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Title: Investigation of TSGA10 gene expression, localization and protein interaction in human and mouse spermatogenesis
Author: Behnam, B.
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2005
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Isolation of the testis-specific human gene TSGA10 was reported in 2001 (Modarressi et al.). In this project, I have carried on the work by further characterising the gene and its product by cloning and sequencing the mouse homologue (TsgalO) (GenBank Accession no. AF5300501). TsgalO was localised by t-XSH to mouse chromosome 1 band B, and RNA in situ hybridization and RT-PCR experiments showed that TsgalO transcripts were developmentally regulated and expressed in pachytene cells of the testis. It was also expressed in some other actively dividing cells such as foetal tissues, primary tumors and astrocytes. Interestingly, the program Pfam (which predicts protein structural motifs) suggests that the TsgalO protein contains a 'myosin like' coiled-coil domain. A Green fluorescent protein (GFP)-TsgalO fusion gene was constructed and cell transfection resulted in the formation of short thick protein filaments. Antibodies raised individually against the N-terminus and C-terminus parts of the TsgalO protein were used to localise TsgalO protein expression by immunohistochemistry. In collaboration with Hossein Modarressi and Frans Van der Hoorn, using immuno-blotting and staining techniques, I discovered that TsgalO encodes a 82 kD precursor protein in spermatids which is incorporated into sperm tails. Upon passage of sperm into the epididymis this is processed to a 27 kD protein which is associated with the fibrous sheath, a major tail structure. Using TsgalO as bait in a yeast two-hybrid experiment,citestis cDNA library was screened for proteins that interacted with TsgalO. Three candidate interacting proteins were found Odf2, a previously described protein of the fibrous sheath, the rat homologue of FU32880, (a hypothetical human protein whose gene is expressed predominantly in spermatocytes, spermatids and mature sperm cells), and the mitochondrial protein cytochrome cl. Using immunocytochemistry I then localised FLJ32880 (newly named as TSGA10 interacting protein, TSGA10IP) in the sperm tail as another fibrous sheath protein. These results support the theory that TsgalO has a role to play in the fibrous sheath of the sperm tail, and also suggest a possible functional involvement with a mitochondrial protein. Antibody was raised against the mouse homologue of TSGA10IP for further study. I suggest that TsgalO plays multiple roles, one in sperm tail formation and possibly a second role in cell cycle regulation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available