STUDIES I N ULTRAVIOLET MUTAGENESIS IN DROSOPHILA

INVOLVING TREATMENT OF INSEMINATED FEMALES

LUOLIN S. BROWNING AND EDGAR ALTENBURG

University of St. Thomas, Houston, Texas
Received May 21, 1964

I N the study of mutagenesis, the employment of ultraviolet light as a mutagenic

agent has certain advantages as compared with X rays. For example, relatively
few of the mutant changes induced by ultraviolet belong to the class of minute
deletions which are difficult to distinguish phenotypically or cytologically from
point mutations and more seem to be actual point mutations than in the case of
X rays, as first clearly shown by STADLER (1941) . Ultraviolet is therefore a very
suitable agent in studies which touch on the intimate nature of the mutagenic
process. Moreover, it is probable that ultraviolet induced mutations are more like
the naturally occurring ones than are the X-ray induced, since chromosomal
rearrangements are comparatively rare among both the ultraviolet induced
(MULLERand MACKENZIE 1939) and the spontaneous, but not the X-ray.
The relative similarity between ultraviolet-induced and spontaneous mutations
may be due in part at least to the fact that a photon of ultraviolet as a rule has
only enough energy to sensitize chemically a single atom or group of atoms,
resulting in a very localized effect, as contrasted to the much greater genetic
damage often resulting from X rays and other high energy radiation. Because
of the localized action of ultraviolet, the percent of fractionals among the muta-
tions induced by ultraviolet might offhand be expected to be relatively high.
However, ultraviolet light (UV) has very low penetrating power, and there-
fore can be effectively used only on genetic material which is close to the outside
surface of the organism, as in the case of microorganisms and the pollen of plants.
I n Drosophila, it is effective on the polar cap (the early germ track, present at
one end of the developing egg). But the statistical error of the mutation rates
resulting from treatment of the polar cap is high because mutagenic hits are
multiplied in step with the multiplication of the germ cells in the course of
development, and unless the experiments are conducted on a very large scale,
a few ‘‘lucky’’hits resulting in large clusters might cause large deviations from
the actual rates. Treatment of the mature sperm cells would circumvent this
statistical error. Moreover, in order to detect fractional mutations, as revealed
by mosaicism among the progeny, it is necessary to apply the treatment to sperm
cells or other post-meiotic stages.
However, as above implied, the sperm cannot be effectively treated with UV
when applied to the surface of a mature Drosophila male. The mutation rate
induced early in the present studies by this method was only between .5 and 1
This investigation was supported by Public Health Servlce Research Grant CA-05960

Genetics 5 0 : 695-699 October, 1964.

696 L. S. BROWNING A N D E. ALTENBURG
percent even after males were treated with UV of 2800 A wavelength and longer
(more penetrating and less toxic than the 2537 A of a germicidal lamp) in doses
high enough to kill 90 percent of them and then progeny used from only those
males which survived the treatment for not more than three days (presumably
the most heavily treated of the fertile survivors), this all being in accordance
with the method of MULLER and MACKENZIE (1939).
A rate as low as this is hardly practical for such studies as the rate of visibles
us. lethals induced by the treatment or the rate of whole-body us. fractional
mutations. But because of its well understood genetic background, Drosophila
still has many advantages for work on UV mutagenesis. It therefore seemed
desirable to devise a more effective technique for studies which involved treat-
ments of the mature sperm cells of Drosophila with UV. We believe this objective
has been achieved in the present studies. Using this technique, we have found
an unexpectedly low rate of fractional mutations relative to whole-body induced
by UV in preliminary experiments on Drosophila.
METHODS
The method of treatment referred to above consists in getting females immediately after
mating, everting their vaginas by pressure applied to the abdomen as in a method of chemical
treatment devised by HERSKOWITZ (1955) and then exposing the everted vaginas to UV. While
the sperm are in the everted vaginas, they are clearly visbile under a microscope by transmitted
light through the thin transparent wall of the vagina-the only tissue that now separates them
from the UV source, When inseminated females are treated, all induced mutations derived from
the male are necessarily of independent origin and the “cluster” problem (with its high statisical
error, above mentioned) is avoided. Moreover, all the treated germ cells are in the same stage
of sensitivity to mutagenic agents, since only mature sperm cells are treated. I t is necessary
to apply the treatment within three minutes after the completion of mating i n order to get the
sperm before they start migrating to the storage organs. Some sperm cells remain in the vagina
longer than three minutes, but the first sperm to leave completely fill the storage organs and
the later ones are lost (LEFEVREand JONSON 1962). In the present studies the treatments were
usually started within a minute after the completion of mating. They lasted for from 1 to 9
minutes, though they probably were not effective for more than a minute or two after being
started.
The source of the UV was a mercury arc lamp (UA 2) which emitted wave lengths ranging
from 2800 to 3000 A in relatively large amounts. This is more penetrating than UV from a
germicidal lamp (which is mostly of wavelength 2537 A), though it is not quite as mutagenic.
The flies were immersed in ice water during the treatments (without apparent injury) in order
to protect them from the heat of the lamp.
The sperm in the inseminated females was derived from Canton-S males and the F, were
tested by the Basc (Muller-5) technique both for complete sex-linked recessive lethals and
fractionals. The complete lethals were detected in the usual way (by the absence of males in
the F, with an X of treated origin). The fractionals could not be detected i n this way, since
F, females with a fractional would be mosaic (partly mutant, partly normal) and if they were
mosaic for their gonad (as well as their soma) they would produce some males with a n X of
treated origin in the F,. The F, females were therefore examined for fractionals by testing
them for gonadal mosaicism. This was done by rearing a set of ten F, females per F, female
and determining whether some of the F, females in the set had a lethal in the X of treated
origin and others not (and so determining whether the gonad of the F, female was mosaic for
a lethal and a nonlethal X of treated origin). In some instances, an F, female might have been
nonlethal at the start but might have produced a lethal gamete spontaneously, in which case one
F, daughter in a set of ten might contain a lethal and the rest not. Therefore, sets of ten with
 DROSOPHILA U V MUTAGENESIS 697
only one lethal female do not necessarily originate from an F, gonadal mosaic. However, if the
number of such cases is clearly in excess of the number expected as a result of spontaneous
mutations, then the excess is ascribable to gonadal mosaicism. If two F, daughters of a set had
a lethal X, it would be unlikely that both lethals arose spontaneously, and still less likely if three
or more of the set had a lethal.

RESULTS

Among a total of 711 F, females derived from treated inseminations, there

were 18 complete sex-linked recessive lethals, or about 2.5 percent. Among 142
of these F, females tested for gonadal mosaicism, only one was considered a
gonadal mosaic. Thus the observed rate of fractionals (as judged by gonadal
mosaicism) relative to the whole-body lethals is 0.7:2.5,making the fractionals
about 22 percent of the total lethals. There were three F, females which produced
only one F, female with a lethal per set of ten. These three were not scored as
gonadal mosaics since the lethal might have been of spontaneous origin in each
case. From the F, female considered a gonadal mosaic, a set of ten F, daughters
were bred, all of which appeared to be lethal. However, when nine of these were
further tested (one culture was lost), six were found to be lethal and three non-
lethal (these three having probably been derived from P,-F, cultures which
happened to have no males either because of a small hatch or because the males
had died before the culture was examined). In the further testing above referred
to, three F, daughters of each of the nine F, females in question were bred in
mass and the number of F, flies recovered in each case was large enough to leave
no doubt as to whether the culture was lethal or nonlethal, though no actual count
was made. There was one semilethal with a visible effect (phenotypically a rudi-
mentary wing) which showed aberrant genetic behavior in that males with the
X of treated origin sometimes had normal wings, though they were semilethal.
This case could be explained as originating in a rearrangement involving the
deletion of a small segment of the X and its insertion into an autosome (the
rearrangement in its entirety appearing normal), followed by the loss of the
insertion (by segregation of the autosome) and expression of the deletion as a
rudimentary wing. However, chromosomal rearrangements are very unusual
following UV treatment. The case could also be explained as a dominant auto-
somal suppressor of rudimentary arising independently of the rudimentary muta-
tion, but such a coincidence would also be unusual.
Table 1 shows the variation in the number of lethals from one experiment to
another. It will be seen that in one experiment (No. 2) nine lethals were re-
covered among 166 F, females, making the rate over 5 percent. In several experi-
ments, no lethals were detected, but in some of these the vaginas were found
no longer to be everted at the end of the treatment, and the sperm may not have
been treated.
The nine lethals in Experiment 2 were derived from four inseminated females,
one of which yielded one of the lethals; a second, two; and the remaining two,
three each. All nine could therefore not have been a cluster of spontaneous origin
from a gonia1 cell.
698 L. S. B R O W N I N G A N D E. A L T E N B U R G

TABLE 1
Lethals recouered from sperm treated in the everted uaginas of inseminated females
~ ~~

Lethal tests
Whole-body Gonadal mosaic
Number of
Experiment treated females F, females Lethals Fl females Lethals

1 3 99 1 14
2 4 166 9 31
3 1 51 2
5 2 77 1 8
6 4 39 6
8 1 14 1
9 3 20 2 16
10 3 25 1 8
11 1 13 1 3
12 3 21 3
13 1 3 2
14 2 23 8
17 4 160 1 42 1

Total 32 711 18 (2.5%) 142 1 (.7%)

DISCUSSION

With further improvements in the present technique (such as increased speed

in starting the treatment of the inseminated females after the completion of
mating and keeping their vaginas everted for the entire duration of the treat-
ment), it would seem that the average rate of recovered lethals could be made at
least as high as that in the experiment that gave the 5 percent rate, since all the
male germ cells are at the same sensitive stage in an inseminated female and
the everted vaginas of different females all seem equally transparent.
Among the lethals induced in Drosophila by treatment of adult males with
quinacrine mustard, the percent that were recovered as gonadal mosaics in early
broods as reported by us (about 80 percent, 1962) and by CARLSON and SOUTHIN
(about 55 percent, 1963) is well above that here recovered after UV treatment,
and if virtually all UV-induced mutations were fractionals, the percent recovered
(as gonadal mosaics) would expectedly be at least as high as in the case of the
chemical mutagen.
In yeast, YAMASAKI, ITO,and MATSUDAIRE (1962) report an increase in
adenine-requiring whole-colony mutations relative to fractionals with increasing
UV dose, the fractionals predominating at the lower doses and the whole-colony
at the higher. They postulate single hits of UV to account for the fractionals and
two hits for the whole-colony. The high coincidence of two independent hits at
the same locus required for the whole-colony mutations would seem rather un-
expected, but the authors postulate that the energy absorbed in a DNA molecule
by a second hit “migrates and with greater probability is trapped in the opposite
site” of the strand changed by the first hit.
 DROSOPHILA UV MUTAGENESIS 699
I n the smut fungus HOLLIDAY (1962) has presented evidence that a high pro-
portion of the mutations induced by UV treatment of spores are complete (whole
colony). His experimental design made it possible to determine that both daughter
cells after the mutagenic treatment carry the mutation (as revealed by somatic
crossing over between the mutation and a more distally located marker). It is
therefore unlikely that the mutations in question are derived from fractionals in
which the nonmutant element failed to survive. HOLLIDAY concludes that UV
often induces stable genetic changes before replication. In this conclusion he is
supported by the evidence herein reported.

SUMMARY

In order to circumvent the problem posed by the low penetrating power of

ultraviolet light (UV), a new technique has been devised for the induction of
mutations by UV in Drosophila. This consists in getting females immediately
after mating, everting their vaginas by pressure applied to the abdomen, and then
exposing the vaginas to UV.
Sperm were treated by the above technique and the progeny tested by the Basc
(Muller-5) technique for recessive sex-linked lethals, both whole-body and frac-
tional. Among 711 F, females there were 18 whole-body lethals, or 2.5 percent;
among 142 of these F, females tested for gonadal mosaicism there was only one
undoubted mosaic, or 0.7 percent. The ratio of whole-body lethals to fractionals
(recovered as gonadal mosaics) is then 2.5:0.7, making the fractionals 22 percent
of the total lethals recovered. Among the lethals induced in Drosophila by treat-
ment of adult males with quinacrine mustard, the percent of fractionals is well
above that here recorded for UV, and if virtually all UV-induced mutations were
fractionals, the percent (recovered as gonadal mosaics) would expectedly be at
least as high as that reported for quinacrine mustard.

LITERATURE CITED

BROWNING, L. S., and E. ALTENBURG, 1962 The relatively high rate of mosaicism among the
mutations induced i n the X-chromosome of Drosophila by monofunctional quinacrine
mustard. (Abst.) Genetics 47: 945.
CARLSON, E. A., and J. L. SOUTHIN,1963 Chemically induced somatic and gonadal mosaicism
i n Drosophila. I. Sex-linked lethals. Genetics 48:663=675.
HERSKOWITZ, I. H., 1955 The production of mutations in Drosophila melanogaster with chem-
ical substances administered in sperm baths and vaginal douches. Genetics 4.0:76-89.
HOLLIDAY, R., 1962 Mutation and replication in Ustilago maydis. Genet. Res. 3 : 472-486.
LEFEVRE, G., and U. B. JONSSON, 1962 Sperm transfer, storage, displacement, and utilization in
Drosophila melanogaster. Genetics 47: 1719-1 736.
MULLER,H. J., and K. MACKENZIE, 1939 Discriminatory effect of ultra-violet rays on mutation
in Drosophila. Nature 142: 168-178.
STADLER, L. J., 1941 The comparison of ultraviolet and X-ray effects on mutation. Cold Spring
Harbor Symp. Quant. Biol. 9: 168-178.
YAMASAKI, T., T. ITO, and Y. MATSUDAIRE, 1962 Studies on the multiplicity of a gene in yeast
cells. 11. Effects of ultraviolet dose. Publication NO. RUP-17 from Rikko University.