Advertisement

Springer Nature is making Coronavirus research free. View research | View latest news | Sign up for updates

Multicopy CUP1 plasmids enhance cadmium and copper resistance levels in yeast

Summary

A 3.3 kb fragment of yeast genomic DNA was isolated by screening a genomic library constructed in the high copy number 2 micron plasmid YEp351 vector for clones capable of enhancing the degree of resistance of Saccharomyces cerevisiae strain MW3070-8B to cadmium. The insert contained two complete copies of the CUP1 gene open reading frame (183 bp), including the upstream promoter sequences (450 bp) with two conserved metal responsive cis-acting elements. Northern analysis showed that addition of cadmium (0.02 μM) or copper (50 μM) to overnight liquid cultures of yeast induced expression of CUP1 transcripts from both chromosomal and plasmid-borne gene copies. The cloned 3.3 kb DNA in a high copy number plasmid restored copper resistance to the sensitive strain LS70-313Δ, deleted for the CUP1 gene (cup1Δ), but failed to restore cadmium resistance. Thus, CUP1 gene expression in yeast appears to be influenced differently by cadmium and copper ions. Resistance to heavy metal poisoning resulted from enhanced gene product levels attributable to amplification of the CUP1 gene as well as to increased transcriptions. Two distinct gene product levels mediate cadmium and copper resistance; a higher gene product level was required to confer cadmium resistance.

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

References

  1. Butt TR, Sternberg EJ, Gorman JA, Clark P, Hamer D, Rosenberg M, Crooke ST (1984) Copper metallothionein of yeast, structure of the gene, and regulation of expression. Proc Natl Acad Sci USA 81:3332–3336

  2. Carter AD, Felber BK, Walling MJ, Jubier M, Schmidt CJ, Hamer DH (1984) Duplicated heavy metal control sequences of the mouse metallothionein-I gene. Proc Natl Acad Sci USA 81:7392–7396

  3. Durnam DM, Palmiter RD (1981) Transcriptional regulation of the mouse metallothionein-I gene by heavy metals. J Biol Chem 256:5712–5716

  4. Fogel S, Welch JW (1982) Tandem gene amplification mediates copper resistance in yeast. Proc Natl Acad Sci USA 79:5342–5346

  5. Fogel S, Welch JW, Cathala G, Karin M (1983) Gene amplification in yeast: CUP1 copy number regulates copper resistance. Curr Genet 7:347–355

  6. Foulkes EC (1982) Biological roles of metallothionein. Elsevier Press, New York

  7. Fürst P, Hu S, Hackett R, Hamer DH (1988) Copper activates metallothionein gene transcription by altering the confirmation of a specific DNA binding protein. Cell 55:705–717

  8. Gorman JA, Clark PE, Lee MC, Debouck C, Rosenberg M (1986) Regulation of the yeast metallothionein gene. Gene 48:13–22

  9. Hamer DH (1986) Metallothionein. Annu Rev Biochem 55:913–951

  10. Hamer DH, Thiele DJ, Lemontt JE (1985) Function and autoregulation of yeast copperthionein. Science 228:685–690

  11. Inouhe M, Hiyama M, Tohoyama H, Joho M, Murayama T (1989) Cadmium-binding protein in a cadmium resistant strain of yeast. Biochim Biophys Acta 993:51–55

  12. Kagi J, Nordberg M (1979) Metallothionein. Birkhauser Verlag, Basel

  13. Karin M, Najarian R, Haslinger A, Valenzuela P, Welch JW, Fogel S (1984) Primary structure and transcription of an amplified genetic locus: the CUP1 locus of yeast. Proc Natl Acad Sci USA 81:337–341

  14. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York

  15. Mayo KE, Palmiter RD (1981) Glucocorticoid regulation of metallothionein-I mRNA synthesis in cultured mouse cells. J Biol Chem 256:2621–2624

  16. Musti AM, Zehner Z, Bostian KA, Paterson BM, Kramer RA (1983) Transcriptional mapping of two yeast genes coding for glyceraldehyde 3-phosphate dehydrogenase isolated by sequence homology with the chicken gene. Gene 25:133–143

  17. Nriagu J (1984) Changing metal cycles and human health. Springer-Verlag, Berlin

  18. Premakumar R, Winge DR, Wiley RD, Rajagopalan KV (1975) Copper-chelatin: isolation from various eukaryotic sources. Arch Biochem Biophys 170:278–288

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

  20. Sherman F, Fink GR, Hicks JB (1986) Laboratory course manual for methods in yeast genetics. Cold Spring Harbor Laboratory Press, New York

  21. Tabor S, Richardson CC (1987) DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci USA 84:4767–4771

  22. Thiele DJ, Hamer DH (1986) Tandemly duplicated upstream control sequences mediate copper-induced transcription of the Saccharomyces cerevisiae copper-metallothionein gene. Mol Cell Biol 6:1158–1163

  23. Thiele DJ, Walling MJ, Hamer DH (1986) Mammalian metallothionein is functional in yeast. Science 231:854–856

  24. Tsuchia K (1978) Cadmium studies in Japan: a review. Elsevier Press, New York

  25. Welch JW, Fogel S, Cathala G, Karin M (1983) Industrial yeasts display tandem gene iteration at the CUP1 region. Mol Cell Biol 3:1353–1361

  26. Welch JW, Fogel S, Buchman C, Karin M (1989) The CUP2 gene product regulates the expression of the CUP1 gene, coding for yeast metallothionein. EMBO J 8:255–260

  27. Winge DR, Nielson KB, Gray WR, Hamer DH (1985) Yeast metallothionein. J Biol Chem 260:14464–14470

Download references

Author information

Correspondence to Seymour Fogel.

Additional information

Communicated by C.P. Hollenberg

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Jeyaprakash, A., Welch, J.W. & Fogel, S. Multicopy CUP1 plasmids enhance cadmium and copper resistance levels in yeast. Molec. Gen. Genet. 225, 363–368 (1991). https://doi.org/10.1007/BF00261675

Download citation

Key words

  • Yeast
  • Metallothionein gene
  • Cadmium resistance