Skip to main content
PMC home page
Genetics logoLink to Genetics
. 2000 Aug;155(4):1521–1534. doi: 10.1093/genetics/155.4.1521

A new genetic method for isolating functionally interacting genes: high plo1(+)-dependent mutants and their suppressors define genes in mitotic and septation pathways in fission yeast.

C F Cullen 1, K M May 1, I M Hagan 1, D M Glover 1, H Ohkura 1
PMCID: PMC1461180  PMID: 10924454

Abstract

We describe a general genetic method to identify genes encoding proteins that functionally interact with and/or are good candidates for downstream targets of a particular gene product. The screen identifies mutants whose growth depends on high levels of expression of that gene. We apply this to the plo1(+) gene that encodes a fission yeast homologue of the polo-like kinases. plo1(+) regulates both spindle formation and septation. We have isolated 17 high plo1(+)-dependent (pld) mutants that show defects in mitosis or septation. Three mutants show a mitotic arrest phenotype. Among the 14 pld mutants with septation defects, 12 mapped to known loci: cdc7, cdc15, cdc11 spg1, and sid2. One of the pld mutants, cdc7-PD1, was selected for suppressor analysis. As multicopy suppressors, we isolated four known genes involved in septation in fission yeast: spg1(+), sce3(+), cdc8(+), and rho1(+), and two previously uncharacterized genes, mpd1(+) and mpd2(+). mpd1(+) exhibits high homology to phosphatidylinositol 4-phosphate 5-kinase, while mpd2(+) resembles Saccharomyces cerevisiae SMY2; both proteins are involved in the regulation of actin-mediated processes. As chromosomal suppressors of cdc7-PD1, we isolated mutations of cdc16 that resulted in multiseptation without nuclear division. cdc16(+), dma1(+), byr3(+), byr4(+) and a truncated form of the cdc7 gene were isolated by complementation of one of these cdc16 mutations. These results demonstrate that screening for high dose-dependent mutants and their suppressors is an effective approach to identify functionally interacting genes.

Full Text

The Full Text of this article is available as a PDF (385.4 KB).

Selected References

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

  1. Abrieu A., Brassac T., Galas S., Fisher D., Labbé J. C., Dorée M. The Polo-like kinase Plx1 is a component of the MPF amplification loop at the G2/M-phase transition of the cell cycle in Xenopus eggs. J Cell Sci. 1998 Jun;111(Pt 12):1751–1757. doi: 10.1242/jcs.111.12.1751. [DOI] [PubMed] [Google Scholar]
  2. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  3. Balasubramanian M. K., Helfman D. M., Hemmingsen S. M. A new tropomyosin essential for cytokinesis in the fission yeast S. pombe. Nature. 1992 Nov 5;360(6399):84–87. doi: 10.1038/360084a0. [DOI] [PubMed] [Google Scholar]
  4. Balasubramanian M. K., Hirani B. R., Burke J. D., Gould K. L. The Schizosaccharomyces pombe cdc3+ gene encodes a profilin essential for cytokinesis. J Cell Biol. 1994 Jun;125(6):1289–1301. doi: 10.1083/jcb.125.6.1289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Balasubramanian M. K., McCollum D., Chang L., Wong K. C., Naqvi N. I., He X., Sazer S., Gould K. L. Isolation and characterization of new fission yeast cytokinesis mutants. Genetics. 1998 Jul;149(3):1265–1275. doi: 10.1093/genetics/149.3.1265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Barbet N., Muriel W. J., Carr A. M. Versatile shuttle vectors and genomic libraries for use with Schizosaccharomyces pombe. Gene. 1992 May 1;114(1):59–66. doi: 10.1016/0378-1119(92)90707-v. [DOI] [PubMed] [Google Scholar]
  7. Brand A. H., Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 1993 Jun;118(2):401–415. doi: 10.1242/dev.118.2.401. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Bähler J., Steever A. B., Wheatley S., Wang Y. l., Pringle J. R., Gould K. L., McCollum D. Role of polo kinase and Mid1p in determining the site of cell division in fission yeast. J Cell Biol. 1998 Dec 14;143(6):1603–1616. doi: 10.1083/jcb.143.6.1603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cerutti L., Simanis V. Asymmetry of the spindle pole bodies and spg1p GAP segregation during mitosis in fission yeast. J Cell Sci. 1999 Jul;112(Pt 14):2313–2321. doi: 10.1242/jcs.112.14.2313. [DOI] [PubMed] [Google Scholar]
  11. Chang F., Woollard A., Nurse P. Isolation and characterization of fission yeast mutants defective in the assembly and placement of the contractile actin ring. J Cell Sci. 1996 Jan;109(Pt 1):131–142. doi: 10.1242/jcs.109.1.131. [DOI] [PubMed] [Google Scholar]
  12. Charles J. F., Jaspersen S. L., Tinker-Kulberg R. L., Hwang L., Szidon A., Morgan D. O. The Polo-related kinase Cdc5 activates and is destroyed by the mitotic cyclin destruction machinery in S. cerevisiae. Curr Biol. 1998 Apr 23;8(9):497–507. doi: 10.1016/s0960-9822(98)70201-5. [DOI] [PubMed] [Google Scholar]
  13. Cheng L., Hunke L., Hardy C. F. Cell cycle regulation of the Saccharomyces cerevisiae polo-like kinase cdc5p. Mol Cell Biol. 1998 Dec;18(12):7360–7370. doi: 10.1128/mcb.18.12.7360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Chong L. D., Traynor-Kaplan A., Bokoch G. M., Schwartz M. A. The small GTP-binding protein Rho regulates a phosphatidylinositol 4-phosphate 5-kinase in mammalian cells. Cell. 1994 Nov 4;79(3):507–513. doi: 10.1016/0092-8674(94)90259-3. [DOI] [PubMed] [Google Scholar]
  15. Cohen-Fix O., Peters J. M., Kirschner M. W., Koshland D. Anaphase initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p. Genes Dev. 1996 Dec 15;10(24):3081–3093. doi: 10.1101/gad.10.24.3081. [DOI] [PubMed] [Google Scholar]
  16. Descombes P., Nigg E. A. The polo-like kinase Plx1 is required for M phase exit and destruction of mitotic regulators in Xenopus egg extracts. EMBO J. 1998 Mar 2;17(5):1328–1335. doi: 10.1093/emboj/17.5.1328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fairhead C., Thierry A., Denis F., Eck M., Dujon B. 'Mass-murder' of ORFs from three regions of chromosome XI from Saccharomyces cerevisiae. Gene. 1998 Nov 26;223(1-2):33–46. doi: 10.1016/s0378-1119(98)00171-1. [DOI] [PubMed] [Google Scholar]
  18. Fankhauser C., Marks J., Reymond A., Simanis V. The S. pombe cdc16 gene is required both for maintenance of p34cdc2 kinase activity and regulation of septum formation: a link between mitosis and cytokinesis? EMBO J. 1993 Jul;12(7):2697–2704. doi: 10.1002/j.1460-2075.1993.tb05931.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fankhauser C., Reymond A., Cerutti L., Utzig S., Hofmann K., Simanis V. The S. pombe cdc15 gene is a key element in the reorganization of F-actin at mitosis. Cell. 1995 Aug 11;82(3):435–444. doi: 10.1016/0092-8674(95)90432-8. [DOI] [PubMed] [Google Scholar]
  20. Fankhauser C., Simanis V. The Schizosaccharomyces pombe cdc14 gene is required for septum formation and can also inhibit nuclear division. Mol Biol Cell. 1993 May;4(5):531–539. doi: 10.1091/mbc.4.5.531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fankhauser C., Simanis V. The cdc7 protein kinase is a dosage dependent regulator of septum formation in fission yeast. EMBO J. 1994 Jul 1;13(13):3011–3019. doi: 10.1002/j.1460-2075.1994.tb06600.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Fenton B., Glover D. M. A conserved mitotic kinase active at late anaphase-telophase in syncytial Drosophila embryos. Nature. 1993 Jun 17;363(6430):637–640. doi: 10.1038/363637a0. [DOI] [PubMed] [Google Scholar]
  23. Fukami K., Furuhashi K., Inagaki M., Endo T., Hatano S., Takenawa T. Requirement of phosphatidylinositol 4,5-bisphosphate for alpha-actinin function. Nature. 1992 Sep 10;359(6391):150–152. doi: 10.1038/359150a0. [DOI] [PubMed] [Google Scholar]
  24. Furge K. A., Cheng Q. C., Jwa M., Shin S., Song K., Albright C. F. Regions of Byr4, a regulator of septation in fission yeast, that bind Spg1 or Cdc16 and form a two-component GTPase-activating protein with Cdc16. J Biol Chem. 1999 Apr 16;274(16):11339–11343. doi: 10.1074/jbc.274.16.11339. [DOI] [PubMed] [Google Scholar]
  25. Furge K. A., Wong K., Armstrong J., Balasubramanian M., Albright C. F. Byr4 and Cdc16 form a two-component GTPase-activating protein for the Spg1 GTPase that controls septation in fission yeast. Curr Biol. 1998 Aug 27;8(17):947–954. doi: 10.1016/s0960-9822(98)70394-x. [DOI] [PubMed] [Google Scholar]
  26. Gilmore A. P., Burridge K. Regulation of vinculin binding to talin and actin by phosphatidyl-inositol-4-5-bisphosphate. Nature. 1996 Jun 6;381(6582):531–535. doi: 10.1038/381531a0. [DOI] [PubMed] [Google Scholar]
  27. Glover D. M., Ohkura H., Tavares A. Polo kinase: the choreographer of the mitotic stage? J Cell Biol. 1996 Dec;135(6 Pt 2):1681–1684. doi: 10.1083/jcb.135.6.1681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Golsteyn R. M., Mundt K. E., Fry A. M., Nigg E. A. Cell cycle regulation of the activity and subcellular localization of Plk1, a human protein kinase implicated in mitotic spindle function. J Cell Biol. 1995 Jun;129(6):1617–1628. doi: 10.1083/jcb.129.6.1617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Golsteyn R. M., Schultz S. J., Bartek J., Ziemiecki A., Ried T., Nigg E. A. Cell cycle analysis and chromosomal localization of human Plk1, a putative homologue of the mitotic kinases Drosophila polo and Saccharomyces cerevisiae Cdc5. J Cell Sci. 1994 Jun;107(Pt 6):1509–1517. doi: 10.1242/jcs.107.6.1509. [DOI] [PubMed] [Google Scholar]
  30. Govindan B., Bowser R., Novick P. The role of Myo2, a yeast class V myosin, in vesicular transport. J Cell Biol. 1995 Mar;128(6):1055–1068. doi: 10.1083/jcb.128.6.1055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Hardy C. F., Pautz A. A novel role for Cdc5p in DNA replication. Mol Cell Biol. 1996 Dec;16(12):6775–6782. doi: 10.1128/mcb.16.12.6775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Janmey P. A., Stossel T. P. Modulation of gelsolin function by phosphatidylinositol 4,5-bisphosphate. Nature. 1987 Jan 22;325(6102):362–364. doi: 10.1038/325362a0. [DOI] [PubMed] [Google Scholar]
  33. Jaspersen S. L., Charles J. F., Tinker-Kulberg R. L., Morgan D. O. A late mitotic regulatory network controlling cyclin destruction in Saccharomyces cerevisiae. Mol Biol Cell. 1998 Oct;9(10):2803–2817. doi: 10.1091/mbc.9.10.2803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Kotani S., Tugendreich S., Fujii M., Jorgensen P. M., Watanabe N., Hoog C., Hieter P., Todokoro K. PKA and MPF-activated polo-like kinase regulate anaphase-promoting complex activity and mitosis progression. Mol Cell. 1998 Feb;1(3):371–380. doi: 10.1016/s1097-2765(00)80037-4. [DOI] [PubMed] [Google Scholar]
  36. Kumagai A., Dunphy W. G. Purification and molecular cloning of Plx1, a Cdc25-regulatory kinase from Xenopus egg extracts. Science. 1996 Sep 6;273(5280):1377–1380. doi: 10.1126/science.273.5280.1377. [DOI] [PubMed] [Google Scholar]
  37. Lane H. A., Nigg E. A. Antibody microinjection reveals an essential role for human polo-like kinase 1 (Plk1) in the functional maturation of mitotic centrosomes. J Cell Biol. 1996 Dec;135(6 Pt 2):1701–1713. doi: 10.1083/jcb.135.6.1701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Lassing I., Lindberg U. Specific interaction between phosphatidylinositol 4,5-bisphosphate and profilactin. Nature. 1985 Apr 4;314(6010):472–474. doi: 10.1038/314472a0. [DOI] [PubMed] [Google Scholar]
  39. Llamazares S., Moreira A., Tavares A., Girdham C., Spruce B. A., Gonzalez C., Karess R. E., Glover D. M., Sunkel C. E. polo encodes a protein kinase homolog required for mitosis in Drosophila. Genes Dev. 1991 Dec;5(12A):2153–2165. doi: 10.1101/gad.5.12a.2153. [DOI] [PubMed] [Google Scholar]
  40. Maundrell K. Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene. 1993 Jan 15;123(1):127–130. doi: 10.1016/0378-1119(93)90551-d. [DOI] [PubMed] [Google Scholar]
  41. McCollum D., Balasubramanian M. K., Pelcher L. E., Hemmingsen S. M., Gould K. L. Schizosaccharomyces pombe cdc4+ gene encodes a novel EF-hand protein essential for cytokinesis. J Cell Biol. 1995 Aug;130(3):651–660. doi: 10.1083/jcb.130.3.651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Minet M., Nurse P., Thuriaux P., Mitchison J. M. Uncontrolled septation in a cell division cycle mutant of the fission yeast Schizosaccharomyces pombe. J Bacteriol. 1979 Jan;137(1):440–446. doi: 10.1128/jb.137.1.440-446.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Moreno S., Klar A., Nurse P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 1991;194:795–823. doi: 10.1016/0076-6879(91)94059-l. [DOI] [PubMed] [Google Scholar]
  44. Mulvihill D. P., Petersen J., Ohkura H., Glover D. M., Hagan I. M. Plo1 kinase recruitment to the spindle pole body and its role in cell division in Schizosaccharomyces pombe. Mol Biol Cell. 1999 Aug;10(8):2771–2785. doi: 10.1091/mbc.10.8.2771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Murone M., Simanis V. The fission yeast dma1 gene is a component of the spindle assembly checkpoint, required to prevent septum formation and premature exit from mitosis if spindle function is compromised. EMBO J. 1996 Dec 2;15(23):6605–6616. [PMC free article] [PubMed] [Google Scholar]
  46. Nakano K., Arai R., Mabuchi I. The small GTP-binding protein Rho1 is a multifunctional protein that regulates actin localization, cell polarity, and septum formation in the fission yeast Schizosaccharomyces pombe. Genes Cells. 1997 Nov;2(11):679–694. doi: 10.1046/j.1365-2443.1997.1540352.x. [DOI] [PubMed] [Google Scholar]
  47. Nigg E. A. Polo-like kinases: positive regulators of cell division from start to finish. Curr Opin Cell Biol. 1998 Dec;10(6):776–783. doi: 10.1016/s0955-0674(98)80121-x. [DOI] [PubMed] [Google Scholar]
  48. Nishimoto T., Basilico C. Analysis of a method for selecting temperature-sensitive mutants of BHK cells. Somatic Cell Genet. 1978 May;4(3):323–340. doi: 10.1007/BF01542846. [DOI] [PubMed] [Google Scholar]
  49. Nurse P., Thuriaux P., Nasmyth K. Genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe. Mol Gen Genet. 1976 Jul 23;146(2):167–178. doi: 10.1007/BF00268085. [DOI] [PubMed] [Google Scholar]
  50. Ohkura H., Hagan I. M., Glover D. M. The conserved Schizosaccharomyces pombe kinase plo1, required to form a bipolar spindle, the actin ring, and septum, can drive septum formation in G1 and G2 cells. Genes Dev. 1995 May 1;9(9):1059–1073. doi: 10.1101/gad.9.9.1059. [DOI] [PubMed] [Google Scholar]
  51. Okazaki K., Okazaki N., Kume K., Jinno S., Tanaka K., Okayama H. High-frequency transformation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of Schizosaccharomyces pombe. Nucleic Acids Res. 1990 Nov 25;18(22):6485–6489. doi: 10.1093/nar/18.22.6485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Qian Y. W., Erikson E., Li C., Maller J. L. Activated polo-like kinase Plx1 is required at multiple points during mitosis in Xenopus laevis. Mol Cell Biol. 1998 Jul;18(7):4262–4271. doi: 10.1128/mcb.18.7.4262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Resnitzky D., Gossen M., Bujard H., Reed S. I. Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system. Mol Cell Biol. 1994 Mar;14(3):1669–1679. doi: 10.1128/mcb.14.3.1669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Rørth P., Szabo K., Bailey A., Laverty T., Rehm J., Rubin G. M., Weigmann K., Milán M., Benes V., Ansorge W. Systematic gain-of-function genetics in Drosophila. Development. 1998 Mar;125(6):1049–1057. doi: 10.1242/dev.125.6.1049. [DOI] [PubMed] [Google Scholar]
  55. Schmidt S., Hofmann K., Simanis V. Sce3, a suppressor of the Schizosaccharomyces pombe septation mutant cdc11, encodes a putative RNA-binding protein. Nucleic Acids Res. 1997 Sep 1;25(17):3433–3439. doi: 10.1093/nar/25.17.3433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Schmidt S., Sohrmann M., Hofmann K., Woollard A., Simanis V. The Spg1p GTPase is an essential, dosage-dependent inducer of septum formation in Schizosaccharomyces pombe. Genes Dev. 1997 Jun 15;11(12):1519–1534. doi: 10.1101/gad.11.12.1519. [DOI] [PubMed] [Google Scholar]
  57. Shirayama M., Matsui Y., Toh-E A. The yeast TEM1 gene, which encodes a GTP-binding protein, is involved in termination of M phase. Mol Cell Biol. 1994 Nov;14(11):7476–7482. doi: 10.1128/mcb.14.11.7476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Shirayama M., Zachariae W., Ciosk R., Nasmyth K. The Polo-like kinase Cdc5p and the WD-repeat protein Cdc20p/fizzy are regulators and substrates of the anaphase promoting complex in Saccharomyces cerevisiae. EMBO J. 1998 Mar 2;17(5):1336–1349. doi: 10.1093/emboj/17.5.1336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Sohrmann M., Fankhauser C., Brodbeck C., Simanis V. The dmf1/mid1 gene is essential for correct positioning of the division septum in fission yeast. Genes Dev. 1996 Nov 1;10(21):2707–2719. doi: 10.1101/gad.10.21.2707. [DOI] [PubMed] [Google Scholar]
  60. Sohrmann M., Schmidt S., Hagan I., Simanis V. Asymmetric segregation on spindle poles of the Schizosaccharomyces pombe septum-inducing protein kinase Cdc7p. Genes Dev. 1998 Jan 1;12(1):84–94. doi: 10.1101/gad.12.1.84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Song K., Mach K. E., Chen C. Y., Reynolds T., Albright C. F. A novel suppressor of ras1 in fission yeast, byr4, is a dosage-dependent inhibitor of cytokinesis. J Cell Biol. 1996 Jun;133(6):1307–1319. doi: 10.1083/jcb.133.6.1307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Van Aelst L., D'Souza-Schorey C. Rho GTPases and signaling networks. Genes Dev. 1997 Sep 15;11(18):2295–2322. doi: 10.1101/gad.11.18.2295. [DOI] [PubMed] [Google Scholar]
  63. Yoshida S., Ohya Y., Nakano A., Anraku Y. Genetic interactions among genes involved in the STT4-PKC1 pathway of Saccharomyces cerevisiae. Mol Gen Genet. 1994 Mar;242(6):631–640. doi: 10.1007/BF00283416. [DOI] [PubMed] [Google Scholar]
  64. Yu F. X., Johnston P. A., Südhof T. C., Yin H. L. gCap39, a calcium ion- and polyphosphoinositide-regulated actin capping protein. Science. 1990 Dec 7;250(4986):1413–1415. doi: 10.1126/science.2255912. [DOI] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES