טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentOrly Laufman
SubjectCloning and Characterization of the Schizosaccharomyces
Pombe Homologue of the Human Protein Translin
DepartmentDepartment of Biology
Supervisor Full Professor Manor Haim


Abstract

Our laboratory has previously identified in human fibroblasts a multisubunit protein that specifically bound single-stranded G-rich microsatellite DNA sequences. This protein was later found to be identical to Translin, an octameric protein that bound single-stranded oligodeoxynucleotides consisting of sequences flanking chromosomal translocations. Translin and another protein that was found to be associated with Translin designated TRAX are highly conserved in eukaryots including the fission yeast Schizosaccharomyces pombe. We decided to use S. pombe as a model organism to study the function of Translin and TRAX because of the advantages of S. pombe as a genetic system. Here I report the cloning and expression of the S. pombe Translin and TRAX open reading frames. Western blot analysis and immunoprecipitation assays proved that these open reading frames constitute active genes. I have also shown that S. pombe Translin and TRAX specifically interact in Yeast Two-hybrid and immunoprecipitation assays. Glycerol gradient sedimentation analysis revealed that the native recombinant S. pombe Translin is an octamer. The octamer was found to be the only functional DNA and RNA binding form of Translin and the octameric form was maintained after binding to nucleic acids. Gel mobility-shift assays revealed that recombinant S. pombe Translin bound specific sequences of single-stranded DNA and RNA but not double-stranded DNA. Among the various DNA sequences assayed, single-stranded d(GT)n repeats had the highest affinity for Translin (Kdis=18nM). Translin bound with even higher affinities G-rich single-stranded RNA sequences (Kdis for binding the oligoribonucleotide (GU)15 was 3nM). These and other data support previous suggestion that Translin may be involved in control of mRNA translation and transport.