effect of various condi- chromosomes— caused by crossing
tions of the environment— such as over— that the structure of the chro-
temperature, electrostatic stresses, etc. mosomes was analyzed into their con-
—in the presence of the different ge- stituent genes in line formation; it was
netic situations, a considerable field is through study of molecular changes
opened up. that molecules were analyzed into
This suggested connection between atoms tied together in definite ways,
chromosome behavior and gene struc- and it has been finally the rather re-
ture is as yet, however, only a pos- cent finding of changes in atoms and
sibility. It must not be forgotten that investigation of the resulting pieces,
at present we can not be sure that the that has led us to the present analysis
synaptic attraction is exerted by the of atomic structure into positive and
genes themselves rather than by local negative electrons having characteris-
products of them, and it is also prob- tic arrangements. Similarly, to under-
whether the chief part of the
lematical
mechanism of autocatalysis resides 3 It may
be that there are still other ele-
within the genes rather than in the ments which have the nature of
in the cell
genes, but as no critical evidence has ever
"protoplasm." Meanwhile, the method been adduced for their existence, it would
is worth following up, simply because be highly hazardous to postulate them.
no MULLER
stand the properties and possibilities of a loss or gain of part of the gene,
of the individual gene, we must study our problem of why the changed gene
the mutations as directly as possible, still seems to be autocatalytic would
and bring the results to bear upon our in the main disappear, but such a situa-
problem. tion is excluded a priori since in that
case the thousands of genes now exist-
(a) The Quality and Quantity ing could never have evolved.
of the Change Although a given gene may thus
In spite of the fact that the drawing change in various ways, it is important
of inferences concerning the gene is to note that there is a strong tendency
very much hindered, in this method, for any given gene to have its changes
on account of the remoteness of the of a particular kind, and to mutate in
gene-cause from its character-effect, one direction rather than in another.
one salient point stands out already. It And although mutation certainly does
is that the change is not always a mere not always consist of loss, it often
loss of material, because clear-cut re- gives effects that might be termed
verse mutations have been obtained in losses. In the case of the mutant genes
corn, Drosophila, Portulaca^ and prob- for bent and eyeless in the fourth
ably elsewhere. If the original muta- chromosome of Drosophila it has even
tion was a loss, the reverse must be a been proved, by Bridges, that the
gain. Secondly, the mutations in many effects are of exactly the same kind,
cases seem not to be quantitative at all, although of lesser intensity, as those
since the different allelomorphs produced by the entire loss of the
formed by mutations of one original chromosome in which they lie, for
gene often fail to form a single linear flies having bent or eyeless in one
series. One case, in fact, is known in chromosome and lacking the homol-
which the allelomorphs even affect ogous chromosome are even more
totally different characters: this is the bent, or more eyeless, than those hav-
case of the truncate series, in which I ing a homologous chromosome that
have found that different mutant genes also contains the gene in question. The
at the same locus may cause either a fact that mutations are usually reces-
shortening of the wing, an eruption sive might be taken as pointing in the
on the thorax, a lethal effect, or any same direction, since it has been found
combination of two or three of these in several cases that the loss of genes—
characters. In such a case we may be as evidenced by the absence of an
dealing either with changes of differ- entire chromosome of one pair— tends
ent types occurring in the same ma- to be much more nearly recessive than
terial or with changes (possibly quan- dominant in its effect.
titative changes, similar in type) oc- The effect of mutations in causing
curring in different component parts a loss in the characters of the organism
of one gene. Owing to the universal should, however, be sharply distin-
applicability of the latter interpreta- guished from the question of whether
tion, even where allelomorphs do not the gene has undergone any loss. It is
form a linear series, it can not be cate- generally true that mutations are much
gorically denied,in any individual more apt to cause an apparent loss in
case, that the changes may be merely character than a gain, but the obvious
quantitative changes of some part of explanation for that is, not because the
the gene. If all changes were thus gene tends to lose something, but be-
quantitative, even in this limited sense cause most characters require for
MULLER 111
proper development a nicely adjusted are usually more genes slightly affect-
train of processes, and so any change ing a given character than those play-
in the genes— no matter whether loss, ing an essential role in its formation.
gain, substitution or rearrangement— is The evidence proves that there are
more likely to throw the develop- still more genes whose change does
mental mechanism out of gear, and not affect the given character at all—
give a "weaker" result, than to inten- no matter what this character may be,
sify it. For this reason, too, the most unless it is life itself— and this raises the
frequent kind of mutation of all is the question as to how many mutations
lethal, which leads to the loss of the are absolutely unnoticed, affecting no
entire organism, but we do not con- character, or no detectable character,
clude from this that all the genes had to any appreciable extent at all. Cer-
been lost at the time of the mutation. tainly there must be many such muta-
The explanation for this tendency for tions, judging by the frequency with
most changes to be degenerative, and which "modifying factors" arise,
also for the fact that certain other which produce an effect only in the
kinds of changes— like that from red to presence of a special genetic complex
pink eye in Drosophila—2LTe more fre- not ordinarily present.
quent than others— such as red to
brown or green eye— lies rather in de- (b) The Localizatioji of the
frequency with which single mutant of the cells in a bloc, that are de-
lethals were found in the same experi- scended from the mutant cell.
ments. Ordinarilv% then, the event that
causes the mutation is specific, affect-
(c) The Co?iditions mider which
ing just one particular kind of gene the Change occurs
of all the thousands present in the cell. But the method that appears to have
That due to a spatial
this specificity is most scope and promise is the experi-
limitation rather than a chemical one mental one of investigating the condi-
is shown by the fact that when the tions under which mutations occur.
single gene changes the other one, of This requires studies of mutation fre-
identical composition, located near by quency under various methods of
in the homologous chromosome of the handling the organisms. As yet, ex-
same cell, remains unaffected. This has tremely little has been done along this
been proved by Emerson in corn, by line. That is because, in the past, a
Blakeslee in Portulaca, and I have mutation was considered a windfall,
shown there is strong evidence for it and the expression "mutation fre-
in Drosophila. Hence these mutations quency" would have seemed a con-
are not caused by some general perva- tradiction in terms. To attempt to
sive influence, but are due to "acci- study it would have seemed as absurd
dents" occurring on a molecular scale. as to study the conditions affecting
When the molecular or atomic mo- the distribution of dollar bills on the
tions chance to take a particular form, You were simply fortunate if
sidewalk.
to which the gene is vulnerable, then you found one. Not even controls,
the mutation occurs. giving the "normal" rate of mutation—
It will even be possible to determine ifindeed there is such a thing— were
whether the entire gene changes at attempted.''^ Of late, however, we may
once, or whether the gene consists of say that certain verv^ exceptional bank-
several molecules or particles, one of ing houses have been found, in front
which may change at a time. This of which the dollars fall more fre-
point can be settled in organisms hav- quently—in other words, specially mu-
ing determinate cleavage, by studies table genes have been discovered, that
of the distribution of the mutant char- are beginning to yield abundant data
acter in somatically mosaic mutants. at the hands of Nilsson-Ehle, Zeleny,
If there is a group of particles in the Emerson, Anderson and others. For
gene, then when one particle changes some of these mutable genes the rate
it will be distributed irregularly of change is found to be so rapid that
among the descendant cells, owing to at the end of a few decades half of
the random orientation of the two
halves of the chromosome on the tend not to persist as such, for the occur-
mitotic spindles of succeeding divi- rence of mutation in one particle after the
sions,* but if there is only one particle other would in time differentiate the gene
into a number of different genes consisting
of one particle each.
This depends on the assumption that if
4 5 Studies of "mutation frequency" had of
the gene does consist of several particles, the course been made in the CEnotheras, but as
halves of the chromosomes, at each division, we now know that these were not studies of
receive a random sample of these particles. the rate of gene change but of the fre-
That is almost a necessary assumption, since quencies of crossing over and of chromo-
a gene formed of particles each one of which some aberrations they may be neglected for
was separately partitioned at division would our present purposes.
MULLER 113
the genes descended from those origi- an influence upon mutation in this
nally present would have become same gene, caused by developmental
changed. After these genes have once conditions— the mutations from white
mutated, however, their previous mu- to red of the mutable gene studied
tability no longer holds. In addition occurring far more frequently in the
to this "banking house method" there cells of the more mature ear than in
are also methods, emplo\'ed by Alten- those of the younger ear. These two
burg and myself, for— as it were— auto- results at least tell us decisively that
matically sweeping up wide areas of mutation is not a sacred, inviolable,
the streets and sifting the collections unapproachable process: it may be
for the valuables. By these special gen- altered. These are the first steps; the
etic methods of reaping mutations way now lies open broad for explora-
mutable genes above— would usually account here the reported findings by
require at least a thousand years— several investigators, of genetic vari-
probably very much more— before half ations caused by treatments with vari-
of them became changed. This puts ous toxic substances and with certain
their stability about on a par with, if other unusual conditions. In most of
not much higher than, that of atoms of these cases, however, the claim has
radium— to use a fairly familiar anal- not been made that actual gene
ogy. Since, even in these latter experi- changes have been caused: the results
ments, many of the mutations prob- have usually not been analyzed gen-
ably occurred within a relatively few etically and were in fact not analyza-
rather highly mutable genes, it is ble genetically; they could just as well
likely that most of the genes have a be interpreted to be due to abnor-
stability far higher than this result malities in the distribution of genes—
suggests. for instance, chromosome abnormal-
The above mutation rates are mere itieslike those which Mavor has re-
first gleanings— we have yet to find cently produced with X-rays— as to be
how difi'erent conditions affect the due to actual gene mutations. But even
occurrence of mutations. There had if they were due to real genie differ-
so far been only the negative findings ences, the possibility has in most cases
that mutation is not confined to one by no means been excluded (1) that
sex (Muller and Altenburg, 1919; these genie differences were present in
Zeleny, 1921), or to any one stage in the stock to begin with, and merely
the liJFe cycle (Bridges, 1919; Muller, became sorted out unequally, through
1920; Zeleny, 1921), Zeleny's finding random segregation; or (2) that other,
that bar-mutation not influenced by
is invisible genie differences were pres-
recency of origin of the gene (1921), ent which, after random sorting out,
and the as yet inconclusive differences themselves caused differences in muta-
found by Altenburg and myself for tion rate between the different lines.
mutation rate at different tempera- Certain recent results by Altenburg
tures (1919), until at this year's meet- and myself suggest that genie differ-
ing of the botanists Emerson an- ences, affecting mutation rate, may be
nounced the definite discovery of the not uncommon. To guard against
influence of a genetic factor in corn either of these possibilities it would
upon the mutation rate in its allelo- have been necessary to test the stocks
morph, and Anderson the finding of out by a thorough course of inbreed-
114 MULLER
ing beforehand, or else to have run at lowed, and those in which many
least half a dozen different pairs of homozygous or else genetically con-
parallel lines of the control and treated trolled lines can be run in parallel,
series, and to have obtained a definite either by parthenogenesis, self-fertil-
difference in the same direction be- ization, balanced lethals or other spe-
tween the two lines of each pair; cial genetic means, and later analyzed,
otherwise it can be proved by the through sexual reproduction, segrega-
theory of "probable error" that the tion and crossing over.
differences observed may have been a
mere matter of random sampling V. OTHER POSSIBILITIES
of bacteria, became multiplied or in- known between the genes and them.
creased, and could be so increased Hence we can not categorically deny
indefinitely; it was self-propagable. It that perhaps we may be able to grind
fulfills, then, the definition of an genes in a mortar and cook them in a
autocatalytic substance, and although beaker after all. Must we geneticists
it may really be of very different become bacteriologists, phv^siological
composition and work by a totally chemists and physicists, simultaneously
different mechanism from the genes with being zoologists and botanists?
in the chromosomes, it also fulfills Let us hope so.
our definition of a gene.'^ But the
resemblance goes further— it has been I have purposely tried to paint
found by Gratia that the substance things in the rosiest possible colors.
may, through appropriate treatments Actually, the work on the individual
on other bacteria, become changed gene, and its mutation, is beset with
(so as to produce a somewhat differ- tremendous difliculty. Such progress
ent effect than before, and attack dif- in it as has been made has been by
ferent bacteria) and still retain its self- minute steps and at the cost of infinite
propagable nature. labor. Where results are thus meager,
That two kinds of sub-
distinct all thinking becomes almost equivalent
stances—the d'Herelle substances and to speculation. But we can not give up
the genes— should both possess this thinking on that account, and thereby
most remarkable property of heritable give up the intellectual incentive to
variation or "mutability," each work- our work. In fact, a wide, unhampered
ing by a totally different mechanism, treatment of all possibilities is, in such
is quite conceivable, considering the cases, all the more imperative, in order
complexity of protoplasm, yet it that we may direct these labors of ours
would seem a curious coincidence in- where they have most chance to count.
deed. It would open up the possibility We must provide eyes for action.
of tu'o totally different kinds of life, The real trouble comes when spec-
working by different mechanisms. On ulation masquerades as empirical fact.
the other hand, if these d'Herelle For those who cry out most loudly
bodies were really genes, fundamen- against "theories" and "hypotheses"—
D'Herelle, F. 1917 Conjpt. rend. Acad., vol. Komplexmutationen beim Weizen. Hered-
165, p. 373. itas, vol. 1, pp. ni-lll.
1918 Compt. rend. Acad., vol. 167, p. 970. Troland, L. T. 1917 Biological Enigmas and
1918 Compt. rend. Soc. Biol., vol. 81, p. the Theory of Enzyme Action. Amer.
1160. Nat., vol. 51, pp. 321-350.
1919 Conipt. rend. Acad., vol. 168, p. 631. Wollstein, Ai. 1921 Studies on the Phenome-
1920 Compt. rend. Soc. Biol., vol. 83, pp. non of d'Herelle with Bacillus dysentaria.
52, 97, 247. Jour. Exp. Med., vol. 34, pp. 467-477.
Emerson, R. A. 1911 The Inheritance of a Zeleny, C. 1920 The Direction and Fre-
Recurring Somatic Variation in Variegated quency of Mutation in a Series of Multiple
Ears of Maize. Anier. Nat., vol. 48, pp. 87- Allelomorphs. Anat. Rec, vol. 20, p. 210.
115. , The
Direction and Frequency
1921
Gratia,A. 1921 Studies on the d'Herelle of Mutation in the Bar-eye Series of Multi-
Phenomenon. Jour. Exp. Med., vol. 34, pp. ple Allelomorphs of Drosophila. Jour. Exp.
115-126. Zool, vol. 34, pp. 203-233.
Sex in Relation to Chromosomes and Genes
CALVIN B. BRIDGES
3A. This type of zygote develops into The interpretation of these inter-
Table I
Sex Type
BRIDGES 119
namely 2X,2A, the female genes out- among the offspring of 3N females. At
weigh the male and the result is a fe- first none were found; but presently
male. If we represent the net effective- it was discovered that very late in the
let us say 80. In a 2X,2A individual the been secured that this type of sex has
ratio of female effectiveness to male the constitution X,3A, which agrees
effectiveness 200:160, or 1.25 to 1;
is with the genetical evidence previously
and on this formulation the sex index secured.
of 1.25 corresponds to the normal fe- It was observed that the intersexes
male. In the X,2A individual the ratio showed considerable variation and
of female to male effectiveness is seemed to form a bimodal class. And
100:160; or the sex index of a normal since the cytological investigation had
male is 0.63. In the 2X,3A intersex the showed that some intersexes had three
ratio is 200:240, and the sex index is and others only two of the small round
0.83, whichintermediate between
is fourth chromosome, it was guessed
the indices for female and male. In the that the more male-like mode corre-
3N female the ratio is 300:240, and the sponded to the full trio of fourth
sex index is 1.25, exactly the same as chromosomes, while the more female-
in the normal female. This identity of like mode corresponded to the cyto-
sex indices for the3N and 2N forms logical type that lacked one fourth
corresponds to the observation that chromosome. An effort has been made
there seems to be no strictly sexual to secure cytological evidence on this
differences between them. The larger point. But this evidence is inconclusive;
size, coarser texture of eye, etc., of the as is also that from an attempt to make
3N can be directly attributed to the a genetic test of the number of fourth
changed volume of the nucleus, and chromosomes present through use of
are not sexual in nature. the fourth-chromosome mutant char-
Another type of egg of the 3N 9 is acter eyeless. At present extra fourth
X-l-X+A; and this, fertilized by a chromosomes are being artificially in-
normal XA sperm, gives a 3X,2A in- serted into the intersexes by continu-
dividual with a sex index of 1.88, ally crossing 3N mothers to males
which is 50 per cent higher than that known to have an extra fourth chro-
of the normal female. This constitu- mosome. Contrariwise, in other lines of
tion corresponds in fact to the "super- intersexes,fourth chromosomes are
females" that occur in these cultures being diminished in numbers by con-
and elsewhere. The superfemales are tinually mating 3N mothers to males
much delayed in development, are known to lack one of the two fourth
rarely able to live and are probably chromosomes. Similarly, the superfe-
completely sterile. maleness of the 3X,2A individuals
Conversely, an X-l-A-l-A egg, fer- might be reduced or be increased by
tilized by the type of sperm that does matings with triplo-IV males or with
not carry an X, gives an X,3A zygote haplo-IV males. For this experiment
with a sex-index of only 0.42. This females being used whose two
are
type of individuals was expected to be X-chromosomes are permanently at-
more male-like than an ordinary male; tached to each other (L. V. Morgan
and such individuals were looked for '22), and hence that give through non-
120 BRIDGES
disjunction a very high proportion of that give the explanation of the origin
3X-superfemales. Present indications, of triploids. In three separate prepara-
from the uncompleted experiments, are tions of ordinary 2N females it was
slightly contradictory, but tend to a found that portion of an ovary was
a
conclusion which is the opposite of that constituted of markedly larger cells;
earlier reported as probable on the and in two of the individuals some of
basis of the slight evidence then avail- the giant cells were in division, and the
able (Bridges '22). When the number chromosomes could be counted as 4N.
of fourth chromosomes is three the Evidently there had been in some
intersexes are more female-like, and oogonial cell a division of the chro-
u^hen the number is two they are more mosomes that had not been followed
male-like. The fourth chromosome has by division of the nucleus and cyto-
a net female tendency, similar to that plasm. The resulting tissue was tetra-
of the X and different from that of the ploid, and any reduced gamete would
other autosomes. By variation in the be 2N. Such a 2N gamete, fertilized
number of fourth chromosomes it is by a normal sperm, would account for
possible to have a fringe of minor sex- each of the twenty-five recurrences
types about each of the major types of of triploidy.
sex difference. Furthermore, in examining sections
The list of sex-types has been en- of two individuals were
intersexes,
larged by the discovery of tetraploids, found which similar cysts of even
in
or 4N individuals. These are females, larger cells were present. In one of
quite identical with normal females in these cysts divisions were occurring;
sex characteristics. The tetraploid and the chromosomes were clearly
arose in a stock of triploids; and was 6N. A 6N cyst in a 3N female would
detected only by the strikingly differ- give, upon reduction, 3N eggs, which,
ent offspring given. A female supposed fertilized by X sperm, would give the
to be 3N was selected from the 3N expected 4N type of female.
stock and outcrossed to a normal male. Soon after this first case of 4N fe-
All the offspring were triploid females male a second similar case was found.
(about 30) or triploid intersexes Also L. V. Morgan found a third case,
(about 20). There were no 2N off- and was able to prove by genetic tests
spring or supersexes. It was seen that that four separate X-chromosomes had
this result might be produced if the been present (in press).
mother were 4N instead of 3N. For The fact that 4N individuals are
in that case all the reduced eggs would females, not modified as to sex, has
be 2N; and these fertilized by X-sperm important bearings on our ideas as to
would give 3N females, and fertilized the way in which genes interact to
by Y-sperm would give 2X,3A inter- produce their effect. The view
sexes. adopted here is that in general the
Before the discovery of this 4N in- effectiveness is in proportion to num-
dividual, an expectation that it would ber of genes, and the significant point
occur had arisen from several facts. is the ratio between sets of genes that
Thus, in the three years following the tend to produce alternative effects. On
discovery of triploidy there had been thisview we find a ready explanation
found no less than twenty-five in- of the fact that such diverse forms as
stances of the new occurrence of tri- 2N, 3N and 4N individuals are pre-
ploidy. This very high frequency was cisely alike in their sexual character-
paralleled by cytological observations istics; for in all these forms the cffec-
BRIDGES 121
tiveness of both contending sets of 40 units. strong race both F and
For a
influences has been doubled, trebled M are higher, for example, 100 and
or quadrupled; and the ratio remains 80, but the arithmetical relation be-
constant. tu^een the values of F and A4 would
But a system of formulation differ- still govern the sex of the individual.
ent from the ratio type has been In a cross between a weak female and
adopted by Goldschmidt in dealing a strong male the ZW
individual re-
with the intersexes produced in the ceived an F of 80 from the mother
I I
male tendency gene {M) is in the out running counter to the very valu-
"Z-chromosome" of which two are able physiological ideas that Gold-
present in the male and one in the schmidt has developed.
female. The female tendency is strictly In the table of sex-types of Dro-
maternally inherited; and hence the sophila the haploid individual is en-
locus of the F genes is in the W-chro- tered with the index 1.25, the same
mosome that descends from mother to as that of the 2N, 3N and 4N females.
daughter. The F gene is supposed to Unfortunately the haploid individual
exert its influence on the cytoplasm of has not been discovered. But it is plain
the developing &2,^\ and hence, al- from the view just given that the ex-
though the male has no W-chromo- pectation for a haploid Drosophila
some, he is supposed to have a definite melmiogaster is that it would be fe-
female tendency that was impressed male in sex character. Accordingly, it
upon the cytoplasm of the tgg and is necessary to assume that the sex-
male. But this system has a difficulty sterility in the triploid forms on ac-
in that the intervals between successive count of the instability of the 3N
indices do not correspond very well group in meiosis, and the consequent
with the observed differences between production of inviable gametes or
the sex grades. Thus the smallest ob- zygotes. Among the most striking con-
served interval in fact, that between firmations of this ratio view of genie
the 3N and 2N individuals, is repre- balance is seen in the mosses, through
sented by a difference of 4 units, while the brilliant work of the Marchals,
the very great interval between the Schweitzer and von Wettstein. For ex-
male and the female is represented by ample, they find in a moss with sepa-
only six units. At that time the 4N type rate sexes that a 2N gametophyte that
was not known; and when addedit is combines 2 female groups of chromo-
to the series, the fit is very poor on somes is a pure female, like the haploid
the algebraic system and very good on female plant (Table II). Likewise the
the ratio system. I repeat that I do not 2N gametophyte that combines two
regard the case of the bee as interpret- male groups of chromosomes is a pure
able on the same basis as Drosophila male plant, like the haploid male plant.
so long as the present account of the But a 2N gametophyte that combines
mechanism for the bee is unchallenged. a male and a female group is no longer
BRIDGES 123
hand, in working with monoecious balance view each of the twelve kinds
mosses where the haploid group is a of chromosomes of Datura might have
hermaphrodite, then all haploid, dip- a distinctive internal unbalance of the
loid, triploid and tetraploid plants sex-controlling genes, similar to the
were hermaphrodite without distinc- unbalance in the fourth chromosome
tion, as they should be from their pos- of Drosophila. In Datura there is a
session of the same ratio of female to full series of forms that differ from the
X',A is a male, the value X' must be Nat., vol. 56, pp. 51-63.
less than that of A, e.g., X'=50. Also, Goldschmidt, R. 1920 Untersuchungen iiber
Intersexualitat. Zeit. bid. Abst. u. Verer.,
since the FM plant is a hermaphrodite f.
3A. We have thus five limiting equa- cum Schwartz. Flora, vol. 116, pp. 1-72.
Wettstein, F. v. 1924 Morphologie und
tions for the three values, X, X', and
Physiologie des Formwechsels der Moose
A. As the table shows, the assigned auf genetischer Grundlage. 2.eit. f. ind.
values of 100, 50 and 80 are possible, Abst. 11. Verer., vol. 33, pp. 1-236.
1^
The Effects of Unequal Crossing Over at the Bar
Locus in Drosophila
A. H. STURTEVANT
ge?ies lying i?i the sa?ne chro?noso?ne are more effective on develop-
ment tha?i the same two genes whe?i they lie i?i differe?it chromo-
so?iiesr This concept, a?id exte?isions of it, is now known as ''posi-
tio?i effecf (a tenn used by Sturtevant in this paper, page 141), and
which implies that where the gene is makes no difference i?i the way
it works. Sturteva?7fs work puts a new e?7Tphasis on the role of the
found that the frequency of reversion zygotes will be expressed as above in order
to avoid circumlocution or indefiniteness.
is variable, but in many stocks is such
"Double-bar over bar" is to be understood
that about 1 in 1600 offspring from a as: "carrying double-bar in one X chromo-
pure bar stock receives a not-bar, or some and bar in the other X chromosome."
Figs. 1-10. (1) Homozygous bar female. (2) Bar male. (3) Bar-over-round female.
(4) Female homozygous for round, that was obtained by reversion. (5) Male that
carries round, obtained by reversion. (6) Double-bar male. (7) Homozygous infra-
bar female. (8) Infrabar male. (9) Infrabar-over-round female. (10) Double-infrabar
male.
126
—
STURTEVANT 127
mozvgous bar. In all cases the mothers
had been heterozygous for forked,^ MUTATIONS AND CROSSING OVER
which lies 0.2 units to the left of bar,
The from homozygous bar
results
and for fused ^ which lies 2.5 units to
were reported by Sturte-
females, that
the right of bar. All six reversions
vant and Morgan, were from females
represented crossovers between forked
-\- R f
and fused, though the total forked of the constitution -7-^5 —. A more
fused crossovers constituted less than J B +
3 percent of the number of individuals efficient type of experiment is that in
examined. Sturtevant and Morgan also which females of the constitution
reported that experiments in which
r T, r are mated to forked bar fused
bar entered only through the males
had failed to give any reversions, males. Table 1 (first row) shows the
though no numerical data were re- results obtained from an extensive
ported. The present paper is based on series of this type. In the second row
the results of a more detailed study of of table 1 are given the results from
the relations first shown by Sturtevant mating a few females of the above
and Morgan (1923). constitution to forked fused males.
Table 1
Type
of
Male
Used
128 STURTEVANT
eight reversions, and also both of the mutant individuals are probably over-
double-bars, occurred in gametes that looked, while it is not likely that any
came from crossing over between reversion is overlooked through diffi-
forked and fused, though the total culty of classification.
forked fused crossovers constituted The double-bar over bar experi-
only 2.4 percent of the population. ments reported by Sturtevant and
The one exceptional case, a wild-type Morgan (1923) can be interpreted in
male, is not above suspicion of having the same way: the reversion is here
arisen through contamination rather due to unequal crossing over just as in
than reversion of bar. That he was homozygous bar. In the earlier cul-
really round-eyed was proven by tests. tures of the experiments previously
Only 4 other exceptions to the rule reported, only the reversions were
that mutation in this locus is accom- classified for forked and for fused.
panied by forked-fused crossing over Two of the reversions were in such
have been met with in the work here incompletely classified cultures. Ta-
reported. These will be discussed sepa- ble 2, including all the double-bar over
rately later. bar data for which complete counts
On the basis of these results we may are available, contains one of the pre-
formulate the working hypothesis that viously reported reversions and one
both reversion and the production of new one. This table includes only the
double-bar are due to unequal crossing male offspring, since the BB derived
over. If we suppose that, in a female from the fathers rendered the classi-
+B+ .
such
,
fication of the females uncertain.
T " lu
,
The process of unequal crossing
a way that the respective points of over might be expected to give rise to
interchange lie to the left of the bar triple-bar from the females that are
locus in one chromosome, but to the double-bar over bar. No individual
right of it in the other one, there will that could be so identified was ob-
result chromosomes of the constitu- tained, though several specimens with
tion jBB+ and -t-f„ (or f-|-
and very small eyes were tested. All those
+ BBfu). The hypothesis is that re- that were fertile proved to be double-
verted round is simply no-bar, and that bar. Apparently triple-bar is either
double-bar is BB,— this being the rea- inviable or sterile. Thisproblem will
son for abandoning Zeleny's name, be discussed again below.
ultra-bar.
Table 2
This hypothesis makes reverted
round and double-bar complementary
crossovers, and they should accord- 9 XfBBf^ $
fBBf,
ingly be produced with equal fre-
quency. Table 1 agrees with Zeleny's B or BB$
more showing that
extensive data in
round is apparently far more frequent
than double-bar; but such a result was
to be expected for two reasons. Dou-
ble-bar is not as viable as round, so that
fewer of the double-bar mutant indi-
viduals would be expected to survive;
and double-bar is not always clearly
distinguishable from bar, so that some
STURTEVANT
have found this tv^pe hard to breed
and have therefore not obtained large
numbers of offspring. It may be re-
called that Zeleny has obtained both
bar and round from such females, but
not in experiments in which forked
and fused were present. In table 3,
showinsf the data I have obtained, only
males are recorded, for the same rea-
son as in table 2, and also because the
females could not be classified for
fused.
Table 3
fBB
9 XfBB S
BBf,
BBS
130 STURTEVANT
Table 5
+ Xffu$
?
fBBf^
STURTEVANT 131
Table 7
X r V y. J f^ 6
132 STURTEVANT
stitution of the other was tested. All tested. Matings of bar-infrabar males
the experiments here described, on to attached-X females (also differing
"bar-infrabar" (figure 10), concern one other sex-linked gene)
in at least
this mutant gene. The stability of this have produced 9042 non-disjunctional
new type in the male has also been sons,— all of them bar-infrabar.
Table 10
B'
fBf,,
9 XfBh$
STURTEVANT 133
culture of this series gave two wild- carried vermilion in her forked fused
tvpe females, one in the first count and X, and the father had also been ver-
another in the last count, ten days milion forked fused. The tested ex-
later. These females give rise to the ceptional female was found to carry
same difficulties as did the wild-type one wild-type X and one forked fused
obtained in the series reported in table X, but did not carry vermilion at all.
7. One of them was tested, and gave Under the circumstances it is open to
unexpected results. The mother had question whether these two excep-
Table 13
1
134 STURTEVANT
firms the sequence of the component shows that the double form obtained
parts of bar-infrabar. It was tested, and there must have had infrabar to the
all the results from double-infrabar left of bar; that is, it was infrabar-bar
reported below were obtained from instead of bar-infrabar. The tests made
descended from it.
flies with appear in table 15.
it
Examination of the data in table 1 These results show, in fact, that the
Table 15
fBJB
9 Xffu $
fu
STURTEVANT 135
Table 17
9 Xff^S
f in
136 STURTEVANT
zygous. It therefore serves to complete
FREQUENCY OF BAR MUTATIONS
the demonstration of the relation of
crossing over (between forked and The data presented in tables 1 to 19
fused) to mutation in the bar locus. have been examined in an attempt to
The experiment of table 19 was, formulate some general statements as
however, planned for another purpose. to the relative frequency of the vari-
It will be seen that in the mother, ous types of mutation in the bar locus.
... BB' It is probable that homozygous double
which was .
„ , eqjial, crossmg over
types, and double over single show the
might give rise to new types, namely, lowest frequencies of mutation, and
double-bar and double-infrabar. The that double type over round shows the
first could not be distinguished, in highest. Both these results might have
somatic appearance, from the unmu- been expected. There is, however, so
tated double types (BB' and B'B); but much variability among crosses of the
the double-infrabar should be readily same general nature that these conclu-
detected. Such an individual would be sions must be accepted with caution.
forked. It may accordingly be con- For example, the two largest series are
cluded that none of the 35 forked those from homozygous bar (20,438
(not-fused) offspring represented offspring) and from homozygous in-
equal crossing over between the halves frabar (16,918 offspring). The me-
of the rvvo double-type bar allelo- chanical conditions should be alike in
morphs present. It therefore seems the two cases, since both represent
probable that crossing over of this homozygous single types. Yet from
kind is not much, if any, more fre- the first there appeared 0.03 percent
quent than is that between the two of reversions, or 1 in each 2920 off-
elements of a double-type allelomorph spring; from the second there were
when the other chromosome carries 0.11 percent, or 1 in 940 offspring. In
round (tables 4, 5, 12, 13, 15, 16). view of such unexplained differences
Several of the above tables agree as this, and in view of the statistical
with a small series of infrabar over difficulty of determining probable
round, heterozygous for forked and errors for such small percentages, it
Table 20
f B f„ crossing over
Table 22
Loci
STURTEVANT 139
in major modifiers, though it is still able in extent, and may be not at all
possible to interpret some of the minor evident,— in which case the type can
differences observed as being due to not be distinguished with certainty
uneliminated diversity in modifiers. from homozygous round. In other
The temperature control used was where the modifiers
stocks, are differ-
not very exact, but maximum-mini- ent,it often happens that infrabar over
in facet number, but the two types can -^ = 73.5 versus—— 50.5
be separated by a peculiarity common D +
to all the larger infrabar and double-
B*
^ = 292.6 ' versus —
B^B^
—= 200.2
infrabar types, namely, a roughened B^ +
appearance of the eye, due to irreg-
B'
ularitiesin the rows of facets. This 7 This value for -^ is different from the
peculiarity is not present in bar eyes, one of table 23. It is based on a series reared
and is almost completely recessive in at the
u same time
•
as the
u ——
B'S' . ,
with which
....
it
Table 23
has been tested by determining the really due to reversion,— if not from
effect on facet number of reverted bar the supposed source, then from some
and reverted infrabar. Round obtained of the allelomorphs within the inbred
bv^ reversion from homozygous bar or stock, since a new reversion may have
infrabar stocks cannot carry a normal occurred during the process of getting
allelomorph on the view advanced in the desired modifiers into the reverted
this paper, unless such an allelomorph round stocks. One of the reverted
is already present in the parent stocks. rounds, called "rev. B," came from the
But such a gene is, almost by defini- homozygous bar experiments of table
tion, not an allelomorph of bar; and in 1; the other, called "rev. B'," came
an\' case cannot be supposed to pro- from the homozygous infrabar experi-
duce the effects here under discussion, ments of table 8. The results of these
—— tests are shown in table 24.
since the ^ and the would both
These data suggest that the rever-
carry it. sions,— especially the more thoroughly
Table 24
Table 25
on
Facet counts fro?/!-—-
144 STURTEVANT
can be shown to have occurred in the observed two other such cases,— both
X chromosomes of males, since it may in D. s'nmdans. The mutant types
be taken as established that crossing dusky and fused (both corresponding
over does not occur between the X to the types of the same names in D.
and the Y of a male. 7}7elaiiogaster) each appeared first in
We have seen earlier in this paper an individual that showed the new
that infrabar arose from bar in a male, character in only one wing; and in
and that its later behavior was not in each case tests showed that some of
agreement with the view that it repre- the X-bearing sperms carried the new
sented a quantitative change in the bar gene, while others did not. In all cases
gene, as it should if due to unequal discussed in the last two paragraphs,
crossing over. I have also obtained genetic tests have established the alle-
yellow, a fused allelomorph, and a lomorphism of the new mutant genes
lozenge allelomorph under the same to the old ones whose names they bear.
circumstances, namely, from mothers There is thus clear evidence that
with attached X's and in experiments mutations have arisen in the follow-
where known sex-linked genes were ing sex-linked loci in the germ-cells
present, so that breaking apart of the of males: yellow (3 times), white
attached X's was known not to have (twice), lozenge (twice), dusky
occurred. (twice), fused (twice), singed (once),
Unpublished data are available for bar (once). It should be noted that all
5 other cases of the same sort, either these loci are among the more mutable
from attached-X or from "high-non- ones of Drosophila.
disjunction" mothers, as follows: rudi- Another class of cases to which the
mentary (C. B. Bridges), a dusky alle- unequal-crossing-over hypothesis is
lomorph (C. B. Bridges), a sable allelo- probably not applicable is that in which
morph (E. M. Wallace), white (L. V. mutation can be shown to have oc-
Morgan), and a new lozenge allelo- curred at some stage other than mat-
morph from lozenge (C. B. Bridges). uration. A number of such instances
In all of these cases, as in that of in- are on record for Drosophila. The
frabar, the mutant type first appeared mosaic males described above are ex-
as a single male. amples, and a longer list of cases for
Muller (1920) reported the occur- autosomal mutant types and for sex-
rence of white as a "somatic" mutation linked mutations in females could
in a male. From a stock in which white easily be compiled. But since there is
was not present he obtained a male evidence that crossing over does very
with one wild-tvpe eye and one white rarely occur at somatic divisions, this
eye. This male also transmitted white evidence can hardly be considered de-
to some (all that were tested) of his cisive. In the case of certain t\'pes of
daughters. In the same paper Muller frequently recurring somatic muta-
described briefly a mosaic male that tions in plants, however, the mutation
was partly yellow, and transmitted the occurs far too often to make an appeal
new character to his offspring. Doctor to somatic crossing over seem plausi-
Bridges informs me that he has a sim- ble. The clearest example of this sort
ilar (unpublished) record for yellow. is the variegated pericarp of maize
Mohr (1923 a) reports a similar case studied by Emerson (1917) and others,
for a singed allelomorph, though here in which a given gene mutates many
some of the X-bearing spemis carried separate times in a single individual
singed, while others did not. I have plant.
STURTEVANT 145
Mutations are known in which there do not cross over with
sister strands
appeared to be no crossing over in the each other; or, if they do, that the
region concerned,— both in females crossing over is rarely, if ever, un-
and in males where crossing over does equal.
not normally occur at all. The previ- It is therefore unlikely that apparent
ously cited cases of mutations in the non-crossover mutations in other loci
X chromosomes of males are examples. are to be referred to crossing over be-
These may seem to furnish conclusive tween sister strands.
evidence that mutation need not be
accompanied by crossing over. There "presence and absence" and
is, however, one possibility that needs
quantitative view of mutation
to be considered in this connection. It hypoth-
will be observed that the
Recent results (not yet published, esis advocated in this paper makes
but soon to appear) obtained with bar, double-bar and round by rever-
triploid females (Bridges and Ander- sion (or infrabar, double-infrabar and
son) and with females having unlike round by reversion) represent quan-
attached X's (Anderson, L. V. Mor- titative variations of the same sub-
gan, and Sturtevant) have shown that stance. In the case of bar and round,
crossing over must normally occur the hypothesis is the same as the origi-
when the homologous chromosomes nal and most special type of quantita-
are doubled: that is, in a "four-strand tive view, the "presence and absence"
stage" (in diploid females). These re- hypothesis. But the present scheme
sults show also that crossing over may differsfrom the earlier ones in that it
occur between only two of the strands isbased on definite evidence for the
at a given level. Now, if it be supposed occurrence of unequal crossing over.
that sister strands may cross over with That is, the mechanism whereby the
each other, there will result chromo- quantitative differences are brought
somes in which no rearrangement of about an essential part of the hy-
is
mutant genes has occurred, since sister pothesis. In the preceding section we
strands come from the division of one have seen that there is definite evi-
chromosome and will be identical in dence to show that unequal crossing
the genes that they carry. Yet it is con- over is not usual in the production of
ceivable that such crossing over might new mutant types. It is especially note-
be unequal, and in such a case might worthy was derived
that this evidence
lead to the production of a new muta- in part from the white locus of Dro-
tion that did not appear to be due to sophila and the variegated locus of
crossing over. maize,— two of the best-known ex-
The data presented in this paper amples of loci that have produced
show that such an eventmust be ex- large series of multiple allelomorphs.
tremely rare in the case of bar, since It is clear,therefore, that the bar case
no clear case was found of bar muta- does not furnish support to the idea
tion (in a female) unaccompanied by that mutations in general are quantita-
evident crossing over between forked tive in nature. Even with respect to
and fused. The few^ exceptional cases multiple allelomorphs, where the
may be accounted for in this way; but, quantitative view has often been urged,
as pointed out when they were de- it is obvious that, at least in the cases
scribed, it seems at least equally prob- of white and variegated, the bar evi-
able that all of them are due to experi- dence does not in any way support
mental errors. We must conclude that that view.
146 STURTEVANT
ARE DEFICIENCIES DUE TO UNEQUAL field(1922) records the occurrence
CROSSING OVER? of a notch (probably corresponding
to that of D. melanogaster) in Dro-
The "section-deficiencies" described sophila obscura; the mutation was first
by Bridges (1917, 1919) and by Mohr detected as two females from a pair
(1919, 1923 b) are probably to be in- mating that gave numerous ofi^spring.
terpreted as due to losses of definite In this case the deficiency must have
sections of chromosomes. It will be originated in the gonial cells of one
observed that bar reversion has here parent, unless the two notch females
been treated as due to the loss of a received their notch chromosomes
very short section; it may accordingly from the father, in which case it is
be described as a deficiency that is just possible that they came from two
too short to show the lethal eff^ect and sperms derived from a single sperma-
other properties of the previously de- tocyte. But in this case the hypothesis
scribed deficiencies. When the case is of unequal crossing over remains as
stated in this way, the question at improbable as before. I have observed
once arises: is it probable that notch two cases in D. melmiogaster that
and other deficiencies have also arisen represent "somatic" (i.e., not occur-
through unequal crossing over? If so, ring at the maturation divisions) oc-
the contrary crossover should be a currences of notch. In one case three
chromosome that was double for a re- notch females were produced from a
gion corresponding to the deficient single mother. The X's of the mother
section. Such a chromosome has never were attached, and the notch daugh-
been identified, but it may be doubted ters, like all their sisters, did not carry
even in heterozygous females, in which to demonstrate that the new type was
case it would not be capable of detec- actually notch; but the numerous
tion. characters of notch make the iden-
There is evidence that deficiencies tification very probable. The other
may arise in other ways than by un- case also occurred in a line in which
equal crossing over. In at least one the females all had attached X's. A
case (Bridges and Morgan 1923) the female, from a line with no notch
section missing from a second chro- ancestry, was notch in the left wing
mosome was found to be present, but but not in the right. The offspring
attached to a third chromosome. In showed that this female was, like her
this case, then, the deficiency can not mother, heterozygous for several sex-
have been due to unequal crossing linked genes. These included scute, 3
over. The first deficiency described, units to the left of notch, and cross-
that for forked and bar (Bridges 1917), veinless, 10 units to the right of it.
occurred first as a single female that Some of the eggs of the mosaic carried
had obtained the deficient X from her notch, but many of them did not.
father. Here the deficiency arose Furthermore, tests showed that scute
(either in a male or very early in the and crossveinless were in opposite
cleavage of a female zygote) at a time chromosomes in both types of eggs;
when crossing over (and bar rever- that is, the mutation to notch occurred
sion) does not normally occur. In the at a cleavage division, and was not
case of notch, also, there is evidence accompanied by crossing over between
that the deficiency may originate at scute and crossveinless. From these
stages other than maturation. Lance- three instances we may conclude that
STURTEVANT 147
the notch deficiency may arise at This result can be explained if it
2.
side by side. But the present case indi- improbable that many mutations in
cates that this interpretation will have other loci are to be explained as due
to be applied with some caution. to unequal crossing over.
"^^
Artificial Transmutation of the Gene
H. J. MULLER
One of the major tunmig points i?i the study of heredity was the
discovery that cha?iges i?j the genes could be i?iduced by artificial
jneans. This short paper by Muller describes the first de?no?istrably
successful attempt to make gefies mutate, and represejits the begin-
7iing of a new epoch in genetic history. Mutagenic methods and ma-
terials are now well know7i and widely used i?i genetic experiments,
and probably represent the most useful know?i tool in research. As a
consequence of this paper, the X-ray machine has become as com-
mon a piece of equipment ifi a genetics laboratory as is the micro-
scope in histology.
It should be noted that Muller recognizes the dangers of X-ray
treatmetit to germifial tissues in this, the first paper o?i i?iduced muta-
tions. Midler's recognition of the dual ?iature of the effects of irradia-
tion is clear cut, for he points out in a paragraph on page 152 that
first there is a direct effect on the cells and tissues, and seco7id there
is a?! ifidirect effect on the desce?idant cells ajid tissues through the
mediatioji of the genes and chro?nosomes. The first of these is tra?7-
siefit (although possibly fatal), while the second is permane?it. It is
this lack of exact proportionality be aging the sperm after treatment, be-
confirmed, then, as Dr. Irving Lang- fore fertilization, causes no noticeable
muir has pointed out to me, we should alteration in the frequency of detec-
have to conclude that these mutations table mutations. Therefore the death
are not caused directly by single rate of the mutant sperm is no higher
quanta of X-ray energy that happen than that of the unaffected ones; more-
to be absorbed at some critical spot. over, the mutations can not be re-
If the transnuting effect were thus garded as secondary effects of any
relatively indirect there would be a semi-lethal physiological changes
greater likelihood of its being influ- which might be supposed to have oc-
enceable by other physico-chemical curred more intensely in some ("more
agencies as well, but our problems highly susceptible") spermatozoa than
would tend to become more com- in others.
plicated. There is, however, some Despite the "negative results" just
danger in using the total of lethal mentioned, however, it is already cer-
mutations produced by X-rays as an tain that differences in X-ray influ-
index of gene mutations occurring in ences, by themselves, are not suflicient
single loci, for some lethals, involving to account for all variations in muta-
changes in crossover frequency, are tion frequency, for the present X-ray
probably associated with rearrange- work comes on the heels of the deter-
ments of chromosome regions, and mination of mutation rate being de-
such changes would be much less likely pendent upon temperature (work as
than "point mutations" to depend on yet unpublished). This relation had
single quanta. A re-examination of the first been made probable by work of
effect of different dosages must there- Altenburg and the writer in 1918, but
fore be carried out, in which the dif- was not finally established until the
ferent types of mutations are clearly completion of some experiments in
distinguished from one another. When 1926. These gave the first definite evi-
CREIGHTON AND MCCLINTOCK 155
dence that gene mutation may be to afforded them by the use of X-rays, of
any extent controllable, but the mag- creating in their chosen organisms a
nitude of the heat effect, being similar series of artificial races for use in the
to that found for chemical reactions study of genetic and "phaenogenetic"
in general, too small, in connection
is phenomena. If, as seems likely on gen-
with the almost imperceptible "nat- eral considerations, the effect is com-
ural" mutation rate, for it, by itself, to mon to most organisms, it should be
provide a powerful tool in the muta- possible to produce, "to order,"
tion study. The result, however, is enough mutations to furnish respec-
enough to indicate that various factors table genetic maps, in their selected
besides X-rays probably do affect the species, and by the use of the mapped
composition of the gene, and that the genes, to analyze the aberrant chro-
measurement of their effects, at least mosome phenomena simultaneously
when in combination with X-rays, will obtained. Similarly, for the practical
be practicable. Thus we may hope that breeder, it is hoped that the method
problems of the composition and be- will ultimately prove useful. The time
havior of the gene can shortly be is not ripe to discuss here such pos-
approached from various new angles, sibilities with reference to the human
and new handles found for their in- species.
vestigation, so that it will be legitimate The writer takes pleasure in ac-
to speak of the subject of "gene physi- knowledging his sincere appreciation
olog\%" at least, if not of gene physics of the cooperation of Dr. Dalton
and chemistry. Richardson, Roentgenologist, of Aus-
In conclusion, the attention of those tin, Texas, in the work of administer-
working along classical genetic lines ing the X-ray treatments.
may be drawn to the opportunity,
\^
A REQUIREMENT FOR THE GENETICAL not on its homologue, the knob renders
Study of crossing-over is the hetero- the chromosome pair visibly hetero-
zygous condition of two allelomorphic morphic.
factors in the same linkage group. The In a previous report ^ it was shown
analysis of the behavior of homologous that in a certain strain of maize an
or partially homologous chromosomes, interchange had taken place between
which are morphologically distin- chromosome 8 and 9. The inter-
guishable at two points, should show changed pieces were unequal in size;
evidence of cytological crossing-over. the long arm of chromosome 9 was
It is the aim of the present paper to increased in relative length, whereas
show that cytological crossing-over the long arm of chromosome 8 was
occurs and that it is accompanied by correspondingly shortened. When a
genetical crossing-over. gamete possessing these two inter-
In a certain strain of maize the sec- changed chromosomes meets a gamete
ond-smallest chromosome (chromo- containing a normal chromosome set,
some 9) possesses a conspicuous knob meiosis in the resulting individual is
at the end of the short arm. Its distribu- characterized by a side-by-side synap-
tion through successive generations is sis of homologous
parts. Therefore, it
similar to that of a gene. If a plant should be possible to have crossing-
possessing knobs at the ends of both over between the knob and the inter-
of its 2nd-smallest chromosomes is change point.
crossed to a plant with no knobs, cy- In the previous report it was also
tological observations show that in the shown that in such an individual the
resulting Fi individuals only one mem- only functioning gametes are those
ber of the homologous pair possesses which possess either the two normal
a knob. When such an individual is chromosomes (N, n) or the two inter-
back-crossed to one having no knob changed chromosome (/, /'), i.e., the
on either chromosome, half of the off- full genom in one or the other ar-
spring are heterozygous for the knob rangement. The functional gametes
and half possess no knob at all. The therefore possess either the shorter.
knob, therefore, is a constant feature
of the chromosome possessing it. 1 A^IcClintock, B., Proc. Nat. Acad. Sci.,
When present on one chromosome and 16:791-796, 1930.
CREIGHTON AND MCCLINTOCK 157
normal, knobbed chromosome (w) or
the longer, interchanged, knobbed
chromosome (/). Hence, when such a
plant is crossed to a plant possessing
the normal chromosome complement, N
the presence of the normal chromo-
some in functioning gametes of the
former will be indicated by the ap-
pearance of ten bivalents in the pro-
phase of meiosis
of the resulting
individuals. The
presence of the inter-
changed chromosome in other gametes
will be indicated in other Fi individ-
uals by the appearance of eight bi-
valents plus a ring of four chromo-
somes in the late prophase of meiosis.
gamete possessing a normal
If a
chromosome number 9 with no knob,
meets a gamete possessing an inter-
changed chromosome with a knob, it
isclear that these two chromosomes
which synapse along their homologous Fig. 1. (above) Diagram of the chromo-
parts during prophase of meiosis in the somes in which crossing over was studied.
resulting individual are visibly differ- (below) Diagram of chromosome t\'pes
ent at each of their two ends. If no found in gametes of a plant with the
crossing-over occurs, the gametes constitution shown above.
formed by such an individual will con- — Knobbed, interchanged chromo-
a
Table 1
morphic for the knob, the genes c and and c-wx, respectively. This cross is
zvx and the interchange. Plant 338 diagrammed as follows:
Wif Vx
The results of the cross are given in ing a knob, whereas all the colorless
table 3. In this case all the colored kernels gave rise to individuals show-
kernels gave rise to individuals possess- ing no knob.
Table 3
Knob-C-ii).Y-interchanged Knobless-t-TFx-normal
Knobless-c-lF.r-normal Knobless-c-ti'x-normal
Plant number
160 CREIGHTON AND MCCLINTOCK
The amount of crossing-over be- they therefore represent non-cross-
tween the knob and the interchange over types. An equal number of non-
point is approximately 39% (table 1), crossover types are expected among
between c and the interchange ap- the normal Wxnjox class. Plants 1, 2, 4,
proximately 33%, between wx and the 1 1 and 12 may be of this type. It is pos-
interchange, 13% (preceding paper). sible but improbable that they have
With information in mind it is
this arisen through the union of a c-Wx
possible to analyze the data given in gamete with a gamete resulting from
table 3. The data are necessarilyfew a double crossover in region 2 and 3.
since the ear contained but few ker- Plants 5 and 8 are single crossovers
nels. The three individuals in class I in region 3, whereas plants 3 and 7
are clearly non-crossover types. In probably represent single crossovers
class II the individuals have resulted in region 2 or 3.
from a crossover in region 2, i.e., be- The foregoing evidence points to
tween c and 'wx. In this case a cross- the fact that cytological crossing-over
over in region 2 has not been accom- occurs and is accompanied by the ex-
panied by a crossover in region 1 (be- pected types of genetic crossing-over.
tween the knob and C) or region 3 Co?wb{sio7is.— Pairing chromosomes,
(between vjx and the interchange). All heteromorphic in two regions, have
the individuals in class III had normal been shown to exchange parts at the
chromosomes. Unfortunately, pollen same time they exchange genes as-
was obtained from only 1 of the 6 in- signed to these regions.
dividuals examined for the presence of The authors wish to express appre-
the knob. This one individual was ciation to Dr. L. W. Sharp for aid in
clearly of the type expected to come the revision of the manuscripts of this
from a gamete produced through and the preceding paper. They are in-
crossing-over in region 2. Class IV is debted to Dr. C. R. Burnham for fur-
more difficult to analyze. Plants 6, 9, nishing unpublished data and for some
10, 13, and 14 are normal and WxWx; of the material studied.
A New Method for the Study of Chromosome
Rearrangements and Plotting of Chromosome
Maps
T. S. PAINTER
(2) In old larvae, homologous chro- that the force which causes homo-
mosomes undergo process of somatic
a logues to unite in salivary glands is the
synapsis. This union is more than a same that operates in meiosis, and
simple apposition, for the elements pair while, so far asis known, these special-
up line for line in the most exact way ized chromosomes never divide, we
and form one apparent structure. If can at least study how aberrant chro-
one of the homologues carries an in- mosomes unite at synapsis, a fact
verted section we get typical inversion which should prove of great value to
figures, such as we would expect in geneticists.
meiosis. If one of the homologues is (3) In salivary glands the two arms
The writer has two articles in press, studies of the autosomes have been
one dealing with the technique and under way for some time, and a num-
the general morphological characters ber of students are at work on various
of the salivary chromosome, the sec- cytological and genetic problems
ond, a detailed study of the X-chro- opened up by the new method of at-
niosome from which the drawing pre- tack.
sented herewith was taken. Similar
\^
CALVIN B. BRIDGES
The nature of the bar gene has of the mutant is that occasionally the
been the subject of extensive investiga- homozygous stock gives rise to a fly
tion and speculation since February, indistinguishable in appearance and ge-
1913, when Tice ^ found this reduced- netic behavior from wild-type.^ More
eye mutant as a single male in the rarely the stock gives rise to an even
progeny of normal-eyed parents. The more extreme reduction in eye-size, a
eye-reduction behaves as a sex-linked type which was called Ultra-Bar by
dominant, with a locus at 57.0, and has Zeleny,^ who found it.
changes were the result of a novel type present in excess of the normal com-
of "unequal" crossing-over, by which plement, forming a duplication. The
the two genes originally present in the insertion point of this duplication is in
two parental chromosomes both the bulbous "turnip" segment, not far
emerged in the same chromosome from the basal end of the X.^
(Bar-double) while the other resultant The exact point of the insertion is
chromosome was without Bar (Bar- ambiguous, for a reason which will
reverted). The change from Bar to appear below. The normal X in this
Bar-double was considered to be a region (see revised map below)
single gene duplication, while the con-
verse change, from Bar to Bar-re- BAR- REVERTED
verted, corresponds to a one-gene
deficiency. Since the Bar-reverted type
proved to be indistinguishable from
the normal unmutated wild-type, the
gene present in Bar and lost in Bar-
reverted must have itself corresponded
to a new addition or one-gene duplica-
tion.^
Sturtevant ^ found the unexpected
relation two Bar genes in the
that
NORMAL BAR-DOUBLE
same chromosome (BB/B-I-) gave a
greater reduction in the size of the
shows in sub-section 16A a heavy
Bar eye than did two Bar genes in
band, which in well-stretched chro-
opposite chromosomes (B/B), an in-
mosomes, or with certain fixations, is
tensification of action which he formu-
a clear doublet, usually with the halves
lated as a "position effect." Dobzhan- united in a capsule, but occasionally
sky '^
interpreted his allelic Baroid completely separate. This is followed
mutant as a position effect due to the
by a very faint dotted line, which can
substitution of material at or near the
be seen only in the most favorable
Bar-locus (in the normal X) by ma-
conditions. Next follows a fairly weak
terial translocated from the right limb
line which is distinctly "dotted" in
of chromosome 2, and the reduction
texture, with the separate dots loosely
in the Bar eye to the interaction be-
connected across the width of the
tween a gene in the X chromosome chromosome. Next follows closely a
and the duplication. fainter, continuous-tex-
still diffuse,
A chance to clear up some of the tured doublet, with the doubleness
puzzles as to the origin and behavior
generally appearing as mere broaden-
of Bar was offered by the salivary
ing. The last line of sub-section 16A
chromosomes. Study of the banding in
is again a very faint dotted singlet.
fairly weak bands, while a very sharp banding and irregularity and oblique-
change in size occurs at the transition ness of the synapses. These disturb-
from 15F to 16A. ances were greatest in the miudle one
In the Bar chromosome the condi- of the three seriations.
tion may
be described observationally These findings enable the Bar "gene"
as the repetition of section 16A, with to be reinterpreted as a section of in-
the exception of the final very faint serted genes— a duplication. The pro-
dotted line. But the whole region of duction of Bar-double and of Bar-
this bulb has undergone changes in the reverted is seen to be the insertion of
Bar chromosome as follows: the "puff" this extra section twice, or conversely,
of the bulbous segment is more pro- its total loss— both presumably by a
nounced and its size is increased; the process of unequal crossing-over. That
banding is more discontinuous by the section of bands should behave as
being broken into blocks and vesicles, a unit in this process is perhaps ac-
and the regularity of synapsis is dis- counted for by the observation of
turbed by oblique junctions. Thus, in oblique synapsis, especially frequent
Bar the heavy doublet following the in Bar-double, where presumably one
last faint dotted line of 15F is more entire sequence synapses with another
segmented than normal and more of a different position in the series of
rarely shows its doubleness clearly. three. The oblique synapses were even
This tendency is more pronounced in more frequent in BB/B + where one
,
the heavy broken line of the repeat series in B+ has a choice of three series
seriation to the right. All the lines of in BB, apparently usually synapsing
the repeat seriation to the right differ with one or the other end series.
from the corresponding lines of the According to this interpretation the
initial seriation by being somewhat less source of the duplication is the ma-
intense, more broken, more diffuse and terial directly adjacent to the repeat.
more confused in their synapsis rela- But whether the point of insertion
tions. preceded the heavy doublet of 16A1
In a forked non-Bar stock recently or the very faint final singlet of 16A5,
derived from the above forked Bar can not be determined. If Bar is itself a
stock by breeding from the rare Bar- repeat, a reason is thereby provided
reversions, the banding was found to for its unique behavior of giving rise
be precisely identical with that of un- to Bar-double and Bar-reverted by
related normals as far as could be ob- oblique synapsis. Perhaps half of the
served in excellent permanent prepara- Bar-reversions carry the original series
tions of well-stretched chromosomes. and the other half the subsequent re-
In a forked Bar-double stock, sim- peat restored to its original position.
ilarly derived from the same f B stock On this interpretation, the "position
by breeding from the very rare "Ultra- effect"— the reinforcement of the ac-
Bar" type of eye, it was found that tion of one Bar gene by another in di-
the extra section observed in Bar was rect sequence next to it— has a visible
present still again, giving a thrice-re- cytological accompaniment in the in-
peated seriation in direct sequence. creased size and puffiness, and the
The changes differentiating Bar from change in the character of the banding
normal were carried further in Bar- of both series in Bar as compared with
double, as follows: The size and puffi- normal and of all three series in Bar-
ness of the bulbous regions was still double as compared to Bar itself. Part
greater, as well as the blockiness of the of this is presumably due to the
166 BEADLE AND TATUM
"rounding-up" tendency of the synap- Study of the Baroid translocation
tic attraction alojig the chromosome in apparently shows that the break in X
addition to the oblique attractions and comes between the two halves of the
the straight-across attractions. heavy doublet of 16A1. The break in
The Bar-eye reduction is thus seen 2R follows directly after the heavy
to be interpretable as the effect of in- capsular doublet of 48C1. Thus a de-
creasing the action of certain genes by monstrable basis is laid to Dobzhan-
doubling or triplicating their number— sky's interpretation of the Baroid eye-
a genie balance effect. But "position reduction as a position effect.^
effects" are never excluded when du- The previously reported finding * of
plications or other rearrangements are the presence of "repeats" as a normal
wedging further
present, either in the part of the chromosomes of D. me-
apart of genes normally closer, or by lauogaster, and the suggestion that un-
the interaction with new neighbors. equal crossing-over is probably the
The respective shares attributable in mechanism of production of some
the total effect to the genic-balance short repeats, thus have received ample
change and to the position-effect verification bv^ these direct observa-
change seems to be at present a matter tions on these processes in the case of
of taste. Bar and its derivatives.
In my
remarks on Sewall Wright''s paper (page 18) I pointed out
that the approach implicit in that work was one of the most promis-
ing aspects of genetic research. The problems facing Wright ajid his
students were enormous, however, and ?nost of them ste?fm?ed fro7n
the difficidties of working with the mouse. It is practically impossible
to tamper with the mouse'' s enviro7iment experimentally ajid it is ,
tempts to determine the physiological to the problem implies the use of the
and biochemical bases of already characters with visible manifestations.
known hereditary traits. This ap- Many such characters involve morpho-
proach, as made in the study of an- logical variations, and these are likely
thocyanin pigments in plants,^ the
^VVinge, O. and Laustsen, O., Compt.
1 The possibility that genes may act rend. Lab. Carlsberg, Serie physiol. 22:337-
through the mediation of enzymes has been 352, 1939.
suggested by several authors. See Troland, 4 See Goldschmidt, R., Physiological Ge-
L. T., A?mr. Nat. 51:321-350, 1917; Wright, netics, McGraw-Hill, pp. 1-375, 1939; and
S., Genetics 12:530-569, 1927; and Haldane, J. Beadle, G. W. and Tatum, E. L., Amer. Nat.
B. S., in Perspectives hi Biochemistry, Cam- 75:107-116, 1941, for discussion and refer-
bridge Univ. Press, pp. 1-10, 1937, for dis- ences.
cussion and references. ^ See Sturtevant, A. H. and Beadle, G. W.,
~ Onslow, Scott-Moncrieff and others, see An Introduction to Genetics, Saunders, pp.
review by Lawrence, W. J. C, and Price, 1-391, 1931; and Beadle, G. W. and Tatum,
J. R., Biol. Rev. 15:35-58, 1940. E. L., loc. cit., footnote 4.
168 BEADLE AND TATUM
to be based on systems of biochemical this mutant would then be unable to
reactions so complex as to make analy- grow on a medium containing sucrose
sisexceedingly difficult. as a sole carbon source but should be
Considerations such as those just able to grow on a medium containing
outlined have led us to investigate the some other normally utilizable carbon
general problem of the genetic control source. In other words, it should be
of developmental and metabolic reac- possible to establish and maintain such
tions by reversing the ordinary proce- a mutant strain on a medium contain-
dure and, instead of attempting to ing glucose and detect its inability to
work out the chemical bases of known utilize sucrose by transferring it to a
genetic characters, to set out to deter- sucrose medium.
mine if and how genes control known Essentially similar procedures can
biochemical reactions. The ascomycete be developed for a great many meta-
Neiirospora offers many advantages bolic processes. For example, ability to
for such an approach and is well suited synthesize growth factors (vitamins),
to genetic studies.^ Accordingly, our amino acids and other essential sub-
program has been built around this stances should be lost through gene
organism. The procedure is based on mutation our assumptions are cor-
if
out the specific and enzymatically yeast extract and glucose. The minimal
controlled reaction involved in the medium contains agar (optional), in-
hydrolysis of this sugar. Assuming this organic salts and biotin, and a disac-
reaction to be genetically controlled, charide, fat or more complex carbon
it should be possible to induce a gene source. Biotin, the one growth factor
to mutate to a condition such that the that wild-type Neurospora strains can-
organism could no longer carry out not synthesize,^ is supplied in the form
sucrose hydrolysis. A strain carrying
"^
In so far as we have carried them, our
investigations on the vitamin requirements
« Dodge, B. O., Jour. Agric. Res. 35:289- of Neurospora corroborate those of Butler,
305, 1927;and Lindegren, C. C, Bull. Torrey E. T., Robbins, W. J., and Dodge, B. O.,
Bot. Club 59:85-102, 1932. Science 9'i:262-26i, 1941.
BEADLE AND TATUM 169
of a commercial concentrate contain- the induced metabolic defect been in-
ing 100 micrograms of biotin per cc.** vestigated. For this reason detailed
Any loss of ability to synthesize an es- accounts of the thiamine-deficient and
sential substance present in the com- p-aminobenzoic acid-deficient strains
plete medium and absent in the minimal will be deferred.
medium is indicated by a strain grow- Qualitative studies indicate clearly
ing on the first and failing to grow on that the pyridoxinless mutant, grown
the second medium. Such strains are on a mediumcontaining one micro-
then tested in a systematic manner to gram or more of synthetic vitamin Be
determine what substance or sub- hydrochloride per 25 cc. of medium,
stances they are unable to synthesize. closely approaches in rate and char-
These subsequent tests include at- acteristics of growth normal strains
tempts to grow mutant strains on the grown on a similar medium with no
minimal medium with ( 1 ) known vita- Be. Lower concentrations of Be give
mins added, (2) amino acids added or intermediate growth rates. A prelimi-
(3) glucose substituted for the more nary investigation of the quantitative
complex carbon source of the minimal dependence of growth of the mutant
medium. on vitamin Be in the medium gave the
Single ascospore strains are individ- results summarized in table 1. Addi-
ually derived from perithecia of N. tional experiments have given results
crassa and N. sitophila x-rayed prior essentially similar but in only approxi-
to meiosis. Among approximately 2000
Table 1
weight increase of this mutant can be at an angle of about 45°. Agar medium
used as an accurate assay for vitamin is poured in so as to fill the tube about
Fig. 1. Growth of normal (top two curves) and pyridoxinless (remaining curves)
strains of Neurospora sitophila in horizontal tubes. The scale on the ordinate is
shifted a fixed amount for each successive curve in the series. The figures at the
right of each curve indicate concentration of pyridoxine (B^) in micrograms per
25 cc. medium.
BEADLE AND TATUM 171
12
10
o o
o
f
0.002 0.004 0.008 0015 003 006 012 025 5 1.0
Fig. 2. The relation between growth rate (cm./day) and vitamin Bg concentration.
In order to ascertain the inheritance were isolated and their positions in the
of the pyridoxinless character, crosses asci recorded. For some unknown rea-
between normal and mutant strains !*> It is planned to investigate further the
were made. The techniques for hy- possibility of using the growth of Neuro-
bridization and ascospore isolation spora strains in the described tube as a basis
have been worked out and described of vitamin assay, but it should be emphasized
that such additional investigation is essential
by Dodge, and by Lindegren.^ The in order to determine the reproducibility
ascospores from 24 asci of the cross and reliability of the method.
172 BEADLE AND TATUM
son, most of these failed to germinate. tiated by growing them on a Be defi-
From seven asci, however, one or more cient medium. On this medium the
spores germinated. These were grown mutant cultures grew very little, while
on a medium containing glucose, malt the non-mutant ones grew normally.
extract and yeast extract, and in this The results are summarized in table
they all grew normally. The normal 2. It is clear from these rather limited
and mutant cultures were differen- data that this inability to synthesize
Table 2
Ascus
AVERY, MACLEOD, MCCARTY 173
discover and maintain x-ray induced apparently differentiated by a single
mutant strains which are character- gene from the ability of the organism
ized by their inability to carry out to elaborate this essential growth sub-
specific biochemical processes. stance.
Follow ing this method, three mutant Note: Since the manuscript of this
strains have been established. In one paper was sent to press it has been
of these the ability to synthesize vita- established that inability to synthesize
min Be has been wholly or largely lost. both thiazole and aminobenzoic acid
In a second the ability to synthesize is inherited as though differen-
also
the thiazole half of the vitamin Bi tiatedfrom normal bv single genes.
molecule is absent, and in the third Work supported in part by a grant
para-aminobenzoic acid is not syn- from the Rockefeller Foundation. The
thesized. It is therefore clear that all authors are indebted to Doctors B. O.
of these substances are essential growth Dodge, C. C. Lindegren and W. S.
factors for Neurospora}^ Malloch for stocks and for advice on
Growth of the pyridoxinless mutant techniques, and to Miss Caryl Parker
(a mutant unable to synthesize vitamin for technical assistance.
Be) is a function of the Be content of
themedium on which it is grown. A
method is described for measuring the 11 The
inference that the three vitamins
growth by following linear progres- mentioned are essential for the growth of
normal strains is supported by the fact that
sion of the mycelia along a horizontal
an extract of the normal strain will serve as a
tube half filled with an agar medium. source of vitamin for each of the mutant
Inability to synthesize vitamin Be is
duties are similar to those at hojne, but the details of his performance
are ?iovel, and slightly cofifusing. One must understand the local
situation before he drives successftdly, and so it is here. The effort
necessary to follow the intricacies of bacteriological detail is re-
warded by a new i?isight into ge?ietic ?nechanis?m and structure.
This will be foimd equally true of the paper by Benzer o?i p. 211.
This paper is of outstaTidijig importance to ge?ietics, of course, for
the precise way m which it shows DNA
to be a primary hereditary
material. For the first time a definable chemical co?npoimd plays a
role i?i changing the hereditary makeup of an organisjn, and, once
it has played this role, shows also that it is capable of reproducing
(p. 190). The experi?ne?2tal data and evidence the authors have mar-
shalled to prove this poi?it are quite impressive, and the reader should
note the multitude of techniques a?id apparatus involved. Genetic
investigations on micro-organisms are exceedingly complex, but are
also exceedingly rewardifig.
were prepared.
Another example of transformation EXPERIMENTAL
which is analogous to the intercon- Transformation of pneumococcal
vertibility of pneumococcal types lies types ill vitro requires that certain cul-
in the field of viruses. Berry and De- tural conditions be fulfilled before it is
dependent upon the content of R anti- encapsulated R strain used in the pres-
bodies, since many sera of high titer ent study was derived from a virulent
were found to be incapable of support- "S" culture of Pneumococcus Type
ing transformation. This fact sug- II.It will be recalled that irrespective
AVERY, MACLEOD, MCCARTY 177
glistening surface of colonies of en- was noted that the stock culture of R36
capsulated S cells. on serial transfers in blood broth under-
goes spontaneous dissociation giving rise
The R strain referred to above as to a number of other R variants which
R36A was derived by growing the parent can be distinguished one from another by
S culture of Pneumococcus Type II in colony form. The significance of this in
broth containing Type II antipneumo- the present instance lies in the fact that
coccus rabbit serum for 36 serial passages of four different variants isolated from
and isolating the variant thus induced. the parent R culture only one (R36A)
The strain R36A has lost all the specific is susceptible to the transforming action
and distinguishing characteristics of the of potent extracts, while the others fail
parent S organisms and consists only of to respond and are wholly inactive in this
attenuated and non-encapsulated R vari- regard. The fact that differences exist in
ants. The change S -* R is often a re- the responsiveness of different R variants
versible one provided the R cells are not to the same specific stimulus emphasizes
too far "degraded." The reversion of the the care that must be exercised in the
R form to its original specific type can selection of a suitable R variant for use
frequently be accomplished by successive in experiments on transformation. The
animal passages or by repeated serial sub- capacity of this R strain (R36A) to re-
culture in anti-R serum. When reversion spond to a variety of different transform-
occurs under these conditions, however, ing agents is shown by the readiness with
the R culture invariably reverts to the which it can be transformed to Types I,
encapsulated form of the same specific III, VI, or XIV, as well as to its original
type as that from which it was derived type (Type 11), to which, as pointed out,
R36A has become relatively it has never spontaneously reverted.
(11). Strain
fixed in the R
phase and has never spon- Although the significance of the fol-
taneously reverted to the Type II S form. lowing fact will become apparent later
Moreover, repeated attempts to cause it on, it must be mentioned here that pneu-
to revert under the conditions just men- mococcal cells possess an enzyme capable
tioned have in all instances been unsuc- of destroying the activity of the trans-
cessful. forming principle. Indeed, this enzyme
has been found to be present and highly
The reversible conversion of S^R active in the autolysates of a number of
within the limits of a single type is different strains. The fact that this intra-
quite different from the transforma- cellular enzyme released during auto-
is
Figs. 1 and 2. (1) Colonies of the R variant (R36A) derived from Pneumococcus
type II. Plated on blood agar from a culture grown in serum broth in the absence of
the transforming substance. X3.5. (2) Colonies on blood agar of the same cells after
induction of transformation during growth in the same medium with the addition
of active glistening, mucoid colonies shown are characteristic of Pneumococcus Type
III and readily distinguishable from the small, rough colonies of the parent strain R
illustrated in Fig. 1. X3.5. (The photograph was 7nade by Mr. Joseph B. Haiilenbeek.)
moved from the collecting cylinder and cess of capsular polysaccharide together
resuspended in approximately 150 cc. of with much of the protein, ribonucleic
chilled saline (0.85 per cent NaCl), and acid, and somatic "C" polysaccharide.
care is taken that all clumps are thor- Quantitative titrations of transforming
180 AVERY, MACLEOD, MCCARTY
activity have shovv^n that not more than polysaccharide in dilutions as high as
10 to 15 per cent of the active material 1:6,000,000. The enzymatic breakdown of
is lost in the washing, a loss which is small the polysaccharide is usually complete
in comparison to the amount of inert sub- within 4 to 6 hours, as evidenced by the
stances which are removed by this pro- loss of serological reactivity. The digest
cedure. is then precipitated in 3 to 4 volumes of
After the final washing, the cells are ethyl alcohol, and the precipitate is re-
extracted in 150 cc. of sahne containing dissolved in 50 cc. of sahne. Deprotein-
sodium desoxycholate in final concentra- ization by the chloroform process is
tion of 0.5 per cent by shaking the mix- again used to remove the added enzyme
ture mechanically 30 to 60 minutes. The protein and remaining traces of pneumo-
cells are separated by centrifugation, and coccal protein. The procedure is repeated
the extraction process is repeated 2 or 3 until no further film of protein-chloro-
times. The desoxycholate extracts pre- form gel is visible at the interface.
pared in this manner are clear and color- —
Alcohol Fractionation. Following de-
less.These extracts are combined and proteinization and enzymatic digestion of
precipitated by the addition of 3 to 4 the capsular polysaccharide, the material
volumes of absolute ethyl alcohol. The isrepeatedly fractionated in ethyl alcohol
sodium desoxycholate being soluble in as follows. Absolute ethyl alcohol is
alcohol remains in the supernatant and is added dropwise to the solution with con-
thus removed at this step. The precipitate stant stirring. At a critical concentration
forms a fibrous mass which floats to the varying from 0.8 to 1.0 volume of alcohol
surface of the alcohol and can be re- the active material separates out in the
moved directly bv lifting it out with a form of fibrous strands that wind them-
spatula. The excess alcohol is drained selves around the stirring rod. This pre-
from the precipitate which is then re- cipitate is removed on the rod and
dissolved in about 50 cc. of saline. The washed in a 50 per cent mixture of al-
solution obtained is usually viscous, cohol and saline. Although the bulk of
opalescent, and somewhat cloudy. active material is removed by fractiona-
Deprotei7iizatio7i a?id Re?fioval of Cap- tion at the critical concentration, a small
sular —
Polysaccharide. The solution is but appreciable amount remains in solu-
then deproteinized by the chloroform tion. However, upon increasing the con-
method described by Sevag (12). The centration of alcohol to 3 volumes, the
procedure is repeated 2 or 3 times until residual fraction is thrown down together
the solution becomes clear. After this with inert material in the form of a floc-
preliminary treatment the material is re- culent precipitate. This flocculent pre-
precipitated in 3 to 4 volumes of alcohol. cipitate is taken up in a small volume of
The precipitate obtained is dissolved in saline (5 to 10 cc.) and the solution again
a larger volume of saline (150 cc.) to fractionated by the addition of 0.8 to 1.0
which is added 3 to 5 mg. of a purified volume of alcohol. Additional fibrous
preparation of the bacterial enzyme capa- material is obtained which is combined
ble of hydrolyzing the Type 111 capsular with that recovered from the original
polysaccharide (13). The mixture is in- solution. Alcoholic fractionation is re-
cubated at 37°C., and the destruction of peated 4 to 5 times. The yield of fibrous
the capsular polysaccharide is determined material obtained by this method varies
by serological tests with Type III anti- from 10 to 25 mg. per 75 liters of culture
body solution prepared by dissociation of and represents the major portion of ac-
immune precipitate according to the tive material present in the original crude
method described by Liu and Wu (14). extract.
The advantages of using the antibody —
Effect of Temperature. As a routine
solution for this purpose are that it does procedure all steps in purification were
not react with other serologically active carried out at room temperature unless
substances in the extract and that it selec- specifically stated otherwise. Because of
tively detects the presence of the capsular the theoretical advantage of working at
AVERY, MACLEOD, MCCARTY 181
low temperature in the preparation of crease in solubility and loss of activity.
biologically active material, the purifica- The activity of the transforming
tion of one lot (preparation 44) was car- principle in crude extracts withstands
ried out in the cold. In this instance all
heating for 30 to 60 minutes at 65 °C.
the above procedures with the exception
Highly purified preparations of active
of desoxycholate extraction and enzyme
material are less stable, and some loss
treatment were conducted in a cold room
maintained at 0-4 °C. This preparation
of activity occurs at this temperature.
proved to have significantly higher activ- A quantitative study of the effect of
ity than did material similarly prepared heating purified material at higher
at room temperature. temperatures has not as yet been made.
Desoxycholate extraction of the heat- Alio way (6), using crude extracts pre-
killed cells at low temperature is less pared from Type III pneumococcal
efficient and yields smaller amounts of cells,found that occasionally activity
the active fraction. It has been demon-
could still be demonstrated after 10
strated that higher temperatures facilitate
minutes' exposure in the water bath to
extraction of the active principle, al-
temperatures as high as 90°C.
though activity is best preserved at low
temperatures. The procedures mentioned above
were carried out with solutions ad-
Analysis of Purified Traiisforjjiifig justed to neutral reaction, since it has
Material been shown that hydrogen ion con-
General Properties.—SaVme solutions centrations in the acid range result in
containing 0.5 to 1.0 mg. per cc. of progressive loss of activity. Inactiva-
the purified substance are colorless and tion occurs rapidly at pH 5 and below.
clear in diffuse light. However, in Qualitative Cheiuical Tests—'Xht
strong transmitted light the solution is purified material in concentrated solu-
not entirely clear and when stirred ex- tion gives negative biuretand Millon
hibits a silky sheen. Solutions at these tests.These tests have been done di-
concentrations are highly viscous. rectly on dry material with negative
Purified material dissolved in physi- results. The Dische diphenylamine re-
ological salt solution and stored at 2- action for desoxyribonucleic acid is
4°C. retains its activity in undimin- strongly positive. The orcinol test
ished titer for at least 3 months. How- (Bial) for ribonucleic acid is weakly
ever, when dissolved in distilled water, positive. However, it has been found
it rapidly decreases in activity and be- that in similar concentrations pure
comes completely inert within a few preparations of desoxyribonucleic acid
days. Saline solutions stored in the of animal origin prepared by different
frozen state in a CO2 ice box ( — 70°C.) methods give a Bial reaction of cor-
retain full potency for several months. responding intensity.
Similarly, material precipitated from Although no specific tests for the
saline solution by alcohol and stored presence of lipid in the purified ma-
under the supernatant remains active terial have been made, it has been
over a long period of time. Partially found that crude material can be re-
purified material can be preserved by peatedly extracted with alcohol and
drying from the frozen state in the ether at — 12°C. without loss of activ-
lyophile apparatus. However, when ity. In addition, as will be noted in the
the same procedure is used for the preparative procedures, repeated alco-
preservation of the highly purified hol precipitation and treatment with
substance, it is found that the material chloroform result in no decrease in bi-
undergoes changes resulting in de- ological activity.
182 AVERY, MACLEOD, MCCARTY
Elementary Chemical
Ayialysis}— results are presented in Table I. The
Four were ana-
purified preparations nitrogen-phosphorus ratios vary from
lyzed for content of nitrogen, phos- 1.58 to 1.75 with an average value of
phorus, carbon, and hydrogen. The 1.67 which is in close agreement with
Table I
Preparation No.
AVERY, MACLEOD, MCCARTY 183
tested. The alkaline phosphatase activ- is compared with their capacity to
ity of these preparations was deter- destroy the transforming principle.
mined by their action on yS-glycero- Analysis of these results shows that
phosphate and phenyl phosphate, and irrespective of the presence of phos-
the esterase activity by their capacity phatase or esterase only those prepara-
to split tributyrin.Since the highly tions shown to contain an enzyme
purified transforming material isolated capable of depolymerizing authentic
from pneumococcal extracts was samples of desoxyribonucleic acid
found to contain desoxyribonucleic were found to inactivate the trans-
acid, thesesame enzymes were tested forming principle.
for depolymerase activity on known Greenstein and Jenrette (18) have
samples of desoxyribonucleic acid iso- shown that tissue extracts, as well as
latedby Mirsky ^ from fish sperm and the milk and serum of several mam-
mammalian tissues. The results are malian species, contain an enzyme sys-
summarized in Table II in which the tem which causes depolymerization of
phosphatase, esterase, and nucleode- desoxv^ibonucleic acid. To this en-
polymerase activity of these enzymes zyme system Greenstein has later
Table II
Table III
Table IV
Transformin
188 AVERY, MACLEOD, MCCARTY
specific transformation been experi- duced in turn are chemically distinct
mentally induced hi vitro by a chem- and biologically specific in their ac-
ically defined substance. tion and that both are requisite in
Although the observations are lim- determining the type specificity of the
ited to a single example, they acquire cell of which they form a part.
broader significance from the work of The experimental data presented in
earlier investigators who demonstrated this paper strongly suggest that nu-
the interconvertibility of various pneu- cleic acids, at least those of the desoxy-
mococcal types and showed that the ribose type, possess different specifi-
specificity of the changes induced is evidenced by the selective ac-
cities as
in each instance determined by the tion of the transforming principle.
particular type of encapsulated cells Indeed, the possibility of the existence
used to evoke the reaction. From the of specific differences in biological
point of view of the phenomenon in behavior of nucleic has pre-
acids
general, therefore, it is of special in- viously been suggested (23, 24) but
terest that in the example studied, has never been experimentally demon-
highly purified and protein-free ma- strated owing in part at least to the
terial consisting largely, if not exclu- lack biological methods.
of suitable
sively, of desoxyribonucleic acid is The techniques used in the study of
capable of stimulating unencapsulated transformation appear to afford a sen-
R variants of Pneumococcus Type II sitive means of testing the validity of
to produce a capsular polysaccharide this hypothesis, and the results thus
identical in type specificity with that far obtained add supporting evidence
of the cells from which the inducing in favor of this point of view.
substance was isolated. Equally strik- If it is ultimately proved beyond
ing is the fact that the substance reasonable doubt that the transforming
evoking the reaction and the capsular activity of the material described is
substance produced in response to it actually an inherent property of the
are chemically distinct, each belonej- nucleic acid, one must still account on
ing to a wholly different class of a chemical basis for the biological spe-
chemical compounds. cificity of its action. At first glance,
The inducing substance, on the basis immunological methods would appear
of its chemical and physical properties, to offer the ideal means of determin-
appears to be a highly polymerized ing the differential specificity of this
and viscous form of sodium desoxy- group of biologically important sub-
ribonucleate. On the other hand, the stances. Although the constituent units
Type III capsular substance, the syn- and general pattern of the nucleic acid
thesis of which is evoked by this trans- molecule have been defined, there is
forming agent, consists chiefly of a as yet relatively little known of the
non-nitrogenous polysaccharide con- possible effect that subtle differences
stituted of glucose-glucuronic acid in molecular configuration may exert
units linked in glycosidic union (22). on the biological specificity of these
The presence of the newly formed substances. However, since nucleic
capsule containing this type-specific acids free or combined with histones
polysaccharide confers on the trans- or protamines are not known to func-
formed cells all the distinguishing tion antigenically, one would not an-
characteristics of Pneumococcus Type ticipate that such differences would be
III. Thus, evident that the induc-
it is revealed by immunological techniques.
ing substance and the substance pro- Consequenth', it is perhaps not surpris-
AVERY, MACLEOD, MCCARTY 189
ing that highly purified and protein- However, from a bacteriological point
free preparations of desoxyribonucleic of view, numerical estimations based
acid,although extremely active in in- on colony counts might prove more
ducing transformation, showed only misleading than enlightening because
faint trace reactions in precipitin tests of the aggregation and sedimentation
with potent Type III antipneumococ- of the R cells agglutinated by the anti-
cus rabbit sera. serum in the medium. Attempts to
From these limited observations it induce transformation in suspensions
would be unwise to draw any conclu- of resting cells held under conditions
sion concerning the immunological inhibiting growth and multiplication
significance of the nucleic acids until have thus far proved unsuccessful, and
further knowledge on this phase of the it seems probable that transformation
ent lots of immune serum and occurred lysis and in the free state is capable of
more frequently in antipneumococcal rapidly and completely destroying the
horse serum than in corresponding sera activity of the transforming agent. It
of immune rabbits. The irregularity would appear, therefore, that during
and broad cross reactions encountered the logarithmic phase of growth when
led these investigators to express some cell division is most active and auto-
doubt as to the immunological signifi- lysis least apparent, the cultural con-
cance of the results. Unless special ditions are optimal for the maintenance
immunochemical methods can be de- of the balance between maximal re-
vised similar to those so successfully activity of the R
cell and minimal
used in demonstrating the serological destruction of the transforming agent
specificity of simple non-antigenic through the release of autolytic fer-
substances, it appears that the tech- ments.
niques employed in the study of trans- In the present state of knowledge
formation are the only ones available any interpretation of the mechanism
at the present for testing possible dif- involved in transformation must of
ferences in the biological behavior of necessity be purelv^ theoretical. The
nucleic acids. biochemical events underlying the
Admittedly there are many phases phenomenon suggest that the trans-
of the problem of transformation that forming principle interacts with the R
require further study and many ques- cell giving coordinated series
rise to a
tions that remain unanswered largely of enzymatic reactions that culminate
because of technical difficulties. For in the synthesis of the Type III capsu-
example, it would be of interest to lar antigen. The experimental findings
know the relation between rate of re- have clearly demonstrated that the in-
action and concentration of the trans- duced alterations are not random
forming substance; the proportion of changes but are predictable, always
transformed to those that remain
cells corresponding in type specificity to
unaffected in the reaction system. that of the encapsulated cells from
190 AVERY, MACLEOD, MCCARTY
which the transforming substance was some specific protein that serves as a
isolated. Once transformation has oc- "pabulum" and enables the R form to
curred, the newly acquired character- manufacture a capsular carbohydrate.
istics are thereafter transmitted in More recently the phenomenon has
series through innumerable transfers been interpreted from a genetic point
in artificial media without any further of view (26, 27). The inducing sub-
addition of the transforming agent. stance has been likened to a gene, and
Moreover, from the transformed cells the capsular antigen which is produced
themselves, substance of identical
a in response to it has been regarded as
activity be recovered in
can again a gene product. In discussing the phe-
amounts far in excess of that originally nomenon of transformation Dobzhan-
added to induce the change. It is evi- sky (27) has stated that "If this trans-
dent, therefore, that not only is the formation is described as a genetic
capsular material reproduced in suc- mutation— and it is difficult to avoid
cessive generations but that the pri- so describing it— we are dealing with
mary factor, which controls the oc- authentic cases of induction of specific
currence and specificity of capsular mutations by specific treatments. ."
. .
acids of this type must be regarded not 4. Evidence is presented that the
able under appropriate cultural condi- 5. Dawson, M. H., and Sia, R. H. P., /. Exp.
Med., 1931, 54:681.
tions of inducing the transformation of
6. Alloway, J. L., /. Exp. Med., 1932, 55:91;
unencapsulated R variants of Pneu-
1933,57:265.
mococcus Type II into fully encap-
7. Berry, G. P., and Dedrick, H. M., J.
sulated cells of the same specific type Bact., 1936, 31:50.
as that of the heat-killed microorgan- 8. Berry, G. P., Arch. Path., 1937, 24:533.
isms from which the inducing material 9. Hurst, E. W., Brit. J. Exp. Path., 1937,
was recovered. 18:23. Hoffstadt, R. E., and Pilcher,
K. S., /. Infect. Dis., 1941, 68:67. Gard-
2. Methods for the isolation and
ner, R. E., and Hyde, R. R., /. Infect.
purification of the active transforming Dis., 1942, 71:47. Houlihan, R. B., Proc.
material are described. Soc. Exp. Biol, and Med., 1942, 51:259.
3. The data obtained by chemical, 10. MacLeod, C. M., and Mirick, G. S., /.
Bact., 1942, 44:277.
enzymatic, and serological analyses
together with the results of prelimi-
11. Dawson, M. H., /. Exp. Med., 1928, 47:
577; 1930, 51:99.
nary studies by electrophoresis, ultra-
12. Sevag, M. G., Biochevi. Z., 1934, 273:419.
centrifugation, and ultraviolet spec- Sevag, M. G., Lackman, D. B., and
troscopy indicate that, within the Smolens, J., /. Biol. Chem., 1938, 124:
limits of the methods, the active frac- 425.
tion contains no demonstrable protein, 13. Dubos, R. J., and Averv, O. T., /. Exp.
Med., 1931, 54:51. Dubos, R. J., and
unbound lipid, or serologically reactive
Bauer, J. H., /. Exp. Med., 1935, 62:271.
polysaccharide and consists princip-
14. Liu, S., and Wu, H., Chinese J. Physiol.,
ally, if not solely, of a highly polymer-
1938, 13:449.
ized, viscous form of desoxyribonu- 15. Martland, M., and Robison, R., Biochetn.
cleic acid. J., 1929, 23:237.
192 LEDERBERG AND TATUM
16. Albers, H., and Albers, E., Z. physiol. 24. Mirsky, A. E., in Advances in enzym-
Cheni., 1935, 232:189. ology and related subjects of bioche?n-
17. Levene, P. A., and Dillon, R. T., /. Biol.
istry, (F. F. Nord and C. H. Werkman,
Others. Sex plays its greatest role in heredity in that it provides for
the recombination of genes into new., ^^experimentar series, which
jnake up the raw 'material itpoji which natural selection acts. Leder-
berg and Tatinn reverse the origi?ial procedure in that they utilize
the phenonie7wn of recombiTiation to demo?istrate the existe?ice of
sex i?i bacteria. Since bacteria reproduce rapidly and abu?ida?nly the ,
Up to this point, many of the papers that have been included have
already demojistrated their sigjiificance by the ways in which they
have affected the directions a?id aijiis of the field of ge?ietics. They
have created new problems, pointed out new pathways, and pro-
vided new methods that proved fruitful i?i genetic analysis and
understanding. Obviously, it is difficult to select such papers from
the literature of the past few years, because a certain amount of
time is always necessary to permit a paper to reveal its basic impor-
tance.
This paper arid the one by Sturteva?it (p. 259) strongly indi-
cate the existence of still another aspect of genetics, the sigjiificance
of which lies in the relationship of man to mankind. The geneticist
has suddefily found hi?/iself in possession of infovDiation of the
utmost importance in our moder?i world, and at the saiJie time finds
himself pai?ifidly lacking in information most urgently needed for
GENETICS CONFERENCE 195
decisions of far-reaching social significance. Biologists, for the most
part, lead rather sheltered lives. Their discoveries are of interest
prificipally to their inmiediate colleagues, although coimnercial as-
pects of many biological have beco7ne increasingly
discoveries
important. This is no longer true of the geneticist. His activities are
of public interest; his debates are reported in the world'' s press. His
discoveries are widely publicized, and are often misinterpreted and
garbled in the transmission. He has moved, ivilly-nilly, into the
public eye. The consequences to society of discoveries that are likely
to affect the hwnan genetic constitution ?nust be i?ivestigated and
determined, and the Jieiv trend for genetic research is clear-cut.
The first of these two papers illustrates another general biological
phenofnenon, that of team research. This is the report of a group
of research workers, not of a single man. Research today in any
area is likely to demand too much specialized k?iowledge and back-
groimd for afiy i?idividual to encompass, a?id as a consequence several
specialists cooperate to bring their pooled knowledge to bear on a
problejfi.
This paper introduces the proble?n of the effects of the atomic
bomb on the genetics of mankind. It is perhaps one of the most
active and most heavily supported areas of research today.
BARBARA MCCLINTOCK
information that has led to the con- going the "mutational" events. The Ds
clusions to be presented. These conclu- locus is composed of this kind of ma-
sions are concerned with the origin of terial.
mutable loci, the events occurring at Various types of alterations are ob-
these loci that result in a change in served as the consequence of events
phenotypic expression, the reasons for occurring at the Ds locus. Some of
changes in the frequency of visible these alterations resemble the effects
mutations at these loci, the factors con- produced by x-rays, ultra-violet light,
trolling the time when mutations will chemicals, etc. They involve chromo-
occur, the production of mutations at some breakage and fusion. The breaks
the ai locus in maize without Dt being are related, however, to events occur-
present, and heterochromatin as the ring at this one specific locus in the
probable controlling factor. chromosome— the Ds locus. The Ds
A fortunate discovery was made designation was given to this locus be-
early in the study of the mutable loci cause the dissociation, now known to
which proved to be of singular im- be related to dicentric and associated
portance in showing the kinds of acentric chromatid formation, was
events that are associated with their recognized before the other events oc-
origin and behavior. A
locus was found curring at Ds had been disclosed. Some
in the short arm of chromosome 9 at of the events occurring at Ds, when
which breaks were occurring in considered without reference to all the
somatic cells. The time and frequency known events, would not by them-
of the breakage events occurring at selves suggest that changed conditions
this Ds (Dissociation) locus appeared at this locus are associated with a
to be the same asthe time and fre- breakage-inducing phenomenon. All
quency of the mutation-producing of them can be explained, however, by
events occurring at some of the mu- the assumption that one kind of altera-
table loci.^ An extensive study of the tion of the inserted chromatin (the
4 The annual reports of the author, ap- chromatin of the Ds locus) takes place,
pearing Yearbooks of the Carnegie //z-
in the and that the various kinds of changes
stitutionof Washington, 41-48, 1942-1949, observed represent consequences of
contain more detailed summaries of some of
this one altered condition. This condi-
the observations that are described in this
papyer. tion is assumed to be a stickiness of the
MCCLINTOCK 203
materials composing the Ds locus, (6) above, and those with less of this
which arises only at precise times in material showing high frequencies of
the development of a tissue. The con- consequence (2) above.
trol of the timing of this changed It is from the transpositions of Ds
condition will be considered shortly. that some of the new mutable loci may
The reasons for assuming the change arise. The mechanism of transposition
curring at this mutable c locus. The of the progeny of the original self-
dicentric chromatid formations were pollinated cultures, other mutable c
not associated with the appearance of loci have arisen from previously nor-
a C phenotype, suggesting that the in- mal C loci. One of these, c-7ii2, shows
serted inhibiting material composing the type (c) expression of variegation,
Ds may be situated proximal to the C which difi^ers markedly from that
locus. Several cases of transposition of shown by c-7nl. A wide range of
Ds from this location to still another quantitative expression, for at least two
location in the short arm of chromo- difi'erent reactions associated with
some 9 were recognized. In each case, aleurone pigment formation, appears as
a restored C action was associated with the consequence of various mutations
a disappearance of Ds activity at the C at this locus. The intermediate alleles,
locus and the appearance of Ds activ- full wild-type alleles and some alleles
ity at the new position. The changes showing even stronger phenotypic ex-
in state of Ds at this mutable c locus pressions than the wild-type from
(event (7) above) are particularly sig- which it arose, are produced by muta-
nificant since it has been determined tions at c-m2.The mutations are often
that a specific change in state of Ds expressed twin sectors, the depth of
as
is often accompanied by a specific color in one sector being greater than
change in the frequency of c to C mu- that in the sister sector. These twin
tations. sectors may reflect a single mutation-
The origin and behavior of this mu- producing event at the c-?n2 locus that
table c locus has been interpreted as involved both sister chromatids. It has
follows: Insertion of the chromatin also been determined that chromosome
composing Ds adjacent to the C locus breakage may occur at this locus.
is responsible for complete inhibition The phenotypic expressions result-
of the action of C
Removal of this ing from mutations of c-uil and c-ml
foreign chromatin can occur. In many are clearly quite different. That this
MCCLINTOCK 205
difference may be related to differ- present in a nucleus, no mutation-pro-
ences in the inserted chromatin is sug- ducing events occur at c-ml, c-ni2 or
gested by the appearance of a mutable "tcx-fnl; nor are any chromosome
wx locus arising from a IVx locus in a breakas^e events detected at Ds, for no
gamete of a plant carrying c-7n2 U'a', ( such events occur. As an example of
starch of endosperm stains blue with this interaction it may be stated that
iodine; wx, recessive allele, starch stains c-uil has been maintained in cultures
red with iodine; located in short arm of having no Ac locus for several genera-
chromosome 9, proximal to C). The tions, and has given completely color-
type of variegation expressed by this less aleurone with no evidence of c to
substance that induced c-7}?2 had oc- appropriate crosses. Thus a series of
curred. Such an event is suspected stable recessive mutations or stable
from the known transposition ca- alleles of a mutable locus may be iso-
pacities of this material. lated and maintained (if the chromo-
In thisreport, Ds, c-ml, c-m2 and some complement is normal, see be-
ixix-vil have been used as illustrations low). When Ac is returned to the nu-
of newly arising mutable loci because cleus, however, instability may again
all of them require an activator and appear.
all respond to the same activator. This The dosage action of Ac may be
activator has been designated Ac. Ex- studied in the diploid plant or in the
tensive studies of Ac have shown that endosperm tissue of the ker-
triploid
it is inherited as a single unit. It shows, nels. When
marked dosage effects are
however, a very important character- produced by a particular state of Ac,
istic not exhibited in studies of the they are registered alike in both the
inheritance of the usual genetic fac- plant and the endosperm tissues; the
tors. This characteristic is the same as higher the dose of Ac, the more de-
that shown by Ds. Transposition oi Ac layed is the time of occurrence of mu-
takes place from one position in the tations at the /ic-controlled mutable
chromosomal complement to another— loci. Ac determines, therefore, not
very often from one chromosome to only the mutation process at these mu-
another. Again, as in D^, changes in table loci but also the time at which the
state may occur at the Ac locus. These mutations occur, the different states of
changes in state are of two main types: Ac giving different times of occur-
either changes that resemble the rence in 1, 2 or 3 doses. The action of
known effects produced by different Ac on the mutable loci it controls has
doses of the Ac locus from which it been described. It is believed that this
was derived, or changes that result in action produces a stickiness of the in-
a decidedly altered time of action and hibiting materials adjacent to the af-
dosage response of Ac. Ac may be fected loci. With reference to Ds, the
detected and its action studied by ob- observed consequences of this sticki-
serving the mutations occuring at the ness have been enumerated. This
mutable loci requiring its presence for physical change probably takes place
mutability to be expressed. It should in the inserted inhibiting materials at
be emphasized that when no Ac is all the .(4^-controlled mutable loci at
206 MCCLINTOCK
the same time in the same cell. This frequency of appearance of visible
latter conclusion rests on the observa- mutations.The study of Ac has indi-
tion that mutations occur concomit- cated the nature of the control of the
antly at two or more /^c-controlled time when the mutations will occur at
mutable loci when these are present these mutable loci. The different
in the same nucleus. The similarity in doses oi Ac together with the changed
the type of inheritance and the be- states of Ac control the time of occur-
havior of Ds and Ac has been indicated rence of these mutations. The changes
above. Another similarity is that in time of occurrence of visible mu-
changes in state, loss or transposition tations are thus reflections of changes
of Ac occur at the same time that in dosage or changes in state of Ac.
changes take place at the Ac-con- The mutable loci that require no ac-
trolled mutable loci. It would appear tivator show the same kinds of expres-
that the changes in the physical prop- sion of variegation as do the activator-
erties of the specific inhibiting chro- requiring mutable loci. It has been
matin at the mutable loci and at Ac shown that the changes occurring at
itself are of the same nature, and that Ac are much the same as those occur-
all are expressions of the primary gene- ring at Ds. Thus, Ac or Ac-Yike. loci,
tic action of the material composing could be responsible for the origin of
Ac. It is suspected that Ds and Ac are new mutable loci when transposed to
composed of the same or similar types a position adjacent to a gene whose
of material. The possible composition inhibited action could be detected by a
of this material will be considered visible change in phenotype. Dosage
shortly. action could be exhibited by such
The study of Ac and the ^^-con- autonomous mutable loci, as well as
trolled mutable loci has made it possi- various "changes in state," reflected
ble to interpret the many patterns of by changes in the phenotype expres-
variegation exhibited by mutable loci. sion and the time and frequency of
The variegated pattern an expression
is occurrence of visible mutations of the
of the time and frequency of occur- affected genes. The study of the be-
rence of visible changes in the pheno- havior of Ds in its several states makes
type. The frequency of appearance of it possible to reinterpret the variega-
a visible mutation need not reflect the tion patterns in Drosophila, which in
frequency of the events that occur at some cases appear to be associated with
a mutable locus, as the study of c-7fil loss of segments of chromosomes and
has clearly revealed. The muta-
visible in other cases appear to be associated
tions reflect only the frequency of one with changes in the degree of action
or several particular consequences of of the genes involved. It also makes it
one primary type of event occurring possible to interpret the reported "po-
to the inhibiting material adjacent to sition-effect" in Oenothera, because
the affected gene. The changes in state the events responsible for the changes
of this inhibiting material that arise as in phenotype and the appearance of
one of the consequences of the pri- duplications and deficiencies in this or-
mary event, lead to changes in the ganism appear to be the same or simi-
relative frequency of the consequences lar to those described for Ds in maize.'^
of this event when it again occurs in The possible composition of Ac may
future cell and plant generations. Such •">
Catcheside, D. G., /. Genet. 38:345-352,
changes in state are reflected either in 1939; Ibid. 48:31-42, 1947; Ibid. 48:99-110,
increases or decreases in the relative 1947.
MCCLINTOCK 207
now be considered. Until recently, the phila appear to affect the functioning
investigation was not focused on this of the centrically placed heterochro-
problem. It is believed, however, that matin. For example, some of the Mi-
this material is probably heterochro- nutes bring about chromosome elimi-
matin. This statement is based, in part, nation and "somatic-crossinijover,"
on the evident homologies in the ex- both of which may well be related to
pression of variegation in maize and adhesions of specific heterochromatin
Drosophila, but is more convincingly that occur at certain times in develop-
suggested by the results of a prelimi- ment. To answer the above questions,
'^
nary experiment focused on the induc- plants homozygous for a] and having
tion of mutations at the ai locus in no Dt locus (designated dt by
maize when the known Dt (Dotted) Rhoades) were crossed by plants sim-
locus is absent. The action of Dt in ilarly constituted with reference to a^
chromosome 9 on the ai locus in chro- and dt but carrying a rearrangement of
mosome 3 is very much the same as the the short arm of chromosome 9 that
action of Ac on the mutable loci it would introduce a chromosome 9 with
controls.® The similarities are too great a newly broken end into many of the
to be dismissed as being due to causally primary endosperm nuclei in the given
unrelated phenomena. The Dt locus cross.^ Breakage-fusion-bridge cycles
activates the ^i locus;mutations to involving such a chromosome 9 with a
higher Ai alleles occur (A^, colored newlv^ broken end would occur during
aleurone; a\, colorless aleurone, reces- the development of the kernels. Some
sive to Ax). Without Dt in the nucleus, of these broken chromosomes 9 would
^1 has been shown to be completely carry a knob, and this knob could then
stable. Dt is located in the heterochro- be subjected to modifications as a
?}iatic knob terminating the short arm consequence of the breakage events.
of chromosome 9. The suspicion is im- If some of these modifications gave rise
mediately aroused: Is Dt action caused to the same conditions that were pres-
by some modification of the hetero- ent at Dt, mutations from a^ to A^
chromatic knob in chromosome 9? If could appear in some of the kernels re-
so, could this modification be produced sulting from the cross. A
large number
anew by subjecting 9
a chromosome of crosses of this type were made. The
to the breakage-fusion-bridge cycle? results were positive with respect to
Would the effective alterations of the inducing mutations of a-i to A^. A small
knob arise directly because of the in- number of from
the kernels resulting
duced changes, or would they be pro- these crossesshowed mutations of a^
duced secondarily by some other in- MO Ax. Often, only a single small Ax
duced structural alteration, either spot was present on the kernel. Sev-
within the short arm of chromosome 9 eral of the kernels, however, had a
or elsewhere, that would upset, in pattern of mutations of ai to Ax that
some way, the normal functioning of was indistinguishable from that pro-
the knob substance and thus bring duced by Dt. These kernels could not
about an alteration in its action? This have arisen by contamination, for
last question is pertinent because some stocks with the known Dt locus had
of the structural alterations in Droso- never been obtained and thus no plants
with this locus could have been pres-
«Rhoades, M. M., Genetics 23:377-397,
1938. Cold Spring Harbor Symposia Quant.
Biol. 9:138-155, 1941; ¥roc. Natl. Acad. Sci. 7 Stern, C, Genetics, 21:625-730, 1936.
31:91-95, 1945. 8 McClintock, B., Ibid. 26:234-282, 1941.
208 MCCLINTOCK
ent in the field. Furthermore, the stock and the relative effectiveness of genie
having ai and dt, originally obtained action. There can be little question that
from Rhoades, had been grown for transpositions of both Ds and Ac oc-
several years. A number of sib crosses cur and that the time of their occur-
were made each year and no muta- rence in the development of a tissue is
tions of ai to Ai were observed in the under precise control. This control is
kernels on these ears. determined by the number of Ac loci
The facts (1) that Dt is located in present and their organization and pos-
the heterochromatic knob of chromo- sibly their position in the chromosome
some 9, (2) that the effect it produces complement. Is this transposition of
can be recreated by subjecting chro- heterochromatin? Is it a reflection of a
mosome 9 to the breakage-fusion- process that normally occurs in nuclei?
bridge cycle, (3) that Ac appeared in Is it responsible for controlling the
stocks that had undergone this cycle, rates and types of exchange that occur
and (4) that Ac and Dt are alike in between nucleus and cytoplasm? Is it
their respective actions, all point to usually an orderly mechanism, which
heterochromatin as the material com- is related to the control of the pro-
posing Ac. The burst of new mutable cesses of differentiation? If so, induced
loci which appeared in the self-pol- disturbances in quantity and organiza-
linated progeny of plants that had been tion of the heterochromatic elements
subjected to the chromosome type of of the chromosome could give rise to
breakage-fusion-bridge cycle becomes a of alterations reflected both
series
comprehensible if it is considered that in chromosome structure and behavior
the alterations in the quantity or struc- and in genie reactions that could
ture of heterochromatic elements dur- markedly alter phenotypic expres-
ing this cycle were primarily respon- is well known that the vari-
sions.^^ it
sible for the initial appearance of these ous knobs and centromeres may co-
mutable loci. This report has shown alesce in the resting nuclei. This co-
that, once suchloci arise, other mut- alescence is also frequently observed
able loci through transposition
arise both in the somatic and the meiotic
of the inhibiting chromatin substances prophases. Are the transpositions and
to other loci which in turn become the changes in state of Ac products of
mutable. this coalescence? This is suspected be-
Why should altered heterochroma- cause of the frequent transpositions of
tin be responsible for initiating such a Ac from one chromosome to another.
chain of events? To answer this ques- It may be considered that these
tion, attention must be centered on the speculations with regard to hetero-
action of heterochromatin in the nor- chromatin behavior and function have
mal nucleus. That it is associated with been carried further than the evidence
the exchange of materials between nu- warrants. This may be true; but it
cleus and cytoplasm has been indi- cannot be denied that one basic kind
cated.^ Changes in quantity, quality or of phenomenon appears to underlie the
structural organization of heterochro- expression of variegation in maize.
matic elements may well alter the kind
and/or degree of particular exchanges 10 This report deals only with the origin
that occur, and in this way control the and behavior of mutable loci arising in these
cultures. A number of other heritable
chromosome organization and the kind changes are also arising. Many are associated
with marked alterations in morphological
» Vanderlyn, L., Bot. Rev. 15:507-582, 1949. characters.
HOROWITZ AND LEUPOLD 209
In many cases, there can be little qucs- sion of variegation in Drosophila. That
tion about the similarities in expression a heterochromatic element likewise is
of variegation in Drosophila and responsible for the origin and behavior
maize. A heterochromatic element has of variegation in maize has not been
repeatedly been found to be basically proved, although it is indicated, as the
associated with the origin and expres- analysis of Dt has shown.
The assumption that a given gene production of but a single enzyme has
is involved, in a primary way, in the been implicit in most speculations on
210 HOROWITZ AND LEUPOLD
the nature of gene action since Cue- pression of a mutation is necessarily
not's time. As a result of the investiga- restricted to a particular structure or
tions of the last ten years stemming function of the organism. The ulti-
from the discovery of nutritional mu- mate effect of a mutation is the result
tants in Nemospora by Beadle and of an enormous magnification of the
Tatum (1941), one is now in a position initial gene change, brought about
to scrutinize this supposition more through a system of reactions which,
closely than was previously possible. originating at the gene, rapidly
Specifically, we are in a better position branches out in various directions and
to trace the consequences of the hypo- coalesces with similar networks deriv-
thesis and of its various alternatives, ing from other loci to form a reticu-
and to appraise the evidence which lum of as yet indeterminate extent and
may have a bearing on it. In this paper complexity. It is impossible to decide
we propose to examine some of the from the end-effects alone whether the
evidence, deriving from studies on gene has one or many primary func-
Neiirospora, and E. coli,^ which re- tions, since on either assumption a
lates to thisproblem. complex pattern of effects is expected
Before considering the experimental in most cases. In the biochemical mu-
findings, it may be useful to define tants of Neiirospora and other micro-
more explicitly the meaning of the one organisms, the end effects would, if
gene-one enzyme hypothesis. The they could be analysed, undoubtedly
concept is that of a gene whose sole prove to be exceedingly numerous. A
activity aside from self-duplication is mutation which induces a deficiency
that of functioning in the synthesis of of an amino acid, for example, must
a particular enzyme or enzyme pre- secondarily affect the synthesis of
cursor. It not thereby implied that
is virtually every protein of the cell, and
genes at other loci may not also func- an exhaustive enumeration of the end
tion directly in the formation of the effects might well include every struc-
enzyme. This is a completely inde- ture and function of the organism.
pendent problem with which we are It turns out, however, that it is pos-
not concerned here, and regarding sible in such a case to prevent the sec-
which there is little evidence in Neu- ondary damage and the consequent
rospora one way or the other; all that death of the mutant by supplying the
can be said with assurance is that if lacking amino acid. When given a suf-
two or more genes do, in fact, co- ficient quantity of the amino acid the
operate in the production of a given mutant becomes normal in growth
enzyme, then their respective contri- rate, morphology, and fertility. It is
butions must be different. Nor does difficult to escape the conclusion that
the one gene-one enzyme hypothesis the sole function of the gene in this
imply that the final phenotypic ex- case is to play some essential role in
the synthesis of the amino acid. When
1 The studies on E. coltreported in this biochemical analysis of the mutant is
paper were supported by a Grant-in-Aid
carried farther, it is discovered that
from the American Cancer Society upon
recommendation of the Committee on the field of action of the gene is even
Growth of the National Research Council; more circumscribed than might have
by a grant from the Rockefeller Foundation; been supposed: it is restricted to sen-
and by a contract between the Office of
sibly a single chemical step of the
Naval Research, Department of the Navy
and the California Institute of Technology synthesis. Apparently a single reaction
(NR 164010). is abolished in the mutant, while all
HOROWITZ AND LEUPOLD 211
Others proceed normally. It is inferred a sequence of reactions; and it is vir-
that the role of the gene is to function tually excluded in those cases where it
in the synthesis of the enzyme which has been possible, by enzyme studies,
catalyses this reaction. to define the reaction precisely.
It has not yet been possible to ana- A second ground for suspicion of
lyse all, or even the majority, of the the one gene-one enzyme interpreta-
known Neurospom mutants in the de- tion rests on the fact that closer study
tail we have just outlined, while in a of the mutants shows that they are
few cases the analysis has been carried not in all cases restored to a fully nor-
still farther by showing that the mu- mal phenotype when supplied with
tants are in fact lacking in particular the required growth substance. While
enzymes (Mitchell and Lein, 1948; all of the lethal consequences of the
pathway. This interesting idea appears that in these cases the gene performs a
rather improbable, however, in view specific function, possibly independent
of the cumulative evidence from series of its vegetative biochemical function,
of mutants which shows that each gene in connection with zygote formation
can be assigned to a particular step in or maturation. Some recent prelimi-
212 HOROWITZ AND LEUPOLD
nary results which have been obtained suspect that the so-called residual ef-
in our laboratory by Mr. Henry fects are to a large extent the irrepar-
Gershowitz, working with certain me- able side-effects of the primary block.
thionine-requiring strains, indicate, (For further discussion, see Emerson,
however, that the sterility can be over- 1950.)
come by supplementing the medium
with a large quantity of the amino acid THE SELECTION PROBLEM
—at least twice as much as is required At the Cold Spring Harbor Sym-
to produce optimal growth of vegeta- posium of 1946, Delbriick raised a
tive cultures. This suggests that the question as to whether incompatibil-
sterility may result from a high meta- ities with the one gene-one enzyme
bolic requirement for the growth fac- hypothesis could be detected even if
tor during the sexual process, or to a they occurred (see Discussion follow-
lowered permeability to it. A fourth, ing paper by Bonner, 1946b). Del-
and relatively rare, residual effect is briick's argument was based on the
failure of the mutant to attain a normal recognized fact that not all of the mu-
growth rate. This also can characterize tations which are produced can be
an entire class of mutants, as in a cer- detected by the methods usually em-
tain group of strains of the cysteine- ployed for this purpose. Principally
methionine series now under investiga- three classes of biochemical mutants
tion in our laboratory. are not recoverable: (1) those requir-
Everything considered, it is perhaps ing a substance which is absent from
surprising that residual effects are not the so-called "complete" medium used
observed more frequently, since in no for recovering nutritional mutants,
event is it possible, even in theory, to (2) those requiring a substance which
avoid all of the consequences of a is unable to diffuse into the cell, and
has been shown in numerous instances which, for the above, or for any other
that metabolic intermediates may ac-
reasons, are incapable ofgrowing on
cumulate behind the block, sometimes complete medium as mutants which
have lost an indispensable junction.
in spectacular quantities (for review,
The point of Delbriick's argument
see Horowitz, 1950). It would be sur-
was that if any gene has more than one
prising if the presence of abnormal
primary function, it is likelv' that at
concentrations of metabolic intermedi-
least one of these an indispensable
is
ates in the cells did not at times pro-
function; in which case mutation of
duce deleterious side-effects. Actually, the gene would not be detected.
evidence has been obtained both in Now the validity of this argument
Neurospora (Bonner, 1946a) and in depends on the relative frequency of
E. colt (Davis, 1950; Umbarger and indispensable functions. If this fre-
Mueller, 1951) that accumulated inter- quency very high, then the probabil-
is
mutations which cause loss of a dis- functions are in no sense natural cate-
pensable function. It occurred to one gories, but depend largely on the com-
of us (Horowitz, 1948, 1950) that the position of the complete
particular
so-called "temperature mutants" of medium which employed. There is
is
Neiirospora might form the basis of thus no reason to assume that the genes
such a method. Temperature mutants governing these functions differ from
are a class in which the mutant pheno- one another in any fundamental way.
type is fully expressed only in a par- In the second place, among the tem-
ticular temperature range. Generally, perature mutants whose specific re-
such mutants exhibit a growth factor quirement is known there is no indica-
requirement when cultured at 35°, tion that any one kind of nutritional
but grow in its absence at 25°; in a requirement is favored over others.
few cases this relationship is reversed Mutation to temperature alleles ap-
—i.e., the growth factor is required at pears to occur at random among genes
the lower, but not at the higher, tem- controlling known biochemical syn-
perature. In three instances it has been theses (Horowitz, 1950).
found that particular temperature mu- With the information that the fre-
tations behave as alleles of mutations of quency of indispensable gene func-
the usual, temperature-independent tions constitutes approximately 50
214 HOROWITZ AND LEUPOLD
per cent of the total, it becomes pos- too small to give an accurate estimate
sible to estimate the intensity of the of the frequency of indispensable
selection which operates against the functions. It is to the last point that
detection of multifunctional genes. we now turn.
With a random distribution of func-
tions, one-half of genes with a single THE FREQUENCY OF INDISPENSABLE
function will be detectable by the FUNCTIONS IN E. COLI
usual methods, one-fourth of bifunc- It was clearly desirable to obtain a
tional genes, and, in general, (Yi)^ of more reliable estimate of the fre-
72-functional genes. The original mini- quency of indispensable functions, but
mal estimate of 84 per cent of unifunc- to even double the existing number
tional genes, based on the observation of temperature mutants in Neurospora
that this fraction of the mutants re- would be a formidable operation. We
sponds to single growth substances can therefore turned to E. coli K-12, with
now be corrected. A sufficiently close the expectation of recovering large
approximation is given by neglecting numbers of temperature mutants by a
genes with more than two functions, modified penicillin technique (Davis,
and we obtain 73 per cent as the cor- 1948; Lederberg and Zinder, 1948).
rected frequency of unifunctional Providentially, thismethod proved to
genes: be unsuited to our purpose: al-
This value is so high, that in spite of out U.V.-treated cells on minimal me-
the uncertainties in its determination dium and incubating them for 48 hours
it may be regarded as strongly sup- at 40°. The plates are transferred to
porting the conclusion that at least the 25° for an additional 5 days, and the
majority of genes controlling biosyn- colonies which come up during this
thetic reactions in Neiirospora are uni- second period— so-called secondary
functional. There are several obvious colonies— are picked off and tested.
sources of uncertainty in the calcula- This procedure was made feasible by
tions. First, they should be based on a visual method devised by Dr. Leu-
the number of genetically different pold which makes it easier to detect a
mutations, rather than on the total few secondary colonies on a plate con-
number of occurrences; this cannot be taining hundreds of primary colonies.
done at the present time. Second, the Altogether 161 temperature mutants
assumption was made that all of the were obtained by this method. Of
unanalysed mutants, 16 per cent of these, only 37, or 23 per cent, were un-
the total, represent
multi-functional able to grow on the Neurospora com-
genes; this is almost certainly incorrect plete medium at 40° and therefore
and biases the calculations against the represent losses of indispensable func-
one gene-one enzyme theory. Finally, tions. The statistics are shown in Ta-
the number of temperature mutants is ble 1.
HOROWITZ AND LEUPOLD
Table 1
Table 2
Amino A(
216 HOROWITZ AND LEUPOLD
Abscissa Colonies/plate
:
4ooo-
2ooo-
chemical mutants which have been fed essential growth factors. The first two
"syntrophicallv" by the wild types. mechanisms are probably mainly re-
Figure 2 shows that the composition sponsible for the rapid decrease in the
of the population of secondary colo- yield of secondary colonies with in-
nies is also influenced by the popula- creasing plate densities. Their intensity
tion density. It is seen that the yield is evidently quite remarkable. They
of temperature mutants per hundred are unspecific forces, however, and
secondary colonies decreases with in- cannot be expected to influence sys-
creasing densities, indicating that the tematically the relative frequencies of
yield of temperature mutants decreases the two types of temperature mutants
even more rapidly than does the yield which we set out to find. The third
of secondary colonies in general. influence, cross-feeding, is much more
At least three selective forces are dangerous in this respect, although its
at work in these populations: ) com- ( 1 direction and intensity are difficult to
petition for food, (2) probably more predict. The effect of cross-feeding on
important, mutual inhibition by-prod- the selective isolation of the two kinds
ucts of metabolism, and (3) superim- of temperature mutants depends on
posed on these but acting in the both the quantity and quality of the
opposite direction, cross-feeding, or output of growth factors bv^ wild type
syntrophism, the mutual exchange of E. coli. For example, let us assume that
HOROWITZ AND LEUPOLD 217
Abscisso Colonies/plate
Ordinate Temperature mutants/
lo^secondory colonies
15-
lo—
5-
5oo
7"
looo
"T"
l5oo
0.8-
0.6-
0.4-
0.2- "1
-r-
5oo loco l5oo
population density. Vertical lines show the range within which the experimental
points would be expected to fall in 95 per cent of similar experiments. The broken
line shows the weighted mean of the distribution.
they are not individually essential to is not attained, the evidence indicates
survival, or else that they are independ- that this is to be accounted for on the
ent of the genes for their production. basis of secondary effects unrelated to
There is a strong suggestion in this the mode of gene action.
result that the mechanism of biosyn- Biochemical analysis of the mutants
thesis of large molecules, especially has indicated in many instances that
proteins, is not an enzymatic mechan- the effect of the mutation is to block
ism in the usual sense. That is to say, sensibly a single step in the pathway
the protein molecule may not be built, of synthesis of the growth factor. In a
cathedral-like, by a process of accre- few cases it has been possible to show
tion; but rather may be made directly that the mutants are lacking in the
from the amino acids or their simple specificenzyme involved.
derivatives by a single catalyst. question of whether the known
The
It is interesting to note that such a biochemical mutants are a highly se-
mechanism provides a simple explana- lected sample from which multifunc-
tion of another our
essential feature of tionalmutants are excluded by virtue
findings; namely, the mutual independ- of the screening procedure employed
ence of the pathways of synthesis of has been attacked by the temperature
different enzymes. On the hypothesis mutant method. Evidence has been
220 HOROWITZ AND LEUPOLD
presented which indicates that this then given by e^'', the first term of a
method makes possible the recovery of Poisson distribution. Of these, the
mutants without regard to the nature fraction (1— i) are recoverable. The
of the induced biochemical deficiency fraction of recoverable unifunctional
or the composition of the (complete) genes is therefore
medium.
Application of this method has
Pa= (l-i)e-<^
shown that the proportion of biochem-
ical mutants not recoverable by the
Similarly, the fraction of all genes, uni-
usual screening tests may be less than
functional and multifunctional, which
50 per cent in Neurospora and less
can be recovered is
than 25 per cent in E. coli. It is calcu-
lated that this rate of loss does not
produce a sufficiently intense selection Pa+b= (l-i)e--
of unifunctional mutations to account
for the high frequency of such muta- The frequency of unifunctional genes
tions actually found. among those recovered is therefore
Finally, it is suggested that the re-
sults can be simply accounted for on
Pa/Pa + b = e-^<l-''
the hypothesis that the synthesis of a
protein molecule is accomplished by a
working directly on the
single catalyst Equating this expression to the ob-
constituent amino acids or their simple served value, 0.84, and substituting 0.5
derivatives. for i, one finds c = 0.34. The cor-
rected frequency of unifunctional
APPENDIX genes is then e"^^** = 0.71.
The corrected value of the fre-
quency of unifunctional genes can be REFERENCES
computed from a Poisson distribution Beadle, G. W. and Tatum, E. L. 1941 Ge-
as follows: netic control of biochemical reactions in
Assume that each gene has one func- Neurospora. Proc. Nat. Acad. Sci. Wash.
27:499-506.
tion to begin with and that there is in
Bonner, D., 1946a Further studies of mutant
addition number of functions, m,
a
Neurospora requiring isoleucine
strains of
randomly distributed among the genes. and valine. /. Biol. Che???. 166:545-554.
The fraction i of all gene functions is
1946b Biochemical mutations in Neuro-
indispensable. Letting spora. Cold Spr. Harbor Syniposiin?!
Quant. Biol. ll:l+-24.
n= the number of genes Davis, B. D. 1948 Isolation of biochemically
Then n -I- m= the number of gene deficient mutants of bacteria by penicillin.
functions, /. Af/ier.Chem. Soc. 70:4267.
m/n = c = the mean number of 1950 Studies on nutritionally deficient bac-
additional functions terial mutants isolated by means of penicil-
lin. Experientia 6:41-50.
per gene,
Doermann, A. H. 1944 A lysineless mutant of
And ic = the mean number of Neurospora and its inhibition by arginine.
indispensable addi- Arch. Biochevi. 5:373-383.
tional functions per Fmerson, S. 1950 Competitive reactions and
gene. antagonisms in the biosynthesis of amino
acids bv Neurospora. Cold Spr. Harbor
The fraction of genes with no addi- Symposiuvi Quant. Biol. 14:40-48.
tional functions— i.e., unifunctional— is Fincham, J. R. S., /. Gen. Microbiol, in press.
ZINDER AND LEDERBERG 221
Horowitz, N. H. 1948 The one gene-one en- Mitchell, H. K. and Lcin, J. 1948 A
Neuro-
zyme hypothesis. Genetics 33:612-613. spora mutant deficient in the enzymatic
synthesis of tryptophan. /. Biol. Chem.
1950 Biochemical genetics of Neurospora.
175:481^82.
Advances in Getietics 3:33-71.
Umbarger, H. E. and Mueller, J. H. 1951
Lederberg, and Zinder, N. 1948 Concentra-
J. Isoleucine and valine metabolism of Es-
tion of biochemical mutants of bacteria cherichia colt. I. Growth studies on amino
with penicillin. /. Avier. Che?n. Soc. 70: acid-deficient mutants. /. Biol. Che?fi. 189:
4267. 277-285.
Genetic investigations with many bacteria. This paper ^ presents the re-
different bacteria have revealed paral-
1 Department of Genetics, paper no. 479.
lelisms and some contrasts with the
This investigation was supported by research
biology of higher forms. The success-
grants (E72) from the National Microbi-
enrichment
ful application of selective ological Institute of the National Institutes of
techniques to the study of gene re- Health, Public Health Service, from the
combination in Escherichia coli (Ta- Rockefeller Foundation and from the Re-
search Committee of the Graduate School
tum and Lederberg, 1947; Lederberg from funds supplied by the Wisconsin
et al., 1951) suggested that a similar Alumni Research Foundation. This work has
approach should be applied to other been submitted by the senior author to the
222 ZINDER AND LEDERBERG
suits of such experiments with Sal- in
g per L: sodium lactate, 2.5;
monella typhijjnirium and other Sal- (NHi)oS04, 5; NaCl, 1; MgS04, 1;
mo7iella serotypes. The mechanism of K2HPO4, methylene blue hydro-
2;
genetic exchange found in these ex- chloride, 0.05; eosin Y, 0.3; and agar,
periments differs from sexual recom- 15. Difco products, penassay broth,
bination in E. coli in many respects so and nutrient agar, were employed as
as to warrant a new descriptive term, "complete" media.
transduction. Unless otherwise stated, all cultures
were incubated at 37C, and plates
MATERIALS AND METHODS
were scored after 24 and 48 hours.
Most of the strains of S. typhimii-
riiim were provided by Lilleengen EXPERIMENTAL RESULTS
(1948) as representative of his 21 Direct crosses: platings of mixed
"phage types", LT-1 through LT-22. cultures. In E. coli., recombinants
Most if not all strains of 5. typh'mni- were detected selectively by plating
riimi are lysogenic (Boyd, 1950), and various auxotrophs together on mini-
these have provided 12 lines of mal agar. Both parents are suppressed
bacteriophage. Other cultures were on this medium and, barring various
obtained from F. KaufTmann, E. K. experimental errors, colony formation
Borman, and P. R. Edwards. All cul- is confined to prototrophic recom-
tures were maintained on nutrient agar binant cells. Such errors can be de-
slants. tected by appropriate controls but are
Specific growth factor dependent best mitigated by the use of double
mutants (auxotrophs) were obtained nutritional mutants (diauxotrophs).
from ultraviolet irradiated cell suspen- These are obtained by the iterated iso-
sions subjected to the penicillin method lation of mutants in previously estab-
for selective isolation (Davis, 1950^; lished auxotroph lines.
Lederberg and Zinder, 1948). Similar Oneof Lilleengen's strains was
mutants have been obtained in Sal- refractory to our techniques of mutant
monella by Plough et al. (1951) and isolation.Two-step mutants with mu-
Bacon et (1951). Other methods for
al. tually complementary nutritional re-
the isolation and characterization of quirements were prepared from each
auxotrophic and fermentation mutants of the remaining twenty types. Of the
have been documented elsewhere two hundred possible pairwise com-
(Lederberg, 1950; Lederberg and Led- binations, including "selfed" crosses,
erberg, 1952). Streptomycin resistant one hundred were tested. Each com-
mutants were selected by plating bination was studied by mixing and
dense, unirradiated cell suspensions plating 10^ washed cells of the two
into agar containing 500 mg per L of parents on a minimal agar plate. Fif-
dihvdrostreptomycin. teen mixture plates and five control
"Complete" indicator medium plates for each parent by itself were
(EMB) was made up from the same inoculated in each test. Fifteen com-
formula as for E. coli (Lederberg, binations yielded prototrophs in con-
1950). The defined eosin methylene trast to barren controls. Strain LA-22
blue medium ("EML agar") contained was the most "fertile", especially with
LA-2 (see table 1). This cross yielded
University of Wisconsin as a dissertation for
about one prototroph per hundred
the degree of Doctor of Philosophy. His
present address is Rockefeller Institute for
thousand parental cells plated. Crosses
Medical Research, New York, New York. in which LA-22 was not involved gave
ZINDER AND LEDERBERG 223
Table 1
Number
224 ZINDER AND LEDERBERG
produced agent (FA), under
a filtrable plate. The decline at high cell den-
stimulation from LA-22, that could sities was probably due to overcrowd-
elicit prototrophs from LA-22. Fil- ing and inhibition of colony forma-
trates of LA-22 cultures, containing tion, and at lower densities to physical
substantial amounts of phage (PLT- separation of cells and agent or to the
22) active on LA-2, also stimulated FA saturation of susceptible cells.
production from LA-2. The role of lO'* cells LA-22 were plated with
of
this phage will be discussed later. serial dilutions of FA. Over a consider-
To help the further exposition of able range a linear relationship was
our experiments, we shall use the term found between the yield of proto-
transduction for genetically unilateral trophs and amount of FA (figure 1).
transfer in contrast to the union of The efi^ect of higher concentrations of
equivalent elements in fertilization. FA will be discussed in a later section.
The working hypothesis that 5^;/- A
unit of FA may be defined as the
monella FA is an agent of genetic content of a filtrate that will elicit a
transduction provides a useful frame single prototroph from an optimum
of reference for our discussion. concentration of LA-22 cells. Filtrates
Assay of FA. Stock FA was pre- from mixed cell preparations usually
pared by growing LA-22 and LA-2 in contain about 2,500 such units per ml.
mixed culture in broth. After 48 hours, Cheffiical reactivity of FA. With
the cells were sedimented and the the development of a standardized
supernatant passed through a sintered assay it was possible to compare the
pyrex filter. The sterility of a filtrate effects of various treatments on FA
was verified by inoculating samples and bacterial cells. The latter are steri-
into broth at the time of preparation lized by shaking with such agents as
and by platings in agar as controls for chloroform, toluene, alcohol, and
particular experiments. This precau- formalin. Of these only formalin in-
tion was taken although complete activates FA. The bacteria are steri-
sterility is not critical to most experi- lized by heating at 56 C for 30 minutes.
ments since more than a million cells Temperatures of 70 C are necessary
of LA-2 per plate are needed to inter- for detectable effects on FA. It is
act with LA-22 to give prototrophs rapidly inactivated only when 100 C
in the "direct crosses". These prepara- is approached.
tions have been stored in the refrigera- FA is quantitatively precipitated
tor for several months without loss of from broth by one to two volumes of
activity. cold alcohol or half saturated ammo-
A standard procedure for assay of nium sulfate. A heavy floe appears in
FA was developed for further work. both cases which, for the most part,
LA-22 was grown on nutrient agar remains water insoluble; FA, however,
plates and harvested in dense saline redisperses.
suspension. The viable count was ob- None of several enzymes tested
tained by plating suitable dilutions on affected FA. They were added di-
nutrient agar. Various dilutions of cells rectly to the active filtrates and in-
were plated with a constant volume cubated for two hours. The tests in-
of an FA
preparation on minimal agar. cluded pancreatin (100 mg/ml), tryp-
Prototrophs appeared at 24 hours and sin (100 jLtg/ml), Taka-diastase (100
were counted after 48 hours. Figure 1 mg/ml), ribonuclease (10 fig/ml), and
shows that a constant response was desoxyribonuclease (20 jxg/ml). The
found with about 10^ to lO^*' cells per failure of desoxyribonuclease to inac-
ZINDER AND LEDERBERG 225
CELL COUNT
Fig. 1. Assay of FA. FA (LT-2) and cells (LA-22) were mixed at various dilutions
, "»
cu^/i
^ I' ' » ^
% » * ^
1 .
» » > r
Sources a?id range of activity of FA. EML agar containing one of the vari-
FA has been defined thus far as a spe- ous sugars so that one unselected fer-
cific product of strain LA-2 with the mentative character could be scored
single capacit\' of transducing a par- directly on the cross plate. Of some
ticular mutant of LT-22. However, 20,000 prototrophs screened, none dif-
other direct crosses involving LA-2 fered from SW-478 except in their
had given prototrophs. To determine nutrition. In addition to mutational
if FA could be obtained from other differences, LA-22 and LA-2 differed
strains, a simplified test was applied, intrinsically in ability to utilize malate,
involving the selection of streptomycin alanine, or succinate as the sole carbon
resistant prototrophs, "SRP" (Leder- source required for growth. All of the
berg, 1951a). SW-435 (LA-22 SO was prototrophs resembled LA-22. With a
228 ZINDER AND LEDERBERG
total of eight unselected markers there there first appeared a thin film of
was no evidence of co-segregation. growth (pink and hence nonferment-
These experiments were repeated with ing) and then small outcroppings or
active filtrates from LT-2 and gave the papillae which fermented the galactose
same result. or xylose. These papillae grow quite
Genetic transfers for each of three large (figures 3 and 4) because of their
markers (one nutritional and two fer- utilization of the sugar when other nu-
mentative) were observed when ex- trients are depleted. The xylose-nega-
periments were set up in such a way as tive mutant gave some due topapillae
to select for them. SW-435 (Aux, spontaneous reversion but not enough
Gal—, Xyl— S"") was plated with FA
, to interfere with the scoring of the
(from LT-2 Prot, Gal + Xyl + S«) on , , test. The galactose negative mutant is
minimal, EMB galactose and EA4B more stable and has only rarely re-
xylose agar. Upon the EMB media verted. The number of papillae on
.-•.•.
•• • • • • •
^
• • • •
Figs. 3 and 4. SW-435 plated on EMB galactose agar with heat inactivated (3) and
active (4) FA.
Table 2
Medium
ZINDER AND LEDERBERG 229
prototroph selections were nonfer- structural differences in the chromo-
menters and the papillae selections somes of the parents. Alternatively,
acted only upon the one sugar and FA might have been considered in
were auxotrophic. All of the trans- terms of a nonspecific mutagen with
duced cells were still streptomycin re- independent action on different fac-
sistant. tors. Further experiments have disqual-
The foregoing experiment was re- ified both of these views beyond
peated on double sugar agar. Individ- reasonable doubt.
ual papillae fermented either galactose LT-7 served as an efficient donor
or xylose and were all auxotrophic. and receptor of FA and was chosen for
Because of a slight difference in tex- the study of the intrastrain transfers
ture it was possible to differentiate the and to test these considerations (see
two kinds of papillae directly on the table 1 for its markers). To be certain
indicator Entire papillae were
plate. of the source of the FA employed, it
picked and transferred to the alterna- was prepared (as described previ-
and to minimal agar. Among
tive sugar ously) without external bacterial oi
the many
tested, no mixed papillae FA was prepared from
viral influences.
were found. Any such could be de- SW-184 (prolineless), SW-188 (me-
tected by this rigid selection. thionineless), and SW-191 (leucine-
From these experiments, we con- less). Each preparation was assayed for
clude that an FA filtrate has many transduction from auxotrophy to
activities, producing many different prototrophy of each of the three LT-7
transductions (but no more than one auxotrophs and LA-22 (control for the
per cell) that result in singly trans- presence of any activity). The prepa-
duced clones. rations had fairly uniform activity on
We have observed no linked segre- LA-22. However, FA from each of
gations such as had been found in E. the three LT-7 auxotrophs could
roll recombination. The singular
D ac- transduce the other two but not its
tivity of FA mig^ht still be reconcilable source culture (table 3). FA thus con-
with a gametic interpretation if the forms to the genotype of the cells
failure to show linkages were due to from which it comes. Several galac-
Table 3
The effect of FA from LT-7 and its derivatives upon LT-7 derivatives
Cdh/FA
230 ZINDER AND LEDERBERG
with each of the three auxotrophs in by FA from their parental wild type.
similar experiments. In the course of FA from these mutants gave diverse
transduction, there was no Hnked seg- results. The mutants were never trans-
regation or association of these three duced by their own FA, but they could
nutritional markers with each other or be transduced by FA from some of the
with fermentative markers. Strepto- other mutants. These interactions pro-
mycin resistance provided still an- vided a basis for grouping the mutants
other marker that remained unaltered with respect to allelism or genie iden-
in cells transduced for other char- tity (table 4).
acters. All of the transductions discussed
Several galactose-negative mutants thus far have been in the direction of
were transducible to galactose-positive mutant to wild type. It is difficult, as
Table 4
The effect of FA fro?n several galactose-negative umtayits upon these sajne jmitants
CtWs/FA
ZINDER AND LEDERBERG 231
It is now
evident that the particular ber of exchanges. After a heat shock
FA for which an assay has been de- at 56 C to destroy any unsedimented
fined is just one of several coexisting cells, the supernatants were assayed
functions of a given filtrate. are We with LA-22 for unadsorbed FA. A4od-
entitled to refer to FA for any of the erate amounts of FA were completely
genetic factors so far studied, and the adsorbed within the time necessary for
range of action of a given filtrate can centrifugation (15 minutes) and were
be designated in the same way as the recovered quantitatively in the pre-
genotype of the culture from which cipitated cells.
it is obtained: e.g., Prot, Gal-|-, Xyl + ,
All tested smooth strains of S.
S' for SW-514 (figure 5), as we'll as typhiiimrhmi adsorbed FA. Cells of
for the FA derived from it. Unless the donor strain adsorbed as efficiently
otherwise qualified, however, FA will as the others, consistently with the
continue to refer to the transduction success of intrastrain transfers. Disin-
assayed on LA-22. fection b\' boiling or ultraviolet ir-
Adsorption of FA. The first step in radiation (to leave an extremely small
transduction must be the adsorption of viable fraction) did not aff^ect adsorp-
FA on competent cells. LA-22 was tion. Rough cultures, selected by ag-
harvested from nutrient agar plates. ing in broth (Page et al., 1951) did not
Aliquots were suspended in one ml of adsorb. These results indicated that the
an active filtrate for various intervals. site of adsorption is heat stable, is not
The cells were sedimented and plated afi^ected by the death of the cell, and
on minimal agar to determine the num- may be related to the somatic antigen.
FA NO FA
)
32 r
28 LEGEND
0--0 SEDIMENT
X — X SUPERNATANT
24 TOTAL RECOVERY
INPUT
20
16
4 -
cept and indicates that the adsorption lO^VS or 1.3 X 10*'. A maximum num-
is irreversible after the fifteen minutes ber of particles per ml is set by the fact
allowed for saturation. that the active filtrate showed no tur-
Since adsorption of FA is so rapid biditv" as might be expected with more
it appears safe to assume that the large than 10^^ particles per ml.
proportion of the individual bacteria So many unverified postulates are
are capable of adsorbing it. canWe required that a detailed discussion of
make an approximate minimum esti- possible models for the kinetics of
mate of the number of adsorbable par- adsorption would be unprofitable here.
ticles per ml of this filtrate by dividing" It may be pointed out, however, that
other. If most or the bacteria are com- from xylose negative to positive. The
petent to be transduced, the frequency order in which these transfers were ac-
of a particular transduction will be the complished made no difference. There
probability that any of the particles was no loss of efficiency with the ite-
adsorbed will have a particular effect. rated transductions as compared to the
Double transductions will occur in the single transduction of SW-351 for
same ratio to single exchanges as the any of the characters.
absolute frequency of the latter, and Specificity of adsorption of FA. The
this is too low (ca lO^^^) for double adsorption experiments had indicated
exchanges to be detected in our ex- a correlation of adsorptive ability and
periments. However, if transduction is immunological specificity. Preliminary
limited to a small proportion of com- experiments with some dozen Salmo-
petent cells, dual transductions would nella serotypes confirmed and nar-
not have independent probabilities, rowed this correlation to the presence
and further assumptions such as mutual of somatic antigen XII. Broth cultures
exclusion w^ould be required to ac- of the serotypes to be tested were
count for the low frequency of ob- sedimented and one ml of FA was
served dual events. added. Adsorption proceeded for fif-
The following picture appears to be teen minutes, and then the reaction
most consistent with the observations tubes were heat shocked at 56 C for
to date. An active filtrate contains a one hour to sterilize the cells. Pre-
population of numerous species of liminary experiments with known ad-
granules, each corresponding to a sorbing cells had shown that FA once
genetic effect although some may be adsorbed was not eluted by this pro-
intrinsically inert. Each bacterium may cedure. The mixtures were assayed on
absorb a limited number of particles, LA-22 for free FA. Some fifty' differ-
in the possible range from one to per- ent serotypes have been tested in this
haps one hundred. Each adsorbed manner. Although some types with
particle has a fixed, independent prob- XII are inert, none of the tv^pes with-
ability of exerting its particular trans- out XII adsorbed. This correlation is
ductive effect. The low frequency of maintained with the ''Sahtwnella coli"
single, and particularly of double types. The XII carrying strains that
transductions, is limited by the total adsorbed were: S. paratyphi B, S.
number of particles that may be ad- typhimiiriiim (25 strains), 5. Stanley,
sorbed as well, perhaps, as by the low S. heidelberg, S. Chester, S. san-diego,
probability that an adsorbed particle S. aborti/s-ovis, S. typhi W, S. typhi V,
will complete its effect. 5. enteritidis, S. moscozv, S. blegdam,
Serial transduction. Dual transduc- S. eastboiirjie, S. sejidai, S. abony, E.
tion has never been observed in a sin- coli 3, E. coli 4, 5. kaapstad, S. salinatis,
gle experiment. That this is due to the S. pullonim, and S. gallinanim. The
considerations described previously following XII types did not adsorb:
rather than some intrinsic limitation is S. paratyphi A and S. abortns-bovis,
shown by serial transfers. Once a cell presumably owing to the absence of
has been transduced it can be grown the XII2 component. The nonadsorb-
out, reexposed to FA, and selected for ing, non-XII types tested were: 5.
other changes. SW-351 (Aux, Gal—, typhiimiriimi (rough variant), 5.
observed on both the experimental and gen was obtained from twelve of
control plates. However, the purple thirty-one tested inocula of 10^ FA
(fermenting) papillae were observed saturated 5. typhicells, "f phases have
A— to A+
by either means, it can a phage which could be assayed on
again produce A+ agent. Mutation in these same cells. When added to A-I-,
free FA has not yet been studied. B — C+ cells (from the same original
,
the FA granules is that they are a the more pertinent ones should suffice
heterogeneous population of species at this time. Wollman and WoUman
each with its own competence— in (1925) reported the acquisition of
other words, each carries a "single Sabnonella immunological specificity
gene" or small chromosome fragment. by E. coli via filter passing material.
Regardless of the nature of the FA Similar material (which can be ob-
particles, some mechanism must be tained by phage been impli-
lysis) has
postulated for the introduction of the cated in the change of penicillin re-
transduced genetic material to the sistant staphylococci and streptococci
fixed heredity of the recipient cell. to relative penicillin sensitivity (Vou-
Muller's (1947) analysis of type trans- reka, 1948; George and Pandalai,
formation in the pneumococcus is 1949). Shigella paradyse?7teriae (Weil
apropos here: ". . . there were, in ef- and Binder, 1947) acquired new im-
fect, still viable bacterial chromosomes, munological specificity when treated
or parts of chromosomes, floating free with extracts of heterologous types.
in the medium used. These might, in Boivin (1947)reported a similar
my opinion, have penetrated the cap- change in E. Unfortunately his
coli.
suleless bacteria and in part taken root strains have been lost and confirmation
there, perhaps, after having undergone is impossible. Bruner and Edwards
a kind of crossing-over with the chro- (1948) in a report of variation of so-
mosomes of the host." matic antigens of Salmonella grown in
In a preliminary report on the 5^/- the presence of specific serum com-
monella recombination system (Leder- mented on the possibility that bacterial
berg et al., 1951) it was suggested that products dissolved in the serum were
FA might be related to bacterial L- responsible for the changes.
forms (Klieneberger-Nobel, 1951). These systems, provocative as they
The occurrence of swollen "snakes", documented for
are, are insufficiently
filtrable granules, and large bodies in detailed comparison with Salmonella
response to certain agents is character- transduction. The transformations in
istic both of FA and L-forms. Except the pneumococcus (Avery et al., 1944;
for the suggestion of viable filter pass- AicCarty, 1946) and Hemophilus in-
ing granules we have not repeated the (Alexander and Leidy, 1951)
fluejizae
reported cycles. The visible agglutin- have been studied more completely.
able granules and the antiserum-in- The genetic "transformation" of the
duced swollen form are not necessary capsular character of the pneumococ-
for FA activity. However, this failure cus depends on a specific bacterial
to fit all of the elements to a simple product (pneumococcus transforming
scheme may be due to a system more principle, FTP). Originally inter-
complex than we are now aware. preted as a directed mutation, it is now
The bacteriological literature has regarded as a variety of genetic ex-
numerous reports of results which change (Ephrussi-Taylor, 1950). Thus
might be interpreted as transduction far transformations have been achieved
(see reviews by Luria, 1947, and for the full capsular character (Grif-
Lederberg, 1948). These experiments fith, 1928), a series of intermediate
have been criticized or neglected be- capsular characters (Ephrussi-Taylor,
cause of difficulties in their reproduc- 1951), Mprotein character (Austrian
tion and quantitization but might now and MacLeod, 1949), and penicillin
be reinvestigated in light of the find- resistance(Hotchkiss, 1951). As in
ings presented. A citation of some of Salmonella each character is trans-
238 ZINDER AND LEDERBERG
formed independently. However, The agent of recombination in E. coli
there are several differences between isalmost certainly the bacterial cell.
the two systems. FA must be evoked The cells apparently mate, forming
while the FTP is extractible from zygotes from which parental and re-
healthy cells. The resistance of FA to combinant cells may emerge following
various chemical treatments has given meiosis, in which linkage is a promi-
only negative evidence of its chemical nent feature (Lederberg, 1947). The
nature. The role of desoxyribonucleic combination of genomes within a sin-
acid in the FTP was verified by its in- gle cell has been confirmed by the
activation by desoxyribonucleic acid- exceptional occurrence of nondisjunc-
ase. Retention of activity by gradocol tions which continue to segregate both
membranes has given comparable esti- haploid and diploid complements
mates for the size (about 0.1 fi) of the (Zelle and Lederberg, 1951). Although
FA particles affecting two different lysogenicity plays a critical role in
characters. On the other hand, while transduction in Sahnonella, all combi-
the particle size of the FTP has been nations of lysogenic and nonlysogenic
variously estimated from an average cultures of E. coli cross with equal
centrifugal mass of 500,000 (Avery facility (Lederberg, E. M., 1951).
et 1944) to an ionizing irradiation
al., Owing to the lack of recombination
sensitive volume equivalent to a mole- of unselected markers, transduction is
cular weight of 18,000,000 with high a less useful tool than sexual recombi-
asymmetry (Fluke et al., 1951), it is nation for certain types of genetic
considerably smaller than the FA par- analysis. However, as FA may corres-
ticle. Pneumococci must be sensitized pond to extracellular genetic material,
by acomplex serum system for ad- such problems as gene reproduction,
sorption of FTP. The low but poorly metabolism, and mutation may be more
determined frequency of transforma- accessible to attack. Sexual systems
tions has been thought to be due to the usually provide for the reassortment
low competence of the bacteria. In of genetic material and given an im-
the absence of adsorption experiments portant source of variation for the
a system similar to Salmojiella has not operation of natural selection in or-
been ruled out. Important information ganic evolution. Both sexual recombi-
is still lacking in both systems and time nation and transduction, because of
may resolve these apparent differences. their low frequency, allow only
The relationship of transduction in limited gene interchange in bacteria.
Sahfiofiella to sexual recombination in Transductive exchange is limited both
E. coli obscure. Transduction has
is in frequency and extent.
not been found in crossable E. coli nor too early to assess the role that
It is
antigenic characters have been trans- ribonucleic nature: its meaning for the
general biochemistry of heredit\'. Cold
duced. The new^ characters are stable
Spring Harbor Symposia Quant. Biol. 12:
after many generations of subcultures. 7-17.
FA is such bacterial dis-
resistant to
Boyd, J. S. K. 1950 The symbiotic bacteri-
infectants as chloroform, toluene, and ophages of Sahnonella typhimurium. J.
alcohol and to such enzymes as pan- Path. B act. 62:501-517.
240 ZINDER AND LEDERBERG
1951 Observations on the relationship Lederberg, J. 1947 Gene recombination and
of symbiotic and lytic bacteriophage. /. linked segregations in Escherichia coli. Ge-
Path.Bact.61>MS-^S7. netics 32:505-525.
Bruner, D. W., and Edwards, P. R. 1948 1948 Problems in microbial genetics.
Changes induced in the O
antigens of Sal- Heredity 2:145-198.
monella. J. Bact. 55:449. 1950 Isolation and characterization of
Burnet, and McKie, Margot 1929 Ob-
F., biochemical mutants of bacteria. Methods
servations on permanently lysogenic strain in Medical Research 3:5-22.
of B. enteritidis Gaertner. Australian J. 195 la Prevalence of Escherichia coli
Exptl. Biol. Med. Sci. 6:276-284. strains exhibiting genetic recombination.
Davis, B. D. 1950a Studies on nutritionally Science 14:68-69.
deficient bacterial mutants isolated by 1951^ Streptomycin resistance: a ge-
means of penicillin. Experientia 6:41-50. netically recessive mutation. /. Bact. 61:
1950^; Nonfiltrability of the agents 549-550.
of recombination in Escherichia coli. /. Lederberg, J., and Lederberg, Esther M. 1952
Bact. 60:507-508. Replica plating and indirect selection of
1951 Aromatic biosynthesis. III. Role bacterial mutants. /. Bact. 63:399-406.
of p-aminobenzoic acid in the formation of Lederberg, and Zinder, N. 1948 Con-
J.,
vitamin B^g- /• Bact. 62:221-230. centration of biochemical mutants of bac-
Dienes, L., and Weinberger, H. J. 1951 The teria with penicillin. /. Am. Chefn. Soc. 70:,
L forms of bacteria. Bact. Revs. 15:245- 4267.
288. Lederberg, J., Lederberg, Esther M., Zinder,
Ephrussi-Taylor, Harriett 1950 Heredity in N. D., and Lively, Ethelyn R. 1951 Recom-
pneumococci. Endeavor 9:34—40. bination analysis of bacterial heredity. Cold
Genetic aspects of transforma-
1951 Spri?7g Harbor Symposia Quant. Biol. 16:
tions of pneumococci. Cold Spring Harbor 413^43.
Syjnposia Quant. Biol. 16:445-456. 1948 Typing Salmonella ty-
Lilleengen, K.
Fleming, A., Voureka, Amalia, Kramer, I. R. phimurium by means of bacteriophage.
H., and Hughes, W. H. 1950 The mor- Acta Path. Microbiol. Scand. Suppl. 77.
phology and motility of Proteus vulgaris Luria, S. E. 1947 Recent advances in bacterial
and other organisms cultured in the pres- genetics. Bact. Revs. 1 1 1-40. :
ence of penicillin. /. Gen. Microbiol. 4: McCarty, M. 1946 Chemical nature and bio-
257-269. logical specificity of the substance induc-
Fluke, D. F., Drew, R. M., and Pollard, C. ing transformations of pneumococcal
1951 The effect of ionizing radiations on types. Bact. Revs. 10:63-71.
the transforming factor of pneumococci. MuUer, H. J. 1947 The gene. Proc. Roy. Soc.
Science 114:480. London, B, 134:1-37.
George, M., and Pandalai, K. M. 1949 Sen- Page, L. A., Goodlow, R. J. and Braun, W.
sitization of penicillin resistant pathogens. 1951 The effects of threonine on popula-
Lancet 256:955-957. tion changes and virulence of Sahftonella
Griffith, F. 1928 The significance of pneu- typhjimtrium. J. Bact. 62:639-647.
mococcal types. /. Hyg. 27:113-159. Plough, H. H., Miller, Helen Y., and Berry,
Hotchkiss, R. D. 1951 Transfer of penicillin Marion E. 1951 Alternative amino acid re-
resistance in pneumococci by the desoxy- quirements in auxotrophic mutants of Sal-
ribonucleate derived from resistant cul- ?Honella typhi?nuriu?f2. Proc. Natl. Acad.
tures. Cold Spring Harbor Sytnposia Sci., U. S. 37:640-644.
Quant. Biol. 16:457-^62. Ryan, Beadle, G., and Tatum, E. 1943
F.,
Kauffmann, F. 1936 Ueber die diphasische The tube method of measurement of
Natur der Typhusbacillen. Z. Hyg. Infec- growth rate of Neurospora. Am. J. Botany
tiojiskrankh. 119:104-118. 30:784-799.
Klieneberger-Nobel, Emma 1951 Filterable Tatum, E. L., and Lederberg, J. 1947 Gene
forms of bacteria. Bact. Revs. 15:77-103. recombination in the bacterium Escheri-
Kristensen, M. 1948 Mutative bacterial fer- chia coli. J. Bact. 53:673-684.
mentation. Acta Path. Microbiol. Scand. Tulasne, R. 1951 Les formes L des bacteries.
25:244-248. Revue hmminol. 15:223-251.
Lederberg, Esther M. 1951 Lysogenicity in Voureka, Amalia 1948 Sensitization of peni-
E. coli K-12. Genetics 36:560. cillin resistant bacteria. Lancet 254:62-65.
WATSON AND CRICK 241
Weil, A. J., and Binder, M. 1947 Experi- Wollman, E., and Wollman, E. 1925 Sur la
mental type transformation of Shigella transmission parahereditaire de caracteres
paradysenteriae (Flexner). Proc. Soc. chez les bacteries. Compt. rend. soc. biol.,
Exptl. Biol., N. Y. 66:349-352. Pam 93: 1568-1569.
White, P. B. 1926Further studies of the Sal- Zelle, M. R., and Lederberg, J. 1951 Single
?nonella group. Med. Res. Council (Bri- cell isolations of diploid heterozygous Es-
tain), Spec. Rept. Ser. no. 103. cherichia coli. J. Bact. 61:351-355.
plausible tautomeric forms (that is, the specific pairing we have postulated
with the keto rather than the enol immediately suggests a possible copy-
configurations) it is found that only ing mechanism for the genetic ma-
specific pairs of bases can bond to- terial.
gether. These pairs are: adenine (pu- Full details of the structure, includ-
rine) with thymine (pyrimidine), and ing the conditions assumed in building
guanine (purine) with cytosine (py- it, together with a set of co-ordinates
L. J. STABLER
so for all other fields of biology, if more of those workers with long
years of experience would set down their accumulated concepts a?id
research ideas as retirement approaches, as did Stadler. All too often
a man's accumulated knowledge dies with him.
mine v\ hether or not a specific chem- can identify individuals differing only
ical reaction will occur, presumably, by the effects of a single gene. The
in some cases at least, by determining prospect of determining the properties
the production of a specific catalyst. of the gene is, therefore, depend-
The great bulk of the substance of ent upon the development of valid
the cell apparently consists of ma- methods for the study of gene muta-
terials produced by the aforemen- tion.
tioned guided reactions. The nature It is appropriate to cite here the
and behavior of these materials, so far monumental contributions of H. J.
as we know them, do not require the Muller to the investigation of this
assumption that they have properties problem. More than 30 years ago he
essentially difiFerent from those of non- recognized clearly the unique signifi-
living matter. cance of gene mutation in the study
The genie substance, on the con- of the physical nature of life (1) and
trary, appears to have properties quite boldly attacked the imposing technical
difiFerent from those with which we problems that blocked its experimental
are familiar from our knowledge of investigation.
the physical science of nonliving mat- The difficulties of analysis that have
ter. Modern physical science gives us been mentioned are not different in
no model to explain the reduplication kind from those involved in other
of the gene-string in each cell genera- problems in which the properties of
tion, or to explain the production of hypothetical elements must be in-
eff^ective quantities of specific enzymes ferred from their effects— for example,
or other agents by specific genes. The in the problems of molecular or atomic
precise pairing and interchange of seg- structure. In such studies, the inves-
ments by homologous gene-strings at tigator proceeds by constructing the
meiosis also suggest novel physical simplest model that will fit the known
properties of this form of matter. facts and then attempting to apply
These facts indicate that a knowledge every significant experimental test of
of the nature and properties of the the predictions that may be made from
genie substance might give clues to the the model. By a series of successive
distinctive physical mechanisms of life. approximations, the model finally
The study of the
difficulties in the evolves to a form that seems to pro-
genie substance are obvious. It cannot vide the most plausible mechanism for
be isolated for chemical analysis or the behavior observed. The study of
pure culture. The possibility of direct the physical nature of the gene from
analv^sis of specific segments or in- purely genetic evidence is closely
dividual genes is, of course, even more comparable to this.
remote. The properties of the genes These difficulties of analysis are
may be inferred only from the results mitigated in some degree by the pos-
of their action. sibility of parallel investigation of cer-
Furthermore, a critical study of the tain problems of mutation through di-
eflFects of a single gene may be made rect observation of the chromosomes.
only by comparing individuals wholly Although the gene-string itself is be-
comparable in genotype except for a low the limit of microscopic visibility,
difference in the one gene concerned. its behavior is such that it provides a
246 STABLER
visible shadow, so to speak, in the tion, which turns out to be about 1.5
chromosome. Some alterations of the ev. Unstable genes are assumed to have
gene-strings are readily detectable by correspondingly lower activation en-
visible alteration of the chromosomes. ergies, and the fact that temperature
The cytogenetic analysis of individual affects their mutation rate less than
mutations provides a wholesome check that of normally stable genes is in
on hypotheses derived from the statis- agreement with expectation on this
tics of mutation frequencies. basis. The energy spent in one ioniza-
An illuminating example of this is tion is about 30 ev, and it is therefore
afforded by certain interpretations of to be expected that irradiation will
the evidence on mutation rate as cause the mutation of any of the genes,
affected by x-ray treatment and by regardless of their relative stability un-
temperature. At an early stage in the der normal conditions. The propor-
study of x-ray-induced mutations, tional increase in mutation rate will,
Delbrueck (2) constructed a tentative therefore, be much less for genes
"atomic physics model" of the gene, distinctly unstable at ordinary tem^
as inferred from the frequency of point peratures than for genes of normal
mutations observed under varying stability. These expectations also are
physical conditions. This has become realized.
widely known through its application This is an impressive picture, but it
and discussion in the engaging little has been evident for many years that
book What Is Lije? {3), published it has no valid relationship to the ex-
several years later by the eminent the- perimental data from which it was
oretical physicist, Erwin Schrodinger. derived. The detailed analysis of in-
In this view, the geneis considered dividual cases among the x-ray-in-
a molecule, and the observed muta- duced mutations has shown clearly
tions are considered to represent its that many of these result not from a
transitions from one stable state to an- structural change in a gene but from
other, as a result of thermal agitation some alteration external to the gene,
or the absorption of radiant energy. such as physical loss or rearrangement
The linear-dosage curve and the con- of a segment of the gene-string. We
stancy of mutation yield, regardless of have no basis for estimating the pro-
variation in the time factor, show that portion of such extragenic mutations
the x-ray-induced mutations result among the total of mutations observed
from single "hits"; the constant pro- and no ground for assuming that this
portionality of mutation yields to ion- proportion is the same among the mu-
ization, regardless of variation in tations observed under the various ex-
wavelength, shows that the unit "hit" perimental treatments.
is an ionization. Calculation of the vol- The basis of the model is the as-
ume within which these hits must sumption that the statistics of observed
occur to account for the mutations mutation are in fact the statistics of
observed provides a basis for estimat- structural alteration of the molecules
ing the average size of the gene-mole- that constitute the gene-string. The in-
cules postulated. This turns out to be vestigations of specific mutations con-
of the order of 1000 atoms. The rela- tradict this assumption and show that
tive frequency of spontaneous muta- the model has no basis in reality.
tions at different temperatures permits It is interesting to reflect that if the
the calculation of the activation energy determiners of heredity had chanced
required for the occurrence of a muta- to be of a lower order of magnitude,
STADLER 247
below the level at which the experi- ("lethals") are probably more fre-
mental study of individual cases is quent than mutations permitting sur-
possible, we might still be constructing vival with modified phenotype ("visi-
more and more refined models of the bles"). In experiments extending over
gene on this pattern. As the predic- the next 10 years (5), he developed
tions made from the model were con- various special techniques by which it
This does not mean that questions parently means only that this hypo-
concerning the undetermined prop- thetical gene does not exist. His con-
STABLER 251
tention that the properties commonly investigation of the mutation of spe-
ascribed to "the classical, corpuscular cific genes. The fact is unpleasant be-
gene" go far beyond the evidence is, I cause the various technical difficulties
think, fully justified. that arise from the very low frequency
Sturtevant is correct if, by the characteristic of mutation are at their
gene, we mean the gene of the opera- worst when the study must be made
tional definition, since this implies no on single genes, particularly on the
unproved properties. If it were true spontaneous mutation of single genes.
that there are no discrete units in the The unpleasant statement is a fact
gene-string, Sturtevant points out, the because, as we have seen, it is hopeless
most direct way of establishing the to identify and exclude the spurious
fact experimentally would still be by or extragenic mutations in experiments
studying the properties and interrela- on mutation rates at miscellaneous un-
tionships of these distinguishable re- specified loci.
^•ions. These are the genes of the op- The chief advantage in focusing the
erational definition. study on the single gene is that this
What is the operational definition makes it possible to substitute the di-
of gene mutation? We
have recog- rect experimental analysis of specific
nized that our studies of gene mutation mutants for the application of general-
have significance for the major prob- assumed to apply to mutations
izations
lem only to the extent that they iden- at all loci. Each mutant studied may
tify and analyze the mutations that add to the background of detailed in-
represent the evolution of new hered- formation available for the diagnosis of
itary units. But it is obvious that no other mutants of the same gene.
operational definition of gene muta- An important further advantage is
tion in this sense can now be formu- that the specific loci selected for study
lated—for these hereditary units are may be loci with unusual technical
not the genes of the operational defini- advantages for the recognition and
tion; they are the hypothetical genes analysis of their mutants. For example,
postulated in our interpretation of the the genes R' and A^ in maize, like
experimental evidence. To say that no other known genes in various species,
operational definition is now possible yield spontaneous mutants that are
isonly to repeat in difl^erent words the clearly distinct from the forms pro-
foregoing statement that "we have no duced by recognizable short defi-
positive criterion to identify mutations ciencies at these loci. This does not
caused by a change within the gene, prove that the spontaneous mutants are
and that the alterations interpreted as not due to still smaller deficiencies, but
gene mutations in experiments are it supplies a convenient screen for
merely the unclassified residue that identifying a large class of deficiencies
cannot be proved to be due to other without further investigation. Another
causes. The major objective in further very useful aid in discriminating be-
investigations must be to develop such tween gene loss and gene alteration is
criterions. available for the recessive allele a. This
allele, although phenotypically dis-
STUDY OF THE MUTATION tinguishable only by the loss of A
OF SPECIFIC GENES action, may be distinguished from gene
The main purpose of this paper {11) deficiency by its response to the mu-
is to emphasize the unpleasant fact that tagenic gene Dotted (Df), in the pres-
significant progress in our understand- ence of which it reverts sporadically to
ing of gene mutation requires the the dominant allele A. The retention
252 STADLER
of the Dt response provides a criterion ally unlimited populations would re-
to exclude gene loss in the interpreta- move the difficulty, but unfortunately
tion of experiments on spontaneous these do not provide the critical gene-
and induced mutation of A. A tech- tic background essential to the study.
nical advantage of a different sort is A technique for determining the
provided by the R alleles. The pheno- spontaneous frequency of mutation of
typic effect of R is such that a large specific genes is practicable in maize
number of alleles may be objectively for mutation rates ranging as low as
distinguished by very slight differ- about one per 1 million gametes (18).
ences of plant color intensity and pat- A test of eight genes, unselected ex-
tern. A gene with equally variable cept for the technical advantage of
allelic forms, if identified only by its showing their effects in the endo-
effect on some all-or-none response, sperm, yielded mutations in all but one
would seem to have only two alleles, of the genes tested, the mutation fre-
and its mutations would not be detec- quencies ranging from about one to
table except for those that crossed the about 500 per 1 million gametes tested
linebetween these two distinguishable (19). The genes that yielded mutations
levels of action.Another advantage of in sufficient numbers to permit the
great practical importance is that both comparisons showed rather wide varia-
R and A are genes affecting endosperm tion in mutation frequency in different
characters and are, therefore, suitable cultures. The gene R, for example,
for the identification of mutations in yielded no mutations in large popula-
large populations. Both are apparently tions in some cultures, but its muta-
genes of such trivial effect physiologic- tion rate in other cultures ranged as
ally that their mutants survive with no high as 0.2 percent. Later studies have
detectable loss of viability. shown that such differences are due
The effective analysis of the diverse in part to differences intrinsic to the
genetic phenomena that may result R allele concerned and in part to dif-
in the origin of a Mendelizing varia- ferences caused by factors modifying
tion maynot be impossible in intensive the mutation rate of R (20). Such fac-
studies of the mutations of suitable se- tors are apparently quite common,
lected genes, despite the fact that it since a study in which only strong ef-
seems hopeless in studies of mutation fects could be detected indicated the
at miscellaneous, unspecified loci. occurrence of such modifiers in three
These considerations are of no ac- of the seven regions marked (21).
count if the frequency of spontaneous The average mutation rates deter-
mutation of the single gene is actually mined are rather low for effective ex-
too low to permit effective experi- perimental investigation of factors af-
mental study. We
cannot safely avoid fecting the mutation rate and even for
this difficulty by selecting for study the extraction of adequate samples of
the genes of unusually high mutation mutants for individual study. How-
frequency, because there is no assur- ever, the fact that mutation rates are
ance that the mechanism responsible so readily affected by diverse modi-
for the behavior of "unstable genes" is fiers makes it feasible to extract
representative of the mechanisms con- strains in which the mutations of spe-
cerned in typical gene mutation. The cific genes may be made frequent
use of microorganisms that permit ef- enough to permit direct experimental
fective screening for mutants in virtu- study.
STADLER 253
adduced tomorrow to show that geno-
DETECTION OF SPURIOUS GENE
types breed true only as a statistical re-
MUTATIONS sult ofsampling in each generation in
The development of criterions for populations of genes genetically fluc-
identifying gene mutations of evolu- tuating over an imperceptible range,
tionary significance is difficult even in there is nothing in our present knowl-
the study of selected genes of the most edge that would contradict this con-
favorable properties. In past studies, clusion.
the problem has been given a disarm- A study of R alleles of diverse origin
ingly simple appearance by various as- showed the common occurrence of
sumptions, some of which were un- minute differences in the level of plant-
warranted, and some of which have color expression (22). Such allelic dif-
been invalidated by later discoveries. ferences would not be expected if the
For example, we tend to feel that only source of variation in this gene
some of the mutations detected in were mutation of the type that we
our experiments must be qualitative study in our experiments, but they
changes in the genes concerned, for would be expected as a result of sub-
surely qualitatively altered genes have liminal mutation.
arisen in the course of evolution. This If subliminal mutations occur, it is
is mainly responsible for the wide- possible that this type of mutation ac-
spread belief that, even though some of counts largely or wholly for the evo-
the apparent gene mutations identified lution of new gene forms in nature.
are demonstrably false, "true" gene Thus it is quite possible that the
mutations must be included in the un- sharply distinct mutations identified
classified residue. in our experiments may be exclusively
This belief is Granting
fallacious. the result of extragenic phenomena.
that qualitatively changed genes must A
second assumption, or group of
have been evolved by mutation at rates assumptions, is concerned with the
high enough to permit experimental possibility of distinguishing gene mu-
investigation, there is no assurance that tation from gene loss. It was originally
the steps in their evolution are repre- supposed that induced recessive "visi-
sented in the mutants that are found in bles" could safely be considered gene
our mutation experiments. When we mutations, on the assumption that all
setout to identify mutants in a muta- genes were essential to survival. This
tion experiment, we must confine our- was contradicted by various instances
selves to mutations of relatively large of cytologically demonstrable defi-
effect, large enough to set the mutant ciencies viable in haploid tissue or in
beyond the range of varying expres- hemizygous individuals, or viable as
sion due to environmental and genetic homozygotes in diploid individuals.
modifiers. If mutant changes occur Such cases were relatively few, but
within the narrower range, we have no since both the cytological and the
way of identifying them. There is no genetic criterions of deficiency ap-
good evidence against the occurrence proach the limit of their range of ef-
of such subliminal mutations. The as- fective application as the deficient
sumption of the high constancy of the segment becomes smaller, there is
gene is backed by evidence only con- reason to suspect that physical loss may
cerning the rarity of the distinct mu- be responsible for observed mutations
tations. If convincing evidence were also in cases in which deficiency can-
254
not be demonstrated. As we have be-
come better acquainted with individual
genes and their functions, the assump-
tion that genes, as a rule, are individu-
ally essential to life has lost its plausi-
bility.
Mutation to an intermediate allele is
sometimes considered evidence against
loss mutation. This involves another
assumption, that of the unitary nature
of the gene— an assumption made con-
sciously and with careful consideration
in the early development of gene
theory, but one that must be seriously
questioned in the light of later evi-
dence. It is only on the hypothesis that
multiple alleles are variant forms of a
single unit that we may exclude the
possibility of their occurrence by loss
mutation. On the hypothesis that they
represent different mutations in a com-
plex of closely linked genes, we could
account for mutation to different levels
by the loss of different segments of the
chain.
The basis for the choice of the uni-
tary hypothesis is perhaps best shown
in the considerations underlying the
classical criterion, of allelism. These
were stated by Morgan in 1919 (23)
as follows:
1 •
2«
A 3»
4«
STADLER 255
theticalgene is unitary by definition. other. They give no cytological or
But the genes identified in our experi- genetic indication of deficiency, and
ments cannot be made unitary by defi- they are wholly normal in develop-
nition. The five genie
elements repre- ment in the haploid gametophyte, as
sented in the diagram are not actually is shown even by the very sensitive
parts of one gene; they are five genes. test of competitive pollen-tube growth
But if certain multiple allelic series in the transmission of the mutant
have a basis of this type, it would be through male germ cells. The cross-
possible to establish the fact experi- over-mutants are wholly indistinguish-
mentally only in the cases most favor- able in appearance and genetic be-
able for analysis. Accordingly, there havior from the noncrossover mutants
might be many cases in which the seg- occurring in the same cultures.
ment of the gene-string identified ex- The occurrence of unequal crossing
perimentally as a single gene might over within the R complex yields some
actually be a cluster of genes of iden- interesting indications of the genetic
tical or similar effect. nature of multiple allelic series and of
The
notion of the compound gene, the possible role of gene losses in re-
or some equivalent unit, may prove to lation to seemingly qualitative muta-
have significance, since there may be tions. In addition to (P) and (S), there
special relationships among the clus- are other phenotypically recognizable
tered elements that mark them off as genie elements of the R complex. In
a group from adjoining unrelated ele- certain R' alleles of dilute pigmenta-
ments. One of these may be interrela- tion, both plant and seed color are de-
tionships in gene action between the pendent upon a single genie element
clustered elements, which could lead (D). In various i?*" alleles of unusually
to the occurrence of position effects strong pigmentation, there appear to
when members of the cluster are sepa- be additional elements determining
rated by crossing over or transloca- certain aspects of plant-color expres-
tion. This may be a basic factor in the sion. In addition, there are various dis-
explanation of position effect in gen- tinguishable aleurone-eolor types such
eral. Another relationship to be ex- as "Stippled," "Marbled," "Navajo-
pected synaptic equivalence, leading
is spot," and so forth, some occurring
to the opportunity of unequal crossing with plant color and some without.
over. It is the latter that concerns us Each of the distinguishable complexes
here. may be regarded as one of a long series
A striking example of minute defi- of multiple alleles of the gene R.
ciencies simulating gene mutations is Let us pause a moment to clear the
provided by the "crossover-mutants" terminology. To avoid confusion I
of R'^ (24). Certain i?'' alleles consist shall refer to the recognized alleles of
of at least two independently mutating R under their customary italicized
genie elements: (P), determining an- designations (R\ R", r, 'R^\ and so
thocyanin pigmentation of certain forth), although the analysis shows
plant tissues and of the pericarp, and that several of these so-called "alleles"
(S), determining anthocyanin pigmen- are actually complexes of two or more
tation of theendosperm and embryo. genes.
The crossover-mutants 7?" and r^ re- The term gejiic eleme?it will be used
sult from unequal crossing over and for any gene-like constituent identi-
must, therefore, involve the loss of fied as a component of one of the R
(P) in the one case and of (S) in the alleles. The use of this term does not,
256 STADLER
in the absence of further evidence, ucts "P P S" and "P S S" were not
necessarily imply that the element is recognizable, but these represented the
unitary.The genie elements are desig- production of potential new alleles
nated by symbols not italicized, such carrying three genie elements instead
as P, S, D, and so forth. of two. By using distinguishable forms
In addition to the crossover mutants of S or P in the original compound, the
there arenumerous noncrossover mu- addition-crossovers may be made rec-
tants. A
noncrossover r^ mutant is ognizable, and by this means it is pos-
presumably of constitution "P s" ible to produce such new synthetic
rather than merely "P." The postu- alleles as R (Stippled-Navajo), and so
lated element (s) is element
a "null" forth. In this manner, it would be ex-
phenotypically but presumably would pected that more complex clusters
function synaptically in the same way would develop by successive steps, un-
as "S." These postulated elements are less the gene is one whose action sets
designated "s," "p," "d," and so forth. a closer limit on the viability of its
P S /
P s'^P P S
PS ^P s s
(1)
P s
9 and in the absence of Dt, the stand- while, if itis capable of reverse muta-
ard a allele has given occasional en- tion, there is ground for the suspicion
dosperm dots apparently due to that it may be due to an expression-ef-
mutation to A. This strongly indicates fect. The only escape from this di-
that the standard ^ is a repressed A, lemma is through the more intensive
and, if so, its reversion under the influ- study of the mutations of specific
ence of Dt must also be due to some genes selected as best suited to detailed
modification of conditions affecting genetic analysis, in the hope of de-
gene expression. veloping more sensitive criterions for
Whether or not there is acceptance the identification of gene mutations.
of my hypothesis that these manifesta-
tions of unstable gene behavior are REFERENCES AND NOTES
brought about by the transposition of 1. Muller, H. J., Am. Naturalist 56:32, 1922.
invisible bits of heterochromatin to the 2. Delbrueck, M., Nachr. Ges. Wiss. Got-
locus of the gene affected, this brilliant t'mgen (Math, physik. Kl., Biol.) 1:223,
1935.
investigation clearly shows that expres-
may be the actual cause of
sion effects 3. Schrodinger, E., What is Life? (Cam-
bridge Univ. Press, New York, 1944).
apparent gene mutations, even when
4. Muller, H. J., Proc. Natl. Acad. Sci., U.
the mutation observed shows no indi-
S. 3:619, 1917.
cation of a change of position or of 5. , Gefietics 13:279, 1928.
any associated chromosomal alteration.
6. , Science 66:84, 1927.
The
resulting difficulty in the analy- 7. ; Z. ind., Suppl. 1:234, 1928.
sisof observed mutations further em- 8. Patterson, T., and Muller, H. Gene-
J. J.,
phasizes the necessity for carrying on tics 15:495, 1930.
loci. If we
think of these results in 10. Stadler, L. J., Sci. Agr. 11:645, 1931.
terms of the generalizing assumptions 11. , Froc. 6th Intern. Congr. Genet.
1:274, 1932.
characteristic of the study of mutation
12. Bridgman, P. W., The Logic of Modern
en masse, we may be inclined to apply
Physics (Macmillan, New York, 1927).
the findings to the nature of gene in-
13. James, W., Pragmatism (Longmans,
stability in general, oreven to the na- Green, London, 1907).
ture of mutant alleles in general. If we 14. Goldschmidt, R., Sci. Monthly 46:268,
think of them against the background 1938.
sively studied gene, we are inclined to 16. Sturtevant, A. H., in Genetics in the
20th Century (Macmillan, New York,
make detailed comparisons of the mu- 1950), pp. 101-110.
tants of this category with those of 17. Given as the presidential address, Amer-
other types and other modes of origin ican Society of Naturalists, annual
in the hope of developing criterions meeting, Boston, Mass., Dec. 30, 1953.
It is a report of the cooperative investi-
that distinguish mutants of different
gations of the Field Crops Research
kinds. Branch, Agricultural Research Service,
Meanwhile, in the study of gene mu- U. S. Department of Agriculture, and
tation, we are for the present in an Department of Field Crops, University
of Missouri (Missouri Agri. Expt. Sta.
anomalous position. A mutant may
J., Ser. No. 1409). The work was aided
meet every test of gene mutation, and by a grant from the U. S. Atomic En-
yet, if it is not capable of reverse ergy Commission.
STURTEVANT 259
18. Stadler, L. J., Genetics 31:377, 1946. 25. Green, M. M., and Green, K. C, Froc.
Natl. Acad. Sci., U. S. 35:586, 1949.
19. ,Spragg Memorial Lectures 3rd j
ser. (Michigan State College, East Lan- 26. Laughnan, J., ibid. 35:167, 1949.
sing, 1942), pp. 3-15. 27. Silow, R. A., and Yu, C. P., /. Genet.
43:249, 1942.
20. , A?n. Naturalist 82:289, 1948.
28. Green, M. M., Proc. Natl. Acad. Sci.,
21. , ibid. 83:5, 1949.
U. S. 40:92, 1954.
22. — , Cold Spring Harbor Symposia 29. Stephens, S. G., Advances in Genetics
16:49, 1951. 4:247, 1951.
23. Morgan, T. H., The Physical Basis of 30. AlcClintock, B., Proc. Natl. Acad. Sci.,
Heredity (Lippincott, Philadelphia, U.S. 36:344, 1950.
1919). 31. , Cold Sprittg Harbor Symposia
24. Lewis, E. B., Genetics 30:137, 1945. 16:13, 1951.
A. H. STURTEVANT
As I vientioned above (p. 194), this paper and the report of the
Genetics Co?ijerence point a 7iew direction for genetic thought. The
Genetics Conference paper indicates a new trend in inethods as well,
that of team research.
This is not to say that the day of the i?idividual is over. As this
paper de777onstrates, thought is still the prerogative of a si7igle T77i7id.
Intellige7it analysis of a proble7n will always depend up07i a7i i7idi-
vidtiaPs experience, abilities, accim7idated knowledge, a7id logical
reasoning. It is good to have 77ien like Stn7'tevant, Limis Paiding, and
Edward Teller concer7ii7ig thcTnselves with the iTnpact of gefietics
on society. Whether they be right or wrong, their argi(7ne7its and
disciissio7is force lis all to think a little 77iore clearly about the prob-
lems we face as himian beings.
Man is one of the most uxsatis- cessive generations is long, at best in-
factory of all organisms for genetic dividual families are too small to estab-
study. The time interval between suc- lish ratios within and the them,
test-matings that a might geneticist
*
want cannot be made. Obviously no
Presidential address at the Pacific Divi-
sion of A.A.A.S., Pullman, Wash., June geneticist would study such a refrac-
22,
1954. tory object, were it not for the impor-
260 STURTEVANT
tance that a knowledge of the subject for little, genetic differences for nearly
has in other fields. all the observed diversity. In these cir-
One consequence of the difficulty of cumstances it is necessary to examine
the material is that the exact mode of what direct evidence we have.
inheritance is known for very few of At the sensory level there is good
the differences among individuals. It evidence for inherited differences.
is important that suspected cases be There can be no question that such
recorded, in order that other workers things as color-blindness, night-blind-
may check them; but there is an un- ness, or sensitivity to the bitter taste
fortunate tendency to accept such of phenylthiourea are simply inherited;
records as demonstrations rather than and one may confidently suppose that
as suggestions. After examining some other such inherited sensory differ-
of the available published evidence, I ences remain to be discovered. As has
am convinced that, even for some of been pointed out by Blakeslee, we all
the standard textbook examples, the live in different worlds by virtue of
evidence for the accepted mode of in- inherited differences in our sensory
heritance is far from conclusive— and reactions to external stimuli. It should
that it would be recognized as at best further be pointed out that these dif-
suggestive, if any organism other than ferences have effects at the highest
man were concerned. mental levels. About 8 percent of
There are enough unambiguous ex- white males are at least partially red-
amples known to make it clear that the green blind; and when such a man
same principles are at work in man as looks at a painting he does not see what
in other higher animals and plants—
all the artist put there or what other peo-
and even without such evidence, ple see. It is clear that this simple and
enough is known about the cytology rather frequent genetic property has
of human tissues to give us confidence inevitable effects on the esthetic life
that no peculiar kind of inheritance is of the individual.
to be expected in man. In fact, much of These remain rather of
trivial sorts
the argument concerning the practical differences; but there another large
is
ally identical. If one studies members no one ever does anything for which
of such pairs that were separated in in- he does not have the necessary genes;
fanc\% any observed differences must but one must never forget that there is
be nongenetic. also a necessary environment. It
The difficulty here is in the measure- scarcely needs argument that human
ment of the properties we are inter- behavior is strongly influenced by eco-
ested in. Such studies of separated iden- nomic status,tradition, and training.
tical twins were begun by Muller and After all, most of the members of this
have been greatly expanded by New- society are in the business of teaching
man, Freeman, and Holzinger. I must or, at least, have spent a good deal of
confess to a feeling, however, that time and energy pursuing academic
these studies tell us more about what work; we are therefore all of us wit-
the psychological tests used are really nesses to the obvious fact that men
measuring than they do about the rela- are teachable— that their behavior can
tive effects of heredity and environ- be strongly modified by environmental
ment. stimuli.
There are then inherited differences This caution about attributing ob-
in the sensory components of human served differences to genetic causes,
mentality and also in components lead- rather than to environmental ones, ap-
ing to severe derangements. In the plies with special force to comparisons
area between these extremes the tech- among racial groups, for here the ef-
nical difficulties of getting clear-cut fects of tradition and of public opinion
evidence are still unsurmounted. But are especially strong.
it seems safe to conclude, from what Another thing that must be avoided
we know of the genetics of complex isthe view that one race (usually that
characteristics in other organisms, that to which one himself belongs) is "bet-
any property as complex and as vari- ter" than another. All that can prop-
able as this must have a large amount erly be concluded is that they are in-
of underlying genetic diversity. herently different. It follows that
) a
262 STURTEVANT
society would do well to insure that as nant growths, perhaps years after the
many people as possible, of as diverse exposure. There is, in fact, no clearly
oppor-
racial origins as possible, get an safe dosage— all high-energy radiation,
tunity to show what they can do to even of low intensity and brief dura-
advance civilization. It may confi- tion, must be considered as potentially
dently be expected that individuals of dangerous to the exposed individual.
various races will have the necessary Let us now turn to the effects of ir-
genetic equipment to make unique radiation on the genes in the germ lines
contributions. of exposed individuals. Here again we
I wish to devote the rest of this are handicapped by the special diffi-
paper to the effects of high-energy culties of dealing with the genetics of
radiationon the genetic properties of man, for the quantitative determina-
man. This is a matter that has been of tion of the genetic effects of irradia-
significance chiefly because of the use tion requires much more refined tech-
of x-rays for diagnostic and therapeu- niques than are possible with man—
tic purposes; but with the develop- point that becomes obvious when one
ment of A-bombs and H-bombs it has tries to evaluate the data available con-
become of far more general impor- cerning the survivors of the Hiroshima
tance, for it is already true that all of bomb. There is sufficient evidence that
us have been subjected to irradiation quantitative results obtained with one
from these sources. organism cannot safely be applied to
There are two possible types of ra- a wholly different kind of organism.
diation damage to be considered— dam- However, there are certain general
age to exposed individual, and
the qualitative results that have now been
damage to the genes in his germ cells. so widely confirmed that we may con-
The first will be more or less im- fidently assert that they apply to all
The
former, tissue effect, appears to 3 The effects of successive exposures
are cumulative.
be substantially absent at low doses,
4) The effects are permanent in the
recovery from moderate effects is pos-
descendants of the affected genes. There
sible, and doses spaced well apart in is no recovery.
time have little or no cumulative ef-
5) The overwhelming majority of
fects. It is on the basis of these effects these mutations is deleterious that is, —
that the "permissible" dose, to which they seriously affect the efficiency of in-
it supposedly safe to expose indi-
is dividuals in later generations in which
viduals, is calculated. But there is rea- they come to expression. These deleteri-
son to suppose that gene mutations, in- ous genetic effects may lead to early
duced in an exposed individual, also death or to any of a wide variety of de-
fects, often gross ones.
constitute a hazard to that individual
—especially in an increase in the prob- There is a store of such undesirable
ability of the development of malig- genes already present in any popula-
STURTEVANT 263
tion. What irradiation does is to add ... it should be noted that after every
to this store. test we have had, and the Russian tests as
well, there is a small increase in natural
follows from these facts that any
It
"background" radiation in some localities
large-scale increase in the amount of
within the continental United States. But
irradiation to which human popula-
currently it is less than that observed
tions are subjected is a serious matter.
after some of the previous continental
Even though we cannot say that a and overseas tests, and far below the
given amount of irradiation will have levels which could be harmful in any way
a quantitatively specified effect, we to human beings. [Bull. Atomic
. . .
persed over the earth, will result in an arguing that the benefits ultimately to
increase in this ultimate harvest of de- be derived from atomic explosions are
fective individuals. Some such defec- outweighed by the biological damage
tives would be present if the bombs they do. It may be that the possible
had never been invented; the point is gains are worth the calculated risk.
that the number due to the bombs will But it must be remembered that the
be added to this irreducible minimum. risk is one to which the entire human
Under these circumstances, I have race, present and future, is being sub-
been disturbed that Chairman Strauss jected. I regret that an official in a
of the Atomic Energy Commission position of such responsibility should
should state, in an official press release have stated that there is no biological
from the White House, on 31 March hazard from low doses of high-energy
1954 irradiation.
^0
Reconstitution of Active Tobacco Mosaic Virus
from Its Inactive Protein and Nucleic Acid
Components*
One
of the most remarkable a?id exciting achievements bio- m
was the reconstitutioji of active
logical research in recent years
tobacco mosaic virus (abbreviated ^''TMV'') from its ina?iimate
co?istitue?it parts by Fraenkel-Conrat and Williams. While the dis-
tinctio?i between "life"" and "jion-life'" is a difficult thing to define
at the level of the virus, this piece of research i?idicates that a sub-
sta?ice considered to be livifig can be artificially cojistructed from
substafices considered to be ?w?i-living. Our bodies perfor?n this
transformatioti daily, of course, but it is the experimejital reproduc-
tion of it that is impressive in this work.
Once more, in this paper, we see the i?}iportance of the introduc-
tion of new weapo?is into the arse?7al of biology. The development
of the electron microscope by the physicists found its greatest use
and significance ifi its application to biological problejtis of ultra-
structure, and it appears likely that it will do much to destroy the
boimdaries betwee?i physics and biology as distinct sciences, just
as the study of the gene has done rmich to eliminate distinctions
between chemistry a?id biology. At certaiji levels of biological in-
vestigation all boundaries disappear, and biology becomes a ques-
tion of physico-chemical relationships.
264
FRAENKEL-CONRAT AND WILLIAMS 265
protein subunits of a molecular weight is precipitated twice more with 0.25-
near 18,000 are arranged in a helical 0.35 saturated ammonium sulfate, dia-
manner to form a rod with a hollow lyzed,brought to pH 7.0-8.0 with
core. The nucleic acid is believed to NaOH, and finally again freed from
occur as strands in the core. Electron heavy particles, such as undegraded
micrographs which support this con- virus, by ultracentrifugation. The pro-
cept have been obtained of the virus tein gives a water-clear solution at
at various stages of disaggregation.^"^'' pH 7; the masked— SH group is still
A protein isolated from infected plants present. The spectrum resembles that
has been found to reaggregate— first to of a mixture of tryptophan, tyrosine,
short pieces of the presumed helix ly- cysteine, and phenylalanine, simulating
ing on end and resembling disks with the composition of the protein, al-
central holes and then to much longer, though the minimum (at 250 m^i) is not
but inactive, rods of the diameter of quite as low (max./min. = 2.4 versus
the virus vet free from nucleic acid.^ 2.9) (Fig. 1); P analyses (0.01-0.03
It has now been possible to achieve the per cent) indicate removal of about
co-aggregation of inactive virus pro- 95-98 per cent of the nucleic acid.
tein subunits and inactive virus nucleic Evidence for the absence of detectable
acid to give nucleoprotein rods which virus particles will be discussed below.
appear to be infective. The nucleic acid fractions from such
Preparation of Frotein and Nucleic alkali-degraded TA4V are not as effec-
Acid Components— TMY was dia- tive for reconstitution as that obtained
lyzed against pH 10-10.5 glycine by the detergent method. - A virus so-
buffer (0.01 M) or pH 10.5 carbonate- lution (1 per cent) containing 1 per
bicarbonate (0.1 M) at 3° C. for 48-72 cent sodium dodecyl sulfate is adjusted
hours. Undegraded virus was separated to pH 8.5 and held at 40° for 16-20
bv cold ultracentrifugation, and the hours. Following this treatment, am-
supernatant was brought to 0.4 satura- monium sulfate is added to 0.35 satura-
tion with ammonium sulfate. The pro- tion, and the protein precipitate is
Table 1
40,000-rpm. pellet
TMV stock preparation
Table 3
Assay Con-
centration Lesions/ Total Particles/Ml. of Particles/Ml./
Preparation (M.g./Ml.) Half-Leaf Particles/All. Length 290-3 10 M^ 10 Lesions
Control TMV 0.1 10 1.6 X 109 7.0 X 108 7 X 108
Reconstituted virus
(25,000 rpm.) 10 10 2.2 X 1011 2.0 X 1010 2 X 1010
SEYMOUR BENZER
m r r tu
42 47 51 41
/o =10% rl region
Table 2
Reversion
Index
Mutant Map Transmission (units
Number Position Coefficient of IQ-**)
r47 0.03 <0.01
rl04
276 BENZER
is very little chance of introducing a infecting a culture of B with equal
wild-type particle present in the multiplicities (three per bacterium) of
stock). If S is used as the host, both rll each type. The yield after lysis con-
mutant and any reversions which arise tains the two parental types and,
if the
can multiply with little selection, as parents are genetically distinct, two
shown by control mixtures. The aver- recombinant types, the double mutant
age fraction of wild-type particles and wild type. In the average yield
present in several lysates is an index from many cells, the recombinant
which can be shown to be roughly types occur in equal numbers. ^^ In all
proportional to the probability of re- cases thus far tested, double rll mu-
version per duphcation of the rll mu- tants, like single mutants, do not pro-
tant. Under the conditions of measure- duce plaques on K. On the assumption
ment the index is of the order of 10- that this is generally true, the propor-
20 times the probability of reversion tion of recombinants in the yield can
per duplication. The plaques appearing be measured simply by doubling the
on K must be tested by picking and ratio of the plaque count on K (which
replating on B. This eliminates the registers only the wild recombinant)
"spurious" plaques produced by par- to the count on B (which registers all
tial reversions and by leaky mutants, types). The percentage of wild type
which show up as r type on B. As may thus measured agrees well with a di-
be seen in Table 2, the reversion in- rect count of plaque types on B.
dices for rll mutants vary over a very In this way, a series of six rll mu-
wide range. One mutant has been tants of T4 (the first sixisolated— not
found which reverts 10 times more selected inany way) have been crossed
frequently than r51, so that the re- with each other and with r47 and r51
version rates cover a known range of (kindly supplied by A. H. Doermann)
over 10-^-fold. in 23 of the 28 possible pairs. The re-
It has not been proved that these sults of these crosses are given in Fig-
apparent reversions constitute a genu- ure 2 and are compatible with the indi-
ine return to the original wild type. cated seriation of the mutants. The dis-
However, the possibility of suppres-
sor mutations distant from the site of
the rll mutation has been ruled out by
backcrosses to the original wild type, ^^^ BA
Krieg ^^ found very few, if any, r-
type recombinants in backcrosses of
several reversions, localizing the re-
verse changes to within a few tenths
of a per cent linkage distance from 321
crosses. Thus, while all rll mutants in a division of the rll region into two
this set fall into a small portion of the segments. If both mutants belong to
phage linkage map, it is possible to the same segment, mixed infection of
seriate them unambiguously, and their K gives the mutant phenotype (very
positions within the region are well few cells lyse). If the two mutants be-
scattered. long to different segments, extensive
Tests for Pseiido-allelisif?.— The lysisoccurs with liberation of both in-
functional relatedness of two closely fecting types (and recombinants).
linked mutations causing similar de- These results are summarized in Fig-
fects may
be tested by constructing ure 3. Thus, on the basis of this test,
diploid heterozv^gotes containing the the two segments of the rll region
two mutations in different configura- correspond to independent functional
tions.^' - The cis form, with both mu- units.
tations in one chromosome, usually be-
haves as wild type, since the second
chromosome supplies an intact func-
tional unit However, the
(or units). octive active presumed
octive
trajis form, containing one of the mu-
tations in each chromosome, may or
may not produce the wild phenorvpe.
If it does, it is concluded that the two inoctive inactive active
mutations in question are located in
separate functional units. Fig. Summary of tests for "position-
3.
In applying this test to the rll mu- effect pseudo-allelism" of rll mutants.
471-479, 1951; Levinthal, C, Genetics 39:169- with any of the mutants located within
184, 1954. a certain span, while behaving nor-
BENZER 279
mally with respect to mutants located in Figure 4 by horizontal lines extend-
outside that span. They are indicated ing over the span.
segment A
'^ segment B .^
1^ 7
r r r r r r I r
47 104 101 103 105 106 1
102
H III HI H I -ttf
/> '\% 1
I
a b c d
Spot tests on numerous other mu- whether all mutations involve more or
tants have shown that mutants of less long pieces of the chromosome. It
varied reversion rates, transmission co- must be remembered that the mutants
efficients, and rates of "partial rever- used in these experiments were selected
sion" occur at scattered positions in for extreme stability against rever-
both segments. sion. This procedure would be ex-
Mapping of "Microclusters.''— The pected to enrich the proportion of
spot test enables us to pick out "micro- mutants containing gross chromosomal
clusters,"i.e., groups of very closely alterations. So far as is known, the
neighboring mutations. Four such anomalous observed
cases could
groups selected for further study are equally well be imagined to be due
indicated in Figure 4, and the results to double (i.e., two near-by "point")
of mapping them are given in Figure 5. mutations, inversions, or deletions of
While some intervals show reasonably the wild-type chromosome. In con-
good additivity properties, there are tinuing these experiments, it would
some mutants which give violently seem well advised to employ only mu-
anomalous results. Thus in microclus- tants for which some reversion is ob-
ter a, r47 gives no wild recombinants served.
than 1
(i.e., less in 10'^) with any of the Discussion.— The set of rll mutants
other three mutants, but two pairs of defines a bounded region of a linkage
the three do show recombination. group in which mutations may occur
These results can be understood if it at various locations, all the mutations
is assumed that each mutation extends leading to qualitatively similar pheno-
over a certain length of the chromo- typic eff'ects. The rll region would
some, and production of wild type re- seem, therefore, to be functionally
quires recombination within the space connected, so that mutations arising
between those lengths. According to anywhere within the region affect the
this interpretation, the mutations same phenotype. This effect is ex-
would cover the lengths indicated by pressed, in case strain B is the host, by
the bars in Figure 5. These anomalies failure to produce lysis inhibition; in
resemble those observed in the spot case S is the host, by no consequence;
tests, only they are more limited in and in case K is the host, by inability
span. to multiply normally. The failure of
This observation raises the question an mutant to mature in K can be
rll
of whether there exist true "point" overcome by the presence of a wild-
mutations (i.e., involving an alteration type phage in the same cell. This could
of only one nucleotide pair) or be understood if the function of the
:;x3
29?]
:X4^
0.10
0.0&
0.0000
0.0000
0.0000
0.0000
BEXZER 281
region in the wild-type "chromosome" By extension of these experiments to
were to control the production of a still more closely linked mutations,
substance or substances needed for one may hope to characterize, in mole-
reproduction of this phage in K cells. cular terms, the sizes of the ultimate
The phenotv'pic test for "pseudo- units of genetic recombination, muta-
allelism" leads to the division of the tion, and "function." Our preliminary
region into two functionally distin- results suggest that the chromosomal
guishable segments. These could be elements separable by recombination
imagined to affect two necessary are not larger than the order of a dozen
sequential events or could go to make nucleotide pairs (as calculated from
up a single substance the two parts of the non-zero recombination
smallest
which must be unblemished in order v^alue)and that mutations involve vari-
for the substance to be fully active. able lengths which mav extend over
For example, each segment might hundreds of nucleotide pairs.
control the production of a specific In order to characterize a unit of
polypeptide chain, the two chains later "function," it is necessary to define
being combined to form an enzyme. what function is meant. The entire rll
While it is not known whether this region is unitary in the sense that mu-
sort of picture is applicable, a model tations anywhere within the region
of this kind is capable of describing cause the rll phenotype. On the basis
the observed properties of the rll mu- of phenotype tests of trans configura-
tants. The map position of a mutation tion heterozygotes, this region can be
would localize a change in the region subdivided into two functionally sep-
(and also in the "enzyme" molecule), arable segments, each of which is esti-
the reversion rate would characterize mated to contain of the order of 4 X
the type of change involved in the 10^ nucleotide pairs. If one assumes
genetic material, and the degree of that each segment has the "function"
phenotypic effect would be an ex- of specifying the sequence of amino
pression of the degree of resultant acids in a polypeptide chain, then the
change in the activity of the enzyme. specification of each individual amino
A "leaky" mutant would be one where acid can as well be considered a uni-
this latter effect was small. While no tary function. It would seem feasible,
obvious correlation has yet been ob- with this system, to extend genetic
served among these three parameters studies even to the level of the latter
of rll mutants, gne may well show up functional elements.
upon more exhaustive study. Swmnary .—\t has been discovered
"Clustering" of similar mutants sep- that the mutations in the rll region of
arable by crossing-over has been ob- phage T4 have a characteristic in com-
served for several characters in phage mon which sets them apart from the
by Doermann and Hill and appears to mutations in all other parts of the
represent the rule. This may well be map. This characteristic is a host-range
the rule in all organisms, simply be- reduction, namely, a failure to produce
cause functional genetic units are com- plaques on a host (K) lysogenic for
posed of smaller recombinational and phage ^. The mutant phage particles
mutational elements. One would ex- adsorb to and K, but normal lysis
kill
pect to see this effect more readily in and phage release do not occur.
phage because the probability of All mutants with this property are
recombination per unit of hereditary located within a sharply defined por-
material is much greater than for tion of the phage linkage map. Within
higher organisms. that region, however, their locations
282 BENZER
are widely scattered. An unambiguous The mutants differ greatly in de-
seriation of the mutants,with roughly gree of residual ability to grow on K.
additive distances, can be accom- There is no evident correlation be-
plished, except for certain anomalous tween map position, reversion rate,
cases. and degree of residual activity of the
The simultaneous presence of a various mutants.
wild-type phage particle in K enables The selective feature of K for wild-
the multiplication of rll mutants to type recombinants offers the possibil-
proceed, apparently by supplying a ity of extending the recombination
function in which the mutant is defi- studies to an analysis of the fine details
cient. A heterozygous diploid in the
of the region.
trajis configuration is simulated bv a
Preliminary studies of this type in-
mixed infection of K with two mu-
dicate that the units of recombination
tant types. The application of the
are not larger than the order of one
phenotype test to pairs of rll mutants
dozen nucleotide pairs and that muta-
leads to the division of the region into
two functionally separable segments.
tions may involve various lengths of