Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

Isolation and Characterization of mas1+ of Schizosaccharomyces pombe, a Homologue of Human CIP29/Hcc-1 Involved in the Regulation of Cell Division

세포분열에 관여하는 인간의 CIP29/Hcc1 유전자와 상동성을 가지는 분열형 효모의 새로운 유전자 mas1+의 특성분석

  • Cha, Jae-Young (Division of Biologycal Science, Wonkwang University) ;
  • Shin, Sang-Min (Division of Biologycal Science, Wonkwang University) ;
  • Ha, Se-Eun (Division of Biologycal Science, Wonkwang University) ;
  • Lee, Jung-Sup (Department of Biotechnology, Chosun University) ;
  • Park, Jong-Kun (Division of Biologycal Science, Wonkwang University)
  • Received : 2011.10.24
  • Accepted : 2011.11.25
  • Published : 2011.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

The regulation of gene expression plays an important role in cell cycle controls. In this study, a novel gene, the $mas1^+$($\underline{mi}$tosis $\underline{as}$sociated protein) gene, a homolog of human CIP29/Hcc1, was isolated and characterized from fission yeast Schizosaccharomyces pombe (S. pombe) using a gene-specific polymerase chain reaction. The isolated gene contained a complete open reading frame capable of encoding 245 amino acid residues with a typical promoter, as judged by nucleotide sequence analysis. It was also found that a PCB ($\underline{p}$ombe cell $\underline{c}$ycle $\underline{b}$ox) is located in the promoter region, which controls M-$G_1$ specific transcription in S. pombe. The quantitative analysis of the $mas1^+$ transcript against $adh1^+$ showed that the pattern of expression is similar to that of the septation index. Cytokinesis of mas1 mutant was greatly delayed at $25^{\circ}C$ and $36^{\circ}C$, and a large number of multi-septate cells were produced. The mas1 mutant had 2C, 4C and 6C DNA contents, as determined by FACS analysis. In addition, the number of multi-septate cells significantly increased. When cells were cultured in nitrogen starvation medium to increase proliferation, the abnormal phenotypes of mas1 mutant dramatically increased. These phenotypes could be rescued by an overexpression of the $mas1^+$ gene. The mas1 protein localized in the nuclei of S. pombe and human HeLa cells, as evidenced by Mas1-EGFP signals. The abnormal growth pattern and the morphology of mas1 mutant were complemented by a plasmid carrying human CIP29/Hcc-1cDNA. In addition, CIP29 /Hcc-1 transcript level increased in active cell proliferation stages in the developing mouse embryos. These results indicate that the $mas1^+$ ishomologous to the human CIP29/Hcc1 gene and is involved in cytokinesis and cell shape control.

세포주기조절에서 유전자 발현의 조절은 매우 중요한 부분이다. 본 연구에서는 인간의 유전자인 CIP29/Hcc1과 상동성을 가지는 분열형 효모의 새로운 유전자 mas1+을 분리하였다. 중합효소연쇄반응을 수행하여 cDNA를 얻고 이 cDNA의 염기서열을 분석한 결과 mas1+의 전체 염기서열은 735 bp로서, 245개의 아미노산을 암호화하고 있다. mas1+의 프로모터에서는 M-G1에 특이적인 전사를 보이는 유전자들에 보존되어 있는 PCB 서열이 발견되었다. 세포주기별 mas1+의 전사 수준을 분석한 결과 격막이 형성된 세포수의 빈도를 나타내는 격막 세포지표 의 양상과 유사하게 발현하는 것을 확인하였다. mas1+ 결손 돌연변이를 $25^{\circ}C$와 $36^{\circ}C$에서 배양한 결과, 세포질 분열과정이 늦어진 다중격막 세포의 빈도가 증가하였다. 이를 FACS로 분석하여 DNA 함량이 2C, 4C와 6C등이 형성됨을 확인하였다. mas1+결손 돌연변이 세포를 질소 결핍 배양액에서 배양한 결과 다중격막 세포의 형성이 확연히 증가하였는데 이는 질소 결핍에 따른 세포분열의 가속화 단계에서 mas1+의 결손이 특히 부정적 영향을 초래함을 시사한다. mas1+ 유전자 결손 돌연변이 세포에 mas1+을 포함한 plasmid를 형질전환한 후 mas1+의 발현을 유도한 결과 정상의 세포 형태로 전환됨을 확인하였다. Mas1 단백질에 EGFP를 융합시켜 발현을 유도한 결과 핵내에서 위치함을 분열형 효모와 인간 배양세포인 HeLa에서 확인하였다. 또한, mas1+ 결손 돌연변이에서 상동성을 가지는 인간 유전자 CIP29/Hcc1을 발현시킨 결과 multi-septate 세포가 줄어들었다. 한편, 생쥐의 배발달 단계에 따른 CIP29 유전자의 전사체 수준은 세포 분열이 활발한 시기에 증가하였다. 이상의 결과들은 Mas1은 인간의 핵단백질인 유전자 CIP29/Hcc1과 구조 기능적으로 상동성을 가지며, 세포주기 중 M-G1에 속하는 세포질 분열에 연관되어 있음을 시사한다.

Keywords

References

  1. Alarcon, V. B. and Y. Marikawa. 2004. Molecular study of mouse peri-implantation development using the in vitro culture of aggregated inner cell mass. Mol. Reprod. Dev. 67, 83-90. https://doi.org/10.1002/mrd.20015
  2. Altschul, S. F., T. L. Madden, A. A. Schäffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389-3402. https://doi.org/10.1093/nar/25.17.3389
  3. Anderson, M., S. S. Ng, V. Marchesi, F. H. MacIver, F. E. Stevens, T. Riddell, D. M. Glover, I. M. Hagan, and C. J. McInerny. 2002. $plo1^{+}$ regulates gene transcription at the M-G1 interval during the fission yeast mitotic cell cycle. EMBO J. 21, 5742-5755.
  4. Aravind, L. and D. Landsman. 1998. AT-hook motifs identified in a wide variety of DNA-binding proteins. Nucleic Acids Res. 26, 4413-4421. https://doi.org/10.1093/nar/26.19.4413
  5. Aravind L., and E. V. Koonin. 2000. SAP-a putative DNA binding motif involved in chromosomal organization. Trends Biochem. Sci. 25, 112-113. https://doi.org/10.1016/S0968-0004(99)01537-6
  6. Barreda, D. R. and M. Belosevic. 2001. Transcriptional regulation of hemopoiesis. Dev. Comp. Immunol. 25, 763-789. https://doi.org/10.1016/S0145-305X(01)00035-0
  7. Baxevanis, A. D. and D. Landsman. 1995. The HMG-1 box protein family: classification and functional relationships. Nucleic Acids Res. 23, 1604-1613. https://doi.org/10.1093/nar/23.9.1604
  8. Biswas, S. and D. Bastia. 2008. Mechanistic insights into replication termination as revealed by investigations of the Reb1-Ter3 complex of Schizosaccharomyces pombe. Mol Cell Biol. 28, 6844-6857. https://doi.org/10.1128/MCB.01235-08
  9. Choong, M. L., L. K. Tan, S. L. Lo, E. C. Ren, K. Ou, S. E. Ong, R. C. Liang, T. K. Seow, and M. C. Chung. 2001. An integrated approach in the discovery and characterization of a novel nuclear protein over-expressed in liver and pancreatic tumors. FEBS Lett. 496, 109-116. https://doi.org/10.1016/S0014-5793(01)02409-7
  10. Downes, K. M. and T. Davies. 1993. Staging of gastrulating mouse embryos by morphological landmarks in the dissecting microscope. Development 118, 1255-1266.
  11. Dufu, K., M. J. Livingstone, J. Seebacher, S. P. Gygi, S. A. Wilson, and R. Reed. 2010. ATP is required for interactions between UAP56 and two conserved mRNA export proteins, Aly and CIP29, to assemble the TREX complex. Genes Dev. 24, 2043-2053. https://doi.org/10.1101/gad.1898610
  12. Fankhauser, C., A. Reymond, L. Cerutti, S. Utzig, K. Hofmann, and V. Simanis. 1995. The S. pombe cdc15 gene is a key element in the reorganization of F-actin at mitosis. Cell 82, 435-444. https://doi.org/10.1016/0092-8674(95)90432-8
  13. Feinberg, P. and B. Volgelstein. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 1, 6-13.
  14. Fukuda, S., D. W. Wu, K. Stark, and L. M. Pelus. 2002. Cloning and characterization of proliferation-associated cytokine- inducible protein. Biochem. Biophys. Res. Commun. 292, 593-600. https://doi.org/10.1006/bbrc.2002.6680
  15. Fukuda, S. and L. M. Pelus. 2005. Growth inhibitory effect of Hcc-1/CIP29 is associated with induction of apoptosis, not just with G2/M arrest. Cell Mol. Life Sci. 62, 1526-1527. https://doi.org/10.1007/s00018-005-5093-4
  16. Futcher, B. 2000. Microarrays and cell cycle transcription in yeast. Curr. Opin. Cell Biol. 12, 710-715. https://doi.org/10.1016/S0955-0674(00)00156-3
  17. Gutz, H., H. Heslot, U. Leupold, and N. Loprieno. 1974. Schizosaccharomyces pombe. In King, R. C. (ed.), Handbook of Genetics. pp. 395-446, Vol. I. Plenum, New York, NY,
  18. Hamamori, Y., B. Samal, J. Tian, and L. Kedes. 1994. Persistent erythropoiesis by myoblast transfer of erythropoietin cDNA. Hum. Gene Ther. 5, 1349-1356. https://doi.org/10.1089/hum.1994.5.11-1349
  19. Harper, J. V. and G. Brooks. 2004. The Mammalian cell cycle: an overview. Methods Mol. Biol. 296, 113-154.
  20. Hashii, Y., J. Y. Kim, A. Sawada, S. Tokimasa, F. Hiroyuki, H. Ohta, K. Makiko, Y. Takihara, K. Ozono, and J. Hara. 2004. A novel partner gene CIP29 containing a SAP domain with MLL identified in infantile myelomonocytic leukemia. Leukemia. 18, 1546-1548. https://doi.org/10.1038/sj.leu.2403450
  21. Hershkovits, G., H. Bangio, R. Cohen, and D. J. Katcoff. 2006. Recruitment of mRNA cleavage/polyadenylation machinery by the yeast chromatin protein Sin1p/Spt2p. Proc. Natl. Acad Sci. 103, 9808-9813. https://doi.org/10.1073/pnas.0602014103
  22. Javerzat, J. P., G. Cranston, and R. C. Allshire. 1996. Fission yeast genes which disrupt mitotic chromosome segregation when overexpressed. Nucleic Acids Res. 24, 4676-4683. https://doi.org/10.1093/nar/24.23.4676
  23. Kang, R., K. M. Livesey, H. J. Zeh, M. T. Loze, and D. Tang. 2010. HMGB1: a novel Beclin 1-binding protein active in autophagy. Autophagy 6, 1209-1211. https://doi.org/10.4161/auto.6.8.13651
  24. Kohli, J., H. Hottinger, P. Munz, A. Strauss, and P. Thuriaux. 1977. Genetic mapping in Schizosaccharomyces pombe by mitotic and meiotic analysis and induced haploidization. Genetics 87, 423-471.
  25. Labazi, M., L. Jaafar, and H. Flores-Rozas. 2009. Modulation of the DNA-binding activity of Saccharomyces cerevisiae MSH2-MSH6 complex by the high-mobility group protein NHP6A, in vitro. Nucleic Acids Res. 37, 7581-7589. https://doi.org/10.1093/nar/gkp649
  26. Lin, Z., K. Yin, D. Zhu, Z. Chen, H. Gu, and L. J. Qu. 2007. AtCDC5 regulates the G2 to M transition of the cell cycle and is critical for the function of Arabidopsis shoot apical meristem. Cell Res. 17, 815-828. https://doi.org/10.1038/cr.2007.71
  27. Maundrell, K. 1993. Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene 123, 127-130. https://doi.org/10.1016/0378-1119(93)90551-D
  28. Moreno, S., A. Klar, and P. Nurse. 1991. Molecular genetic analysis of the fission yeast Schizosaccharomyces pombe. Methods Enzymol. 194, 795-823. https://doi.org/10.1016/0076-6879(91)94059-L
  29. Peng, L., J. Arensburg, J. Orly, and A. H. Payne. 2002. The murine 3beta-hydroxysteroid dehydrogenase (3beta-HSD) gene family: a postulated role for 3beta-HSD VI during early pregnancy. Mol. Cell Endocrinol. 187, 213-221. https://doi.org/10.1016/S0303-7207(01)00689-X
  30. Qiu, X., B. E. Dul, and N. C. Walworth. 2010. Activity of a C-terminal plant homeodomain (PHD) of Msc1 is essential for function. J. Biol. Chem. 285, 36828-36835. https://doi.org/10.1074/jbc.M110.157792
  31. Ratsima, H., A. M. Ladouceur, M. Pascariu, V. Sauve, Z. Salloum, P. S. Maddox, and D. D'Amours. 2011. Independent modulation of the kinase and polo-box activities of Cdc5 protein unravels unique roles in the maintenance of genome stability. Proc. Natl. Acad. Sci. USA 108, 914-923. https://doi.org/10.1073/pnas.1106448108
  32. Ribar, B., A. Banrevi, and M. Sipiczki. 1997. sep1+ encodes a transcription-factor homologue of the HNF-3/forkhead DNA-bindingdomain family in Schizosaccharomyces pombe. Gene 202, 1-5. https://doi.org/10.1016/S0378-1119(97)00390-9
  33. Romig, H., F. O. Fackelmayer, A. Renz, U. Ramsperger, and A. Richter. 1992. Characterization of SAF-A, a novel nuclear DNA-binding protein from HeLa cells with high affinity for nuclear matrix/scaffold attachment DNA elements. EMBO J. 11, 3431-3440.
  34. Ruvolo, P. P., X. Deng, and W. S. May. 2001. Phosphorylation of Bcl2 and regulation of apoptosis. Leukemia 15, 515-522.
  35. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  36. Suto, K. A., Nagata, H. Murakami, and H. Okayama. 1999. A double-strand break repair component is essential for S phase completion in fission yeast cell cycling. Mol. Biol. Cell 10, 3331-3343. https://doi.org/10.1091/mbc.10.10.3331
  37. Utzig, S., C. Fankhauser, and V. Simanis. 2000. Periodic accumulation of cdc15 mRNA is not necessary for septation in Schizosaccharomyces pombe. J. Mol. Biol. 302, 751-759. https://doi.org/10.1006/jmbi.2000.4065
  38. Zilahi, E., E. Salimova, V. Simanis, and M. Sipiczki. 2000. The S. pombe sep1 gene encodes a nuclear protein that is required for periodic expression of the cdc15 gene. FEBS Lett. 481, 105-108. https://doi.org/10.1016/S0014-5793(00)01990-6