Sequence organization of the nuclear DNA of Schizophyllum commune

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Biochimica et Biophysica Acta, 607 (1980) 385--396 © Elsevier/North-HollandBiomedicalPress

BBA 99661 SEQUENCE ORGANIZATION OF THE NUCLEAR DNA OF S C H I Z O P H Y L L UM COMMUNE

J.J.M.DONS * and J.G.H. WESSELS ** Department of Developmental Plant Biology, Biological Centre, University of Groningen, Kerklaan 30, 9751 NN Haren (The Netherlands)

(Received July 20th, 1979) Key words: Repetitive DNA; Nuclear DNA; Sequence organization; Genome complexity; (Schizophyllum commune)

Summary Several methods were used to characterize the organization of repetitive DNA in the fungus Schizophyllum commune. They all failed to show interspersion of repetitive sequences among single copy sequences. Saturation hybridization showed that 2.2% of the double-stranded nuclear DNA coded for rRNA. The size of the ribosomal cistron (11.9 • 10 6 daltons) was determined by restriction enzyme analysis. From these values it was calculated that about 6% of the nuclear D N A consisted of ribosomal cistrons, which approx, equals the amount of repetitive D N A present. Thus, this simple sequence organization in Schizophyllum c o m m u n e is fundamentally differentfrom organization patterns in higher eukaryotes.

Introduction

In most eukaryotic organisms studied, a large fraction of the nuclear genome consists of short repetitivesequences interspersed between longer single copy sequences. This 'short period interspersion pattern' was first described for Xenopus leavis D N A [1] and proved to be of general validity,both in animals [2--5] and in plants [6--9]. Such structural organization fits well in the Britten-Davidson model for gene regulation [10,11], which suggests that the interspersed repetitive sequences have a coordinate regulatory function in the differentialtranscriptionduring development. * Present address: D e p a r t m e n t of Biochemistry, University of Groningen, Groningen, The Netherlands. ** To w h o m correspondence should be addressed. Abbreviations: Pipes, piperazine-/~r,N'-his(2-ethanesulfonic acid); SDS, sodium d o d e e y l sulphate.

386 In contrast to this sequence organisation it was found that DNA of some insects like Drosophila melanogaster [12] was characterized by a 'long period interspersion pattern'. As the number of investigated organisms increased, more exceptions were found. No short period interspersion pattern was found in birds [13,14], Nematodes [15,16] and in the Syrian hamster [17]. The genome size of fungi is very small compared to that of higher plants and animals and the amount of repetitive DNA is relatively small [18,19]. Short period interspersion was found for the genome of the cellular slime mold Dictyostelium discoideum [20] but not in the Oomycete Achlya bisexualis [21]. In these organisms only a part of the repetitive DNA consisted or ribosomal cistrons [21]. For the Ascomycetes Saccharomyces cerevisiae [22] and Aspergillus nidulans [23] it was suggested that nearly the entire, small repetitive fraction of the genome could be accounted for by rRNA cistrons. In the Basidiomycete SchizophyUum commune about 7% of the nuclear DNA belongs to the repetitive class [19]. The results presented here show that no short period interspersion pattern exists and that most of the repetitive DNA accommodates the rRNA cistrons. Materials and Methods

Materials. Radiochemicals: Sodium [32P]orthophosphate (carrier free) and L-[3H]leucine were obtained from The Radiochemical Centre, Amersham, U.K. Enzymes: $1 nuclease from Aspergillus oryzae (EC 3.1.30.7) was obtained from Sigma, U.S.A. Restriction enzymes Eco RI and Hind III were a gift from Mr. B. Wierenga (Department of Biochemistry, University of Groningen). Calf thymus DNA from Serva, Heidelberg, F.R.G., Hind III digested k DNA from Boehringer, Mannheim, F.R.G. DNA from phage SPP1 was a gift from Dr. S. Bron (Department of Genetics, University of Groningen). Agarose A50 (100--200 mesh) and hydroxyapatite (HTP, DNA grade) were obtained from BioRad Laboratories, Richmond, U.S.A. and Heparine from Leo Pharmaceutical Products, Ballerup, Denmark. Preparation of nuclear [32P]DNA. Basidiospores obtained after mating S. commune strain 1-40 (A41 B41) with 1-50 (A51 B51) were germinated in an inorganic phosphate-free medium and labelled with sodium [32P]orthophosphate [19]. Nuclear DNA, free of mitochondrial DNA, was isolated and purified by hydroxyapatite chromatography as described [19]. The specific activity of the nuclear DNA was 35 000 cpm • pg-1. Preparation and sizing of DNA fragments. Short DNA fragments (approx. 500 basepairs) were prepared by two passages through a refrigerated French Pressure Cell at 703 kg • cm -2. Longer fragments were prepared by shearing in a Virtis 60 homogenizer at 0°C: fragments of 7.7 kilo-basepairs by shearing for 30 min at 3500 rev./min fragments of 2.4 kilo-basepairs by a second shearing for 30 min at 10 000 rev./min. Shearing was carried out in 0.15 M NaC1/ 0.015 M trisodium citrate and the DNA fragments concentrated by precipitation with 2 vols. ethanol. After centrifugation (15 min, 9000 × g, --10°C) the pellet was dried in vacuo, dissolved in 6 mM Pipes, pH 6.7, 0.18 M NaCI and chromatographed on a Agarose A50 column. DNA fragments were recovered from the exclusion volume.

387 The length of the DNA fragments was determined by horizontal slab gel electrophoresis in agarose 0.7% in 0.09 M Tris-borate buffer, pH 8.0, 2.5 mM EDTA. Gels were run at room temperature for 20 h at 1 V . cm -1. After staining with ethidium bromide (1/~g • ml-1), the position of the fragments was compared with those of Hind III digested XDNA and Eco RI digested DNA from phage SPP1. Alternatively lanes were cut in slices and radioactivity determined. Reassociation analysis. Reassociation of [32p] DNA fragments was performed at 64°C in 0.12 M sodium phosphate buffer (an equimolar mixture of NaH2PO4 and Na2HPO4, pH 6.8) and monitored by hydroxyapatite chromatography [19]. When $1 nuclease digestion was used, reassociation was performed in 6 mM Pipes, pH 6.7, 0.18 M NaC1 or in 10 mM Tris-HC1, pH 7.5, 0.18 M NaC1, 1 mM EDTA and 0.25% (w/v) sodium dodecyl sulphate (SD8). Computer analyses for multicomponent reassociation kinetics were made according to programs described by Kells and Straus [24] and Monahan et al. [25]. $1 nuclease digestion and sizing of resistant regions. DNA fragments were denatured for 10 rain at 105°C and reassociated at 64°C in 6 mM Pipes, pH 6.7, 0.18 M NaC1 to the desired Cot value. Reassociation was stopped by chilling in ice-water. Single-stranded DNA was digested with $1 nuclease essentially according to Goldberg et al. [2] in 0.03 M sodium acetate, 0.16 M NaC1, 1 mM ZnSO4 and 5 mM ~-mercaptoethanol, adjusted to pH 4.4 by adding I M acetic acid. After digestion with $1 nuclease (3 U/~g DNA) at 37°C for 45 min, the reaction was stopped by chilling and addition of sodium phosphate buffer to a final concentration of 0.15 M. The digest was passed over hydroxyapatite at 60°C and double-stranded fragments were eluted with 0.3 M sodium phosphate buffer. The size distribution of $1 resistant duplexes was determined on agarose A50 columns run in 0.12 M sodium phosphate buffer. L-[3H]leucine was used as inclusion marker. Isolation of RNA. Ribosomal RNA: Mycelium was grown in liquid minimal medium [19] for 48 h at 24°C and harvested on nylon cloth. 30--40 g (wet wt.) was added to 100 ml 50 mM Tris-HC1, pI-I 8.8, 10 mM MgC12, 10 mM NaC1, 8 mg • m1-1 heparine and 1% (v/v) diethylpyrocarbonate (buffer A) and homogenized at 4°C for 30 s at maximum speed in a Waring blendor. The homogenate was diluted with 50 ml of buffer A and passed twice through a special grind-mill [26]. After centrifugation (10 rain, 2 7 0 0 0 × g , 4°C) the supernatant was filtered through glass wool and 10 ml layered on a double cushion of 1.5 ml 2.5 M sucrose and 1 ml 0.5 M sucrose in 10 mM Tris-HC1, pH 7.5, 10 mM MgC12, 10 mM NaC1 and 4 rag. m1-1 heparine (buffer B). A turbid layer containing (poly)ribosomes was visible on the 2.5 M sucrose cushion after centrifugation (2 h, 280 000 ×g, 4°C). This band was removed and diluted with 2 vols. of buffer B. Dissociation of the (poly)ribosomes was achieved by a treatment with 0.03 M EDTA for 30 rain at 4°C [27]. Ribosomal subunits were collected by centrifugation (15 h, 280 000 X g, 4°C) on a cushion of 2.5 M sucrose in buffer B, omitting MgC12 and stored at --70°C. rRNA was isolated using the same procedure as used for isolation of total RNA. SDSpolyacrylamide gel electrophoresis showed that the rRNA preparation contained all four rRNA species (25 S, 1 8 S, 5.8 S and 5 S) whereas no tRNA could be detected. 5 S RNA was present in minor quantities, possibly due to

388

loss of the small 5 S RNP particle, which is released from the major ribosomal subunit during EDTA treatment. Total RNA was isolated from mycelium as described [28]. To obtain [32p]rRNA, germinating basidiospores were labelled with sodium [32p]orthophosphate as described [19]. RNA/DNA saturation hybridization. Nuclear [ 32p] DNA fragments (approx. five kilo-basepairs) were denatured in 10 mM Tris-HC1, pH 7.5 at 100°C for 10 min and quickly cooled in an ice/salt mixture. Denatured DNA (0.9 ~g • ml -I) was then incubated with different concentrations (up to 35 ~g. m1-1) rRNA or total RNA in 10 mM Tris-HC1, pH 7.5, 0.18 M NaC1, 0.25% (w/v) SDS and 1 mM EDTA for 30 min at 64°C. The reaction was stopped by chilling and the hybridization mixture was diluted 10 times with 33 mM sodium acetate, pH 4.4, 36 mM NaC1, 3 mM ZnC12 and 5.6% (v/v) glycerol. The nucleic acid concentration was adjusted to 20 pg. m1-1 with RNA (from S. commune), S1 nuclease was added (20 U . m1-1) and single strands digested for 45 min at 37°C. The amount of S1 resistant hybrids was determined by precipitation with trichloroacetic acid [28]. The thermal stability of the hybrids was determined by incubating hybridization mixtures at different temperatures for 10 min, followed by S1 nuclease digestion. Restriction fragmentation of nuclear DNA. High molecular weight DNA (about 25 • 106 daltons) was isolated from protoplasts by a gentle lysis procedure [19]. Large amounts of DNA were isolated directly from mycelium as described [19]. Such DNA was heterogenous in size and was run on a preparative agarose gel (0.8%). High molecular weight DNA was recovered from the gel by electrophoresis into hydoxyapatite according to the procedure of Tabak and Flavell [29].. Nuclear DNA and mitochondrial DNA were separated in a preparative CsC1 gradient [19]. The nuclear DNA band was isolated as a whole or divided into a light and heavy part and dialyzed extensively against 0.1 M Tris-HC1, pH 7.5, 0.05 M NaC1. Digestions with restriction enzymes were generally performed in a final volume of 0.09 ml containing 2--5 ~g DNA and 8 U Eco RI in 0.1 M TrisHC1, pH 7.5, 0.05 M NaC1, 5 mM MgC12 and 1 mM dithiothreitol or 16 U Hind III in 7 mM Tris-HC1, pH 7.4, 0.06 M NaC1, 7 mM MgC12 and 1 mM dithiothreitol. After incubation for 2 h at 37°C, fragments were separated by gelelectrophoresis in 0.8% agarose as described above. Eco RI fragments of DNA from phage SPP1 and Hind III fragments of ~DNA were used as molecular weight markers. Gels were stained with ethidiumbromide (2 pg. m1-1) and photographed using a ultraviolet-transilluminator (ultraviolet Products Inc. U.S.A.) and Kodak wratten gelatin filter No. 9. Hybridization of [32p]rRNA to blotted DNA fragments. DNA restriction fragments in agarose gels were transferred to sheets of nitrocellulose (Sartorius 0.45 #m, SM 11306) as described by Southern [30]. The nitrocellulose sheets were incubated with 80 ~g. m1-1 [32p]rRNA (spec. act. 20--30.103 cpm~g-1) in 50% (v/v) formamide, 0.9 M NaCI/0.09 M trisodium citrate for 5 h at 40°C. The sheets were rinsed for 0.5 h in the same buffer and left overnight in 0.3 M NaC1/0.03 M trisodium citrate at 4°C. After five additional washings

389

with 0.3 M NaC1/0.03 M trisodium c'itrate, the sheets were dried and subjected to autoradiography {Kodak X-ray film XR 1). Results Reassociation kinetics o f long DNA fragments By comparing reassociation kinetics of DNA fragments of various lengths qualitative information can be obtained about interspersion. If repetitive sequences are interspersed between single copy sequences, one might expect that the reassociation rate of long fragments measured by hydroxyapatite binding increases extremely due to the apparent binding of single stranded DNA sequences adjacent to reassociated repetitive sequences. This phenomenon is observed in organisms having a short period interspersion pattern [2,4, 20]. Fig. 1 shows the reassociation kinetics of nuclear DNA from S. commune

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with different fragment lengths, assayed by binding to hydroxyapatite. A least square analysis [24] of the data of the reassociation of short fragments yielded three components. After correction for the terminal fraction unreassociated (11.4%) the following genome fractions (F) and rate constants (kpure) were calculated, with values previously [19] found in parenthesis: zero time binding DNA: F = 0.039 (0.023), k is not defined; repetitive DNA: F = 0.054 (0.071), k = 44 (39) M -~ • s-l; single copy DNA: F = 0.906 (0.906), k = 0.068 (0.063) M -~ • s -1. In view of the present study it is important to note that some variation in the calculated amounts of repetitive D N A occurs (5.4 and 7.1%). The reassociation rates of long D N A fragments (2.4 kilo-base pairs) and 7.7 kilo-basepairs were increased compared to the reassociation of the short frag-

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F i g . 2. R e a s s o c i a t i o n k i n e t i c s o f l a r g e s i z e d n u c l e a r D N A as d e t e r m i n e d b y h y d r o x y a p a t i t e a n d $ 1 n u c l e ase d i g e s t i o n . N u c l e a r [ 3 2 p ] D N A ( 5 k i l o b a s e p a i r ) w a s r e a s s o c i a t e d i n 1 0 m M T r i s - H C l , p H 7.5, 0 . 1 8 M NaC1, 1 m M E D T A a n d 0 . 2 5 % ( w / v ) S D S a t 6 4 ° C . S a m p l e s w e r e t a k e n at d i f f e r e n t t i m e s a n d t h e a m o u n t o f d u p l e x e s m e a s u r e d e i t h e r b y h y d r o x y a p a t i t e c h r o m a t o g r a p h y (e, 3 0 ~tg • rn1-1 ; ©, 2 6 0 /~g • m1-1 ) o r b y t h e S1 n u c l e a s e a s s a y ( A 3 0 # g • m 1 - 1 ; ~, 2 6 0 p g • m l - ! ) . T h e l i n e ( ) r e p r e s e n t s t h e b e s t fit [25] yielding two components e a c h r e a s s o c i a t i n g w i t h s e c o n d o r d e r k i n e t i c s : C/C 0 = 1 / ( 1 + k C o t ) . T h e k i n e t i c p a r a m e t e r s f o u n d w e r e : R e p e t i t i v e D N A F = 0 . 0 5 7 w i t h k = 3 1 0 M -1 • s -1 a n d s i n g l e c o p y D N A F = 0 . 8 8 2 w i t h /~ = 0 . 1 5 1 M -1 • s - 1 . T h e s e p a r a m e t e r s w e r e u s e d t o c a l c u l a t e a c u ~ e ( . . . . . . ) representing the reassociation of both components w h e n m e a s u r e d w i t h S1 n u c l e a s e a c c o r d i n g t o n o n - s e c o n d o r d e r k i n e t i c s : C/C 0 = [ 1 / ( 1 + k C 0 t ] 0 . 4 5 .

391

ments (0.5 kilo-basepairs). An acceleration of the reassociation is to be expected from the known influence of fragment length on reassociation rate. According to the relationship k2 = kl (MW2/MWl) in [31] the expected Cotln values of the 2.4 kilo-basepairs and 7.7 kilobasepairs would be approx. 9 and 5, respectively, close to the observed values of 13 and 8. Also the more rapidly reassociating fraction (repetitive DNA) did n o t increase considerably with increasing fragment length indicating that most, if not all of the single copy DNA reassociated independently of the repetitive DNA. Up to a sequence length of about eight kilo-basepairs there seemed to be no interspersion. A similar conclusion could be drawn from a comparison of reassociation kinetics of long fragments (five kilo-basepairs) as measured by both the hydroxyapatite m e t h o d and the S1 nuclease assay. Reassociation assayed on hydroxyapatite showed (Fig. 2) the typical two c o m p o n e n t curve of repetitive DNA and single copy DNA, calculated by assuming second order kinetics as described in the familiar form C/Co = 1/(1 + kCot). Using the parameters of the best fit curve, a second curve was calculated on basis of non-second order kinetics C/Co = [1/(1 + kCot)] 0.4s, described for reassociation measured with S1 nuclease [32]. This curve fits well with the data points obtained from resistance to $1 nuclease (Fig. 2). Since both curves closely follow the theoretical reassociation kinetics it can be concluded that during reassociation of long fragments no single strands were attached to renatured sequences apart from those that could be expected on reaction kinetical grounds. $1 nuclease digestion of repetitive sequences An alternative m e t h o d to detect the presence of short interspersed sequences is the sizing of duplexes excised by digestion with a single strand specific nuclease (e.g. Refs. 2 and 3). The experiments were performed using DNA fragments which after shearing were excluded from a Agarose A50 column. These fragments with an average length of 7.7 kilo-basepairs could be bound completely to hydroxyapatite in 0.15 M sodium phosphate buffer at 60°C. After reassociation to a low Cot value (0.145) single-stranded DNA was digested with S1 nuclease and duplex structures were bound to hydroxyapatite. Both the unbound fraction and the bound fraction (after elution with 0.3 M sodium phosphate buffer) were analyzed on a Agarose A50 column. The unbound fraction consisted of completely degraded DNA as shown by its co-elution with the inclusion marker. 4.5% of the DNA was present in the bound fraction and almost all of this DNA was excluded from the A50 column (Fig, 3). By comparing this profile with the elution pattern of reassociated DNA bound to hydroxyapatite without prior S1 nuclease treatment it was evident that only a small fraction of the duplexes was included in the column due to shortening by S1 nuclease. About 17% of the DNA was smaller than 1000 nucleotide pairs. However these degraded duplexes showed a continuum of sizes and there was no distinct class of small duplexes. Agarose slab gel electrophoresis showed that the excluded duplexes had an average fragment length of 6.8 kilo-basepairs. Therefore reassociation of repetitive DNA took place over almost the total length of the fragments. For comparison, the procedure described above was also applied to long fragments of calf t h y m u s DNA (10 kilo-basepairs) reassociated to a Cot value of

392

2. The agarose A50 profile of S1 nuclease treated duplexes was characteristic for DNA containing short interspersed repetitive sequences (Fig. 3). The fragments in the included peak were on the average 300--400 basepairs long. About 57% of the S1 resistant DNA was smaller than 1000 nucleotide pairs, which agrees with earlier reports on calf thymus DNA [3,33].

Hybridization of repetitive sequences The amount of nuclear DNA coding for ribosomal RNA was determined by RNA/DNA hybridization. By increasing the RNA concentration a saturation

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393 level was reached (Fig. 4). rRNA hybridized to 1.1% of the nuclear DNA. Assuming asymmetric transcription this means that 2.2% of the DNA accounts for the sequences present in mature ribosomal RNA species. The thermal stability of the hybrids was determined by incubation in 0.18 M NaC1 at different temperatures, followed by S1 nuclease treatment. A Tmi * of 91°C was found, which is about 4°C below the Tmi of native DNA fragments. This decrease is expected because the G + C content of the ribosomal cistrons is 45%, which is 12% lower than the overall G + C content of nuclear DNA from S. commune [19]. Using total RNA a saturation level of about 1.4% was reached (Fig. 4). Substracting the contribution of the ribosomal RNAs leaves about 0.3% of the DNA which hybridizes fast. Most likely these sequences contain the tRNA genes. In other fungi tRNA saturation levels ranging from 0.08% (Saccharomyces cerevisiae, [34]) to 0.3% (Neurospora crassa, [35]) have been reported. Length o f the ribosomal cistron of 8. commune High molecular weight nuclear DNA was digested with restriction endonuclease Eco RI and Hind III. The fragments were separated by agarose gel electro-

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Fig. 4. H y b r i d i z a t i o n l d n e t i c s o f r e p e t i t i v e s e q u e n c e s . N u c l e a r [ 3 2 p ] D N A w a s i n c u b a t e d f o r 3 0 r a i n a t 6 4 ° C w i t h d i f f e r e n t c o n c e n t r a t i o n s p u r i f i e d r R N A (e ~) o r t o t a l R N A (o o). H y b r i d f o r m a t i o n w a s a s s a y e d w i t h S1 n u e l e a s e . T h e c u r v e s r e p r e s e n t t h e b e s t fit a s s u m i n g p s e u d o - f i r s t o r d e r k i n e t i c s [25].

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Fig. 5. R e s t r i c t i o n f r a g m e n t a t i o n p a t t e r n s of n u c l e a r D N A f r o m S. c o m m u n e . E l e c t r o p h o r e s i s of u n d i g e s t e d D N A is s h o w n in lane 2. T h e n u c l e a r D N A b a n d f r o m a CsC1 g r a d i e n t waS d i v i d e d i n t o a light a n d h e a v y p a r t a n d b o t h w e r e d i g e s t e d w i t h Eco RI (lane 3a a n d 4a, r e s p e c t i v e l y ) . A f t e r b l o t t i n g , b o t h w e r e h y b r i d i z e d w i t h [ 3 2 p ] r R N A . T h e a u t o r a d i o g r a p h s are p r e s e n t e d in 3b a n d 4b. L a n e 5a s h o w s t h e H i n d I I I r e s t r i c t i o n p a t t e r n of t o t a l n u c l e a r D N A . T h e a u t o r a d i o g r a p h a f t e r [ 3 2 p ] r R N A h y b r i d i z a t i o n is s h o w n in lane 5b. As m o l e c u l a r w e i g h t m a r k e r s w e r e used: lane 1: H i n d I I I d i g e s t e d k D N A ; lane 6: u n d i g e s t e d ( u p p e r b a n d ) a n d Eco R I d i g e s t e d D N A f r o m p h a g e SPP1.

phoresis and the molecular weights determined b y comparison with restriction fragments of phage DNAs (Fig. 5, lane 1 and 6). The molecular weight of undigested DNA was a b o u t 2 5 . 1 0 6 (lane 2). By Eco RI digestion three prominent bands were generated with molecular weights o f 6.1, 3.5 and 2.4 • 106. These bands were found in DNA taken from the light side of the nuclear DNA band after CsC1 centrifugation (lane 3a) b u t not in the heavy DNA fraction (lane 4a). Since the ribosomal cistrons of S. c o m m u n e do have a lower G + C content than the overall nuclear DNA and thus band at a lower density in CsC1 gradients [19], the three Eco RI fragments were most likely generated by digestion of the reiterated rRNA cistrons. This was confirmed b y their specific hybridization with [32p]rRNA as shown by the autoradiograms (3b, 4b). From the sum of the molecular weights of the restriction fragments the size o f the ribosomal cistron of S. c o m m u n e could be calculated as 1 2 . 0 . 1 0 6 daltons. The size of the largest fragment almost equals the sum of the two smaller fragments. However, this large fragment is n o t a partial since a second digestion of this fragment after isolation [29] did n o t reveal smaller fragments. Moreover, from restriction analysis of nuclear DNA with another enzyme, Hind III {lane 5a, 5b), the size of the ribosomal cistron was calculated as 11.8 • 106 (fragments of 5.7, 4.2 and 1 . 9 . 106), in good agreement with Eco RI digestion.

395 Discussion The nuclear genome of the Basidiomycete S. c o m m u n e lacks extensive short period interspersion of repetitive and single c o p y DNA sequences. This conclusion is based on the results of methods which have proved to be useful in the detection of such a sequence organization in a great number of eukaryotic organisms. The fraction of double-stranded DNA that b o u n d to hydroxyapatite at low Cot values did n o t increase significantly when longer DNA fragments were used. Reassociation assayed by hydroxyapatite chromatography or by resistance to S1 nuclease digestion both followed normal reassociation kinetics. Both results indicated that there is no substantial a m o u n t of unreassociated tails b o u n d to renatured repetitive DNA. Moreover, a direct analysis of S1 nuclease resistant duplexes obtained after a short reassociation of long fragments did not reveal the presence of a class of short repetitive sequences. The nuclear genome size of S. c o m m u n e has been determined as 22.8 • 109 daltons [19]. Saturation hybridization with r R N A showed that 2.2% (or 0.5 • 109 daltons) of the nuclear DNA coded for rRNA. The molecular weights of 25 S and 18 S rRNA of S. c o m m u n e have been estimated as 1.32 and 0.73 • 106, respectively [37]. Taking into account the two small r R N A species 5.8 S (Mr 50 000) and 5 S (Mr 40 000), the total molecular weight of ribosomal RNA is 2 . 1 4 . 1 0 6 . From this value and the hybridization percentage the multiplicity of the ribosomal cistrons was calculated as approx. 120. The ribosomal cistron was cleaved b y Eco RI and Hind III into three fragments with a total weight of 11.9 • 106 daltons. Since the total weight of the ribosomal RNA species is 2.14 • 106 daltons, a b o u t 65% of the cistron consisted o f transcribed or non-transcribed spacer DNA. This is a rather high value compared to the ribosomal cistron of S a c c h a r o m y c e s cerevisiae [38] b u t is n o t exceptional since a range from 5 • 106 daltons up to 25 • 106 daltons [39] was found for the size of the cistron in different organisms. The total a m o u n t of nuclear DNA consisting of ribosomal cistrons could be calculated as 1 . 4 . 1 0 9 daltons, which represents a b o u t 6% of the nuclear genome. This value is close to the a m o u n t of repetitive DNA found (5.4 and 7.1%). This leads to the conclusion that nearly all of the repetitive DNA o f S. c o m m u n e codes for rRNA, except of course for the t R N A genes (up to 0.6% o f double-stranded nuclear DNA) and possibly for a small number of reiterated genes, like the histone genes. In S. c o m m u n e the incompatibility genes are considered as regulatory genes that coordinately control the occurrence of metabolic pathways related to sexual morphogenesis [40]. The apparent absence of interspersion of repetitive sequences in the genome makes it less likely that these regulatory genes operate according to the Britten-Davidson model of regulation. Acknowledgements We are very grateful to Mr. J. Groffen for carrying o u t the restriction enzyme analysis. We wish to thank Mrs. R. Janssen, Mr. B. Zantinge and Dr. O.M.H. de Vries for their contribution. Part of this work was supported by the Netherlands Foundation for Fundamental Biological Research ( B I O N ) w i t h

396

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