Summary
A procedure is described for the detection of specific DNA sequences in Saccharomyces cerevisiae. This method allows a rapid screening of a large number of yeast colonies. The yeast cells of each colony, grown on nitrocellulose filters, are converted, in situ, to protoplasts by snail enzyme, and are then lysed and their DNAs are denatured and fixed on the filter. The presence of the specific DNA sequence is detected directly on the filter by hybridization with a radioactive cRNA.
We have used successfully this technique to detect the presence or the absence of specific mt DNA sequences, in ρ+, ρ- and ρ0 strains, and to detect the presence or the absence of the 2 μm DNA sequences in different strains.
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Borst, P.: Structure of petite mt DNA; prospects for mitochondrial gene isolation, mapping and sequencing. In: The biogenesis of mitochondria (Kroon, A.M. and Saccone, C., eds.), pp. 147–156. New York: Academic Press 1974
Borst, P., Bos, J.L., Grivell, L.A., Groot, G.S.P., Heyting, C., Moorman, A.F.M., Sanders, J.P.M., Talen, J.L., Van Kreijl, C.F., Van Omnen, G.J.B.: The physical map of yeast mitochondrial DNA anno 1977. In: Mitochondria 1977 (Bandlow, W., Schweyen, R.J., Wolf, K., Kaudewitz, F., eds.), pp. 213–254. Berlin: Walter de Gruyter 1977
Burgess, R.R., Travers, A.A.: DNA-dependant RNA polymerase. In: Procedures in nucleic acid research (Cantoni, G.I. and Davies, D.R., eds.), Vol. 2, pp. 851–863. New York: Harper and Row 1971
Cameron, J.R., Philippsen, P., Davis, R.W.: Analysis of chromosomal integration and deletions of yeast plasmids. Nucl. Acid Res. 4, 1429–1448 (1977)
Clark-Walker, G.D.: Size distribution of circular DNA from petitemutant yeast lacking: ϱ DNA. Europ. J. Biochem. 32, 263–267 (1973)
Clewell, D.B.: Nature of Col E1 plasmid replication in the presence of chloramphenicol. J. Bact. 110, 667–676 (1972)
Conaughy, M., Laird, C.E., McCarthy, B.J.: Nucleic acid reassociation in Formamide. Biochemistry 8, 3289–3295 (1969)
Di Franco, A., Sanders, J.P.M., Heyting, C., Borst, P., Slonimski, P.P.: Restriction enzyme analysis and physical mapping of mitochondrial DNA from petite mutants carrying a genetic marker for oligomycin or paromomycin resistance. In: The genetic function of mitochondrial DNA (Saccone, C. and Kroon, A.M., eds.), pp. 291–304. Amsterdam: North Holland 1976
Duell, E.A., Inoue, S., Utter, M.F.: Isolation and properties of intact mitochondria from spheroplasts of yeast. J. Bact. 88, 1762–1773 (1964)
Ephrussi, B., Hottinguer, H., Chimenes, A.M.: Action de l'acriflavine sur les levures. La mutation “petite colonie”. Ann. Inst. Pasteur 76, 351–368 (1949)
Faye, G., Fukuhara, H., Grandchamp, C., Lazowska, J., Michel, F., Casey, J., Getz, G.J., Locker, J., Rabinowitz, M., Bolotin-Fukuhara, M., Coen, D., Deutsch, J., Dujon, B., Netter, P., and Slonimski, P.P.: Mitochondrial nucleic acids in the petite colonie mutants: Deletions and repetitions of genes. Biochimie 55, 779–792 (1973)
Goldring, E.S., Grossman, L.I., Krupnick, D., Cryer, D.R., Marmur, J.: The petite mutation in yeast. J. molec. Biol. 52 323–335 (1970)
Grunstein, M., Hogness, D.S.: Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc. nat. Acad. Sci. (Wash.) 72, 3961–3965 (1975)
Guérineau, M., Grandchamp, C., Paoletti, C., Slonimski, P.P.: Characterization of a new class of circular DNA molecules in yeast. Biochem. biophys. Res. Commun. 42, 550–557 (1971)
Guérineau, M., Grandchamp, C., Slonimski, P.P.: Circular DNA of a yeast episome with two inverted repeats: structural analysis by a restriction enzyme and electron microscopy. Proc. nat. Acad. Sci. (Wash.) 73, 3030–3034 (1976)
Guérineau, M., Slonimski, P.P., Avner, P.R.: Yeast episome: oligomycin resistance associated with a small covalently closed nonmitochondrial circular DNA. Biochem. biophys. Res. Commun. 61, 462–469 (1974)
Guerry, P., Le Blanc, D.J., Falkow, S.: General method for the isolation of plasmid deoxyribonucleic acid. J. Bact. 116, 1064–1066 (1973)
Hollenberg, C.P., Borst, P., Van Bruggen, E.F.J.: A 25μm closed circular duplex DNA molecule in wild-type yeast mitochondria. Structure and genetic complexity. Biochim. biophys. Acta (Amst.) 209, 1–15 (1970)
Hollenberg, C.P., Degelman, A., Kusterman-Kuhn, B., Royer, H.D.: Characterization of 2μm DNA of Saccharomyces cerevisiae by restriction fragment analysis and integration in an Escherichia coli plasmid. Proc. Nat. Acad. Sci. (Wash.) 73, 3030–3034 (1976)
Jones, K.W., Murray, K.: A procedure for detection of heterologous DNA sequences in lambdoid phage by in situ hybridization. J. molec. Biol. 96, 455–460 (1975)
Kotylak, Z., Slonimski, P.P.: Mitochondrial mutants isolated by a new screening method based upon the use of the nuclear mutation opl. In: Mitochondria 77 (Bandlow, W., Schweyen, R.J., Wolf, K., Kaudewitz, F. eds.) pp. 83–89. Berlin: Walter de Gruyter 1977
Kramer, R.A., Cameron, J.R., Dawis, R.W.: Isolation of bacteriophage λ containing yeast ribosomal RNA genes: screening by in situ RNA Hybridization to plaques. Cell 8, 227–232 (1976)
Lazowska, J., Michel, F., Faye, G., Fukuhara, H., Slonimski, P.P.: Physical and genetic organisation of petite and grande yeast mitochondrial DNA. II. DNA-DNA hybridization studies and buoyant density determinations. J. molec. Biol. 85, 393–410 (1974)
Lewin, A., Morimoto, R., Merten, S., Martin, N., Berg, P., Christianson, T., Levens, D., Rabinowitz, M.: Physical mapping of mitochondrial genes and transcripts in Saccharomyces cerevisiae. In: Mitochondria 77 (Bandlow, W., Schweyen, R.J., Wolf, K., Kaudewitz, F.), pp. 271–289. Berlin: Walter de Gruyter 1977
Livingston, D.M.: Inheritance of the 2μm DNA plasmid from Saccharomyces. Genetics 86, 73–84 (1977)
Livingston, D.M., Klein, H.L.: Deoxyribonucleic acid sequence organization of a yeast plasmid. J. Bact. 129, 472–481 (1977)
Michaelis, G., Douglas, S., Tsai, M.J., Criddle, R.S.: Mitochondrial DNA and suppressiveness of petite mutants in Saccharomyces cerevisiae. Biochem. Genetics 5, 487–495 (1971)
Nagley, P., Linnane, A.W.: Mitochondrial DNA deficient petite mutants of yeast. Biochem. biophys. Res. Commun. 39, 989–996 (1970)
Sanders, J.P.M., Borst, P., Weijers, P.J.: the organization of genes in yeast mitochondrial DNA. Molec. gen. Genet. 143, 53–64 (1975)
Slonimski, P.P., Tzagoloff, A.: Localisation in yeast mitochondrial DNA of mutations expressed in a deficiency of cytochrome oxidase and/or coenzyme OH2-cytochrome c reductase. Europ. J. Biochem. 61, 27–41 (1976)
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Communicated by W. Gajewski
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Blanc, H., Dujon, B., Guerineau, M. et al. Detection of specific DNA sequences in yeast by colony hybridization. Molec. gen. Genet. 161, 311–315 (1978). https://doi.org/10.1007/BF00331006
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DOI: https://doi.org/10.1007/BF00331006