Current Genetics (1984) 8 : 499-506
© Springer-Verlag 1984
Comparison of the lignin-degrading white rot fungi Phanerochaete
chrysosporium and Sporotrichumpulverulentum at the DNA level
Ute Raeder and Paul Broda
Department of Biochemistry and Applied Molecular Biology, University of Manchester Institute of Science and Technology, P. O.
Box 88, Manchester M60 1QD, UK
Summary. DNA-hybridisation studies showed a close
relationship between Phanerochaete chrysosporium
ME446, most used in lignin degradation studies, and
Sporotrichum pulverulentum Novobranova, the other
standard lignin degrading strain. Two other strains of
P. chrysosporium were both less related. We show that
P. chrysosporium ME446 and S. pulverulentum Novo-
branova both have a GC-content of 59% for chromosomal
DNA with the rRNA genes present as an AT-rich satel-
lite; the mitochondrial DNA has a GC-content of 33%.
The genome size estimated for P. chrysosporium ME446
is about 4 - 5 x 107 bp.
Key words: Lignin degradation - Phanerochaete chry-
sosporium - DNA homology
Introduction
The white rot fungi Phanerochaete chrysosporium and
Sporotrichum pulverulentum are studied because of
their ability to degrade lignin ( F e n n and Kirk 1982;
Ander and Eriksson 1976). S. pulverulentum lacks a
reliable mating system. On the basis of morphological
comparison it has been described as an imperfect form
of P. chrysosporium (Burdsall and Esyln 1974; Eriksson
and Petterson 1975). As part of a project to study the
molecular genetics of lignin degradation by these fungi
we have compared S. pulverulentum with three strains of
P. chrysosporium at the DNA level to learn more about
their relationship.
Gene libraries were prepared from S. pulverulentum
and one of the strains ofP. chrysosporium. DNA of ran-
Offprints requests to. U. Raeder
~ ~ ~
domly chosen clones from these libraries, containing
chromosomal sequences, were hybridised to restricted
total DNA of all four strains by the Southern procedure.
Mitochondrial DNA and the ribosomal RNA genes from
the four strains, which were enriched as AT-rich satellite
DNAs, were also compared.
Materials and methods
Culture of fungal strains. The strains listed in Table 1 were
grown on agar plates containing 2% malt extract (Oxoid) at 37 ° C.
For DNA preparation liquid medium containing 1.5% malt ex-
tract was inoculated with a spore suspension (about 0.3 ml of a
5 ml spore suspension made from one heavily sporulated plate
per 400 ml of medium). The cultures were grown in 2 1conical
flasks for one day at 37 °C shaken at 100 rpm.
DNA preparation. DNA was prepared using a modification of the
"gentle extraction method" described by Specht et al. (1982), as
follows: pellets from 8-10 x 400 ml culture were collected in a
sieve, washed three times in 50 mM EDTA and dried in several
portions onto filter papers (Whatman #1, in a Buchner funnel,
connected to a waterpump). The resulting "cakes" were peeled
off, frozen in liquid nitrogen and lyophilised overnight. The
lyophilised material (about 4 5 g) was ground with mortar and
pestle until it had the consistency of fine sand. It was resuspended
in 100 ml 50 mM TrisHC1 pH8, 100 mM EDTA pH8, 150 mM
NaC1, 2% SDS, 20% toluene and shaken at 60 rpm for three days
at room temperature on a rotary shaker. The suspension was
filtered (squeezed) through 2 layers of nylon stocking to remove
particles. The filtrate was shaken with 1 volume of phenol : chlo-
roform:isoamylalcohol (25 : 24 : 1, phenol was saturated with
50 mM TrisHC1 pH 8, 100 mM EDTA pH 8, 150 mM NaC1) for
10 rain. The phases were separated by centrifugation for 1 h at
10,000 rpm in a Sorvall SS34 rotor (9,200 x g). The aqueous
phase was taken off carefully~ mixed with 2.5 volumes cold
ethanol and centrifuged for 20 rain at 10,000 rpm in a Sorvall
GSA rotor (13,300 x g). The pellet was dried and resuspended
in 6 ml 30 mM TrisHC1 pH 7.8, 36 mM NaH2PO4, 2 mM EDTA
(final pH about 5). The solution was incubated first with 1 ml
RNAseA (5 mg/mL Sigma Type IIIA, preincubated for 15 rain at
500
Table 1. Fungal strains
Strain ATCC
# #
S. pulverulentum Novobranova 24,725 a
P. chrysosporium Burdsall Lombard ME446 34,541
P. chrysosporium Burdsall Nicot Elphick
P. chrysosporium Burdsall White
a Listed in the American Type Culture Collection as P. chrysosporium
b Catalogue of the Culture Collection of the Commonwealth Mycological Institute
100 °C in 50 mM Naacetate pH 6.5) for 2 h at 37 °C and then
with 2 ml protease (10 mg/ml, Sigma pancreatic protease Type I,
preincubated for 2 h at 37 °C in 50 mM TrisHC1 pH 7.5) over-
night at 37 °C. The solution was extracted with 1 volume phenol.
chloroform, isoamylalcohol and subsequently with ether. Resid-
ual ether was removed by shaking the solution gently at 45 °C.
The aqueous layer was brought to a volume of 10 ml with 50 mM
TrisHC1 pH 8. 10 g of CsCI and 200 /A of ethidium bromide
(10 mg/ml) were added successively. The solution was centrifuged
for 65 h at 38,000 rpm in a Beckman Ti50 rotor (95,200 x g) at
17 °C. Fluorescing bands were taken off the gradient and ethidi-
um bromide was e~racted several times with isopropanol
(saturated with buffer and CsC1). The solution was diluted 1 : 3
with sterile water. DNA was precipitated with 2.5 volumes of
ethanol, spun down, dried and resuspended in 10 mM TrisHC1
pH 8, 1 mM EDTA. For CsCI gradients containing bisbenzimide
(Hoechst dye #33,258 from Calbiochem Behring) 2 - 3 mg of
DNA (isolated as above) in 8.3 m150 mM TrisHC1 pH 8, 10 mM
EDTA, were mixed with 9.6 g CsC1 and 1 ml bisbenzimide
(1 mg/ml, made up in water). This solution (which contained
some yellow, threadlike precipitate) was centrifuged for 65 h at
33,000 rpm in a Beckman Ti50 rotor (71,000 x g) at 17 °C. DNA
was recovered as described above for CsCl-ethidium bromide
gradients. In order to remove carbohydrate which sometimes
cobanded with some of the DNA bands in the gradients, the
DNA (after ethanol precipitation and resuspension in 25 mM
Naacetate pH 7) was loaded on a small DE52 column (Whatman,
0,1-5 ml). Carbohydrate was washed off in 2 3 column volumes
25 mM Naacetate. DNA was eluted with 2 column volumes 2 M
Naacetate and precipitated with ethanol. For exact spectropho-
tometrical measurement of DNA concentrations and for the
GC-content estimation experiment DNA was purified on a second
CsCl-ethidium bromide gradient.
Construction of gene libraries of Sp and PcA. Bacterial strains:
E. coli LE392: supE, supF, thyA, metE, (Brunel et al. 1979);
E. coli MM21: supE, supF, thyA, metE, rpsL, (Collb) (Brunel
et al. 1979); E. coli BHB2688: (kimm434, ci857, b2, recl3,
Eam7, Sam7)/h (Hohn 1979); E. coli BHB2690: (kimm434,
c1857, b2, red3, Dam7, Sam7)/h (Hohn 1979). The vector
hgtWES-T5 "622, used for making the gene libraries, is a deriva-
tive of the EK2 replacement vector hgtWES, XB' (Leder et al.
1977). hgtWES-T5 - 622 was constructed by Davison et al. (1979)
and given to us by J. Davison. EcoRI restriction fragments of
abotlt 3 15 kb can be cloned in this vector. The vector was
amplified on E. coli LE392. Hybrid phages were selected on
E. coli MM21 (Davison et al. 1979). In vitro packaging mixes
were prepared from lysates of E. coli BHB2688 and E. coli
BHB2690 (Maniatis et al. 1982). Fungal DNA fragments were
prepared in the following way: the minimal time necessary to
U. Raeder and P. Broda: DNA of lignin-degrading fungi
CCCCMIb Geographical Abbreviation
source used
- - Sp
- - PcA
74,691 France PcF
110,120 Papua New Guinea PcP
restrict 50 ~g of fungal DNA completely with EcoRI was esti-
mated in a time course experiment. Five aliquots of 50 /~g of
total fungal DNA were partially restricted with EcoRI for the
estimated time using 1/2, 1/4, 1/8, 1/16 and 1/32 dilution of the
amount of enzyme necessary for complete restriction. 50 /~g of
DNA were restricted completely with EcoRI. DNA from these
restrictions were pooled and size fractionated on a 10 40%
sucrose gradient (prepared in 30 mM TrisHCl pH8, 1 M NaC1,
10 mM EDTA in a Beckman SW41 rotor run at 25,000 rpm,
76,800 x g, for 24 h at 4 °C). All DNA fragments larger than
8 kb were recovered. DNA of kgtWES-T5:622 was restricted
with EcoRI, the two large fragments were separated on a 10 40%
sucrose gradient from the small replacement fragment and re-
covered. Vector DNA (the large fragments, about 1 ~g) and the
size fractionated fungal DNA (about 0.5 #g) were ligated and
packaged in vitro. The resulting phages were titered on E. coli
MM21. About 4 x 104 phages were recovered for each gene
library. The average insert size as determined by restriction was
about 10 kb. These gene libraries are not complete since they
do not contain any EcoRI fragments of more than 15 kb length;
this constraint would exclude about 30% of a genome with 59%
GC content (see below) and randomly distributed EcoRI restric-
tion sites.
Nick translation and Southern hybridisation. As probes about
1 #g of fungal hybrid clone DNA or mitochondrial DNA were
labelled by nick translation (Manlatis et al. 1982) with about
10 /~Ci 32pdATP (Amersham PB204, 3,000 Ci/mmole) and
2.5 U DNA Polymerase I (Boehringer) in the presence of 0.01 pg
DNAse I for 1 h at 18 °C. The incorporation rates were between
30% and 60%. Labelled DNA was separated from unincorporated
dNTPs on Sephadex G50 coloumns. To prepare the nitrocellu-
lose-filters (Millipore HAWP) 20 t~g of total or ehomosomal
EcoRI restricted DNA or 1-5 #g of restricted mitochondrial
DNA were run on 0.6% agarose gels, denatured and neutralised.
The DNA was blotted overnight to the filters (Southern 1975).
The filters were washed in 6 x SSC, baked for 2 h under vacuum
at 80 °C, wetted in 6 x SSC and prehybridised in 6 x SSC, 0.5%
SDS, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% BSA0 100
~g/ml denatured, sonicated calf thymus DNA for 2 - 4 h at 68 °C
(200 t~l/cm2 filter). Hybridisation was done in a solution with
the same content as the prehybridisation solution but with
10 mM EDTA and 1 ~g of heat-denatured labelled probe (50
~zl/cm2) with gentle agitation for 14-18 h. Hybridisation tem-
perature was 68 °C except for the heterologous hybridisation
with labelled mitochondrial DNA of Aspergillus nidulans (a gift
from P. Sims) which was carried out at 55 °C (Kiick and Esser
1982). After hybridisation the filters were washed in 2 x SSC,
0.5% SDS for 5 min, in 2 x SSC, 0,1% SDS for 15 min both at
room temperature and in 0.1 x SSC, 0.5% SDS for 2 h, followed
U. Raeder and P. Broda: DNA of lignin-degradingfungi
by another 1/2 h, both at hybridisation temperature. Total
1251 labelled RNA of strain PcA (kindly provided by R. Haylock),
was hybridised in 5 x SSPE, 0.1% SDS, 0.1% Ficoll, 0.1% poly-
vinylpyrrolidone, 0.1% BSA, 100 g/ml denatured sonicated calf
thymus DNA as described above at 68 °C. Autoradiography was
done with Fuji X-ray film (for 32p) or Kodak XAR-5 X-ray film
(for 1251) and an intensifier screen at -80 °C.
GC content estimation of DNA from strains Sp and PcA. Marker
DNAs from Staphylococcus aureus (33% GC), kc1857 (50% GC)
and Streptomyces coelicolor (71% GC, a gift from K. Kendall)
(20 #g each) were run separately and together with fungal DNA
(40 #g of total DNA or 20/~g of mitochondrial DNA) in CsC1-
bisbenzimide gradients. The gradients were prepared with
8.3 ml DNA solution in 50 mM TrisHCl pH 7.5 10 mM EDTA,
10.8 g CsC1 (for total DNA) or 10.2 g CsC1 (for mitochondrial
DNA) and 1 ml bisbenzimide solution (1 mg/ml). They were run
in a Beckman Ti50 rotor at 33,000 rpm (71,000 x g) for 65 h at
17 °C.
Genome size estimation of strain PcA. Constant amounts of total
fungal DNA (6.7 /~g) alone, and mixed with various amounts of
DNA of ~.gtWES-T5.622 (1/5000, 1/2500, 1/1000, 1/500 of the
amount of fungal DNA) in 20/A 0.5 M NaOH, 1.25 M NaC1 were
spotted onto a nitrocellulose filter (2 cm x 2 cm for each spot, 3
replicates). During application the filter was fixed at its edges,
not touching a surface below, so as to avoid sample losses. The
filter was left to dry in this position for 20 min, floated on 0.5 M
TrisHCl pH 8, 1.5 M NaC1 until completely wet, submerged in
this solution for 5 rain, rinsed in 6 x SSC and baked under vacu-
um for 2 h at 80 °C. Two filters were prepared. As probes DNA
of two clones of the PcA gene library containing chromosomal
DNA were labelled by nick translation, separated from unincor-
porated dNTPs and hybridised to the filter bound DNAs at
68 °C. The filters were washed as described above. As a back-
ground control labelled DNA of ~gtWES-T5.622 was hybridised
to filter-bound fungal DNA. After hybridisation and washing the
filters were checked for non-specific binding of radioactivity by
autoradiography. The individual filter squares (2 cm x 2 cm)
were cut out, dissolved overnight in a scintillation mix (Packard
"Filter count", 5 ml per square) and the radioactivity was mea-
sured in a liquid scintillation counter. The proportions of radio-
activity in the vector part (v, in %) and the fungal part (f, in %)
of the probe was measured in the following way: an aliquot of
the labelled probes was precipitated with ethanol, resuspended,
restricted with EcoRI and run on an agarose gel. The bands
(from vector and insert) were cut out of the gel and transferred
to Eppendorf tubes. Water was added to a final volume of 200/~1
each. The agarose pieces were dissolved by boiling. The dissolved
agarose solution was mixed with 5 ml "Filter count" and the
radioactivity was measured in a liquid scintillation counter.
The data were evaluated in the following way: a straight line
of slope a and intercept/~ was fitted by least squares analysis to
the graph of cpm hybridised to the filters plotted against the
proportion of vector DNA on the filters. The intercept/3 is thus
a measure of hybridisation of fungal DNA alone and was corrected
by substracting a background due to hybridisation of vector
DNA to filter-bound fungal DNA alone (117 cpm background
compared to intercept values of 8,093 cpm and 9,150 cpm). The
slope c~corresponds to the additional hybridisation due to vector
DNA added to the filters. In order to compare the molarities of
fungal and vector sequences on the filters, c~and ~ must be scaled
according to the differing amounts of radioactivity present in
the vector and the fungal sequences of the probe; this gives a/v
and ~/f. The genome size of the fungus (Gf) can then be calcu-
lated as
501
c~.f
Gf = x Gv
/3.v
where Gv is vector genome size (in this case 35.1 kb, the length
of the part of kgtWES-T5.622, present in the clone used as
probe).
Results and discussion
Isolation o f mitochondrial DNA and enrichment o f
ribosomal DNA in CsCl-bisbenzimide gradients
From 5 g lyophilised fungal tissue several milligrams of
high molecular weight DNA were isolated. Restriction
and electrophoresis on agarose gels showed that a
relatively high proportion of DNA of the four strains is
repetitive.
In order to isolate mitochondrial DNA, total DNA
was fractionated in CsC1 gradients containing bisbenz-
imide. This compound binds to adenine residues in the
DNA, thereby reducing the buoyant density in CsCI
(Miiller and Gautier 1975). This "gradient stretching"
effect has been used by others to separate AT-rich mito-
chondrial DNA from chromosomal DNA (S. Oliver, per-
sonal communication) and to separate GC-rich ribosomal
DNA ofSaccharomyces cerevisiae (Petes et al. 1977).
In shallow CsC1 bisbenzimide gradients (71,800 x g in
a Beckman Ti50 rotor) total DNA of the four strains was
separated into three bands: the lowest and main band
containing chromosomal DNA, above this a relatively
weak "satellite" band and one strong band about 2 cm
above.
DNA from the uppermost band was restricted and
identified as mitochondrial DNA by heterologous South-
ern hybridisation using Aspergillus nidulans mitochon-
drial DNA as labelled probe (Fig. 3). In CsCl-ethidium
bromide gradients (the last step in the DNA preparation
procedure) a very faint fluorescing band inside a carbo-
hydrate band about 2 cm below the main band was visi-
ble in all four strains. DNA from this band was separated
from carbohydrates on a DE52 column, restricted, label-
led by nick translation and run on an agarose gel. The
autoradiographs show the same restriction patterns as
for mitochondrial DNA. This correspondence and the
position of this DNA in CsCl-ethidium bromide gradients
suggest that this DNA is covalently closed circular mito-
chondrial DNA. It may however also be possible that a
fraction of the mitochondrial DNA is trapped by carbo-
hydrate and bands together with it, regardless of its con-
formation.
Restriction of the DNA of the "satellite" bands in
CsCl-bisbenzimide gradients shows that these bands con-
rain repetitive DNA, which was readily visible as distinct
bands in EcoRI restricted total DNA of the four strains.
502
Fig. 1. a Autoradiograph of a Southern hybridisation of 125I-
labelled total RNA of PcA to EcoRI-restricted DNA of PcA (not
containing mitochondrial DNA). b, c Agarose gel electrophoresis
of satellite DNA of Sp (S), PcA (A), PcF (F) and PcP (P) restricted
with EcoRI, BamHI and HindllI.
We show in the next section that these satellite bands
contain the genes for ribosomal RNA.
Mitochondrial and satellite DNA account for about
15-19% and less than 5% of total DNA respectively.
Contamination of mitochondrial DNA with chromosomal
DNA is less than 0.02% as judged from signal intensities
in Southern hybridisation experiments. The proportion
of mitochondrial DNA isolated in CsCl-ethidium bromide
gradients, was estimated to be less than 0.1% of total
DNA.
Satellite DNA
Restriction digestions of satellite DNAs of the four
strains show that they are enriched for repetitive DNA.
Restriction with EcoRI shows the same main pattern for
Sp and PcA on the one hand and PcF and PcP on the
other (Fig. 1). The largest strong EcoRI band of Sp/PcA
is a double band. The approximate sizes of the strong
EcoRI bands (for Sp/PcA: 2 x 3.3 kb, 2.2 kb, 1.1 kb;
for PcF/PcP: 6.7 kb, 3.3 kb) add up to about 10 kb for
all strains. As these repetitive DNAs band separately in
CsCl-bisbenzimide gradients and as there is no band of
10 kb or smaller visible in total, high molecular weight
DNA run on agarose gels, the repeat must be tandemly
arranged in all four strains. Restriction with BamHI and
HindlII shows bands of approximately 10 kb for all four
U. Raeder and P. Broda: DNA of lignin-degrading fungi
strains, indicating the presence of a 10 kb repetitive
sequence with a single target size for these two enzymes.
In addition there are two smaller bands visible in BamHI-
restricted satellite DNA of PcP and in HindIII-restricted
satellite DNA of Sp, PcA and PcP. The sizes of these
fragments (about 5.6 kb and 4.3 kb in all cases) again
add up to approximately 10 kb (Fig. 1). This suggests
that at least in Sp, PcA and PcP there are two sets of the
10 kb repeat which are similar but have slight sequence
divergence.
The size and tandem arrangment of the 10 kb repeats
were consistent with their containing the ribosomal
RNA genes. In order to test this hypothesis, we used
total RNA as a probe for ribosomal RNA genes, as it
consists mainly of ribosomal RNA. 125I-labelled total
RNA of PcA was hybridised to Southern transfers of
EcoRI-restricted chromosomal and satellite DNA of PcA.
Figure 1 shows tht the predominantly hybridising bands
were the 3.3 kb and 2.2 kb bands of the 10 kb repeat
(with less hybridisation to the 1.1 kb band). There was
also hybridisation to a 6.7 kb band which corresponds to
a minor band in the EcoRI-restricted satellite DNA;
this could be due to heterogeneity of the ribosomal
RNA repeat, as revealed more strikingly in the HindlII
digests described above. We assume that the very similar
satellite sequences of the other three strains also contain
ribosomal RNA genes.
The occurrence of ribosomal RNA genes as an AT-rich
satellite in appropriate gradients may be a more general
property of basidiomycetes. Garber and Yoder (1983)
have recently reported similar results with Cochliobolus
heterostrophus.
Comparison o f t he mitochondrial DNAs
of the four strains
Restriction digests of the mitochondriai DNAs of the
four strains show similar fragment patterns for Sp and
PcA. The patterns of PcF and PcP are different from
those of Sp and PcA and from each other (Fig. 2). The
differences in the restriction pattems can be either a
reflection of general sequence divergence or of extensive
DNA rearrangments as observed in intraspecific compari-
sons of mitochondrial DNAs of many species, especially
lower eukaryotes and plants (Collins and Lambowitz
1983). To decide between these possibilities, labelled
mitochondriai DNA of each strain was hybridised under
stringent conditions to BamHI restricted mitochondrial
DNA of all four strains (Fig. 3). The autoradiographs
show that mitochondrial DNAs of Sp and PeA cross-
hybridised perfectly as expected. Both hybridised only
weakly to mitochondrial DNAs of PcF and PeP. Mito-
chondriai DNAs of PcF and PcP hybridised more strong-
ly to each other than to mitochondrial DNAs of Sp and
U. Raeder and P. Broda: DNA of lignin-degrading fungi 503

PcA, but the former pair is not as closely related as the

latter pair. This means that the restriction differences
between Sp/PcA compared to PcF and PcP can be ex-
plained b y general sequence divergence.
Labelled mitochondrial DNA o f Aspergillus nidulans
was hybridised under non-stringent conditions (55 °C)
to BamHI-restricted mitochondrial DNA o f the four
strains and to EcoRI-restricted mitochondrial DNA of
Saccharomyces cerevisiae (a gift from S. Oliver). Figure
3 shows that the mitochondrial restriction bands which
hybridised most strongly, and therefore contain the
most conserved sequences, have the same size for Sp and
PcA on one hand and PcF and PcP on the other. Hybrid-
isation to mitochondriai DNA o f S. cerevisiae is much
weaker.
An interesting observation was that rnitochondrial
DNA of each o f the four strains hybridised also to bands
o f the 10 kb repeat o f the respective strain under strin-
gent conditions. Even mitochondrial DNA o f A . nidulans
hybridised to bands of the 10 kb repeat of PcA under
non-stringent conditions (Fig. 3). This might be due to
homology between nuclear and mitochondrial ribosomal
DNA.

Fig. 2a, b. Mitochondrial DNA of Sp (S), PcA (A), PcF (F), and
PcP (P) restricted with PstI (a) and BamHI (b). Lane m contains
HindlII-restricted size marker hc1857 (23.8 kb, 9.4 kb, 6.6 kb,
4.3 kb). Lane a contains EcoRI-restricted DNA of PcA (not con-
taining mitochondrial DNA). Lane Sc contains EcoRI-restricted
mitochondrial DNA of Saccharomyces cerevisiae.
Comparison of homology between the chromosomal
DNAs of the four strains
We have tested DNA homology between the four strains
b y Southern hybridisation o f labelled random chromoso-

Fig. 3. a Autoradiograph of a Southern hybridisation (at 55 °C) of labelled mitochondrial DNA of Aspergillusnidulansto EcoRI-restricted
DNA of PcA (lane a: not including mitochondrial DNA), to BamHI-restricted mitochondrial DNA of Sp (S), PeA (A), PcF (F), PcP (P)
and to EcoRI-restricted mitochondrial DNA of Saccharomyces cerevisiae (Sc). b - d Autoradiographs of Southern hybridisations (at
68 °C) of labelled mitochondrial DNA of PcA (b), PcF (c) and PcP (d) to BamHI-restricted mitochondrial DNA of Sp (S), PcA (A),
PcF (F), PcP (P) and to EcoRI-restricted satellite DNA of PcA (sA), PcF (sF) and PeP (sP). The filters for these hybridisations were
prepared from gels like those shown in Fig. 2(b) and in Fig. 1 (for EcoRI-restricted satellite DNA). Note that the amount of mitochon-
drial DNA on these gels is smaller for PcF and PcP than for Sp and PcA.
504
real sequences (obtained by cloning) of one strain to
constant amounts of restricted total DNA of all four
strains. The previous identification of mitochondrial and
ribosomal sequences allowed us to exclude such se-
quences from this comparison. The hybridisations were
carried out under stringent conditions. Provided that the
genome size and the copy number of the sequences
which correspond to the probe sequence are similar for
the tested strains, the strength of the hybridisation signal
on the autoradiograph is an indication of DNA homology.
Gene libraries of Sp and PcA were prepared by cloning
random EcoRI fragments (> 8 kb) into XgtWEST-T5 -622.
Phages from randomly picked plaques (ten from each
library) were propagated and DNA was prepared. The
DNAs were restricted with EcoRI and run on agarose
gels to check for the presence of inserts: two out of the
twenty DNAs did not appear to contain inserts. DNA
from such a gel was blotted onto nitrocellulose filters
and hybridised with labelled mitochondrial DNA of PcA.
Three of the eighteen clones with inserts hybridised. As
mitochondrial DNAs of Sp and PcA are almost identical
and were found to hybridise also with the ribosomal
DNA repeat, it was assumed that the DNAs of the fif-
teen clones which did not hybridise do not contain
mitochondrial or ribosomal DNA. Eleven of these fifteen
clones (six from the Sp library and five from the PcA
library) covering about 0.1% of each genome (see
"genome size estimation" below) were labelled by nick
translation. Each probe was hybridised under stringent
conditions to EcoRI-restricted total DNA of the four
strains. Figure 4 shows typical autoradiographs obtained
in this experiment.
The DNA of all eleven clones hybridised with similar
efficiency to Sp DNA and PeA DNA, indicating high
DNA homology between these two strains. Five (three
of Sp and two of PcA) hybridised to similar sized frag-
ments in total DNA of both Sp and PcA (e.g. Fig. 4a,
d). These hybridising fragments correspond in size and
number to the cloned fragments. From the six other
clones which hybridised to differently sized fragments in
total DNA of Sp and PcA, four (two of Sp and two of
PeA) hybridised to more EcoRI fragments in total DNA
of the respective strains (two or three) than were original-
ly present in the clone (one or two; e.g. Fig. 4b, c, e).
This indicates the presence of more than one (probably
two) slightly different copies of the tested sequence
(or part of it) in the genome. This could suggest that the
two fungi Sp and PcA are diploid. None of the clones
tested contained middle or highly repetitive sequences;
Wu et al. (1983) made similar observations with another
basidiomycete Coprinus cinerus.
In contrast the signals obtained from hybridisation of
Sp-derived and PcA-derived clones to EcoRI-restricted
total DNA of PcF and PcP were much weaker. This, like
the results with the mitochondrial DNAs, strongly sug-
U. Raeder and P. Broda: DNA of lignin-degradingfungi
gests a lower degree of DNA homology of PcF and PcP
compared to Sp and PcA. However we cannot exclude
that PcF and PcP have a very much larger genome size
than Sp and PcA. Some similarities between the Southern
hybridisation patterns between PcF and PcP (not shown)
indicate that PcF and PcP are more closely related to
each other than either of them is to Sp or PcA. This is
also consistent with the similarities of the 10 kb repeats
and the mitochondrial DNAs, described above.
Estimation of the GC content of DNA
from strains Sp and PcA
Bisbenzimide in CsCl-gradients increases the separation
effect of DNAs with different GC-contents by binding to
adenine-residues in the DNA and thereby reducing the
buoyant density in CsC1 (Miiller and Gautier 1975).
Experiments with marker DNAs of different GC-contents
(33%, 50%, 71%) showed a linear relationship between
GC-content and position in the CsCl-bisbenzimide gradi-
ents. Gradients prepared by running in a Ti50 rotor at
71,800 x g at 17 °C separated the marker DNAs by 0.78
ram/l% difference in the GC-content. Chromosomal and
mitochondrial DNAs of Sp and PcA were run separately
in such gradients with internal markers and gradients
with marker DNAs were run in parallel. The positions of
the fluorescing bands were plotted against GC-content of
the markers.
The GC-contents were estimated to be 59% for chro-
mosomal DNA and 33% for mitochondrial DNA for
both Sp and PcA. These values are comparable with
those reported for other basidiomycetes (Storck and
Alexopoulos 1970; Villa and Storck 1968).
Estimation of the genome size of strain PeA
We have estimated the genome size of PcA as a multiple
of the known genome size of XgtWES-T5-622. This ap-
proach depends on the following concept: in a mixture
of two genomes (here PcA and XgtWES-T5"622), where
the amount of each DNA is proportional to its genome
size, all single copy sequences are present in equimolar
amounts. A labelled clone, consisting of a fungal single
copy sequence and the vector sequence (with a known
distribution of radioactivity in the two parts), was hy-
bridised to a constant amount of filter-bound fungal
DNA alone and mixed with various amounts of vector
DNA. By measuring the amounts of hybridisation and
considering the distribution of radioactivity in the fungal
and the vector part of the probe, the ratio of filter-bound
fungal DNA: vector DNA was determined at which
U. Raeder and P. Broda: DNA of lignin-degrading fungi 505

Fig. 4a-e. Autoradiographs of Southern hybridisations (at 68 °C) of labelled DNA of random chromosomal clones of Sp (a, b, c) and
PcA (d, e) to EcoRI-restricted total DNA of Sp (S), PcA (A), PcF (F) and PcP (P) (20 #g per lane). The clones used in hybridisations
(a, b, c, d) contain one fungal EcoRI fragment; the clone used in hybridisation (e) contains two fungal EcoRI fragments. Lane m con-
tains 3 ng of HindlII-restricted DNA of ~.c1857.

CPM '~

5O0OO"

10000

• ,! ,IP
1:o l:s-~ "~ ." ~ 1:.~ R
¢1

Fig. 5. a Graphical representation of the data obtained in the genome size estimation experiment. R is the ratio of the amounts of filter-
bound fungal DNA: vector DNA. The amount of hybridisation (cpm) of the two different probes used in this experiment are marked
with (o) and (x) (three replicates each). The two straight lines were fitted to the two sets of values using least squares analysis, b Auto-
radiograph of one of the filters after hybridisation with increasing amounts of filter-bound vector DNA from the bottom row (R = 1 : 0)
to the top row (R = 1 : 1/500).

equimolar amounts of the fungal and the vector part
would hybridise. Provided that the vector and the fungal
sequences hybridise with the same efficiency (tilter
driven hybridisation), this ratio of equimolar hybridisa-
tion is the ratio o f equimolar presence of fungal and
vector single copy sequences and corresponds to the
ratio of the sizes o f the two genomes.
Two o f the PcA clones from the cross-hybridisation
experiment were chosen for this experiment. They are
believed to contain single copy sequences for the follow-
ing reasons: (1) they do not contain mitochondrial or
ribosomal DNA; (2) their intensities o f hybridisation are
similar to those o f the other nine clones; (3) t h e y hy-
bridise only to those EcoRI fragments in restricted total
DNA that correspond to the EcoRI fragments in the
clones.
Figure 5 is a graphical representation o f the results.
Evaluation o f the data showed that equimolar hybridisa-
506
tion would occur at a ratio o f fungal to vector DNA of
1,161 : 1 and 1,347: 1. F r o m this the size of the haploid
genome is calculated as 4.1 or 4.7 x 107 bp. This is com-
parable with the genome sizes of other basidiomycetes
such as SchizophyUum commune (complexity of 3.3 x
107 bp; Dons et al. 1979)) and Coprinus lagopus (3.5 x
10 v pb; Dutta 1974), which were determined by other
methods.
Conclusions
(1) The two lignin degraders P. chrysosporium MFA66
and S. pulverulentum Novobranova are very similar
at the DNA level. This confirms their taxonomic clas-
sification as members o f the same species (Burdsall
and Eslyn 1974).
(2) Two other strains classified as P. chrysosporium
(Catalogue of the Culture Collection of the Com-
monwealth Mycological Institute #74,691 and
#110,120), which were originally included in this
comparison as indicators for intraspecific variations,
are both less related to S. pulverulentum Novobra-
nova and P. chrysosporiurn MFA46, but may be
more related to each other.
(3) The genome size ( 4 - 5 x 107 bp, estimated for P.
chrysosporium ME446) and the DNA GC-contents
(59% for chromosomal DNA and 33% for mitochon-
diral DNA) o f S. pulverulentum Novobranova and
P. chrysosporium ME446 are similar to those of
other basidiomycetes.
(4) The ribosomal DNA repeat which was present as an
AT-rich satellite to bulk chromosomal DNA was
found to occur in two sets in three of the four
strains.
U. Raeder and P. Broda: DNA of lignin-degrading fungi
Acknowledgements. This work was part of a programme sup-
ported jointly by BP Venture Research and the Agriculture and
Food Research Council. We wish to thank Olwen Birch for ex-
cellent technical assistance and John CuUum for helpful com-
ments on the manuscript.
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Communicated by B. S. Cox
Received March 1 / May 14, 1984