Electrophoretic karyotype of the ascomycete Podospora anserina

Current Genetics

Curr Genet (1990)18:481-483

© Springer-Verlag 1990

Short communication

Electrophoretic karyotype of the ascomycete Podospora anserina Heinz D. Osiewacz, Annette Clairmont, and Marion Huth Deutsches Krebsforschungszentrum, Institut fiir Virusforschung, Angewandte Tumorvirologie, Projektgruppe: Molekularbiologie der Alterungsprozesse, Im Neuenheimer Feld 506, W-6900 Heidelberg, Federal Republic of Germany Received August 16, 1990

Summary. F r a c t i o n a t i o n o f i n t a c t c h r o m o s o m e s o f the a s c o m y c e t e Podospora anserina b y c o n t o u r - c l a m p e d h o m o g e n o u s electric field ( C H E F ) gel electrophoresis resuited in the r e s o l u t i o n o f five d i s t i n c t c h r o m o s o m a l b a n d s . Two o f these b a n d s m i g r a t e d as d o u b l e t s . U s i n g c h r o m o s o m a l s t a n d a r d s f r o m Schizosaccharomyces pombe we e s t i m a t e d the size o f the P. anserina g e n o m e to a b o u t 33.4 m e g a b a s e s (Mb). By h e t e r o l o g o u s h y b r i d i z a tion o f f r a c t i o n a t e d c h r o m o s o m e s the r D N A locus was identified o n one o f the two c h r o m o s o m e s m i g r a t i n g at a b o u t 4.9 M b .

Key words: Podospora anserina - E l e c t r o p h o r e t i c k a r y o t y p e - C o n t o u r - c l a m p e d h o m o g e n e o u s electric field ( C H E F ) electrophoresis

Introduction Podospora anserina is a n a s c o m y c e t e t h a t has b e e n used in a g r e a t v a r i e t y o f b i o c h e m i c a l , genetic a n d m o l e c u l a r studies (for reviews see Esser 1974, 1985; O s i e w a c z 1990). T h e h a p l o i d c h r o m o s o m e n u m b e r o f P. anserina is seven ( F r a n k e 1962) c o r r e s p o n d i n g to the seven l i n k a g e g r o u p s d e f i n e d b y genetic analysis. A large n u m b e r o f well defined genes h a v e been m a p p e d on these l i n k a g e g r o u p s (for review see Esser 1974). H o w e v e r , until n o w n o t h i n g is k n o w n a b o u t the size o f the P. anserina g e n o m e . H e r e we r e p o r t the r e s o l u t i o n o f i n t a c t c h r o m o s o m e s b y p u l s e d field gel e l e c t r o p h o r e s i s , the l o c a l i z a t i o n o f a n u c l e a r gene o n one o f the s e p a r a t e d c h r o m o s o m e s a n d the first e s t i m a t i o n o f the size o f the g e n o m e o f this ascomycete.

Materials and methods Intact chromosomes of P. anserina were isolated from mycelial spheroplasts. For preparation of spheroplasts, cultures of

Offprint requests to. H. D. Osiewacz

P. anserina were grown on solid cornmeal medium (CCM; Esser 1974) for 2 days at 25°C. After this incubation period agar plugs with mycelium were transferred to 200 ml liquid cornmeal medium and incubated for 2 days at 25 °C with agitation. The mycelium was harvested by filtering the culture through sterile cheesecloth. After the mycelium was suspended in 50 ml spheroplast buffer (25 mM Tris-Maleate, 5 mM CaC12 x2 H20, 0.6 M sucrose; pH 7.0), Novozyme 234 (Novo Laboratories) was added at a final concentration of 5 mg/ml. The hyphae were mechanically disrupted using a Waring blendor and incubated for 2 h at 33 °C with shaking. After incubation with the lytic enzyme, the mycelial debris was removed by filtration through sterile glass wool. Spheroplasts were collected by centrifugation (3500 rpm, 4°C, SS34) and washed once with 10 ml spheroplast buffer. After centrifugation, spheroplasts were disolved in I ml spheroplast buffer and diluted to 7.75 x 10s spheroplasts/ml using 0.6 M KC1. Subsequently, the spheroplasts were embedded in 1% molten low-melting agarose (Sigma). After solidification of the agarose, the plugs were incubated in lysis buffer (0.4 M EDTA, 10 mM Tris, pH 7.5; 1% N-lauryl sarcosyt; 5 mg/ml Proteinase K) for 16 h at 50 °C followed by three washing steps in 8 ml 0.5 M EDTA, pH 9.5. Finally the agarose plugs were stored at 4°C in 0.5 M EDTA, pH 9.5. Pulsed field gel electrophoresis of intact chromosomes were performed with a contour-homogenous electric field (CHEF) gel electrophoresis system from BioRad according to the instructions of the manufacturer. Agarose gels, using different concentrations of chromosomal grade agarose from BioRad, were run in 0.5 x TBE (Tris-Borate-EDTA). Switching intervals and running times were modified in order to optimize chromosomal resolution. After electrophoresis, gels were stained with ethidium bromide for 30 min, destained for 1 h and photographed. Chromosomes were transferred to nitrocellulose or nylon (Gene screen or Hybond) membranes using a Pharmacia vacuum blotting apparatus (VacuGene XL) according to the manufacturers recommendation. After transfer was complete, UV-crosslinking of DNA to the membrane was obtained using a Stratagene crosslinker. Hybridization experiments were carried out with radiolabelled plasmid DNA. This DNA was labelled using a oligomer primed labelling kit (Gibco, BRL) and (c~-32p)dCTP. After prehybridization of filters in 50% formamide, 5 x SSPE, 0.2% SDS and 100 gg/ml salmon sperm DNA for 4 h at 37 °C in a hybridization oven (Biometra, G6ttingen, FRG), the buffer was changed and hybridization was carried out for 18 h at 37°C in the same buffer containing the denaturated radiolabelled probe. Filters were washed in 5 x SSPE and 0.2% SDS for 10min at 22°C, 10min at 37°C, 5min at 54°C and 5min at 57 °C. After these washes the filters were subjected to autoradiography.

482

Results In initial experiments, pulsed field gel electrophoresis with Podospora chromosomes was carried out at 45V with switching intervals every 60 min. Gel concentration was 0.5% and gels were run for 7 days. These conditions resulted in the resolution of two bands of about 4.0 and 5.0 megabases, Mb (data not shown). Because of this poor resolution, electrophoresis conditions were modified. Electrophoresis using a 0.5% agarose gel, with switching intervals of 50 rain for 48 h, 45 min for 12 h and 37 rain for 37 h resulted in resolution of three diffuse bands (Fig. 1 a). Using a 0.5% agarose gel and switching intervals of 50 rain for 72 h, 45 min for 68 h and 37 rain for 24 h, three bands could again be resolved (Fig. 1 b). The intensity of the lowest band indicated that several chromosomes were comigrating in this band. In order to improve the quality of chromosomal bands and chromosome separation a higher agarose concentration (0.7%) was used and the running time of the gel was extended. As may be seen from Fig. 1 c, five chromosomal bands were resolved under these conditions. The relative intensities of ultraviolet fluorescence of the bands after ethidium bromide staining suggested that the band with a molecular weight of 4.9 Mb and 4.1 Mb, respectively, represent doublets. Thus, seven chromosomes corresponding to the seven genetically determined linkage groups could be identified. Using the three chromosomes of Schizosaccharornycespombe as standards, the size of the Podospora chromosomes could be estimated as follows: chromosome VII = 6.3 Mb; chromosome VI = 5.2 Mb; chromosomes V and I V = 4 . 9 Mb each; chromosomes III and I I = 4 . 1 Mb each and chromosome I = 3 . 9 M b . F r o m these calculations, the size of the whole genome of P. anserina was estimated to be 33.4 Mb. However, because the largest chromosome of P. anserina migrates slower than the largest chromosome o f S. pombe, and no size markers of higher molecular weight were available, the size of this chromosome could be determined only approximately. A more accurate size determination of this chromosome, as well as of those chromosomes migrating as doublets, may be possible after the isolation of single (or double) bands from pulsed field gels, digestion of the corresponding chromosomes with rare cutting restriction endonucleases and electrophoresis of the corresponding fragments.

Fig. 1. Separation of P. anserina chromosomes by pulsed field gel electrophoresis (a-d) and hybridization of the resolved chromosomes with radiolabelled rDNA from Saccharornyces carlsbergensis (d). Chromosomal DNA of P. anserina was prepared as described. Chromosomes of S. cerevisiaeand S. pombewere used as size markers (BioRad). The approximate length of the separated chromosomes is indicated in megabases (Mb). White dots indicate fragments with low fluorescence. Electrophoretic conditions: a 0.5% agarose, 45V, buffer temperature 15 °C, switching intervals 50 min, 48 h; 45 min, 12 h; 37 min, 37 h; b 0.5% agarose, 45V, buffer temperature 15°C, switching intervals 50min, 72h; 45min, 68h; 37 min, 24 h; c 0.7% agarose, 45V, buffer temperature 15 °C, switching intervals 50 rain, 72 h; 45 min, 72 h; 37 rain, 72 h; d 0.7% agarose, 45V, buffer temperature 15 °C, switching intervals 50 min, 72 h; 45 rain, 72 h; 37 min, 76 h

Nevertheless, the size estimate for the genome of P. anserina lies in the order of magnitude of the size published for three closely related filamentous fungi: Neurospora crassa, 47 Mb (Orbach et al. 1988); Aspergillus nidulans, M Mb (Brody and Carbon 1989) and Cephalosphorium acremonium, 22.5 Mb (Skatrud and Queener 1989). Finally, in initial hybridization experiments using the r D N A unit from Saccharomyces carlsbergensis (Verbeet et al. J983) we were able to localize the corresponding gene on one of the Podospora chromosomes comigrating to about 4.9 Mb (Fig. 1 d). In addition, specific hybridization was obtained with the chromosomes of S. cerevisiae and S. pombe.

483 In conclusion, the data presented show that pulsed field gel electrophoresis, in combination with hybridization procedures, opens new potentials for a detailed characterization of nuclear genomes. Using this methodology we should in the future be able to m a p specific genes which cannot be located by conventional m a p p i n g techniques because the corresponding mutants are not available. Finally, the development of preparative pulsed field gel electrophoresis techniques m a y allow for the enrichment of particular D N A fractions (e.g., pre-characterized chromosomes) for further molecular analysis.

References

Acknowledgements. This work was supported by a grant of the

Brody H, Carbon J (1989) Proc Natl Acad Sci USA 86:6260-6263 Esser K (1974) In: King RC (ed) Handbook of genetics, vol I. Plenum Press, New York, pp 531-551 Esser K (1985) In: Bergener M, Ermini M, Stfihelin HB (eds) The 1984 Sandoz Lectures in Gerontology. Thresholds in aging. Academic Press, London, pp 3 20 Franke G (1962) Z Vererbungsl 93:109-117 Osiewacz HD (1990) Mutat Res 237:1-8 Orbach MJ, Vollrath D, Davis RW, Yanofsky C (1988) Mol Cell Biol 8:1469-1473 Skatrud PL, Queener SW (1989) Gene 78:331-338 Verbeet MP, Kloodwijk J, van Heerrikhuizen H, Fontijn R, Vreugdenhil E, Plana RJ (1983) Gene 23:53-63

Deutsche Forschungsgemeinschaft (Bonn-Bad Godesberg) to H.D.O. We wish to thank Prof. H. zur Hausen for support and encouragement and Prof. T. E. Elthon (Lincoln, USA) for critical reading the manuscript.

C o m m u n i c a t e d by K. Wolf