Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1987 May;84(9):2838–2842. doi: 10.1073/pnas.84.9.2838

Temperature-sensitive mutations in the yeast DNA polymerase I gene.

M Budd, J L Campbell
PMCID: PMC304755  PMID: 3554248

Abstract

Seven mutations that yield thermolabile DNA polymerases have been isolated in the DNA polymerase I gene, POL1, of Saccharomyces cerevisiae. Strains carrying the mutant genes are temperature sensitive for growth. The pol1 mutants were identified by a method that has general applicability for identification of both temperature-sensitive and null mutations. A plasmid containing a mutagenized pol1 gene was transformed into a strain in which the only functional copy of the POL1 gene was carried on an unstable plasmid. The genes conferring temperature-sensitive growth were detected after elimination of the unstable plasmid containing the wild-type gene. DNA polymerase I isolated from each of the mutants is defective at both 23 degrees C and 36 degrees C. DNA synthesis is deficient in vivo at 36 degrees C in all the mutants, while RNA synthesis is normal in all but one of the mutants. The terminal phenotype of pol1 temperature-sensitive mutants is dumbbell-shaped cells in which the nucleus has migrated to, but apparently not entered, the isthmus separating the mother and the daughter. The POL1 gene is located on chromosome XIV approximately 2 centimorgans away from met4.

Full text

PDF
2841

Images in this article

2840Image
on p.2840
2840Image
on p.2840
2840Image
on p.2840

Selected References

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

  1. Bisson L., Thorner J. Thymidine 5'-monophosphate-requiring mutants of Saccharomyces cerevisiae are deficient in thymidylate synthetase. J Bacteriol. 1977 Oct;132(1):44–50. doi: 10.1128/jb.132.1.44-50.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boeke J. D., LaCroute F., Fink G. R. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet. 1984;197(2):345–346. doi: 10.1007/BF00330984. [DOI] [PubMed] [Google Scholar]
  3. Broach J. R., Strathern J. N., Hicks J. B. Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. Gene. 1979 Dec;8(1):121–133. doi: 10.1016/0378-1119(79)90012-x. [DOI] [PubMed] [Google Scholar]
  4. Busby S., Irani M., Crombrugghe B. Isolation of mutant promoters in the Escherichia coli galactose operon using local mutagenesis on cloned DNA fragments. J Mol Biol. 1982 Jan 15;154(2):197–209. doi: 10.1016/0022-2836(82)90060-2. [DOI] [PubMed] [Google Scholar]
  5. Campbell J. L. Eukaryotic DNA replication. Annu Rev Biochem. 1986;55:733–771. doi: 10.1146/annurev.bi.55.070186.003505. [DOI] [PubMed] [Google Scholar]
  6. Carle G. F., Olson M. V. Separation of chromosomal DNA molecules from yeast by orthogonal-field-alternation gel electrophoresis. Nucleic Acids Res. 1984 Jul 25;12(14):5647–5664. doi: 10.1093/nar/12.14.5647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Conrad M. N., Newlon C. S. Saccharomyces cerevisiae cdc2 mutants fail to replicate approximately one-third of their nuclear genome. Mol Cell Biol. 1983 Jun;3(6):1000–1012. doi: 10.1128/mcb.3.6.1000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dumas L. B., Lussky J. P., McFarland E. J., Shampay J. New temperature-sensitive mutants of Saccharomyces cerevisiae affecting DNA replication. Mol Gen Genet. 1982;187(1):42–46. doi: 10.1007/BF00384381. [DOI] [PubMed] [Google Scholar]
  9. Eki T., Murakami Y., Enomoto T., Hanaoka F., Yamada M. Characterization of DNA replication at a restrictive temperature in a mouse DNA temperature-sensitive mutant, tsFT20 strain, containing heat-labile DNA polymerase alpha activity. J Biol Chem. 1986 Jul 5;261(19):8888–8893. [PubMed] [Google Scholar]
  10. Game J. C. Yeast cell-cycle mutant cdc21 is a temperature-sensitive thymidylate auxotroph. Mol Gen Genet. 1976 Aug 2;146(3):313–315. doi: 10.1007/BF00701257. [DOI] [PubMed] [Google Scholar]
  11. Hartwell L. H. Macromolecule synthesis in temperature-sensitive mutants of yeast. J Bacteriol. 1967 May;93(5):1662–1670. doi: 10.1128/jb.93.5.1662-1670.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hartwell L. H. Sequential function of gene products relative to DNA synthesis in the yeast cell cycle. J Mol Biol. 1976 Jul 15;104(4):803–817. doi: 10.1016/0022-2836(76)90183-2. [DOI] [PubMed] [Google Scholar]
  13. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Johnson L. M., Snyder M., Chang L. M., Davis R. W., Campbell J. L. Isolation of the gene encoding yeast DNA polymerase I. Cell. 1985 Nov;43(1):369–377. doi: 10.1016/0092-8674(85)90042-x. [DOI] [PubMed] [Google Scholar]
  15. Johnston L. H., Game J. C. Mutants of yeast with depressed DNA synthesis. Mol Gen Genet. 1978 May 3;161(2):205–214. doi: 10.1007/BF00274189. [DOI] [PubMed] [Google Scholar]
  16. Johnston L. H., Nasmyth K. A. Saccharomyces cerevisiae cell cycle mutant cdc9 is defective in DNA ligase. Nature. 1978 Aug 31;274(5674):891–893. doi: 10.1038/274891a0. [DOI] [PubMed] [Google Scholar]
  17. Jong A. Y., Kuo C. L., Campbell J. L. The CDC8 gene of yeast encodes thymidylate kinase. J Biol Chem. 1984 Sep 10;259(17):11052–11059. [PubMed] [Google Scholar]
  18. Kuo C., Nuang H., Campbell J. L. Isolation of yeast DNA replication mutants in permeabilized cells. Proc Natl Acad Sci U S A. 1983 Nov;80(21):6465–6469. doi: 10.1073/pnas.80.21.6465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Scherer S., Davis R. W. Replacement of chromosome segments with altered DNA sequences constructed in vitro. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4951–4955. doi: 10.1073/pnas.76.10.4951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Schwartz D. C., Cantor C. R. Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell. 1984 May;37(1):67–75. doi: 10.1016/0092-8674(84)90301-5. [DOI] [PubMed] [Google Scholar]
  21. Sclafani R. A., Fangman W. L. Yeast gene CDC8 encodes thymidylate kinase and is complemented by herpes thymidine kinase gene TK. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5821–5825. doi: 10.1073/pnas.81.18.5821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Shortle D., Haber J. E., Botstein D. Lethal disruption of the yeast actin gene by integrative DNA transformation. Science. 1982 Jul 23;217(4557):371–373. doi: 10.1126/science.7046050. [DOI] [PubMed] [Google Scholar]
  23. Shortle D., Novick P., Botstein D. Construction and genetic characterization of temperature-sensitive mutant alleles of the yeast actin gene. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4889–4893. doi: 10.1073/pnas.81.15.4889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  25. Williamson M. S., Game J. C., Fogel S. Meiotic gene conversion mutants in Saccharomyces cerevisiae. I. Isolation and characterization of pms1-1 and pms1-2. Genetics. 1985 Aug;110(4):609–646. doi: 10.1093/genetics/110.4.609. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES