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ISOLATION OF DNA FROM FUNGAL MYCELIA AND SINGLE SPORES
34 ISOLATION OF DNA FROM FUNGAL MYCELIA AND SINGLE SPORES Steven B. Lee and John W. Taylor
Previous methods for rapid isolation of total D N A for the compara tive study of many fungi have focused on obtaining a high yield of D N A for restriction enzyme analysis (Lee et al. 1988; Taylor and Natvig 1987; Biel and Parrish 1986; Zolan and Pukkila 1986). Fungal genetic studies have also required tedious methods to separate nu clear, mitochondrial, and ribosomal D N A fractions as well as other extranuclear fractions (Lambowitz 1979; Cramer et al. 1983; Hud speth et ah 1980) to study specific D N A molecules by cloning or use in D N A hybridization as probes (Kwok et al. 1986; Anderson et al. 1987; Bruns et al. 1988; Lee and Taylor, manuscript in preparation). T h e introduction and application of P C R to fungal genetic studies has changed these requirements for isolating D N A (Bruns et al. 1989). Our previous concern for a high yield is of little consequence since single-copy genomic sequences can be routinely amplified from 1 /xg of total D N A (Saiki et al. 1988). If the target sequence is present in multiple copies, as with nuclear ribosomal D N A or mito chondrial DNA, even less starting material is required (0.1 to 10.0 ng of total DNA). Moreover, the separation of nuclear and organelle
282
PCR Protocols: A Guide to Methods and Applications Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
34. Isolation of DNA from Fungal Mycelia and Single Spores
283
1 2 3 4 5 6
Figure 1 PCR-amplified 16s rDNA from ten thousandfold dilutions of miniprepped total DNA isolated from fresh Thraustotheca, Dictyuchus, Apodachlya, Aqualinderella, and Pythium mycelium using the DNA protocol given in this chapter, lanes 1-5. Distilled water control, lane 6. Primers NS5 and NS6 are listed in Chapter 38. For each sample, 5 f i \ from a total 100-/xl reaction was run on a 2% NuSieve, 1% agarose gel at 75 V for 2 hours. The minigel was then stained in ethidium bromide (0.5 ptg/ml), and DNA was visualized under UV.
genomes is unnecessary because target amplification is primer di rected, and primers can be designed to amplify specific genomic or organellar fractions (Chapter 38). We have simplified our previous method of total D N A isolation (Lee et al. 1988) and have used this method to amplify multicopy genes from small quantities of fresh and freeze-dried mycelia and herbarium samples (Bruns et al., in press). Thousandfold to tenthousandfold dilutions of miniprep total DNAs have been used suc cessfully in amplification reactions (see steps 10 and 11 in proce dure) yielding double-stranded and single-stranded product (see Fig. 1). This method has worked for members of fungi in every group ex Oomycetes, Zygo amined to date, including the Chythdiomycetes, and Basidiomycetes. mycetes, Deuteromycetes, Ascomycetes
284
Part Three. Genetics and Evolution
DNA Isolation Protocol
Solutions Lysis buffer
50 mM Tris-HCI (pH 7.2) 50 mM EDTA 3% SDS 1% 2-mercaptoethanol Chloroform : TE-saturated phenol (1 : 1, v : v) [TE: 10 mM Tris-HCI (pH 8.0), 1 mM EDTA] 3 M NaOAc (pH 8.0) Isopropanol Ethanol (70%) TE (for resuspending pellet): 10 mM Tris-HCI, 0.1 mM EDTA Procedure 1. Fill a 1.5-ml Eppendorf microcentrifuge tube one-third up the conical portion with ground lyophilized mycelium (20 to 60 mg dry) or fresh mycelium (0.1 to 0.3 g wet [see Note]) ground in liquid nitrogen. In either case the mycelium is ground by hand with a mortar and pestle. 2. Add 400 fx\ lysis buffer and stir with a dissecting needle and/or vortex so the mixture is homogeneous. If the mixture is too viscous, add more lysis buffer (up to 700 /xl). 3. Incubate at 65°C for 1 hour. 4. Add 400 /xl chloroform : phenol and vortex briefly, but be care ful as the caps may loosen during vortexing. (If more than 400 /xl of lysis buffer was used in step 2, add an equal amount of chloroform : phenol.) 5. Microcentrifuge at 10,000 x g for 15 minutes at room tempera ture or until aqueous (top) phase is clear. 6. Remove 300 to 350 /xl of the aqueous phase containing the DNA to a new tube. Be careful not to take any cellular debris from the interface. 7. Add 10 fx\ of 3 M NaOAc to the aqueous phase followed by 0.54 volumes of isopropanol. Invert gently to mix. DNA clots that precipitate may or may not be visible depending on the amount of starting material.
34. Isolation of DNA from Fungal Mycelia and Single Spores
285
8. Microcentrifuge as above (step 5) for 2 minutes at room tem perature. Pour off the supernatant. Rinse the pellet o n c e with 70% ethanol. Invert the tubes for 1 minute and drain them on a paper towel. 9. Place the tubes in a vacuum oven at 50°C for 15 minutes or until dry. 10. Resuspend the pellet in 100 to 500 /xl TE (10 mM Tris-HCI, 0.1 mM EDTA) or distilled water. The amount of TE used depends on the amount of DNA in the pellet. The final concentration of total DNA should be in the range of 0.1 to 10 /xg//xl. 11. For PCR amplification, 1 /xl of the DNA sample should be di luted in distilled water or TE to a final concentration of 0.1 to 10 ng DNA in each PCR amplification. W e have found a 1 : 1000 dilution works well for double-stranded amplification and a 1 : 10,000 dilution works for asymmetric amplification of single strands. Note: Do not despair if you are unable to get this much starting material. W e have started with a colony of 2 cm in diameter scraped from an agar petri dish and have success fully amplified rDNA from the extracted DNA.
Notes D N A isolated by this method from basidiocarps stored in herbaria for 3 0 years has been successfully amplified with P C R (Bruns et al., in press). Amplification from such samples will allow compari sons of genetic variability present in samples collected many years ago with that of contemporary samples. Recent reports of PCRamplified sequences from even older samples of human D N A (Paabo et al. 1988; Chapter 20) and formalin-fixed samples (Impraim et al. 1987) indicate that extraction and amplification of D N A from an cient collections of fungi may be possible. Gardes, Fortin, Bruns, Taylor, and White (unpublished) have used PCR to amplify fungal D N A from ectomycorrhizal roots by us ing fungal-specific primers. We anticipate that with taxon-specific primers, this method will be extended to other heterogenous D N A samples such as plant tissues containing obligate fungal pathogens and natural soil samples. Many fungi that cannot be cultured, e.g.,
Part Three. Genetics and Evolution
286
endomycorrhizal fungi and some lichens, would be amenable to comprehensive molecular studies by isolating and amplifying D N A by using these techniques.
Amplification from Single Spores Recent reports of amplification from a single human sperm (Li et al. 1988; see also Chapter 36) and a single human hair (Higuchi et al. 1988) have encouraged us to try to amplify sequences from single Neurospora ascospores. Using a modified version of our D N A isola tion method, PCR amplification of rDNA sequences from a single tetrasperma was possible (Fig. 2). Amplifica spore of Neurospora tion and analysis of specific D N A fragments from single spores
Figure 2
P C R - a m p l i f i e d r D N A f r o m s i n g l e a s c o s p o r e s o f Neurospora
tetrasperma.
Single ascospores w e r e c r u s h e d b e t w e e n t w o siliconized b a k e d slides. Slides
3
siliconized in silicoat ( C H C I ,
5 % dichlorodimethlysilane) and baked
were
at 1 8 0 ° C f o r
2 h o u r s . T h e spore a n d c o n t e n t s w e r e t h e n w a s h e d off t h e slides w i t h sterile-distilled
2
H 0
directly into t h e P C R m i x t u r e . P C R was p e r f o r m e d w i t h 7 0 cycles o f amplifica
t i o n : 9 7 ° C d e n a t u r e / 5 1 ° C a n n e a l / 7 3 ° C e x t e n s i o n . F o r e a c h s a m p l e , 5 /xl f r o m a t o t a l 1 0 0 - j u , I r e a c t i o n w a s r u n o n a 2 % N u S i e v e , 1 % a g a r o s e g e l at 7 5 V f o r 2 h o u r s . T h e m i n i g e l w a s t h e n s t a i n e d i n e t h i d i u m b r o m i d e ( 0 . 5 /xg/rr\\)
and D N A was visualized
u n d e r UV. Lanes 1 - 3 e a c h c o n t a i n e d a s i n g l e a s c o s p o r e , lane 4 c o n t a i n e d 1 0 as
2
c o s p o r e s , a n d lane 5 c o n t a i n e d sterile H 0 .
3 4 . Isolation of DNA f r o m Fungal Mycelia and Single Spores
287
would be especially useful for studies of recombination frequencies and genetic linkage with species whose spores cannot be germinated. Literature Cited Anderson, J. B., D. M. Petsche, and M. L. Smith. 1987. Restriction fragment poly morphisms in biological species of Armillaha mellea. Mycologia 7 9 : 6 9 - 7 6 . Biel, S. W., and F. W. Parrish. 1986. Isolation of D N A from fungal mycelia and sclerotia without use of density gradient ultracentrifugation. Anal. Biochem. 1 5 4 : 2 1 - 2 5 . Bruns, T. D., J. D. Palmer, D. S. Shumard, L. I. Grossman, and M. E. S. Hudspeth. 1 9 8 8 . Mitochondrial D N A s of Suillus: three fold size change in molecules that share a c o m m o n gene order. Curr. Genetics 1 3 : 4 9 - 5 6 . Bruns, T. D., R. Fogel, T. J. White, J. D. Palmer. 1989. Accelerated evolution of a falsetruffle from a mushroom ancestor. Nature (London) 3 3 9 : 1 4 0 - 1 4 2 . Bruns, T. D., R. Fogel, and J. W. Taylor. Amplification and sequencing of D N A from fungal herbarium specimens. Mycologia, in press. Cramer, C. L., J. L. Ristow, T . } . Paulus, and R. H. Davis. 1 9 8 3 . Methods for mycelial breakage and isolation of mitochondria and vacuoles of Neurospora. Anal. Bio chem. 1 2 8 : 3 8 4 - 3 9 2 . Higuchi, R., C. H. von Beroldingen, G. F. Sensabaugh, and H. A. Erlich. 1988. D N A typing from single hairs. Nature (London) 3 3 2 : 5 4 3 - 5 4 6 . Hudspeth, M. E. S., D. S. Shumard, C. J. R. Braford, and L. I. Grossman. 1 9 8 0 . Rapid purification of yeast mitochondrial D N A in high yield. Biochim. Biophys. Acta 610:221-228. Impraim, C. C., R. K. Saiki, H. A. Erlich, and R. L. Teplitz. 1987. Analysis of D N A ex tracted from formalin-fixed, paraffin embedded tissues by enzymatic amplifica tion and hybridization with sequence-specific oligonucleotides. Biochem. Bio phys. Res. Commun. 1 4 2 : 7 1 0 - 7 1 6 . Kwok, S., T. J. White, and J. W. Taylor. 1986. Evolutionary relationships between fungi, red algae, and other simple eucaryotes inferred from total D N A hybridiza tion to a cloned Basidiomycete ribosomal DNA. Exper. Mycol. 1 0 : 1 9 6 - 2 0 4 . Lambowitz, A. M. 1979. Preparation and analysis of mitochondrial ribosomes. Meth ods Enzymol. 5 9 : 4 2 1 - 4 3 3 . Lee, S. B., M. G. Milgroom, and J. W. Taylor. 1988. A rapid, high yield mini-prep method for isolation of total genomic D N A from fungi. Fungal Gen. Newsl. 35:23-24. Li, H., U. B. Gyllensten, X . Cui, R. K. Saiki, H. A. Erlich, and N. Arnheim. 1988. A m plification and analysis of D N A sequences in single human sperm and diploid cells. Nature (London) 3 3 5 : 4 1 4 - 4 1 7 . Paabo, S., and A. C. Wilson. 1988. P C R Reveals cloning artefacts: the case of the ex tinct Quagga. Nature (London) 3 3 4 : 3 8 7 - 3 8 8 . Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T. Horn, K. B. Mullis, and H. A. Erlich. 1988. Primer-directed enzymatic amplification with a thermo stable D N A Polymerase. Science 2 3 9 : 4 8 7 - 4 9 1 . Taylor, J. W , and D. Natvig. 1987. Isolation of fungal DNA. In Zoospohc fungi in teaching and research (ed. M. S. Fuller and A. Jaworski], p. 2 5 2 - 2 5 8 . South eastern Publishing Corporation, Athens, Georgia. Zolan, M. E., and P.J. Pukkila. 1986. Inheritance of D N A methylation in Coprinus cinereus. Mol. Cell. Biol. 6 : 1 9 5 - 2 0 0 .
Previous methods for rapid isolation of total D N A for the compara tive study of many fungi have focused on obtaining a high yield of D N A for restriction enzyme analysis (Lee et al. 1988; Taylor and Natvig 1987; Biel and Parrish 1986; Zolan and Pukkila 1986). Fungal genetic studies have also required tedious methods to separate nu clear, mitochondrial, and ribosomal D N A fractions as well as other extranuclear fractions (Lambowitz 1979; Cramer et al. 1983; Hud speth et ah 1980) to study specific D N A molecules by cloning or use in D N A hybridization as probes (Kwok et al. 1986; Anderson et al. 1987; Bruns et al. 1988; Lee and Taylor, manuscript in preparation). T h e introduction and application of P C R to fungal genetic studies has changed these requirements for isolating D N A (Bruns et al. 1989). Our previous concern for a high yield is of little consequence since single-copy genomic sequences can be routinely amplified from 1 /xg of total D N A (Saiki et al. 1988). If the target sequence is present in multiple copies, as with nuclear ribosomal D N A or mito chondrial DNA, even less starting material is required (0.1 to 10.0 ng of total DNA). Moreover, the separation of nuclear and organelle
282
PCR Protocols: A Guide to Methods and Applications Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
34. Isolation of DNA from Fungal Mycelia and Single Spores
283
1 2 3 4 5 6
Figure 1 PCR-amplified 16s rDNA from ten thousandfold dilutions of miniprepped total DNA isolated from fresh Thraustotheca, Dictyuchus, Apodachlya, Aqualinderella, and Pythium mycelium using the DNA protocol given in this chapter, lanes 1-5. Distilled water control, lane 6. Primers NS5 and NS6 are listed in Chapter 38. For each sample, 5 f i \ from a total 100-/xl reaction was run on a 2% NuSieve, 1% agarose gel at 75 V for 2 hours. The minigel was then stained in ethidium bromide (0.5 ptg/ml), and DNA was visualized under UV.
genomes is unnecessary because target amplification is primer di rected, and primers can be designed to amplify specific genomic or organellar fractions (Chapter 38). We have simplified our previous method of total D N A isolation (Lee et al. 1988) and have used this method to amplify multicopy genes from small quantities of fresh and freeze-dried mycelia and herbarium samples (Bruns et al., in press). Thousandfold to tenthousandfold dilutions of miniprep total DNAs have been used suc cessfully in amplification reactions (see steps 10 and 11 in proce dure) yielding double-stranded and single-stranded product (see Fig. 1). This method has worked for members of fungi in every group ex Oomycetes, Zygo amined to date, including the Chythdiomycetes, and Basidiomycetes. mycetes, Deuteromycetes, Ascomycetes
284
Part Three. Genetics and Evolution
DNA Isolation Protocol
Solutions Lysis buffer
50 mM Tris-HCI (pH 7.2) 50 mM EDTA 3% SDS 1% 2-mercaptoethanol Chloroform : TE-saturated phenol (1 : 1, v : v) [TE: 10 mM Tris-HCI (pH 8.0), 1 mM EDTA] 3 M NaOAc (pH 8.0) Isopropanol Ethanol (70%) TE (for resuspending pellet): 10 mM Tris-HCI, 0.1 mM EDTA Procedure 1. Fill a 1.5-ml Eppendorf microcentrifuge tube one-third up the conical portion with ground lyophilized mycelium (20 to 60 mg dry) or fresh mycelium (0.1 to 0.3 g wet [see Note]) ground in liquid nitrogen. In either case the mycelium is ground by hand with a mortar and pestle. 2. Add 400 fx\ lysis buffer and stir with a dissecting needle and/or vortex so the mixture is homogeneous. If the mixture is too viscous, add more lysis buffer (up to 700 /xl). 3. Incubate at 65°C for 1 hour. 4. Add 400 /xl chloroform : phenol and vortex briefly, but be care ful as the caps may loosen during vortexing. (If more than 400 /xl of lysis buffer was used in step 2, add an equal amount of chloroform : phenol.) 5. Microcentrifuge at 10,000 x g for 15 minutes at room tempera ture or until aqueous (top) phase is clear. 6. Remove 300 to 350 /xl of the aqueous phase containing the DNA to a new tube. Be careful not to take any cellular debris from the interface. 7. Add 10 fx\ of 3 M NaOAc to the aqueous phase followed by 0.54 volumes of isopropanol. Invert gently to mix. DNA clots that precipitate may or may not be visible depending on the amount of starting material.
34. Isolation of DNA from Fungal Mycelia and Single Spores
285
8. Microcentrifuge as above (step 5) for 2 minutes at room tem perature. Pour off the supernatant. Rinse the pellet o n c e with 70% ethanol. Invert the tubes for 1 minute and drain them on a paper towel. 9. Place the tubes in a vacuum oven at 50°C for 15 minutes or until dry. 10. Resuspend the pellet in 100 to 500 /xl TE (10 mM Tris-HCI, 0.1 mM EDTA) or distilled water. The amount of TE used depends on the amount of DNA in the pellet. The final concentration of total DNA should be in the range of 0.1 to 10 /xg//xl. 11. For PCR amplification, 1 /xl of the DNA sample should be di luted in distilled water or TE to a final concentration of 0.1 to 10 ng DNA in each PCR amplification. W e have found a 1 : 1000 dilution works well for double-stranded amplification and a 1 : 10,000 dilution works for asymmetric amplification of single strands. Note: Do not despair if you are unable to get this much starting material. W e have started with a colony of 2 cm in diameter scraped from an agar petri dish and have success fully amplified rDNA from the extracted DNA.
Notes D N A isolated by this method from basidiocarps stored in herbaria for 3 0 years has been successfully amplified with P C R (Bruns et al., in press). Amplification from such samples will allow compari sons of genetic variability present in samples collected many years ago with that of contemporary samples. Recent reports of PCRamplified sequences from even older samples of human D N A (Paabo et al. 1988; Chapter 20) and formalin-fixed samples (Impraim et al. 1987) indicate that extraction and amplification of D N A from an cient collections of fungi may be possible. Gardes, Fortin, Bruns, Taylor, and White (unpublished) have used PCR to amplify fungal D N A from ectomycorrhizal roots by us ing fungal-specific primers. We anticipate that with taxon-specific primers, this method will be extended to other heterogenous D N A samples such as plant tissues containing obligate fungal pathogens and natural soil samples. Many fungi that cannot be cultured, e.g.,
Part Three. Genetics and Evolution
286
endomycorrhizal fungi and some lichens, would be amenable to comprehensive molecular studies by isolating and amplifying D N A by using these techniques.
Amplification from Single Spores Recent reports of amplification from a single human sperm (Li et al. 1988; see also Chapter 36) and a single human hair (Higuchi et al. 1988) have encouraged us to try to amplify sequences from single Neurospora ascospores. Using a modified version of our D N A isola tion method, PCR amplification of rDNA sequences from a single tetrasperma was possible (Fig. 2). Amplifica spore of Neurospora tion and analysis of specific D N A fragments from single spores
Figure 2
P C R - a m p l i f i e d r D N A f r o m s i n g l e a s c o s p o r e s o f Neurospora
tetrasperma.
Single ascospores w e r e c r u s h e d b e t w e e n t w o siliconized b a k e d slides. Slides
3
siliconized in silicoat ( C H C I ,
5 % dichlorodimethlysilane) and baked
were
at 1 8 0 ° C f o r
2 h o u r s . T h e spore a n d c o n t e n t s w e r e t h e n w a s h e d off t h e slides w i t h sterile-distilled
2
H 0
directly into t h e P C R m i x t u r e . P C R was p e r f o r m e d w i t h 7 0 cycles o f amplifica
t i o n : 9 7 ° C d e n a t u r e / 5 1 ° C a n n e a l / 7 3 ° C e x t e n s i o n . F o r e a c h s a m p l e , 5 /xl f r o m a t o t a l 1 0 0 - j u , I r e a c t i o n w a s r u n o n a 2 % N u S i e v e , 1 % a g a r o s e g e l at 7 5 V f o r 2 h o u r s . T h e m i n i g e l w a s t h e n s t a i n e d i n e t h i d i u m b r o m i d e ( 0 . 5 /xg/rr\\)
and D N A was visualized
u n d e r UV. Lanes 1 - 3 e a c h c o n t a i n e d a s i n g l e a s c o s p o r e , lane 4 c o n t a i n e d 1 0 as
2
c o s p o r e s , a n d lane 5 c o n t a i n e d sterile H 0 .
3 4 . Isolation of DNA f r o m Fungal Mycelia and Single Spores
287
would be especially useful for studies of recombination frequencies and genetic linkage with species whose spores cannot be germinated. Literature Cited Anderson, J. B., D. M. Petsche, and M. L. Smith. 1987. Restriction fragment poly morphisms in biological species of Armillaha mellea. Mycologia 7 9 : 6 9 - 7 6 . Biel, S. W., and F. W. Parrish. 1986. Isolation of D N A from fungal mycelia and sclerotia without use of density gradient ultracentrifugation. Anal. Biochem. 1 5 4 : 2 1 - 2 5 . Bruns, T. D., J. D. Palmer, D. S. Shumard, L. I. Grossman, and M. E. S. Hudspeth. 1 9 8 8 . Mitochondrial D N A s of Suillus: three fold size change in molecules that share a c o m m o n gene order. Curr. Genetics 1 3 : 4 9 - 5 6 . Bruns, T. D., R. Fogel, T. J. White, J. D. Palmer. 1989. Accelerated evolution of a falsetruffle from a mushroom ancestor. Nature (London) 3 3 9 : 1 4 0 - 1 4 2 . Bruns, T. D., R. Fogel, and J. W. Taylor. Amplification and sequencing of D N A from fungal herbarium specimens. Mycologia, in press. Cramer, C. L., J. L. Ristow, T . } . Paulus, and R. H. Davis. 1 9 8 3 . Methods for mycelial breakage and isolation of mitochondria and vacuoles of Neurospora. Anal. Bio chem. 1 2 8 : 3 8 4 - 3 9 2 . Higuchi, R., C. H. von Beroldingen, G. F. Sensabaugh, and H. A. Erlich. 1988. D N A typing from single hairs. Nature (London) 3 3 2 : 5 4 3 - 5 4 6 . Hudspeth, M. E. S., D. S. Shumard, C. J. R. Braford, and L. I. Grossman. 1 9 8 0 . Rapid purification of yeast mitochondrial D N A in high yield. Biochim. Biophys. Acta 610:221-228. Impraim, C. C., R. K. Saiki, H. A. Erlich, and R. L. Teplitz. 1987. Analysis of D N A ex tracted from formalin-fixed, paraffin embedded tissues by enzymatic amplifica tion and hybridization with sequence-specific oligonucleotides. Biochem. Bio phys. Res. Commun. 1 4 2 : 7 1 0 - 7 1 6 . Kwok, S., T. J. White, and J. W. Taylor. 1986. Evolutionary relationships between fungi, red algae, and other simple eucaryotes inferred from total D N A hybridiza tion to a cloned Basidiomycete ribosomal DNA. Exper. Mycol. 1 0 : 1 9 6 - 2 0 4 . Lambowitz, A. M. 1979. Preparation and analysis of mitochondrial ribosomes. Meth ods Enzymol. 5 9 : 4 2 1 - 4 3 3 . Lee, S. B., M. G. Milgroom, and J. W. Taylor. 1988. A rapid, high yield mini-prep method for isolation of total genomic D N A from fungi. Fungal Gen. Newsl. 35:23-24. Li, H., U. B. Gyllensten, X . Cui, R. K. Saiki, H. A. Erlich, and N. Arnheim. 1988. A m plification and analysis of D N A sequences in single human sperm and diploid cells. Nature (London) 3 3 5 : 4 1 4 - 4 1 7 . Paabo, S., and A. C. Wilson. 1988. P C R Reveals cloning artefacts: the case of the ex tinct Quagga. Nature (London) 3 3 4 : 3 8 7 - 3 8 8 . Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T. Horn, K. B. Mullis, and H. A. Erlich. 1988. Primer-directed enzymatic amplification with a thermo stable D N A Polymerase. Science 2 3 9 : 4 8 7 - 4 9 1 . Taylor, J. W , and D. Natvig. 1987. Isolation of fungal DNA. In Zoospohc fungi in teaching and research (ed. M. S. Fuller and A. Jaworski], p. 2 5 2 - 2 5 8 . South eastern Publishing Corporation, Athens, Georgia. Zolan, M. E., and P.J. Pukkila. 1986. Inheritance of D N A methylation in Coprinus cinereus. Mol. Cell. Biol. 6 : 1 9 5 - 2 0 0 .