|M.Sc Thesis||Department of Biology|
|Supervisor:||Prof. Kassir Yona|
|Full Thesis text - in Hebrew|
Meiosis has a central role in the sexual reproduction in nearly all eukaryotes, and is critical for generating genetic diversity while conserving a functional genome. In S.cerevisiae the meiotic pathway is characterized by the activation of a transcriptional cascade which depends on Ime1.
Ime1 possesses putative phosphorylation sites which are recognized by three distinct kinases: Cdc28, Ime2 and Rim11. The first aim of the research was to investigate if phosphorylation, by any of these three proteins, has an influence on the transient transcription pattern of IME1, using two approaches: (i) Constitutive localization of Ime1 in the nucleus by fusing it with Gal4(bd) had no effect on the transient transcription pattern of IME1. Additional work is required in order to verify the continues localization of the protein in the nucleus. (ii) Mutating putative phosphorylation sites. In this approach truncation of the N terminal domain of Ime1 and a Serine to Alanine mutation at residue 306 had no effect on IME1 transcription pattern. However site directed mutations in putative phosphorylation sites of Cdc28 and/or Rim11 exhibited unexpected IME1 transcription pattern: The S302A mutation resulted in no induction in the level of IME1 RNA that is normally observed between 4-10 hours after transferring the culture to sporulation medium. The Y359F mutation resulted in premature reduction in the level of IME1 RNA (after 6 hours in sporulation medium IME1 RNA was not observed). Further work is required in order to reach more decisive conclusions regarding the influence of these sites on the transient transcription pattern of IME1.
Acetylation of Lysine residues in the N- terminal tail of Histones is often linked with gene activation and is catalyzed by Histone acetyl transferases (HATs). Gcn5, which functions as a HAT, together with two additional proteins- Ada2 and Ada3, comprise the catalytic core in four different HAT complexes in yeast. The second aim of the research was to elucidate the role of Ada2 in regulating the transcription of meiosis specific genes. To this end we used two approaches: (i) Using ChIP analysis we found that there is a transient enrichment of Ada2 on IME2 promoter (an early meiosis-specific gene) prior to its transcriptional induction. (ii) By deleting ADA2 from the genome we found that these cells arrested in G1 with a 2C DNA content with low levels of IME1 RNA, prior to the transcription of IME2 and NDT80 (an early-middle gene essential for entry into M1) .