Tez özetleri Astronomi ve Uzay Bilimleri Anabilim Dalı 2

Deletion Of Gua1 Gene With Knock-Out Technique In Schizosaccharomyces pombe
In this study, we aimed to deletion of gua1 gene that encodes inosine monophosphate dehydrogenase (IMPDH) in eukaryotic model organism Schizosachharomyces pombe. IMPDH catalyzes the first step in de novo biosynthesis of guanosine monophosphate (GMP).
Knock-out cassette was constructed with PCR (Polymerase Chain Reaction)-based gene targeting technique in Schizosaccharomyces pombe and this knock-out cassette was transformed to Schizosaccharomyces pombe. In this study, we used wild type (972h^-), ura4h^- mutant strain and diploid strain of Schizosaccharomyces pombe. The pFA6-kanMX4 plasmid was isolated from DH5α strain of E.coli that contains pFA6-kanMX4 plasmid and this plasmid was used as template for knock-out cassette. Cassette contains 653 and 285 bp sequences which are nearby gua1 gene in S.pombe genome and kanamycin resistance gene obtained from pFA6 plasmid. 653 and 285 bp products were obtained after the first PCR. Template in the first PCR was S.pombe genome. On the other hand, the knock-out cassette was produced after the second PCR. After transformation with lithium acetate method we aimed that this cassette replace with the sequences of gua1 gene via homologous recombination.
The cells cultured in YEA medium with geneticin after transformation. Thus, we could select 972h^-, ura4h^- mutant and diploid colonies which integrated knock-out cassette to genome via homologous recombination. Haploidizations of diploid colonies were performed and then wild type (972h^-), ura4h^- and haploid colonies were selected in YEA (yeast extract agar) with geneticin, YEA, MMA (minimal medium agar), MMA with guanine and MMA with guanine and uracil. In this step we determined mutant colonies in gua1 gene. Finally colony PCR and sequencing techniques were performed to control whether the deletion is made in the right place or not. After colony PCR expected result was obtained in one single transformant colony.