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. 1986 Dec 1;5(12):3391–3399. doi: 10.1002/j.1460-2075.1986.tb04655.x

Functional analysis of the yeast plasmid partition locus STB

James A H Murray 1, Gianni Cesareni 1
PMCID: PMC1167338  PMID: 16453734

Abstract

Derivatives of the yeast 2μ plasmid with the cis-acting locus STB (also called REP3) are stably maintained if two plasmid-encoded proteins are present in trans. There are conflicting reports of both the extent of STB and its possible involvement in plasmid partition or copy number control. We have resolved the controversy by constructing 2µ derivatives with a conditional STB function, and showing that when STB is inactivated plasmids become concentrated in a small fraction of the population although the total number of plasmids remains unaltered. Moreover we show that STB consists of two functionally distinct domains which we call STB-proximal and STB-distal relative to the origin of replication. Although STB-proximal is sufficient for proper partitioning, this function is severely disrupted by active transcription from neighbouring sequences. STB-distal is important to protect STB-proximal and ORI from such transcription, and can be effeciently replaced by a 94-bp terminator fragment in an orientation-dependent manner. We find that STB-distal contains an additional element which depresses transcription from upstream promoters. We also describe the phenomenon of replicaton inhibition which we believe can exlain the anomalous instability of some yeast plasmids.

Keywords: 2μ plasmid, partition, replication inhibition, Saccharomyces cerevisiae, conditional mutuants

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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. Adams C. W., Hatfield G. W. Effects of promoter strengths and growth conditions on copy number of transcription-fusion vectors. J Biol Chem. 1984 Jun 25;259(12):7399–7403. [PubMed] [Google Scholar]
  3. Baldari C., Cesareni G. Plasmids pEMBLY: new single-stranded shuttle vectors for the recovery and analysis of yeast DNA sequences. Gene. 1985;35(1-2):27–32. doi: 10.1016/0378-1119(85)90154-4. [DOI] [PubMed] [Google Scholar]
  4. Botstein D., Falco S. C., Stewart S. E., Brennan M., Scherer S., Stinchcomb D. T., Struhl K., Davis R. W. Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene. 1979 Dec;8(1):17–24. doi: 10.1016/0378-1119(79)90004-0. [DOI] [PubMed] [Google Scholar]
  5. 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]
  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. Chlebowicz-Sledziewska E., Sledziewski A. Z. Construction of multicopy yeast plasmids with regulated centromere function. Gene. 1985;39(1):25–31. doi: 10.1016/0378-1119(85)90103-9. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. 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]
  11. 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]
  12. Gentz R., Langner A., Chang A. C., Cohen S. N., Bujard H. Cloning and analysis of strong promoters is made possible by the downstream placement of a RNA termination signal. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4936–4940. doi: 10.1073/pnas.78.8.4936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Guarente L. Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. Methods Enzymol. 1983;101:181–191. doi: 10.1016/0076-6879(83)01013-7. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. 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.5.9.2466-2475.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Lambert P. F., Reznikoff W. S. Use of transcriptional repressors to stabilize plasmid copy number of transcriptional fusion vectors. J Bacteriol. 1985 Apr;162(1):441–444. doi: 10.1128/jb.162.1.441-444.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Petes T. D., Williamson D. H. Replicating circular DNA molecules in yeast. Cell. 1975 Mar;4(3):249–253. doi: 10.1016/0092-8674(75)90172-5. [DOI] [PubMed] [Google Scholar]
  25. Reed K. C., Mann D. A. Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 1985 Oct 25;13(20):7207–7221. doi: 10.1093/nar/13.20.7207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Seiki M., Ogasawara N., Yoshikawa H. Identification of a suppressor sequence for DNA replication in the replication origin region of the Bacillus subtilis chromosome. Proc Natl Acad Sci U S A. 1982 Jul;79(14):4285–4289. doi: 10.1073/pnas.79.14.4285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Stueber D., Bujard H. Transcription from efficient promoters can interfere with plasmid replication and diminish expression of plasmid specified genes. EMBO J. 1982;1(11):1399–1404. doi: 10.1002/j.1460-2075.1982.tb01329.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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.5.10.2770-2780.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tanaka M., Hiraga S. Negative control of oriC plasmid replication by transcription of the oriC region. Mol Gen Genet. 1985;200(1):21–26. doi: 10.1007/BF00383307. [DOI] [PubMed] [Google Scholar]
  31. 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.5.9.2190-2196.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Wolffe A. P., Jordan E., Brown D. D. A bacteriophage RNA polymerase transcribes through a Xenopus 5S RNA gene transcription complex without disrupting it. Cell. 1986 Feb 14;44(3):381–389. doi: 10.1016/0092-8674(86)90459-9. [DOI] [PubMed] [Google Scholar]
  34. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  35. Yarger J. G., Armilei G., Gorman M. C. Transcription terminator-like element within a Saccharomyces cerevisiae promoter region. Mol Cell Biol. 1986 Apr;6(4):1095–1101. doi: 10.1128/mcb.6.4.1095-1101.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Zaret K. S., Sherman F. DNA sequence required for efficient transcription termination in yeast. Cell. 1982 Mar;28(3):563–573. doi: 10.1016/0092-8674(82)90211-2. [DOI] [PubMed] [Google Scholar]

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