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. 1987 Dec 20;6(13):4205–4212. doi: 10.1002/j.1460-2075.1987.tb02768.x

Antagonistic controls regulate copy number of the yeast 2 mu plasmid.

J A Murray 1, M Scarpa 1, N Rossi 1, G Cesareni 1
PMCID: PMC553905  PMID: 2832156

Abstract

The endogenous 2 mu plasmid of yeast encodes a site-specific recombinase FLP that can cause an amplification of plasmid copy number. Using strains overexpressing 2 mu plasmid proteins from chromosomal constructs to disrupt the normal balance of gene products, we show here that copy number is controlled by regulating the transcript level of FLP. Expression of FLP is negatively regulated over a 100-fold range by the joint action of the plasmid-encoded REP1 and REP2 proteins, which also have a role in plasmid partition. We also show that the product of the fourth plasmid open reading frame D increases FLP expression by relieving the repression caused by REP1 and REP2. This is the first demonstration of a function for this gene, which we call RAF. The transcription of RAF is also repressed by REP1 and REP2 acting together, but requires a higher level for complete inhibition than that required to repress FLP. Copy number is therefore negatively regulated by REP1-REP2 concentration both by direct repression of FLP and indirectly, by control of the positive element, the anti-repressor RAF. We propose that these antagonistic regulatory mechanisms amplify the signal produced by a small fall in copy number.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams B. G. Induction of galactokinase in Saccharomyces cerevisiae: kinetics of induction and glucose effects. J Bacteriol. 1972 Aug;111(2):308–315. doi: 10.1128/jb.111.2.308-315.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baldari C., Murray J. A., Ghiara P., Cesareni G., Galeotti C. L. A novel leader peptide which allows efficient secretion of a fragment of human interleukin 1 beta in Saccharomyces cerevisiae. EMBO J. 1987 Jan;6(1):229–234. doi: 10.1002/j.1460-2075.1987.tb04743.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beggs J. D. Transformation of yeast by a replicating hybrid plasmid. Nature. 1978 Sep 14;275(5676):104–109. doi: 10.1038/275104a0. [DOI] [PubMed] [Google Scholar]
  4. Brand A. H., Breeden L., Abraham J., Sternglanz R., Nasmyth K. Characterization of a "silencer" in yeast: a DNA sequence with properties opposite to those of a transcriptional enhancer. Cell. 1985 May;41(1):41–48. doi: 10.1016/0092-8674(85)90059-5. [DOI] [PubMed] [Google Scholar]
  5. Broach J. R., Atkins J. F., McGill C., Chow L. Identification and mapping of the transcriptional and translational products of the yeast plasmid, 2mu circle. Cell. 1979 Apr;16(4):827–839. doi: 10.1016/0092-8674(79)90098-9. [DOI] [PubMed] [Google Scholar]
  6. Broach J. R. Construction of high copy yeast vectors using 2-microns circle sequences. Methods Enzymol. 1983;101:307–325. doi: 10.1016/0076-6879(83)01024-1. [DOI] [PubMed] [Google Scholar]
  7. Broach J. R., Guarascio V. R., Jayaram M. Recombination within the yeast plasmid 2mu circle is site-specific. Cell. 1982 May;29(1):227–234. doi: 10.1016/0092-8674(82)90107-6. [DOI] [PubMed] [Google Scholar]
  8. Broach J. R., Hicks J. B. Replication and recombination functions associated with the yeast plasmid, 2 mu circle. Cell. 1980 Sep;21(2):501–508. doi: 10.1016/0092-8674(80)90487-0. [DOI] [PubMed] [Google Scholar]
  9. Clark-Walker G. D., Miklos G. L. Localization and quantification of circular DNA in yeast. Eur J Biochem. 1974 Jan 16;41(2):359–365. doi: 10.1111/j.1432-1033.1974.tb03278.x. [DOI] [PubMed] [Google Scholar]
  10. Dente L., Cesareni G., Cortese R. pEMBL: a new family of single stranded plasmids. Nucleic Acids Res. 1983 Mar 25;11(6):1645–1655. doi: 10.1093/nar/11.6.1645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  12. Futcher A. B. Copy number amplification of the 2 micron circle plasmid of Saccharomyces cerevisiae. J Theor Biol. 1986 Mar 21;119(2):197–204. doi: 10.1016/s0022-5193(86)80074-1. [DOI] [PubMed] [Google Scholar]
  13. Futcher A. B., Cox B. S. Copy number and the stability of 2-micron circle-based artificial plasmids of Saccharomyces cerevisiae. J Bacteriol. 1984 Jan;157(1):283–290. doi: 10.1128/jb.157.1.283-290.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Futcher A. B., Cox B. S. Maintenance of the 2 microns circle plasmid in populations of Saccharomyces cerevisiae. J Bacteriol. 1983 May;154(2):612–622. doi: 10.1128/jb.154.2.612-622.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Guarente L., Yocum R. R., Gifford P. A GAL10-CYC1 hybrid yeast promoter identifies the GAL4 regulatory region as an upstream site. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7410–7414. doi: 10.1073/pnas.79.23.7410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hartley J. L., Donelson J. E. Nucleotide sequence of the yeast plasmid. Nature. 1980 Aug 28;286(5776):860–865. doi: 10.1038/286860a0. [DOI] [PubMed] [Google Scholar]
  17. Hartwell L. H., Mortimer R. K., Culotti J., Culotti M. Genetic Control of the Cell Division Cycle in Yeast: V. Genetic Analysis of cdc Mutants. Genetics. 1973 Jun;74(2):267–286. doi: 10.1093/genetics/74.2.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hollenberg C. P. Mapping of regions on cloned Saccharomyces cerevisiae 2-mum DNA coding for polypeptides synthesized in Escherichia coli minicells. Mol Gen Genet. 1978 Jun 1;162(1):23–34. doi: 10.1007/BF00333847. [DOI] [PubMed] [Google Scholar]
  19. Holm C. Clonal lethality caused by the yeast plasmid 2 mu DNA. Cell. 1982 Jun;29(2):585–594. doi: 10.1016/0092-8674(82)90174-x. [DOI] [PubMed] [Google Scholar]
  20. Jayaram M., Li Y. Y., Broach J. R. The yeast plasmid 2mu circle encodes components required for its high copy propagation. Cell. 1983 Aug;34(1):95–104. doi: 10.1016/0092-8674(83)90139-3. [DOI] [PubMed] [Google Scholar]
  21. Jayaram M., Sutton A., Broach J. R. Properties of REP3: a cis-acting locus required for stable propagation of the Saccharomyces cerevisiae plasmid 2 microns circle. Mol Cell Biol. 1985 Sep;5(9):2466–2475. doi: 10.1128/mcb.5.9.2466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jazwinski S. M., Edelman G. M. Replication in vitro of the 2-micrometer DNA plasmid of yeast. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1223–1227. doi: 10.1073/pnas.76.3.1223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jong A. Y., Scott J. F. DNA synthesis in yeast cell-free extracts dependent on recombinant DNA plasmids purified from Escherichia coli. Nucleic Acids Res. 1985 Apr 25;13(8):2943–2958. doi: 10.1093/nar/13.8.2943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kieny M. P., Lathe R., Lecocq J. P. New versatile cloning and sequencing vectors based on bacteriophage M13. Gene. 1983 Dec;26(1):91–99. doi: 10.1016/0378-1119(83)90039-2. [DOI] [PubMed] [Google Scholar]
  25. Kikuchi Y. Yeast plasmid requires a cis-acting locus and two plasmid proteins for its stable maintenance. Cell. 1983 Dec;35(2 Pt 1):487–493. doi: 10.1016/0092-8674(83)90182-4. [DOI] [PubMed] [Google Scholar]
  26. Livingston D. M., Kupfer D. M. Control of Saccharomyces cerevisiae 2microN DNA replication by cell division cycle genes that control nuclear DNA replication. J Mol Biol. 1977 Oct 25;116(2):249–260. doi: 10.1016/0022-2836(77)90215-7. [DOI] [PubMed] [Google Scholar]
  27. MORTIMER R. K., JOHNSTON J. R. Life span of individual yeast cells. Nature. 1959 Jun 20;183(4677):1751–1752. doi: 10.1038/1831751a0. [DOI] [PubMed] [Google Scholar]
  28. Marczynski G. T., Jaehning J. A. A transcription map of a yeast centromere plasmid: unexpected transcripts and altered gene expression. Nucleic Acids Res. 1985 Dec 9;13(23):8487–8506. doi: 10.1093/nar/13.23.8487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mead D. J., Gardner D. C., Oliver S. G. The yeast 2 micron plasmid: strategies for the survival of a selfish DNA. Mol Gen Genet. 1986 Dec;205(3):417–421. doi: 10.1007/BF00338076. [DOI] [PubMed] [Google Scholar]
  30. Messing J., Gronenborn B., Müller-Hill B., Hans Hopschneider P. Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3642–3646. doi: 10.1073/pnas.74.9.3642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Morris C. E., Klement J. F., McAllister W. T. Cloning and expression of the bacteriophage T3 RNA polymerase gene. Gene. 1986;41(2-3):193–200. doi: 10.1016/0378-1119(86)90098-3. [DOI] [PubMed] [Google Scholar]
  32. Murray A. W., Szostak J. W. Pedigree analysis of plasmid segregation in yeast. Cell. 1983 Oct;34(3):961–970. doi: 10.1016/0092-8674(83)90553-6. [DOI] [PubMed] [Google Scholar]
  33. Murray J. A. Bending the rules: the 2-mu plasmid of yeast. Mol Microbiol. 1987 Jul;1(1):1–4. doi: 10.1111/j.1365-2958.1987.tb00519.x. [DOI] [PubMed] [Google Scholar]
  34. Murray J. A., Cesareni G. Functional analysis of the yeast plasmid partition locus STB. EMBO J. 1986 Dec 1;5(12):3391–3399. doi: 10.1002/j.1460-2075.1986.tb04655.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Murray J. A. HCC ligation: rapid and specific DNA construction with blunt ended DNA fragments. Nucleic Acids Res. 1986 Dec 22;14(24):10118–10118. doi: 10.1093/nar/14.24.10118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Parent S. A., Fenimore C. M., Bostian K. A. Vector systems for the expression, analysis and cloning of DNA sequences in S. cerevisiae. Yeast. 1985 Dec;1(2):83–138. doi: 10.1002/yea.320010202. [DOI] [PubMed] [Google Scholar]
  37. Rubin G. M. Preparation of RNA and ribosomes from yeast. Methods Cell Biol. 1975;12:45–64. doi: 10.1016/s0091-679x(08)60951-6. [DOI] [PubMed] [Google Scholar]
  38. Rüther U. pUR 250 allows rapid chemical sequencing of both DNA strands of its inserts. Nucleic Acids Res. 1982 Oct 11;10(19):5765–5772. doi: 10.1093/nar/10.19.5765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Scott J. R. Regulation of plasmid replication. Microbiol Rev. 1984 Mar;48(1):1–23. doi: 10.1016/b978-0-12-048850-6.50006-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sigurdson D. C., Gaarder M. E., Livingston D. M. Characterization of the transmission during cytoductant formation of the 2 micrometers DNA plasmid from Saccharomyces. Mol Gen Genet. 1981;183(1):59–65. doi: 10.1007/BF00270139. [DOI] [PubMed] [Google Scholar]
  41. Sollazzo M., Frank R., Cesareni G. High-level expression of RNAs and proteins: the use of oligonucleotides for the precise fusion of coding-to-regulatory sequences. Gene. 1985;37(1-3):199–206. doi: 10.1016/0378-1119(85)90273-2. [DOI] [PubMed] [Google Scholar]
  42. Spratt B. G., Hedge P. J., te Heesen S., Edelman A., Broome-Smith J. K. Kanamycin-resistant vectors that are analogues of plasmids pUC8, pUC9, pEMBL8 and pEMBL9. Gene. 1986;41(2-3):337–342. doi: 10.1016/0378-1119(86)90117-4. [DOI] [PubMed] [Google Scholar]
  43. Sutton A., Broach J. R. Signals for transcription initiation and termination in the Saccharomyces cerevisiae plasmid 2 micron circle. Mol Cell Biol. 1985 Oct;5(10):2770–2780. doi: 10.1128/mcb.5.10.2770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Taketo M., Jazwinski S. M., Edelman G. M. Association of the 2-micron DNA plasmid with yeast folded chromosomes. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3144–3148. doi: 10.1073/pnas.77.6.3144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Twigg A. J., Sherratt D. Trans-complementable copy-number mutants of plasmid ColE1. Nature. 1980 Jan 10;283(5743):216–218. doi: 10.1038/283216a0. [DOI] [PubMed] [Google Scholar]
  46. Veit B. E., Fangman W. L. Chromatin organization of the Saccharomyces cerevisiae 2 microns plasmid depends on plasmid-encoded products. Mol Cell Biol. 1985 Sep;5(9):2190–2196. doi: 10.1128/mcb.5.9.2190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Volkert F. C., Broach J. R. Site-specific recombination promotes plasmid amplification in yeast. Cell. 1986 Aug 15;46(4):541–550. doi: 10.1016/0092-8674(86)90879-2. [DOI] [PubMed] [Google Scholar]
  48. Wu L. C., Fisher P. A., Broach J. R. A yeast plasmid partitioning protein is a karyoskeletal component. J Biol Chem. 1987 Jan 15;262(2):883–891. [PubMed] [Google Scholar]
  49. Zakian V. A., Brewer B. J., Fangman W. L. Replication of each copy of the yeast 2 micron DNA plasmid occurs during the S phase. Cell. 1979 Aug;17(4):923–934. doi: 10.1016/0092-8674(79)90332-5. [DOI] [PubMed] [Google Scholar]

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