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Stanična stijenka kvasca Saccharomyces cerevisiae određuje morfološke karakteristike stanice, kao što su oblik i čvrstoća, te doprinosi integritetu stanice tijekom rasta, diobe i stresnih okolišnih uvjeta. Građena je najvećim dijelom od polisaharida glukana i hitina te proteina. Cilj ovoga rada bio je uspostava semi-kvantitativne western blot metode za analizu proteina stanične stijenke. Prvo su konstruirani haploidni sojevi kvasca oba tipa parenja kojima je gen BGL2 označen hemaglutininskom sekvencom. Potom su iz ovih sojeva i haploidnih sojeva divljeg tipa procesom parenja konstruirani diploidni sojevi kojima su jedna, odnosno obje kopije gena BGL2 označene. Između proteinskih ekstrakata konstruiranih diploidnih sojeva detektirana je jasna razlika u intenzitetu signala vrpce proteina stanične stijenke Bgl2-HA, čime je pokazano da je provedena semi-kvantitativna western blot metoda pogodna za relativnu kvantifikaciju proteina Bgl2-HA. Cell wall of yeast Saccharomyces cerevisiae defines morphological features of the cell, such as shape and rigidity, and is a key factor in cell integrity during growth, mitosis and exposure to stressful environmental factors. Major components of the cell wall are polysaccharides glucan and chitin, as well as proteins. The goal of this thesis was to develop a semi-quantitative western blot method for analysis of cell wall proteins. First, haploid yeast strains of both mating types in which BGL2 gene was tagged by hemagglutinin sequence were constructed. These strains and wild type haploid strains were then mated to construct a set of diploid strains in which one or both copies of BGL2 gene were tagged. Protein extracts from this set of diploid strains displayed a distinct difference in signal intensity of cell wall protein Bgl2-HA band, thereby showing that developed semi-quantitative western blot method is suitable for relative quantification of Bgl2-HA protein. |
