Nucleic Acids Research, Vol. 19, No. 18 5083
Microisolation of yeast nucleic acids on the microtitre plate
without using lytic enzymes
Martin Pospf&ek and Zdena Palkova*
Department of Genetics and Microbiology, Faculty of Natural Sciences, Charles University, Vinicna 5,
12844 Prague 2, Czechoslovakia
Submitted January 31, 1991
Current methods for DNA isolation from yeast start with
protoplasts prepared by removing cell wall by some lytic enzyme
(1, 2). The following method is based on the yeast autolysis.
Autolysis is the final stage of the fungal life cycle (3) and it can
be used for cell disruption and protein isolation (4).
METHOD
1) Yeast cells growing on agar plate are transferred to microtitre
wells, 2) perfectly dried at 45°C (~2 h), 3) resuspended in 40 /xl
TESP (20 raM Tris-HCl, pH 8; 50 mM EDTA-NaOH, pH
8; 2% SDS; 0.05% pronase E) and 4) slowly dried at 37°C
overnight. 5) The sample is dissolved in 20 y\ H2O, mixed with
2 fd BFB (50% glycerol; 130 mM EDTA-NaOH, pH 8; 0.12%
bromophenol blue) and 6) 10 /il of it is electrophoresed in 0.8%
agarose gel in TBE (89 mM Tris; 2 mM EDTA; 90 mM boric
acid) at 1.6 V/cm for 45 min and then at 4.2 V/cm for 100 min.
NOTES: a) If centrifugation of microtitre plate is available, then
washing of cells with 96% ethanol between the steps 1 and 2
(especially when the liquid culture is used) and centrifugation
between the steps 5 and 6 usually give better results, b) After
the step 5, the samples can be stored at -20°C for several weeks.
RESULTS
The method is suitable for isolation of nucleic acids from more
than 107 cells (Figure 1A) growing on a solid or liquid medium.
The method was successfully used for isolation of nucleic acids
from 34 species of 22 yeast genera. Examples are shown in
Figure IB. The method permits the analysis of cytoplasmic
dsRNA or dsDNA from various yeast strains (Figure 1C, ID).
dsRNAs were discovered in Endomyces magnusii and
Wickerhamia fluorescens (Figure 1C, 1 and 2) (manuscript in
preparation). Nuclear plasmids were detected by Southern blotting
(Figure IE).
To make the DNA digestible with restriction endonucleases,
additional purification steps, e.g. these according to (6) were
required (Figure IF).
REFERENCES
1. Ausubel.F.M.. Brent,R., Kingston.R.E., Moore.D.D.. SeidmanJ.G .
Smith J.A. and Struhl.K. (1988) Current Protocols in Molecular Biology.
Greene Publishing Associates'and Wiley Interscience.
2. Pt*rez-Ortfn,J.E. and Estruch.F. (1988) Curr. Microbiol. 17, 19-22.
3. Takeo.K., Yamamura.M. and Kamihara.T. (1969) FEMS Microbiol. Lett.
61, 297-300.
4. Scopes,R.K. (1982) Protein Purification: Principles and Practice. SpringerVerlag, New York.
5. Boras,!., Posfai.G. and Venetianer.P. (1984) Gene 30, 257-260.
6. Cellular and Molecular Biology-of Yeast - Laboratory Manual (1991) EMBO
Practical Course, Heidelberg. EMBL. 21-22.
* To whom correspondence should be addressed
1 2
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Figure 1. Electrophoresis of samples of yeast nucleic acids as obtained by the
autolysis method of microextraction. The arrows show the position of chromosomal
DNA. PANEL A: Analysis of microisolates from Saccharomyces cerevisiae
DMUP12^-20. Lanes 1,2,3,4; 1.107, 5.107, 1.10s, 5.108 cells, respectively.
PANEL B: Microisolates from various yeast species. Lanes are as follows: 1,
Schizosaccharomyces pombe LP-36; 2, Candida tropicalis DMUP2-9-2; 3,
Filobasidium capsuligenum CCY71-1-1; 4, Schwanniomyces occidenlalis
CCY47-M; 5, Pichia membranaefaciens CCY39-M; 6, Hansenula anomala
CCY38-F-F; 7, Trichosporon cutaneum CCY30-5-10. PANEL C: Microisolates
from various yeast species containing cytoplasmic dsRNA. Lane: 1, E.magnusii
DMUP4-1-1; 2, W.fluorescens CCY61-I-1; 3, S.cerevisiae DMUP12^-30; 4,
S.cerevisiae DMUP12-4-20. PANEL D: Microisolates from two different strains
of Kluyveromyccs laais containing cytoplasmic dsDNA. Lane: 1, DMUP8-4-11
(pGKL2); 2,1F01267 (pGKLl, pGKL2). PANEL E: Detection of nuclear plasmids
in microisolates from S.cerevisiae GRF18. I. Gel stained with ethidium bromide,
II. Autoradiography (after hybridization with 32P-radiolabeled plasmid pHC624
(5); (1)). Plasmid conformations: 1 = linear, oc = open circie, ccc = closed
circle. Lanes are as follows: 1. GRF18; 2. Yepl3 (replicating plasmid) (1); 3.
GRF18 transformed with Yepl3; 4. GRF18 transformed with pUCL (integrating
plasmid; pUC18 (1) with LEU2 inserted in Pstl site). PANEL F: Digestion of
DNA from S.cerevisiae GRF18 transformed with Yepl3 with restriction enzymes.
The autolysed cells (after the step 4) were dissolved in 100 n\ H2O and purified
according to (6), starting with heating the sample at 60°C for 10 min. I. Gel
stained with ethidium bromide, 0. Autoradiography (after hybridization with a P radiolabeled plasmid pHC624 (5); (1)). Lanes are as follows: 1. DNA digested
with Hindlll; 2. Non-digested DNA; 3, DNA digested with Pstl.