Skip to main content
SpringerLink
Log in

Phleomycin resistance encoded by the ble gene from transposon Tn 5 as a dominant selectable marker in Saccharomyces cerevisiae

  • Published:
Molecular and General Genetics MGG Aims and scope Submit manuscript

Summary

Phleomycin, a water-soluble antibiotic of the bleomycin family is as effective against Saccharomyces cerevisiae cells as against Escherichia coli cells. The ble gene of transposon Tn5, which confers resistance to phleomycin, was inserted in place of the iso-1-cytochrome C (CYC1) gene on an autonomously replicative multicopy E. coli-yeast shuttle plasmid. Higher resistance levels are obtained in S. cerevisiae when the region immediately upstream from the initiation codon conforms to the nucleotide sequence stringencies observed in almost every yeast gene. The expected regulation pattern of the whole CYC1 promoter confers different phleomycin resistance levels to the cell under varying physiological conditions. Partial deletions in the CYC1 promoter lead to changes in the resistance level of cells which are mostly accounted for by the removal of known positive and negative regulatory elements. Some of the vector constructions allow direct selection of phleomycin-resistant transformants on rich media.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Arcangioli B, Lescure B (1985) Identification of proteins involved in the regulation of yeast Iso-1-cytochrome C expression by oxygen. EMBO J 4:2627–2633

    Google Scholar 

  • Beacham IR, Schweitzer BW, Warrick HM, Carbon J (1984) The nucleotide sequence of the yeast ARG4 gene. Gene 29:271–279

    Google Scholar 

  • Berdy J (1980) Bleomycin type antibiotics In: Handbook of antibiotic compounds, Vol IV. CRC Press, pp 459–491

  • Berg DE, Berg CM (1983) The prokaryotic transposable element Tn5. Biotechnology 1:417–435

    Google Scholar 

  • Berg DE, Davies J, Allet B, Rochaix JD (1975) Transposition of R factor genes to bacteriophage λ. Proc Natl Acad Sci USA 72:3628–3632

    Google Scholar 

  • Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523

    Google Scholar 

  • Boy-Marcotte E, Jacquet M (1982) A Dictyostelium discoideum DNA fragment complements a Saccharomyces cerevisiae ura3 mutant. Gene 20:433–440

    Google Scholar 

  • Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41:459–472

    Google Scholar 

  • Collis CM, Hall RM (1985) Identification of a Tn5 determinant conferring resistance to phleomycins, bleomycins and tallysomycins. Plasmid 14:143–151

    Google Scholar 

  • Davison J, Heuterspreute M, Merchez M, Brunel F (1984) Vectors with restriction-site banks. I. pJRD158, a 3903-bp plasmid containing 28 unique cloning sites. Gene 28:311–318

    Google Scholar 

  • Dobson MJ, Tuite MF, Roberts NA, Kingsman AJ, Kingsman SM (1982) Conservation of high efficiency promoter sequences in Saccharomyces cerevisiae. Nucleic Acids Res 10:2625–2637

    Google Scholar 

  • Ernst JF, Chan RK (1985) Characterization of Saccharomyces cerevisiae mutants supersensitive to aminoglycoside antibiotics. J Bacteriol 163:8–14

    Google Scholar 

  • Faye G, Leung DW, Tatchell K, Hall BD, Smith M (1981) Deletion mapping of sequences essential for in vivo transcription of iso-1-cytochrome C gene. Proc Natl Acad Sci USA 78:2258–2262

    Google Scholar 

  • Genilloud O, Garrido MC, Moreno F (1984) The transposon Tn5 carries a bleomycin-resistance determinant. Gene 32:225–233

    Google Scholar 

  • Grigg GW, Hall RM, Hart NK, Havulak DR, Lamberton JA, Lane A (1985) Amplification of the antibiotic. Effects of the Bleomycins, Phleomycins and Tallysomycins: Its Dependence on the nature of the Variable Basic Groups. J Antibiot 38:99–110

    Google Scholar 

  • Gritz L, Davies J (1983) Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae. Gene 25:179–188

    Google Scholar 

  • Guarente L (1984) Yeast promoters: positive and negative elements. Cell 36:799–800

    Google Scholar 

  • Guarente L, Mason T (1983) Heme regulates transcription of the CYC1 gene of S. cerevisiae via an upstream activation site. Cell 32:1279–1286

    Google Scholar 

  • Guarente L, Lalonde B, Gifford P, Alani E (1984) Distinctly regulated tandem upstream activation sites mediate catabolite repression of the CYC1 gene of S. cerevisiae. Cell 36:503–511

    Google Scholar 

  • Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580

    Google Scholar 

  • Heuterspreute M, Davison J (1984) Restriction site bank vectors II. DNA sequences analysis of plasmid pJRD158. DNA 3:259–268

    Google Scholar 

  • Hertzberg RP, Caranfa MJ, Hecht SM (1985) DNA methylation diminishes bleomycins-mediated strand scission. Biochem 24:5285–5289

    Google Scholar 

  • Ito H, Fukada Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkaline cations. J Bacteriol 153:163–168

    Google Scholar 

  • Jimenez A, Davies J (1980) Expression of a transposable antibiotic resistance element in Saccharomyces. Nature 287:869–871

    Google Scholar 

  • Kaster KR, Burgett SG, Ingolia TD (1984) Hygromycin B resistance as dominant selectable marker in yeast. Curr Genet 8:353–358

    Google Scholar 

  • Klebe RJ, Harriss JV, Sharp ZD, Douglas MG (1983) A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast. Gene 25:333–341

    Google Scholar 

  • Kozak M (1984) Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic Acids Res 12:857–872

    Google Scholar 

  • Kross J, Henner WD, Hecht SM, Haseltine WA (1982) Specificity of deoxyribonucleic acid cleavage by bleomycin, phleomycin and tallysomycin. Biochemistry 21:4310–4318

    Google Scholar 

  • McKenzie T, Hoshino T, Tanaka T, Sueoka N (1986) The nucleotide sequence of pUB110. Some salient features in relation to replication and its regulation. Plasmid 15:93–103

    Google Scholar 

  • Maniatis T, Fritch E, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Mazodier P, Giraud E, Gasser F (1983) Genetic analysis of the streptomycin resistance encoded by Tn5. Mol Gen Genet 192:155–162

    Google Scholar 

  • Mazodier P, Cossart P, Giraud E, Gasser F (1985) Completion of the nucleotide sequence of the central region of Tn5 confirms the presence of three resistance genes. Nucleic Acids Res 13:195–205

    Google Scholar 

  • Peden KW (1983) Revised sequence of the tetracycline resistance gene of pBR322. Gene 22:277–280

    Google Scholar 

  • Pratviel G, Bernadou J, Meunier B (1986) DNA breaks generated by the bleomycin-iron III complex in the presence of KHSO5, a single oxygen donor. Biochem Biophys Res Commun 136:1013–1020

    Google Scholar 

  • Rao RN, Rogers SG (1979) Plasmid pKC7: a vector containing ten restriction endonuclease sites suitable for cloning DNA segments. Gene 7:79–82

    Google Scholar 

  • Rothstein SJ, Reznikoff WS (1981) The functional differences in the inverted repeats of Tn5 are caused by a single base pair nonhomology. Cell 23:191–199

    Google Scholar 

  • Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    Google Scholar 

  • Semon D, Movva NR, Smith TF, El Alama M, Davies J (1987) Plasmid-determined bleomycin resistance in Staphylococcus aureus. Plasmid (in press)

  • Sharp PM, Tuohy T, Mosurski K (1986) Codon usage in yeast: cluster analysis clearly differentiates highly and lowly expressed genes. Nucleic Acids Res 14:5125–5143

    Google Scholar 

  • Sherman F, Stewart JW (1982) Mutations altering initiation of translation of yeast iso-1-cytochrome C; contrasts between the eukaryotic and prokaryotic initiation process. In: Strathern JN, Jones EW, Broach JR (eds) The molecular biology of the yeast Saccharomyces: metabolism and gene expression. Cold Spring Harbor Laboratory, New York, pp 301–333

    Google Scholar 

  • Ueda K, Kobayashi S, Sakai H, Komano T (1985) Cleavage of stem and loop structure DNA by bleomycin. J Biol Chem 260:5804–5807

    Google Scholar 

  • Umezawa H (1974) Chemistry and mechanism of action of bleomycin. Fed Proc 33:2296–2302

    Google Scholar 

  • Umezawa H (1976) Structure and action of bleomycin. Prog Biochem Pharmacol 11:18–27

    Google Scholar 

  • Webster TD, Dickson RC (1983) Direct selection of S. cerevisiae resistant to the antibiotic G418 following transformation with a DNA vector carrying the kanamycin-resistance gene of Tn903. Gene 26:243–252

    Google Scholar 

  • Zitomer RS, Montgomery DL, Nichols DL, Hall BD (1979) Transcriptional regulation of the yeast cytochrome C gene. Proc Natl Acad Sci USA 76:3627–3631

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratoire de Microbiologie et Génétique appliquées du C.N.R.S., Université Paul Sabatier, 118 route de Narbonne, F-31062, Toulouse Cédex, France

    Anne Gatignol, Michel Baron & Gérard Tiraby

Authors
  1. Anne Gatignol
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michel Baron
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gérard Tiraby
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Communicated by C.P. Hollenberg

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gatignol, A., Baron, M. & Tiraby, G. Phleomycin resistance encoded by the ble gene from transposon Tn 5 as a dominant selectable marker in Saccharomyces cerevisiae . Mol Gen Genet 207, 342–348 (1987). https://doi.org/10.1007/BF00331599

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00331599

Key words

Search

Navigation