2011 YÜksek lisans tez özetleri

Studies on directed translocation between non-homolog chromosomes in Schizosaccharomyces pombe

Eukaryotic organisms have so many and complex molecular mechanisms that underlie many diseases and take role in chromosomal rearrangement in translocation occasion. There are so many studies on molecular mechanism of translocation. However first directed translocation was achieved by Yeast Molecular Laboratory in “International Centre for Genetic Engineering and Biotechnology” (ICGEB) in Italy. In ICGEB Yeast Molecular Laboratory, non-homolog directed translocation was performed with DNA locus was chosen randomly before in Saccharomyces cerevisiae and the method was called “Bridge Induced Translocation” (BIT).

The main goal of this project is to generate directed translocation for elucidating molecular events after translocation by using Schizosaccharomyces pombe as a model organism which is used frequently in molecular studies because of its similarities to mammalian cells. Obtaining a model translocant S. pombe will be an important work for medical science because S. pombe has a lot of homolog genes that are responsible for human diseases. So cellular and molecular differences, which are appeared after chromosomal rearrangement, can be contrasted between transformant and parental strains by directed translocation. And also the effects mechanisms of translocations can be clarified.

In this study, wild type of S. pombe was used. A special cassette was prepared with kanamycin and the cassette contained homology to two different chromosomes (chromosome 1 and chromosome 3) with two different sequences (approximately 90bp-800bp) for generating translocation. These homolog sequences are responsible to generate translocation and they are breakpoints of translocation. In content of this thesis, linear DNA cassette was produced by PCR and transported to S .pombe. Then kanamycin resistance features were controlled. Subsequently, colony PCR was performed to control for correct integration.

Model translocant S. pombe, what the aim of the project is, could not be obtained. One side of translocation cassette integrated to just one chromosome (Chromosome 3) and another side of translocation cassette which aims to integrate to chromosome 1, could not be found where it is. Also after hybridization of transformant cells’ chromosomes with the prob that designed specific to marker gene, in some transformant colonies (50% of colonies), translocation and knock-out cassettes were detected on two chromosomes, one of chromosomes is targeted chromosome but another one, chromosome 2, isn’t targeted. This result is a new research subject to understand chromosome 2’s structure and chemicals features that allow the unspecific cassette integration. In the literature no studies were found about this interesting result. Directed translocation studies with S. pombe is just beginning studies of this subject and in the future, creating model translocant S. pombe experiments will be continued by using different chromosomes, different locations and different homology lengths.