Skip to main content
PMC home page
Genetics logoLink to Genetics
. 1990 Dec;126(4):799–812. doi: 10.1093/genetics/126.4.799

Kem Mutations Affect Nuclear Fusion in Saccharomyces Cerevisiae

J Kim 1, P O Ljungdahl 1, G R Fink 1
PMCID: PMC1204279  PMID: 2076815

Abstract

We have identified mutations in three genes of Saccharomyces cerevisiae, KEM1, KEM2 and KEM3, that enhance the nuclear fusion defect of kar1-1 yeast during conjugation. The KEM1 and KEM3 genes are located on the left arm of chromosome VII. Kem mutations reduce nuclear fusion whether the kem and the kar1-1 mutations are in the same or in different parents (i.e., in both kem kar1-1 X wild-type and kem X kar1-1 crosses). kem1 X kem1 crosses show a defect in nuclear fusion, but kem1 X wild-type crosses do not. Mutant kem1 strains are hypersensitive to benomyl, lose chromosomes at a rate 10-20-fold higher than KEM(+) strains, and lose viability upon nitrogen starvation. In addition, kem1/kem1 diploids are unable to sporulate. Cells containing a kem1 null allele grow very poorly, have an elongated rod-shape and are defective in spindle pole body duplication and/or separation. The KEM1 gene, which is expressed as a 5.5-kb mRNA transcript, contains a 4.6-kb open reading frame encoding a 175-kD protein.

Full Text

The Full Text of this article is available as a PDF (8.2 MB).

Selected References

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

  1. Adams A. E., Pringle J. R. Relationship of actin and tubulin distribution to bud growth in wild-type and morphogenetic-mutant Saccharomyces cerevisiae. J Cell Biol. 1984 Mar;98(3):934–945. doi: 10.1083/jcb.98.3.934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boeke J. D., Garfinkel D. J., Styles C. A., Fink G. R. Ty elements transpose through an RNA intermediate. Cell. 1985 Mar;40(3):491–500. doi: 10.1016/0092-8674(85)90197-7. [DOI] [PubMed] [Google Scholar]
  3. Boeke J. D., Trueheart J., Natsoulis G., Fink G. R. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 1987;154:164–175. doi: 10.1016/0076-6879(87)54076-9. [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. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  6. Byers B., Goetsch L. Behavior of spindles and spindle plaques in the cell cycle and conjugation of Saccharomyces cerevisiae. J Bacteriol. 1975 Oct;124(1):511–523. doi: 10.1128/jb.124.1.511-523.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Byers B., Goetsch L. Duplication of spindle plaques and integration of the yeast cell cycle. Cold Spring Harb Symp Quant Biol. 1974;38:123–131. doi: 10.1101/sqb.1974.038.01.016. [DOI] [PubMed] [Google Scholar]
  8. Carle G. F., Olson M. V. An electrophoretic karyotype for yeast. Proc Natl Acad Sci U S A. 1985 Jun;82(11):3756–3760. doi: 10.1073/pnas.82.11.3756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Conde J., Fink G. R. A mutant of Saccharomyces cerevisiae defective for nuclear fusion. Proc Natl Acad Sci U S A. 1976 Oct;73(10):3651–3655. doi: 10.1073/pnas.73.10.3651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Curran B. P., Carter B. L. Alpha-factor enhancement of hybrid formation by protoplast fusion in Saccharomyces cerevisiae II. Curr Genet. 1986;10(12):943–945. doi: 10.1007/BF00398292. [DOI] [PubMed] [Google Scholar]
  11. Delgado M. A., Conde J. Benomyl prevents nuclear fusion in Saccharomyces cerevisiae. Mol Gen Genet. 1984;193(1):188–189. doi: 10.1007/BF00327435. [DOI] [PubMed] [Google Scholar]
  12. Dutcher S. K., Hartwell L. H. Genes that act before conjugation to prepare the Saccharomyces cerevisiae nucleus for caryogamy. Cell. 1983 May;33(1):203–210. doi: 10.1016/0092-8674(83)90349-5. [DOI] [PubMed] [Google Scholar]
  13. Dutcher S. K., Hartwell L. H. The role of S. cerevisiae cell division cycle genes in nuclear fusion. Genetics. 1982 Feb;100(2):175–184. doi: 10.1093/genetics/100.2.175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Elder R. T., Loh E. Y., Davis R. W. RNA from the yeast transposable element Ty1 has both ends in the direct repeats, a structure similar to retrovirus RNA. Proc Natl Acad Sci U S A. 1983 May;80(9):2432–2436. doi: 10.1073/pnas.80.9.2432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Gallwitz D., Sures I. Structure of a split yeast gene: complete nucleotide sequence of the actin gene in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1980 May;77(5):2546–2550. doi: 10.1073/pnas.77.5.2546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hartwell L. H., Smith D. Altered fidelity of mitotic chromosome transmission in cell cycle mutants of S. cerevisiae. Genetics. 1985 Jul;110(3):381–395. doi: 10.1093/genetics/110.3.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  19. Hoffman C. S., Winston F. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene. 1987;57(2-3):267–272. doi: 10.1016/0378-1119(87)90131-4. [DOI] [PubMed] [Google Scholar]
  20. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  21. Hoyt M. A., Stearns T., Botstein D. Chromosome instability mutants of Saccharomyces cerevisiae that are defective in microtubule-mediated processes. Mol Cell Biol. 1990 Jan;10(1):223–234. doi: 10.1128/mcb.10.1.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Huffaker T. C., Hoyt M. A., Botstein D. Genetic analysis of the yeast cytoskeleton. Annu Rev Genet. 1987;21:259–284. doi: 10.1146/annurev.ge.21.120187.001355. [DOI] [PubMed] [Google Scholar]
  23. Huffaker T. C., Thomas J. H., Botstein D. Diverse effects of beta-tubulin mutations on microtubule formation and function. J Cell Biol. 1988 Jun;106(6):1997–2010. doi: 10.1083/jcb.106.6.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Johnston M., Davis R. W. Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Aug;4(8):1440–1448. doi: 10.1128/mcb.4.8.1440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kataoka T., Powers S., McGill C., Fasano O., Strathern J., Broach J., Wigler M. Genetic analysis of yeast RAS1 and RAS2 genes. Cell. 1984 Jun;37(2):437–445. doi: 10.1016/0092-8674(84)90374-x. [DOI] [PubMed] [Google Scholar]
  27. Koerner T. J., Hill J. E., Myers A. M., Tzagoloff A. High-expression vectors with multiple cloning sites for construction of trpE fusion genes: pATH vectors. Methods Enzymol. 1991;194:477–490. doi: 10.1016/0076-6879(91)94036-c. [DOI] [PubMed] [Google Scholar]
  28. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  29. Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
  30. Meluh P. B., Rose M. D. KAR3, a kinesin-related gene required for yeast nuclear fusion. Cell. 1990 Mar 23;60(6):1029–1041. doi: 10.1016/0092-8674(90)90351-e. [DOI] [PubMed] [Google Scholar]
  31. Ng R., Abelson J. Isolation and sequence of the gene for actin in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3912–3916. doi: 10.1073/pnas.77.7.3912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Oakley B. R., Morris N. R. Nuclear movement is beta--tubulin-dependent in Aspergillus nidulans. Cell. 1980 Jan;19(1):255–262. doi: 10.1016/0092-8674(80)90407-9. [DOI] [PubMed] [Google Scholar]
  33. Ohashi A., Gibson J., Gregor I., Schatz G. Import of proteins into mitochondria. The precursor of cytochrome c1 is processed in two steps, one of them heme-dependent. J Biol Chem. 1982 Nov 10;257(21):13042–13047. [PubMed] [Google Scholar]
  34. Polaina J., Conde J. Genes involved in the control of nuclear fusion during the sexual cycle of Saccharomyces cerevisiae. Mol Gen Genet. 1982;186(2):253–258. doi: 10.1007/BF00331858. [DOI] [PubMed] [Google Scholar]
  35. Rose M. D., Fink G. R. KAR1, a gene required for function of both intranuclear and extranuclear microtubules in yeast. Cell. 1987 Mar 27;48(6):1047–1060. doi: 10.1016/0092-8674(87)90712-4. [DOI] [PubMed] [Google Scholar]
  36. Rose M. D., Misra L. M., Vogel J. P. KAR2, a karyogamy gene, is the yeast homolog of the mammalian BiP/GRP78 gene. Cell. 1989 Jun 30;57(7):1211–1221. doi: 10.1016/0092-8674(89)90058-5. [DOI] [PubMed] [Google Scholar]
  37. Rose M. D., Novick P., Thomas J. H., Botstein D., Fink G. R. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene. 1987;60(2-3):237–243. doi: 10.1016/0378-1119(87)90232-0. [DOI] [PubMed] [Google Scholar]
  38. Rose M. D., Price B. R., Fink G. R. Saccharomyces cerevisiae nuclear fusion requires prior activation by alpha factor. Mol Cell Biol. 1986 Oct;6(10):3490–3497. doi: 10.1128/mcb.6.10.3490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
  40. Schatz P. J., Solomon F., Botstein D. Genetically essential and nonessential alpha-tubulin genes specify functionally interchangeable proteins. Mol Cell Biol. 1986 Nov;6(11):3722–3733. doi: 10.1128/mcb.6.11.3722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schmitt H. D., Wagner P., Pfaff E., Gallwitz D. The ras-related YPT1 gene product in yeast: a GTP-binding protein that might be involved in microtubule organization. Cell. 1986 Nov 7;47(3):401–412. doi: 10.1016/0092-8674(86)90597-0. [DOI] [PubMed] [Google Scholar]
  42. Segev N., Botstein D. The ras-like yeast YPT1 gene is itself essential for growth, sporulation, and starvation response. Mol Cell Biol. 1987 Jul;7(7):2367–2377. doi: 10.1128/mcb.7.7.2367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sheir-Neiss G., Lai M. H., Morris N. R. Identification of a gene for beta-tubulin in Aspergillus nidulans. Cell. 1978 Oct;15(2):639–647. doi: 10.1016/0092-8674(78)90032-6. [DOI] [PubMed] [Google Scholar]
  44. Spindler K. R., Rosser D. S., Berk A. J. Analysis of adenovirus transforming proteins from early regions 1A and 1B with antisera to inducible fusion antigens produced in Escherichia coli. J Virol. 1984 Jan;49(1):132–141. doi: 10.1128/jvi.49.1.132-141.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Stearns T., Hoyt M. A., Botstein D. Yeast mutants sensitive to antimicrotubule drugs define three genes that affect microtubule function. Genetics. 1990 Feb;124(2):251–262. doi: 10.1093/genetics/124.2.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Thomas J. H., Neff N. F., Botstein D. Isolation and characterization of mutations in the beta-tubulin gene of Saccharomyces cerevisiae. Genetics. 1985 Dec;111(4):715–734. doi: 10.1093/genetics/111.4.715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Toda T., Cameron S., Sass P., Zoller M., Scott J. D., McMullen B., Hurwitz M., Krebs E. G., Wigler M. Cloning and characterization of BCY1, a locus encoding a regulatory subunit of the cyclic AMP-dependent protein kinase in Saccharomyces cerevisiae. Mol Cell Biol. 1987 Apr;7(4):1371–1377. doi: 10.1128/mcb.7.4.1371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Uno I., Matsumoto K., Ishikawa T. Characterization of cyclic AMP-requiring yeast mutants altered in the regulatory subunit of protein kinase. J Biol Chem. 1982 Dec 10;257(23):14110–14115. [PubMed] [Google Scholar]
  50. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  51. Vollrath D., Davis R. W., Connelly C., Hieter P. Physical mapping of large DNA by chromosome fragmentation. Proc Natl Acad Sci U S A. 1988 Aug;85(16):6027–6031. doi: 10.1073/pnas.85.16.6027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES