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Adding hemagglutinin gene sequence behind NCW2 into the genome of Saccharomyces cerevisiae yeast

By Miroslava Bernat

Abstract

Stanična stijenka kvasca Saccharomyces cerevisiae kompleksna je izvanstanična organela čije su glavne komponente ß-1,3 i ß-1,6-glukan te manoproteini pričvršćeni za glukansku mrežu. Struktura stanične stijenke mijenja se u različitim fazama životnog ciklusa kvasca kao što su mitoza, mejoza i sporulacija. Za protein Ncw2 pokazano je da sudjeluje u organizaciji ß-glukanske mreže u staničnoj stijenci kvasca, ali nije istražena prisutnost u mejotičkim i stanicama koje sporuliraju. Za identifikaciju i detekciju proteina razvijene su različite molekularno genetičke metode, a jedna od njih je dodavanje oznake na kraj proteina. Cilj rada bio je dodati oznaku na protein kako bi se detektirala njegova prisutnost tijekom mejoze i sporulacije. Konstruiran je soj kvasca kojemu je hemaglutininska sekvenca (HA) dodana iza gena NCW2, što omogućuje zajedničku translaciju proteina Ncw2 i dodane oznake. Takav konstrukt je detektiran koristeći antitijela za hemaglutininsku oznaku. Obilježavanje proteina ovim načinom bilo je uspješno, te je pokazana njegova stabilna ekspresija u mejotičkim i sporulirajućim stanicama.Saccharomyces cerevisiae cell wall is complex extracellular organelle whose major components are ß-1,3 i ß-1,6-glucan and mannoproteins attached to glucan network. It is known that cell wall structure changes during different phases of yeast life cycle such as mitosis, meiosis and sporulation. It has been shown that Ncw2 protein takes part in ß-glucan network reorganization however his presence during meiosis and sporulation still has not been determined. The goal of this work was to tag Ncw2 which will enable us to detect his presence in meiotic and sporulating cells. The construct was made in which at genome level hemagglutinin sequence (HA) is downstream of ORF allowing a translational fusion of protein of interest to the HA tag. This technique enables us to track Ncw2 using specific antibodies. We have confirmed that this system provides efficient tagging and stable expression. Finally, we found that Ncw2 is present in mitotic, meiotic and sporulating cells

Topics: protein Ncw2, Saccharomyces cerevisiae, stanična stijenka, cell wall, Ncw2 protein, yeast Saccharomyces cerevisiae, BIOTEHNIČKE ZNANOSTI. Nutricionizam., BIOTECHNICAL SCIENCES. Nutrition.
Publisher: University of Zagreb. Faculty of Food Technology and Biotechnology. Department of Chemistry and Biochemistry. Laboratory for Biochemistry.
Year: 2017
OAI identifier: oai:repozitorij.unizg.hr:pbf_2344

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