In der Spermatogenese von Drosophila melanogaster wird das Chromatin-assoziierte Protein Mst77F in seiner Translation, in seiner Kernlokalisation und in seiner Chromatin-kompaktierenden Funktion über distinkte Bereiche kontrolliert.

In der Spermiogenese von Drosophila wird im Kanustadium der Wechsel von einer auf Histonen- zu einer auf Protaminen basierenden Chromatinstruktur von einer großen Menge an DNA Brüchen begleitet. Die Analyse der Induktion wie auch der Reparatur dieser Brüche, liegt im Fokus des ersten Teils dieser Di...

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Bibliographic Details
Main Author: Kaiser, Sophie
Contributors: Renkawitz-Pohl, Renate (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2014
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During Drosophila spermatogenesis the replacement of histones by protamines at the canoe stage is accompanied by numerous DNA breaks. The induction and repair of these DNA breaks are analyzed in the first part of this thesis. First I analyzed the influence of endonucleases in setting DNA breaks, but neither Tengl1-4, nor Squash or Squash-like are expressed during the switch and therefore are not required for the induction of DNA breaks. In parallel to the DNA breaks high amounts of UbcD6, the Drosophila homolog of yeast Rad6, are detectable. A specific role of the enzyme in DNA repair can be considered. But unfortunately it is not possible to express the fusion protein UbcD6 eGFP in canoe stage nuclei. Therefore we started analysing other components of the Rad6 postreplication repair. However, the transcripts of the components are transcribed only in early stages of spermatogenesis. Thus, the repair of DNA breaks via UbcD6 seems to base on an alternative repair mechanism. The major part of this thesis deals with the linker histone-like protein Mst77F, a chromatin component of mature sperm. It was shown that the transcription of Mst77F depends on tTAFs und that Mst77F mRNA underlies translational repression. In addition, I could show that, in contrast to the majority of testis-specific genes, the 5' UTR is not sufficient to mediate translational repression of the corresponding mRNAs. Interestingly, analyses of transgenic flies clearly highlight distinct regions within the ORF essential for translational repression and activation of Mst77F mRNAs. Furthermore, analyzing ectopic expression of Mst77F suggests that translation of Mst77F may be specific for germ cells and that in spermatocytes Mst77F transcript expression is controlled by a special transcriptional machinery. Mst77F is a chromatin component of the mature sperm and surprisingly, after ectopic expression of Mst77F eGFP in a wild-type background, about 20 % of late spermatid nuclei look abnormal. It can be demonstrated that the phenotype depends on special domains in the protein structure, a C terminal domain with several nuclear location signals and one N terminal coiled coil domain. The coiled coil domain is a typical protein protein interaction domain which allows a kind of crosslinking of the proteins. This fits very well with in vitro data, indicating a role of Mst77F in compaction of the chromatin. We hypothesize that Mst77F affords, in case of sperm maturation, chromatin condensation in specific regions.