Poliaminler, nükleik asitlerle etkileştiği ve hücre döngüsü ile stres tepkisi gibi pek çok hücresel işleve katıldığı bilinen, korunmuş polikatyonik moleküllerdir. Poliaminlerin hücre içine girişi ve hücre dışına çıkışı, poliamin homeostazında görevli olduğu bilinen poliamin taşıyıcı proteinleri tarafından yürütülmektedir. Schizosaccharomyces pombe (Lindner) caf5+ geni de henüz işlevsel olarak karakterize edilmemiş bir spermin ailesi taşıyıcısıdır. Bu çalışmanın amacı ters genetik yöntemlerle caf5+ geninin poliaminlerle ilişkili olduğu bilinen hücresel işlevler üzerindeki önemini anlamaktır. Normal koşullarda yaşayabilir durumda olan, caf5+ geni delesyon mutantları (caf5Δ) pek çok farklı hücresel işlev üzerinden taranmışlardır. Sonuçlar, caf5+ geni delesyon mutantlarının hücre boyunun daha kısa olduğunu ve optimum koşullar altında daha hızlı bölündüklerini göstermektedir. caf5Δ hücreler, aynı zamanda yüksek dozda UV ışınlarına karşı hassasiyet göstermişler ancak ozmotik stres koşullarında ve bir başka DNA hasarı ajanı olan hidroksiüre'ye karşı herhangi bir hassasiyet göstermemişlerdir. DNA ve septum boyamaları sonucunda bu mutantların mitoz ve mayoz bölünmenin farklı fazlarını başarılı bir şekilde tamamlayabilir olduğu bulunmuştur. Özetle, caf5+ geni normal hücre büyümesinde, hücre döngüsünde ve UV ile indüklenen stres tepkisinde rol oynamaktadır.

FUNCTIONAL CHARACTERIZATION OF SPERMINE FAMILY TRANSPORTER caf5+ IN Schizosaccharomyces pombe (Lindner)

Polyamines are well conserved polycationic molecules that are known to interact with nucleic acids and contribute to multiple functions including cell cycle and stress response. The transport of polyamines in and out of the cell is driven by polyamine transporters that play a significant role in polyamine homeostasis. Schizosaccharomyces pombe (Lindner) caf5+ gene codes for a spermine family transporter that is yet to be characterized functionally. This study aims to understand the contribution of caf5+ on different processes previously associated with polyamines, by reverse genetics. Deletion mutants of caf5+, which are viable in normal conditions, were scanned for multiple cellular processes. The results showed that caf5+ deletion caused shorter cell length and slightly faster growth rate at the optimum conditions. caf5Δ cells also showed sensitivity to high doses of UV irradiation, while no sensitivity was observed against osmotic stress or another DNA damaging agent hydroxyurea. The mutants could successfully go through different phases of mitosis and meiosis as observed by DNA and septum staining. In summary, caf5+ gene is involved in normal growth and cell cycle progression, as well as stress response upon UV irradiation.

Keywords:

cell size, cell cycle, stress response, Schizosaccharomyces pombe polyamine,

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1. Aouida, M., Leduc, A., Poulin, R. & Ramotar, D. 2005. AGP2 encodes the major permease for high affinity polyamine import in Saccharomyces cerevisiae. The Journal of Biological Chemistry, 280: 24267-24276.
2. Bähler, J., Wu, J.Q. & Longtine, M.S. 1998. Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast, 14(10): 943-951.
3. Balasundaram, D., Tabor, C.W. & Tabor, H. 1993. Oxygen toxicity in a polyamine-depleted spe2 delta mutant of Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences, 90: 4693-4697.
4. Benko, Z., Fenyvesvolgyi, C., Pesti, M. & Sipiczki, M. 2004. The transcription factor Pap1/Caf3 plays a central role in the determination of caffeine resistance in Schizosaccharomyces pombe. Molecular Genetics and Genomics, 271: 161-170. 5. 5. Cohen, S.S. 1998. A Guide to the Polyamines, New York: Oxford University Press, 624 pp. 6. Chattopadhyay, M.K., Tabor, C.W. & Tabor, H. 2002. Absolute requirement of spermidine for growth and cell cycle progression of fission yeast (Schizosaccharomyces pombe). Proceedings of the National Academy of Sciences, 99(16): 10330-10334.
7. Choi, S.H., Kim, S.W., Choi, D.H., Min, B.H. & Chun, B.G. 2000. Polyamine-depletion induces p27Kip1 and enhances dexamethasone-induced G1 arrest and apoptosis in human T lymphoblastic leukemia cells. Leukemia Research, 24: 119-127.
8. Erez, O. & Kahana, C. 2002. Deletions of SKY1 or PTK2 in the Saccharomyces cerevisiae trk1∆ trk2∆ mutant cells exert dual effect on ion homeostasis. Biochemical and Biophysical Research Communications, 295(5): 1142-1149.
9. Fredlund, J.O. & Oredsson, S.M. 1996. Normal G1/S transition and prolonged S phase within one cell cycle after seeding cells in the presence of an ornithine decarboxylase inhibitor. Cell Proliferation, 29: 457-465.
10. Fukuda, W., Hidese, R. & Fujiwara, S. 2015. Long-chain and branched polyamines in thermophilic microbes. In: Kusano T, Suzuki H (eds) Polyamines: a universal molecular nexus for growth, survival, and specialized metabolism, 1st edn. Springer, Tokyo, 15-26.
11. Gesteland, R.F., Weiss, R.B. & Atkins, J.F. 1999. Recoding: reprogrammed genetic decoding. Science, 257: 1640-1641.
12. Güngör, I. & Örs Gevrekci, A. 2016. The roles of SPBC409.08 and SPAC9.02c hypothetical genes in cell cycle and stress response, in Schizosaccharomyces pombe. Cellular and Molecular Biology, 62(4): 42-47.
13. Hamana, K. & Matsuzaki, S. 1992. Polyamines as a chemotaxonomic marker in bacterial systematics. Critical Reviews in Microbiology, 18: 261-283.
14. Hirano, T., Funahashi, S.I., Uemura, T. & Yanagida, M. 1986. Isolation and characterization of Schizosaccharomyces pombe cut mutants that block nuclear division but not cytokinesis. The EMBO journal, 5(11): 2973-2979.
15. Jantaro, S., Mäenpää, P., Mulo, P. & Incharoensakdi, A. 2003. Content and biosynthesis of polyamines in salt and osmotically stressed cells of Synechocystis sp. PCC 6803. FEMS Microbiology Letters, 228(1): 129-135.
16. Katz, A.M., Tolokh, I.S., Pabit, S.A., Baker, N., Onufriev, A.V. & Pollack, L. 2017. Spermine condenses DNA, but not RNA duplexes. Biophysical Journal, 112(1): 22-30.
17. Kay, D.G., Singer, R.A. & Johnston, G.C. 1980. Ornithine decarboxylase activity and cell cycle regulation in Saccharomyces cerevisiae. Journal of Bacteriology, 141(3): 1041-1046.
18. Krüger, A., Vowinckel, J., Mülleder, M., Grote, P., Capuano, F., Bluemlein, K. & Ralser, M. 2013. Tpo1-mediated spermine and spermidine export controls cell cycle delay and times antioxidant protein expression during the oxidative stress response. EMBO Reports, 14: 1113-1119.
19. Lee, J., Lee, B., Shin, D., Kwak, S.S., Bahk, J.D., Lim, C.O. & Yun, D.J. 2002. Carnitine uptake by AGP2 in yeast Saccharomyces cerevisiae is dependent on Hog1 MAP kinase pathway. Molecules and Cells, 13(3): 407-412.
20. Michael, A.J. 2016. Polyamines in eukaryotes, bacteria and archaea. The Journal of Biological Chemistry, 291(29): 14896-14903.
21. Moreno, S., Klar, A., Nurse, P. 1991. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods in Enzymology, 194: 795-823.
22. Nakamura, T., Kishida, M., Shimoda, C. 2000. The Schizosaccharomyces pombe spo6+ gene encoding a nuclear protein with sequence similarity to budding yeast Dbf4 is required for meiotic second division and sporulation. Genes to Cells, 5(6): 463-479.
23. Nakamura, T., Nakamura-Kubo, M., Nakamura, T. & Shimoda, C. 2002. Novel fission yeast Cdc7-Dbf4-like kinase complex required for the initiation and progression of meiotic second division. Molecular and Cellular Biology, 22(1): 309-320.
24. Örs Gevrekci, A. 2017. The role of predicted spermidine family transporters in stress response and cell cycle in Schizosaccharomyces pombe. Turkish Journal of Biology, 41: 419-427.
25. Gevrekci, A.Ö. 2017. The role of polyamines in microorganisms. World Journal of Microbiology and Biotechnology, 33: 204-210.
26. Ray, R.M., Zimmerman, B.J., McCormack, S.A., Patel, T.B. & Johnson, L.R. 1996. Polyamine depletion arrest cell cycle and induces inhibitors p21Waf7Cip1, p27Kip1, and p53 in IEC-6 cells. American Journal of Physiology, 276: 684-691.
27. Rom, E. & Kahana, C. 1994. Polyamines regulate the expression or ornithine decarboxylase antizyme in vitro by inducing ribosomal frame-shifting. Proceedings of the National Academy of Sciences, 91: 3959-3963.
28. Ucisik-Akkaya, E., Leatherwood, J.K. & Neiman, A.M. 2014. A genome-wide screen for sporulation-defective mutants in Schizosaccharomyces pombe. G3: Genes, Genomes, Genetics, 4(6): 1173-1182.
29. Valdés-Santiago, L., Cervantes-Chávez, J.A. & Ruiz-Herrera, J. 2009. Ustilago maydis spermidine synthase is encoded by a chimeric gene, required for morphogenesis, and indispensable for survival in the host. FEMS Yeast Res., 9(6): 923-35.
30. Valdés-Santiago, L., Guzmán-de-Peña, D. & Ruiz-Herrera, J. 2010. Life without putrescine: disruption of the gene-encoding polyamine oxidase in Ustilago maydis odc mutants. FEMS Yeast Research, 10(7): 928-940.
31. Wallace, H.M. & Keir, H.M. 1981. Uptake and excretion of polyamines from baby hamster kidney cells (BHK-21/C13): the effect of serum on confluent cell cultures. Biochimica et Biophysica Acta, 676: 25-30.
32. Wallace, H.M., Mackarel, A.J. 1998. Regulation of polyamine acetylation and efflux in human cancer cells. Biochemical Society Transactions, 26: 571-575.

Trakya University Journal of Natural Sciences-Cover

ISSN: 2147-0294
Yayın Aralığı: Yılda 2 Sayı
Başlangıç: 2000
Yayıncı: Trakya Üniversitesi

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