Expressional, functional and genomic studies of proteasome subunits during spermatogenesis of Drosophila melanogaster
Date of Award
Doctor of Philosophy (PhD)
John M. Belote
Proteasome subunits, Spermatogenesis
Genetics and Genomics | Life Sciences
Most regulated proteolysis in eukaryotic cells is carried out by a large, multisubunit protease called the 26S proteasome, which is composed of a 20S core particle and two 19S regulatory caps at each end. In Drosophila melanogaster , 6 subunits of the 20S core and 5 subunits of the 19S cap have multiple isoforms encoded by separate genes. The additional isoforms are invariably testis-specific. In the work reported here, I characterized the expression patterns of previously studied subunits Prosα3, Prosα3T and Prosα6T in more detail. In addition, I examined the expression patterns of several other proteasome subunits that had not been studied before, using eGFP-tagged reporter proteins. The housekeeping subunits, Prosα3, Prosα6, and Prosβ5 are expressed in early stages of spermatogenesis in both nuclei and cytoplasm, but gradually fade away in elongating spermatids, and are not detectable in mature sperm. In contrast, testis-specific Prosα3T, Prosα6T, Prosβ5R1 and Prosβ5R2 are expressed after meiosis, and persist in the mature sperm. Another interesting expression pattern is that some of these subunits localize to numerous punctate "speckles" trailing the actin cone complex in spermatid bundles undergoing individualization.
The functions of the Prosα6T subunit was studied by generating a null mutation Prosα6T 1 . Most of the spermatogenesis stages of the male-sterile Prosα6T 1 look normal under phase-contrast microscopy, but no mature sperm are produced. The Individualization Complexes (IC) becomes disorganized as they move along the tail bundles. Spermatid nuclei are either scattered or curled. Histone removal is delayed in mutant spermatid nuclei, but protamine incorporation is not affected. The expression analysis and the mutational study indicate that Prosα6T plays an essential role in Drosophila individualization.
In the second part of my study, I identified the proteasome subunit genes in 11 Drosophila species and three non- Drosophila insect species by database searches. The results showed that proteasome subunit gene duplication is a common phenomenon and that many additional genes are the results of retro transposition.
Zhong, Lei, "Expressional, functional and genomic studies of proteasome subunits during spermatogenesis of Drosophila melanogaster" (2007). Biology - Dissertations. Paper 18.