Determination of the efficient enzyme concentration for lytic digestion of vegetative cells but not spores in Schizosaccharomyces pombe

Abstract

Received: 2016-05-24 | Accepted: 2016-05-30 | Available online: 2017-06-20 http://dx.doi.org/10.15414/afz.2017.20.01.20-22 The fact that lytic enzymes such as b-glucuronidase are capable of cell wall lysis, while the integrity of the spore is not affected, is used in the spore viability tests to investigate meiotic processes. Meiosis as a fundamental feature in all living organisms comprises of a complex tightly linked and mutually dependent processes most of which are scientific targets of many research institutions. The fission yeast Shizosaccharomyces pombe is a powerful tool for studies on eukaryotic meiosis. Mating of yeast strains of opposite mating types on nitrogen free medium results in spores production. Whereas not all cells undergo meiosis, some cells persist in vegetative stage even in the absence of nitrogen, this leads to generation of a mixed suspension of vegetative cells and spores. Thus, in order to separate spores from vegetative cells obtained mixture was exposed to lytic enzyme action. This treatment kills vegetative cells without affecting spores. To obtain the best and reproducible results of spore recovery and viability, different lytic conditions were analysed. Obtained results show, that use of b-glucuronidase as lytic enzyme for random spore analyses in the fission yeast is dose and time dependent. Keywords: b-glucuronidase, cell cycle, spore viability, Schizosaccharomycesn pombe References BAHALUL, M., KANETI, G. and KASHI, Y. (2010) Ether–zymolyase ascospore isolation procedure: an efficient protocol for ascosppores isolation in Saccharomyces cerevisiae yeast. Yeast, vol.27, no.12, pp.999–1003. doi: http://dx.doi.org/10.1002/yea.1808 DAWES, I.W., HARDIE, I.D. (1974) Selective killing of vegetative cells in sporulated cultures by exposure to diethyl ether. Mol Gen Genet, vol.131, no. 4, pp. 281–289. doi: http://dx.doi.org/10.1007/BF00264859 DONOVAN, D.M., KERR, D.E. and WALL, R.J. (2005) Engineering disease resistant cattle. Transgenic Res, vol. 14, pp. 563–567. doi: http://dx.doi.org/10.1007/s11248-005-0670-8 KHARE, A. K., SINGH, B., and SINGH, J. (2011) A fast and inexpensive method for random spore analysis in Schizosaccharomyces pombe . Yeast, vol. 28, pp. 527–533. doi: http://dx.doi.org/10.1002/yea.1855 MORENO, S., KLAR, A. and NURSE, P. (1991) Molecular genetic analysis of the fission yeast Schizosaccharomyces pombe . Methods Enzymol, vol .  194, pp. 795–823. http://dx.doi.org/10.1016/0076-6879(91)94059-L PARENTI-CASTELLI G, et al. (1974) Effect of soluble and membrane proteins upon diethyl ether extraction of aqueous phospholipid dispersions. Lipids, vol . 9, pp.221–228. doi: http://dx.doi.org/10.1007/BF02532197 SALAZAR, O. and ASENJO, J.A. (2007) Enzymatic lysis of microbial cells Review Biotechnology Letters , vol. 29, no. 7, pp. 985-994. doi: http://dx.doi.org/10.1007/s10529-007-9345-2 SMITH, G.R. (2009) Genetic Analysis of Meiotic Recombination in Schizosaccharomyces pombe Methods Mol Biol ., vol. 557, pp. 65–76. doi: http://dx.doi.org/10.1007/978-1-59745-527-5_6 YANG, Y.G, et al. (2000) The application of a novel lytic system to the recovery of recombinant proteins in E. coli. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) Acta biochimica et biophysica Sinica, vol. 32, no. 3, pp. 211–216. ŽUKAITE, V. and BIZIULEVICIUS, G.A. (2000) Acceleration of hyaluronidase production in the course of batch cultivation of Clostridium perfringens can be achieved with bacteriolytic enzymes. Lett Appl Microbiol , vol. 30, pp. 203–206. doi: http://dx.doi.org/10.1046/j.1472-765x.2000.00693.x