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Isolation of DNA from fungal mycelia and sclerotia without use of density gradient ultracentrifugation
ANALYTICAL
BIOCHEMISTRY
154,2
1-25 ( 1986)
Isolation of DNA from Fungal Mycelia and Sclerotia without Use of Density Gradient Ultracentrifugation SUSAN W. BIEL] Food
AND F. W. PARRISH
and Feed Qunlit], Reseurch Unit. Ckited Sam Depurtrnem qf &riculwe. Southern Regional Rewarch Center. New Orkuns. Loui.riuna 70179 Received
.4RS,
June 25. 1985
A rapid method for extracting total DNA from /I.$pergi/lkls flaws and Aspergik~ parasiticus has been developed. The procedure can be completed in 2 h and yields 200 to 350 fig of DNA from 0.5 to I.0 g wet wt of mycelia and 150 rg from 0.5 g of sclerotia. DNA samples had an OD2&OD2s0 of 1.6 to 1.8. Most of the DNA was at least 50 kb pairs in size and showed little degradation. DNA prepared by this method was used for restriction endonuclease digestion and Southern blotting. A DNA fragment containing the repeat unit of the ribosomal RNA genes of .,I. //UWY has been identified. B 1986 Academic FVCSS. IK KEY WORDS: .4s(ler~i/llrs./lu1’I1.s; mycelial DNA: sclerotial DNA: ribosomal RNA genes.
Rapid methods of plasmid and chromosomal DNA extraction such as those of Birnboim and Daly ( 1 ), Holmes and Quigley (2). and Kado and Lui (3) have facilitated the study of genes at the molecular level in bacteria. The most commonly used methods of total DNA isolation from filamentous fungi still require ultracentrifugation of DNA in CsCl or sucrose gradients (4-h). When separation of nuclear and mitochondrial components or very pure nucleic acids is required, ultracentrifugation is still appropriate; however, it is not needed to obtain rest.riction endonuclease digestible DNA. Ultracen;.ifugation is time consuming and requires expensive chemicals and equipment. A fast, simple method of DNA isolation is useful when the object is to study restriction site polymorphism through restriction endonuclease digestion and Southern blotting (7). A rapid method of DNA isolation is also useful for identifying the presence of methylated DNA sequences or locating the fragments of a particular DNA sequence before cloning. We wished to develop a simple, rapid I Present address: siana State University.
Department of Microbiology. Baton Rouge. La. 70803.
method of extracting DNA that would have a good yield but would not require large amounts of fungal material. For our studies of .4spcrgillus ,flu~us and Aspergillzu parusitic~ls the procedure needed to be useful for isolation of DNA from mycelia and sclerotia. The procedure makes use of freeze-thaw and mechanical disruption of cell walls and lysis by sodium dodecyl sulfate (SDS)’ in a TrisHCI, EDTA buffer. After centrifugation at 4°C to remove cell debris and high-molecularweight carbohydrates. components of the lysis buffer were removed from the DNA by Sephadex G-50 chromatography. Fractions were precipitated with isopropanol to identify those which contained nucleic acids. The procedure can be completed in 2 h. Mycelia weighing from 0.5 to 1.O g typically yielded 200 to 350 pg of DNA, sclerotia (0.5 g) usually less than 200 pg. DNAs from mycelia and sclerotia were digested with BarnHI, &&RI, HindIII, s&I. Sinai. S&I, X/zol. b1/joI, IZpuII, and Al@. Another rapid method for isolation of DNA ’ Abbreviations used: SDS. sodium dodecyl sulfate; TE, Tri-HCI (10 mM) + EDTA (I mM): TES, NaCl(0.9 M) + EDTA (0.006 M) + Tri-HCI (0.09 M); SSC. NaCl (0.15 M) + sodium citrate (0.015 M).
Loui-
71
0003-2697/86
$3.00
Copynghl ‘C’ 1986 by Academic Press. Inc All n&s of reprcductmn in an> form reserved
22
BIEL
AND
PARRlSH
urated phenol:chloroform ( 1: 1) was added and thoroughly mixed with the crude lysate. The lysate was centrifuged for 20 min at 15,0001: at 4°C to pellet cell debris and high-molecularweight carbohydrates and to separate the aqueous and phenol phases. The aqueous phase was removed carefully to avoid contamination with cell debris and phenol. Sephadex G-50 was hydrated in TE and poured into a 5.0-ml disposable syringe as described MATERIALS AND METHODS by Maniatis et al. (9). Both DNA and RNA Growth of fitngi. To produce mycelia for are excluded from the Sephadex G-50 matrix. DNA isolation, fungi were grown shaking at RNA may be removed from the samples by 150 rpm at 28°C in 500 ml of yeast extract digestion with ribonuclease A (RNase) when (2%), sucrose (6%) medium for 24 to 36 h. applications require DNA that is free of RNA. Flasks were inoculated with lo7 to lo8 spores Lysate (0.5 ml) was layered on the 5.0-ml colfrom a spore suspension in 0.0 1% SDS. Myumn and eluted with TE. Fractions of 0.5 ml Celia1 pellets were collected by filtration were collected in microcentrifuge tubes. Most through filter paper or miracloth. of the restriction endonuclease digestible DNA Sclerotia were obtained by plating spores was recovered in the first 3.0 ml eluted. Fracon potato dextrose agar (Difco)3 and growing tions containing DNA were identified either cultures at 28°C for 2 weeks. When good by agarose gel electrophoresis or by precipiquantities of sclerotia had formed, they were tation of the DNA. Precipitation aids in the scraped from the agar surface with a loop and purification of the DNA and is a quick method suspended in a beaker of sterile water con- ofdetermining whether large amounts of DNA taining 0.0 I % SDS. Sclerotia were separated are present in a sample; therefore, samples from spores and mycelial fragments by allowwere routinely precipitated, Fractions were ing sclerotia to settle and pouring off the fluid precipitated by addition of 0.1 vol of 1 M sothat contained floating spores and mycelial dium acetate, pH 8.0, and 1.O vol of isoprofragments. Several washings resulted in scle- panol. Large quantities of high-molecularrotia that were free of other material. weight DNA formed a visible fibrous mass Mycelia and sclerotia can be stored at immediately, without chilling of the samples. -70°C until needed. Chilling of samples was avoided because preDNA isolation. One-half to one gram of cipitation of SDS and salts can occur at low mycelia was frozen in liquid nitrogen and temperatures. DNA was collected by centrifground to a fine powder in a mortar and pestle. ugation, dried, and resuspended in sterile waThe powder was transferred to a 30-ml cen- ter. The same r .ocedure was used for the trifuge tube and suspended in 2 ml of lysis preparation of DNA from 0.5 g of sclerotia. buffer consisting of 50 mM Tris-HCI, pH 7.2 Sclerotial samples were precipitated twice if or 8.0, 5 mM EDTA, and 1.5 or 3.0% SDS. substantial amounts of a brown sclerotial pigProteinase K was added to a final concentrament were extracted with the DNA. Precipition of 250 pg/ml. The mixture was incubated tation seems to aid in removal of this pigment. at 4, 37, or 65°C for 20 min. Two milliliters Restriction endonuclease digestion of DNA of TE ( 10 mM Tris-HCl, 1 mM EDTA)-satwas performed as per manufacturer’s recommendations. Agarose gel electrophoresis was in 0.7% gels unless other percentages are spec’ Names of companies or manufacturers are included ified; running and gel buffers were 40 mM for the convenience of the reader and do not imply endorsement or preferential treatment by USDA. Tris-acetate, 2 tnM EDTA. Gels were stained from mycelia has been reported (8). From the data presented (8) the yield appears to be substantially lower than that obtained with our method. In our laboratory DNA prepared using the method reported here has been used to identify a 7.7-kb-pair fragment that contains the ribosomal RNA genes of A. flavus.
ISOLATION
OF DNA FROM MYCELIA
with 1 Fug ethidium bromide/ml water after electrophoresis. Prehybridizations and hybridizations were performed in 6X TES (0.9 M NaCl, 0.006 M EDTA, 0.09 M Tris-HCl, pH 7.5), 5X Denhardt’s solution, 0.5% SDS, and 250 1.18denatured, sonicated salmon sperm DNA per milliliter hybridization solution. Biotin-labeled probe was added to a final concentration of 200 rig/ml ‘of solution. Filters were prehybridized for 2 to 5 h and hybridized overnight at 65°C. The probe consisted of pBR322 containing the 6.4-kb-pair /find111 fragment from Srmharom~~ces cerevisiae ribosomal RNA genes (rDNA). A h phage clone of the S. cerrvisiac rDNA was cut with HilzdIII and ligated into the Hind111 site of pBR322 to create this probe. Since the arms of this phage contained no ffindII1 sites, the subclone of rDNA could easily be recognized by its size and restriction pattern. pBR.322 itself was used as a probe to determine if ‘-t. ~‘WIG contained any pBR322 homologous sequences. The probes were nicktranslated in the presence of the dTTP analog biotin 1 I-UTP ( 10). Hybridized filters were washed once in 2X SSC (1 X SSC is 0.15 M NaCl. 0.015 M sodium citrate, pH 7.0), 0.1% SDS at room temperature: twice in 0.5X SSC, 0.1 %I SDS at room temperature: and twice in 0.5X SSC, 0.1% SDS at 45°C for 15 min each. followed by a room-temperature rinse in 2X SSC. Hybridized DNA was detected using biotin-strept,svidin-labeled alkaline phosphatase (Bethesda Research Laboratories). RESULTS
AND
DISCUSSION
The measurements in Fig. 1 were taken on DNA prepared from 0.7 g wet wt of mycelia. One-half milliliter of lysate was loaded on a 5.0-ml column. Use of more than 0.5 ml of lysate usually overloaded the column and resulted in fractions containing enzyme inhibitory substances from the lysis procedure. Ten 0.5-ml fractions were collected from each column. Fractions eluted from the column were treated with SO wg RNase/ml of fraction, incubated at room temperature for 1 h (9). precipitated. and resuspended in 0.5 ml sterile,
AND SCLEROTIA
23
22or 1 17 N” A
15i
n
C :
132-
: 7
110-
R F
88 -
;
0644 _
0
12346678
0 FRACTION
10
NUMBER
FIG. I. The DNA concentration was determined based on the optical density at 260 nm and the relationship of I OD unit = 50 &ml of DNA (9), and reported as Fg/ml. Concentrations were determined after RNase treatment as described in the text. Results shown were obtained on different columns.
distilled water. The optical density at 260 and 280 nm was determined on a Varian spectrophotometer. The elution profiles shown are typical of those observed when the column length and diameter are those of a 5.0-ml disposable syringe. The procedure described was used with buffers of pH 7.2 and 8.0 and SDS concentrations of 1.5 and 3.0%. Samples were incubated at 4, 37, and 65°C. The time of incubation was also varied from 10 min to 1 h. A 20-min incubation yielded as much DNA as incubation for 1 h. Since the purpose was to develop a rapid method, incubation times of 20 min were routinely used. Buffers of pH 7.2 or 8.0 and SDS concentrations of 1.5 or 3.0% did not alter the account of DNA obtained. Lysis at low temperatures may be somewhat less effective but probably not significantly so. The most important aspect of successful use of the procedure was sample preparation. Disruption
BIEL AND PARRISH
24
of the cell walls by grinding was the most variable aspect of the procedure. It was important that the powdered material was thoroughly dispersed in the lysis buffer as the powder tends to form clumps. The total amount of DNA obtained from mycelia was typically between 200 and 350 pg in 12 different extractions. Sclerotial preparations sometimes contain a brown pigment; however, this pigment did not inhibit restriction endonuclease digestion. The OD260/OD280 of the DNA ranges from 1.6 to 1.8. Figure 2a shows uncut samples of mycelial and sclerotial DNA. The DNA is quite uniform in size. Samples of mycelial and sclerotial DNA and a X DNA standard were subjected to electrophoresis in a 0.3% agarose gel to de-
M
s
b
a
M s
6.7
2.3 2.n
II.56
FIG. 2. (a) Lane 1 contains uncut X DNA which was heated to 65°C for 5 min before loading. Lane 2 contains uncut A. ~QVUS mycelial (M) DNA, and lane 3 contains uncut A. /laws sclerotial (S) DNA. The samples were loaded on a 0.3% agarose gel and subjected to electrophoresis at 10 V/cm for 3 h. (b) Mycelial (M, lanes 2 to 4) and sclerotial (S. lanes 5 to 7) DNA from A. flaws was digested with SalI, MboI, and SmaI. Lane 1 is a Hind111 digest of h DNA. Size in kilobase pairs of the h fragments is indicated.
termine the size of the fungal DNA. The heavy band of the mycelial DNA comigrated with the uncut X DNA, indicating that it was at least 50 kb pairs in size. The sclerotial DNA was slightly smaller. Figure 2b shows restriction endonuclease digested mycelial and sclerotial DNA. Mycelial ( 1.3 pg) and sclerotial ( 1.5 pg) DNAs were digested with &II, MhoI, and Srnal. The reduction in size of the DNAs due to Mhol digestion clearly demonstrated that both samples could be cut with restriction endonculeases. The banding patterns in digestions of sclerotial DNA were not as prominent as those seen in mycelial DNA. This result could be due to greater degradation of sclerotial DNA during extraction or to differences in the copy number or methylation of genes in mycelial versus sclerotial cells. The last two possibilities are currently being investigated. Restriction endonuclease digested DNAs from mycelia and sclerotia have been subjected to electrophoresis in agarose gels and transferred to nitrocellulose paper by the method of Southern (7). Subsequent hybridization to pBR322 containing the 6.4-kb-pair riboHilzdIII fragment of the S. cerrvisiae somal RNA genes allowed identification of DNA fragments belonging to the ribosomal RNA genes of A. Javus. When mycelial DNA was hybridized to pBR322 alone, no sequences homologous to pBR322 were found when the filters were washed at the stringency that allowed detection of rDNA homology. Figures 3a and b show an X/lo1 digest of mycelial DNA and the corresponding Southern blot. In both mycelial and sclerotial samples, a single 7.7kb-pair fragment representing one repeat unit of the rDNA was detected. The 7.7-kb-pair fragment can be seen directly in the agarose gel of digested mycelial DNA due to the presence of multiple copies of the ribosomal RNA genes. Digestion with X/z01 was complete as judged by the lack of bands larger than 7.7 kb pairs in the Southern blot. The rDNA repeat unit of ‘4. nidz~luns has also been identified on a 7.7-kb-pair fragment ( 1 1). Digestion of A. .fluv~ls DNA with E&RI,
ISOLATION a.
1
2
OF
b
DNA
FROM
MYCELIA
AND
25
SCLEROTIA
ACKNOWLEDGMENTS
12
The authors thank Dr. Maynard for the gift of X phage containing
Olsen and George Carle S. wrevisiae rDNA.
REFERENCES I. Birnboim.
H. C.. and Daly.
J. (1979)
NzI&~c.
.4&/s
Re,. 7, 1513-1519.
FIG. 3. (a) Lane I contains A. fluvus mycelial DNA digested with .IlhoI. The single bright band in the gel picture corresponds to the 7.7-kb-pair band in the Southern blot and identifies one complete repeat unit of the rDNA. Lane 2 contains a Hind111 digest of the plasmid containing S. crrevisiae rDNA. The 6.4-kb-pair band is the S. cerevisiue rDNA. and the 4.4-kb-pair band is pBR322. (b) Southern blot of the gel in (a) probed with the plasmid containing S. cwevisiae rDNA. Only one band appears in the mycelial DNA, indicating complete digestion with X/zoI. pBR322 itselfdid not hybridize to blots of,4../la~us mycelial DNA.
HirzdIII. P.srI. and BarnHI, followed by Southern blotting of the gel, indicates that there is significant restriction site polymorphism between the rDNA sequences of ‘4. niddmc ( 1 1) and .A. f1mr.s.
2. Holmes, D. S., and Quigley, M. (I 9X I ) .4nal. Bioch~~n 114, 193-197. 3. Kado. C. I.. and Lui, S.-T. ( I98 I ) J. Bucteriol. 145(3), 1365-1373. 4. Garber, R. C., and Yoder. 0. C. (1983) .4nal Biochem 135,416-422. 5. Tilburn. J., Scazzocchio, C.. Taylor. G. G.. ZabickyZissman. J. H., Lockington, R. A.. and Davies. R. W. (I 983) G‘er~e 26, 205-22 I 6. Specht. C. A.. DiRusso. C. C., Novotny. C. P.. and Ullrich. R. C. (1982) .4nal Bkherrz. 119, 15% 163. 7. Southern, E. ( 1975) J ,Zlo/. Biol. 98, 503-5 17. 8. Yelton. M. M., Hamer. J. E., and Timberlake. W. E. (I 984) Proc. Nut/. .-Icud. SC;. W.J 81, l470- 1474. 9. Maniatis, T.. Fritsch. E. F., and Sambrook. J. (1982) Molecular Cloning. A Laboratory Manual, pp. 464465. 468. Cold Spring Harbor Laboratories, Cold Spring Harbor, N. Y. IO Langer, P. R.. Waldrop, A. A., and Ward. D. C. ( I98 I ) Proc Null .-lead. Ski. US.4 78, 6633-6637. II Lockington. R. A., Taylor, G. G., Winther. M., Scazzocchio. C., and Davies. R. W. (1982) (ic~nr 20, 135-137.
BIOCHEMISTRY
154,2
1-25 ( 1986)
Isolation of DNA from Fungal Mycelia and Sclerotia without Use of Density Gradient Ultracentrifugation SUSAN W. BIEL] Food
AND F. W. PARRISH
and Feed Qunlit], Reseurch Unit. Ckited Sam Depurtrnem qf &riculwe. Southern Regional Rewarch Center. New Orkuns. Loui.riuna 70179 Received
.4RS,
June 25. 1985
A rapid method for extracting total DNA from /I.$pergi/lkls flaws and Aspergik~ parasiticus has been developed. The procedure can be completed in 2 h and yields 200 to 350 fig of DNA from 0.5 to I.0 g wet wt of mycelia and 150 rg from 0.5 g of sclerotia. DNA samples had an OD2&OD2s0 of 1.6 to 1.8. Most of the DNA was at least 50 kb pairs in size and showed little degradation. DNA prepared by this method was used for restriction endonuclease digestion and Southern blotting. A DNA fragment containing the repeat unit of the ribosomal RNA genes of .,I. //UWY has been identified. B 1986 Academic FVCSS. IK KEY WORDS: .4s(ler~i/llrs./lu1’I1.s; mycelial DNA: sclerotial DNA: ribosomal RNA genes.
Rapid methods of plasmid and chromosomal DNA extraction such as those of Birnboim and Daly ( 1 ), Holmes and Quigley (2). and Kado and Lui (3) have facilitated the study of genes at the molecular level in bacteria. The most commonly used methods of total DNA isolation from filamentous fungi still require ultracentrifugation of DNA in CsCl or sucrose gradients (4-h). When separation of nuclear and mitochondrial components or very pure nucleic acids is required, ultracentrifugation is still appropriate; however, it is not needed to obtain rest.riction endonuclease digestible DNA. Ultracen;.ifugation is time consuming and requires expensive chemicals and equipment. A fast, simple method of DNA isolation is useful when the object is to study restriction site polymorphism through restriction endonuclease digestion and Southern blotting (7). A rapid method of DNA isolation is also useful for identifying the presence of methylated DNA sequences or locating the fragments of a particular DNA sequence before cloning. We wished to develop a simple, rapid I Present address: siana State University.
Department of Microbiology. Baton Rouge. La. 70803.
method of extracting DNA that would have a good yield but would not require large amounts of fungal material. For our studies of .4spcrgillus ,flu~us and Aspergillzu parusitic~ls the procedure needed to be useful for isolation of DNA from mycelia and sclerotia. The procedure makes use of freeze-thaw and mechanical disruption of cell walls and lysis by sodium dodecyl sulfate (SDS)’ in a TrisHCI, EDTA buffer. After centrifugation at 4°C to remove cell debris and high-molecularweight carbohydrates. components of the lysis buffer were removed from the DNA by Sephadex G-50 chromatography. Fractions were precipitated with isopropanol to identify those which contained nucleic acids. The procedure can be completed in 2 h. Mycelia weighing from 0.5 to 1.O g typically yielded 200 to 350 pg of DNA, sclerotia (0.5 g) usually less than 200 pg. DNAs from mycelia and sclerotia were digested with BarnHI, &&RI, HindIII, s&I. Sinai. S&I, X/zol. b1/joI, IZpuII, and Al@. Another rapid method for isolation of DNA ’ Abbreviations used: SDS. sodium dodecyl sulfate; TE, Tri-HCI (10 mM) + EDTA (I mM): TES, NaCl(0.9 M) + EDTA (0.006 M) + Tri-HCI (0.09 M); SSC. NaCl (0.15 M) + sodium citrate (0.015 M).
Loui-
71
0003-2697/86
$3.00
Copynghl ‘C’ 1986 by Academic Press. Inc All n&s of reprcductmn in an> form reserved
22
BIEL
AND
PARRlSH
urated phenol:chloroform ( 1: 1) was added and thoroughly mixed with the crude lysate. The lysate was centrifuged for 20 min at 15,0001: at 4°C to pellet cell debris and high-molecularweight carbohydrates and to separate the aqueous and phenol phases. The aqueous phase was removed carefully to avoid contamination with cell debris and phenol. Sephadex G-50 was hydrated in TE and poured into a 5.0-ml disposable syringe as described MATERIALS AND METHODS by Maniatis et al. (9). Both DNA and RNA Growth of fitngi. To produce mycelia for are excluded from the Sephadex G-50 matrix. DNA isolation, fungi were grown shaking at RNA may be removed from the samples by 150 rpm at 28°C in 500 ml of yeast extract digestion with ribonuclease A (RNase) when (2%), sucrose (6%) medium for 24 to 36 h. applications require DNA that is free of RNA. Flasks were inoculated with lo7 to lo8 spores Lysate (0.5 ml) was layered on the 5.0-ml colfrom a spore suspension in 0.0 1% SDS. Myumn and eluted with TE. Fractions of 0.5 ml Celia1 pellets were collected by filtration were collected in microcentrifuge tubes. Most through filter paper or miracloth. of the restriction endonuclease digestible DNA Sclerotia were obtained by plating spores was recovered in the first 3.0 ml eluted. Fracon potato dextrose agar (Difco)3 and growing tions containing DNA were identified either cultures at 28°C for 2 weeks. When good by agarose gel electrophoresis or by precipiquantities of sclerotia had formed, they were tation of the DNA. Precipitation aids in the scraped from the agar surface with a loop and purification of the DNA and is a quick method suspended in a beaker of sterile water con- ofdetermining whether large amounts of DNA taining 0.0 I % SDS. Sclerotia were separated are present in a sample; therefore, samples from spores and mycelial fragments by allowwere routinely precipitated, Fractions were ing sclerotia to settle and pouring off the fluid precipitated by addition of 0.1 vol of 1 M sothat contained floating spores and mycelial dium acetate, pH 8.0, and 1.O vol of isoprofragments. Several washings resulted in scle- panol. Large quantities of high-molecularrotia that were free of other material. weight DNA formed a visible fibrous mass Mycelia and sclerotia can be stored at immediately, without chilling of the samples. -70°C until needed. Chilling of samples was avoided because preDNA isolation. One-half to one gram of cipitation of SDS and salts can occur at low mycelia was frozen in liquid nitrogen and temperatures. DNA was collected by centrifground to a fine powder in a mortar and pestle. ugation, dried, and resuspended in sterile waThe powder was transferred to a 30-ml cen- ter. The same r .ocedure was used for the trifuge tube and suspended in 2 ml of lysis preparation of DNA from 0.5 g of sclerotia. buffer consisting of 50 mM Tris-HCI, pH 7.2 Sclerotial samples were precipitated twice if or 8.0, 5 mM EDTA, and 1.5 or 3.0% SDS. substantial amounts of a brown sclerotial pigProteinase K was added to a final concentrament were extracted with the DNA. Precipition of 250 pg/ml. The mixture was incubated tation seems to aid in removal of this pigment. at 4, 37, or 65°C for 20 min. Two milliliters Restriction endonuclease digestion of DNA of TE ( 10 mM Tris-HCl, 1 mM EDTA)-satwas performed as per manufacturer’s recommendations. Agarose gel electrophoresis was in 0.7% gels unless other percentages are spec’ Names of companies or manufacturers are included ified; running and gel buffers were 40 mM for the convenience of the reader and do not imply endorsement or preferential treatment by USDA. Tris-acetate, 2 tnM EDTA. Gels were stained from mycelia has been reported (8). From the data presented (8) the yield appears to be substantially lower than that obtained with our method. In our laboratory DNA prepared using the method reported here has been used to identify a 7.7-kb-pair fragment that contains the ribosomal RNA genes of A. flavus.
ISOLATION
OF DNA FROM MYCELIA
with 1 Fug ethidium bromide/ml water after electrophoresis. Prehybridizations and hybridizations were performed in 6X TES (0.9 M NaCl, 0.006 M EDTA, 0.09 M Tris-HCl, pH 7.5), 5X Denhardt’s solution, 0.5% SDS, and 250 1.18denatured, sonicated salmon sperm DNA per milliliter hybridization solution. Biotin-labeled probe was added to a final concentration of 200 rig/ml ‘of solution. Filters were prehybridized for 2 to 5 h and hybridized overnight at 65°C. The probe consisted of pBR322 containing the 6.4-kb-pair /find111 fragment from Srmharom~~ces cerevisiae ribosomal RNA genes (rDNA). A h phage clone of the S. cerrvisiac rDNA was cut with HilzdIII and ligated into the Hind111 site of pBR322 to create this probe. Since the arms of this phage contained no ffindII1 sites, the subclone of rDNA could easily be recognized by its size and restriction pattern. pBR.322 itself was used as a probe to determine if ‘-t. ~‘WIG contained any pBR322 homologous sequences. The probes were nicktranslated in the presence of the dTTP analog biotin 1 I-UTP ( 10). Hybridized filters were washed once in 2X SSC (1 X SSC is 0.15 M NaCl. 0.015 M sodium citrate, pH 7.0), 0.1% SDS at room temperature: twice in 0.5X SSC, 0.1 %I SDS at room temperature: and twice in 0.5X SSC, 0.1% SDS at 45°C for 15 min each. followed by a room-temperature rinse in 2X SSC. Hybridized DNA was detected using biotin-strept,svidin-labeled alkaline phosphatase (Bethesda Research Laboratories). RESULTS
AND
DISCUSSION
The measurements in Fig. 1 were taken on DNA prepared from 0.7 g wet wt of mycelia. One-half milliliter of lysate was loaded on a 5.0-ml column. Use of more than 0.5 ml of lysate usually overloaded the column and resulted in fractions containing enzyme inhibitory substances from the lysis procedure. Ten 0.5-ml fractions were collected from each column. Fractions eluted from the column were treated with SO wg RNase/ml of fraction, incubated at room temperature for 1 h (9). precipitated. and resuspended in 0.5 ml sterile,
AND SCLEROTIA
23
22or 1 17 N” A
15i
n
C :
132-
: 7
110-
R F
88 -
;
0644 _
0
12346678
0 FRACTION
10
NUMBER
FIG. I. The DNA concentration was determined based on the optical density at 260 nm and the relationship of I OD unit = 50 &ml of DNA (9), and reported as Fg/ml. Concentrations were determined after RNase treatment as described in the text. Results shown were obtained on different columns.
distilled water. The optical density at 260 and 280 nm was determined on a Varian spectrophotometer. The elution profiles shown are typical of those observed when the column length and diameter are those of a 5.0-ml disposable syringe. The procedure described was used with buffers of pH 7.2 and 8.0 and SDS concentrations of 1.5 and 3.0%. Samples were incubated at 4, 37, and 65°C. The time of incubation was also varied from 10 min to 1 h. A 20-min incubation yielded as much DNA as incubation for 1 h. Since the purpose was to develop a rapid method, incubation times of 20 min were routinely used. Buffers of pH 7.2 or 8.0 and SDS concentrations of 1.5 or 3.0% did not alter the account of DNA obtained. Lysis at low temperatures may be somewhat less effective but probably not significantly so. The most important aspect of successful use of the procedure was sample preparation. Disruption
BIEL AND PARRISH
24
of the cell walls by grinding was the most variable aspect of the procedure. It was important that the powdered material was thoroughly dispersed in the lysis buffer as the powder tends to form clumps. The total amount of DNA obtained from mycelia was typically between 200 and 350 pg in 12 different extractions. Sclerotial preparations sometimes contain a brown pigment; however, this pigment did not inhibit restriction endonuclease digestion. The OD260/OD280 of the DNA ranges from 1.6 to 1.8. Figure 2a shows uncut samples of mycelial and sclerotial DNA. The DNA is quite uniform in size. Samples of mycelial and sclerotial DNA and a X DNA standard were subjected to electrophoresis in a 0.3% agarose gel to de-
M
s
b
a
M s
6.7
2.3 2.n
II.56
FIG. 2. (a) Lane 1 contains uncut X DNA which was heated to 65°C for 5 min before loading. Lane 2 contains uncut A. ~QVUS mycelial (M) DNA, and lane 3 contains uncut A. /laws sclerotial (S) DNA. The samples were loaded on a 0.3% agarose gel and subjected to electrophoresis at 10 V/cm for 3 h. (b) Mycelial (M, lanes 2 to 4) and sclerotial (S. lanes 5 to 7) DNA from A. flaws was digested with SalI, MboI, and SmaI. Lane 1 is a Hind111 digest of h DNA. Size in kilobase pairs of the h fragments is indicated.
termine the size of the fungal DNA. The heavy band of the mycelial DNA comigrated with the uncut X DNA, indicating that it was at least 50 kb pairs in size. The sclerotial DNA was slightly smaller. Figure 2b shows restriction endonuclease digested mycelial and sclerotial DNA. Mycelial ( 1.3 pg) and sclerotial ( 1.5 pg) DNAs were digested with &II, MhoI, and Srnal. The reduction in size of the DNAs due to Mhol digestion clearly demonstrated that both samples could be cut with restriction endonculeases. The banding patterns in digestions of sclerotial DNA were not as prominent as those seen in mycelial DNA. This result could be due to greater degradation of sclerotial DNA during extraction or to differences in the copy number or methylation of genes in mycelial versus sclerotial cells. The last two possibilities are currently being investigated. Restriction endonuclease digested DNAs from mycelia and sclerotia have been subjected to electrophoresis in agarose gels and transferred to nitrocellulose paper by the method of Southern (7). Subsequent hybridization to pBR322 containing the 6.4-kb-pair riboHilzdIII fragment of the S. cerrvisiae somal RNA genes allowed identification of DNA fragments belonging to the ribosomal RNA genes of A. Javus. When mycelial DNA was hybridized to pBR322 alone, no sequences homologous to pBR322 were found when the filters were washed at the stringency that allowed detection of rDNA homology. Figures 3a and b show an X/lo1 digest of mycelial DNA and the corresponding Southern blot. In both mycelial and sclerotial samples, a single 7.7kb-pair fragment representing one repeat unit of the rDNA was detected. The 7.7-kb-pair fragment can be seen directly in the agarose gel of digested mycelial DNA due to the presence of multiple copies of the ribosomal RNA genes. Digestion with X/z01 was complete as judged by the lack of bands larger than 7.7 kb pairs in the Southern blot. The rDNA repeat unit of ‘4. nidz~luns has also been identified on a 7.7-kb-pair fragment ( 1 1). Digestion of A. .fluv~ls DNA with E&RI,
ISOLATION a.
1
2
OF
b
DNA
FROM
MYCELIA
AND
25
SCLEROTIA
ACKNOWLEDGMENTS
12
The authors thank Dr. Maynard for the gift of X phage containing
Olsen and George Carle S. wrevisiae rDNA.
REFERENCES I. Birnboim.
H. C.. and Daly.
J. (1979)
NzI&~c.
.4&/s
Re,. 7, 1513-1519.
FIG. 3. (a) Lane I contains A. fluvus mycelial DNA digested with .IlhoI. The single bright band in the gel picture corresponds to the 7.7-kb-pair band in the Southern blot and identifies one complete repeat unit of the rDNA. Lane 2 contains a Hind111 digest of the plasmid containing S. crrevisiae rDNA. The 6.4-kb-pair band is the S. cerevisiue rDNA. and the 4.4-kb-pair band is pBR322. (b) Southern blot of the gel in (a) probed with the plasmid containing S. cwevisiae rDNA. Only one band appears in the mycelial DNA, indicating complete digestion with X/zoI. pBR322 itselfdid not hybridize to blots of,4../la~us mycelial DNA.
HirzdIII. P.srI. and BarnHI, followed by Southern blotting of the gel, indicates that there is significant restriction site polymorphism between the rDNA sequences of ‘4. niddmc ( 1 1) and .A. f1mr.s.
2. Holmes, D. S., and Quigley, M. (I 9X I ) .4nal. Bioch~~n 114, 193-197. 3. Kado. C. I.. and Lui, S.-T. ( I98 I ) J. Bucteriol. 145(3), 1365-1373. 4. Garber, R. C., and Yoder. 0. C. (1983) .4nal Biochem 135,416-422. 5. Tilburn. J., Scazzocchio, C.. Taylor. G. G.. ZabickyZissman. J. H., Lockington, R. A.. and Davies. R. W. (I 983) G‘er~e 26, 205-22 I 6. Specht. C. A.. DiRusso. C. C., Novotny. C. P.. and Ullrich. R. C. (1982) .4nal Bkherrz. 119, 15% 163. 7. Southern, E. ( 1975) J ,Zlo/. Biol. 98, 503-5 17. 8. Yelton. M. M., Hamer. J. E., and Timberlake. W. E. (I 984) Proc. Nut/. .-Icud. SC;. W.J 81, l470- 1474. 9. Maniatis, T.. Fritsch. E. F., and Sambrook. J. (1982) Molecular Cloning. A Laboratory Manual, pp. 464465. 468. Cold Spring Harbor Laboratories, Cold Spring Harbor, N. Y. IO Langer, P. R.. Waldrop, A. A., and Ward. D. C. ( I98 I ) Proc Null .-lead. Ski. US.4 78, 6633-6637. II Lockington. R. A., Taylor, G. G., Winther. M., Scazzocchio. C., and Davies. R. W. (1982) (ic~nr 20, 135-137.