Modaressi, Mohammad Hussein; (2001) The identification and characterisation of genes expressed in adult human testis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

By the identification and isolation of cDNAs expressed specifically in the human testis I aimed to identify novel genes involved in spermatogenesis and/or spermiogenesis. Using Differential Display Reverse Transcriptase-PCR (DDRT-PCR) on RNA from a range of tissues which were selected to be logically related to testis functions I isolated a number of short cDNA fragments. Tissue-specific expression was confirmed by using RT-PCR with gene-specific primers. Three genes were examined in detail: Entire transcripts were sequenced by primer walking along cDNA templates generated by 5'RACE and the size of full-length cDNAs was confirmed by Northern analysis. The chromosomal locations of the genes were determined by two methods. Firstly by PCR amplification using gene specific primers of a panel of rodent somatic cell hybrids that each contain a single human chromosome Secondly, genomic clones (BAC or a PAC) were hybridised in situ to human metaphase chromosomes (FISH). Using the BAC clones, some genomic gene structure was determined. In this way I have isolated and characterised three novel genes from testis, TSGA10, GNG2 and T2G3. Testis Specific Gene A10 (TSGA10) is not expressed in a variety of tissues functionally related to testis nor is it expressed in either testes of two infertile patients or in foetal testis. The TSGA10 gene (GenBank Accession no. AF254756) consists of 3038 nucleotides spread over 19 exons and contains an open reading frame of 698 amino acids and one alternative splice site. RT-PCR results using RNA prepared from foetal and tumour tissues showed that transcripts from this gene are also present in squamous cell carcinoma, parotid and thyroid tumours, benign prostate hypertrophy and almost all foetal tissues though with different levels of expression in different tissues. Several ESTs with almost 100% homology to the TSGA10 transcript exist in the databases but all are probably derived from testis apart from three which were isolated from tumour tissues. This pattern of expression suggests that TSGA10 has a role in actively dividing cells and possibly in cell division and mitosis. The mouse homologue of this gene was also cloned and characterised. The gene GNG2 is a G protein subunit. The gene T2G3 is another novel gene. Both have been characterised to a lesser extent than TSGA10.

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