Configuración del PID Compact del Step 7

It has been demonstrated by DDRT-PCR, RT-PCR and by Northern blotting that the gene TSGAIO is expressed in normal testis and also in other actively dividing tissues (either foetal or malignant). It is not expressed in a variety of tissues functionally related to testis nor is it expressed in the testes of two infertile patients nor foetal testis. It should be noted that testis development in a foetus at the second and third trimester of pregnancy has already been completed and descent is in process with little cell division occurring (see chapter I, sections 1.1.2 and 1.1.3). Thirteen human ESTs homologous to TSGAIO have been reported. Nine are

certainly or probably derived from testis and three of them, which represent more than two exons of the gene, are derived from a poorly differentiated

adenocarcinoma, an acute myelogenous leukemia and a germ cell tumour. At the present time there are about 6.85 million EST sequences present in databases (January 2001) and table III.5 shows the results of the Blast search of these with

TSGAIO. Most of the matches seem to be real ESTs derived from mRNA and not

libraries. The observation that the sources of the ESTs, both human and other species, which show highly significant matches to human TSGAIO are almost all germinal cells in testis, or tumours or embryonic tissues, may suggest that there is a link between expression of this gene and cell division.

Mammalian spermatogenesis provides an excellent model system for studying cellular division and differentiation. First, the mitotic division of germ cells is similar to many mitotically active undifferentiated tissues such as those in tumours or foetus. The formation of different organs and tissues is based primarily on differential gene expression. While some ‘ housekeeping’ genes that contribute to basic structural or metabolic cellular functions are expressed ubiquitously

throughout the body, other tissue-specific genes that contribute to specialised function in differentiated cell types are expressed in a regulated fashion. It is likely that, due to the structural and functional changes that take place in male germ cells during spermatogenesis, many germ cell-specific gene products are involved in this differentiation process. This process is seen to occur in parallel with changes in the expression of total RNA and stage specific proteins (Hoog, 1995, Monesi, 1965, Boitani et al, 1980 ).

There may be a parallel in the expression of TSGAIO and c-kit. In

melanogenesis, c-kit (see chapter I, section 1.3.1) is expressed in melanoblasts from the time they leave the neural crest and expression continues during embryonic development and in the melanocytes of postnatal animals (Besmer et a l, 1993). The c-kit receptor tyrosine kinase belongs to the PDGF/CSF-l/c-kit receptor subfamily (see chapter I, section 1.3.1). The kit-ligand, KL, also called steel factor, is

synthesised from two alternatively spliced mRNAs as transmembrane proteins that can either be proteolytically cleaved to produce soluble forms of KL or can function as cell-associated molecules. In gametogenesis c-kit is expressed in primordial germ cells and in spermatogonia (see chapter I, section 1.3.1). Studies in white spotting and steel mice have shown that functional SCF and c-Kit are critical in the survival and development of stem cells involved in hematopoiesis, pigmentation and reproduction (review, Linnekin, 1999). Downstream of c-Kit, multiple signal transduction components are activated, such as the Ras-Raf-MAP kinase cascade

which is one of the known cascades in tumours and cell division (see chapter I, section 1.3.2). There is some evidences that may support the links between TSGAIO

and c-Kit. Firstly, the site for phosphorylation by the tyrosine kinase of c-kit is

similar to one of the sites on the predicted TSGAIO protein (explained later in the section). Secondly, one of the matches with 99% identity to the 3' end of TSGAIO ( figure III.21 and table III.5) is an EST (accession no. N33757) from melanocyte and c-kit is also involved in pigmentation. Thirdly, the MAP kinase pathway and many components downstream of c-kit are related to cell division in which TSGAIO is probably involved. Fourthly, The proto-oncogene c-kit is expressed in a broad spectrum of human cancers (Matsuda et a l, 1993) and TSGAIO expression was also shown in at least six different tumours (three from EST database and others by using RT-PCR ). Finally, the role of c-kit during reproduction and its expression in testicular germ cells has been demonstrated (see chapter I, section 1.3.2), and experiments have proved the presence of the TSGAIO transcript in testis and mouse and rat spermatocyte libraries.

In summary, the pattern of expression of TSGAIO strongly suggests that has a role in spermatogenesis. This conclusion is strengthened by consideration of the

TSGAIO homologues in other species. Genbank contains a partial cDNA which is a

rat homologue of TSGAIO, and is claimed to be present in Rattus norvegicus

spermatocytes. In addition, the mouse homologue of TSGAIO was isolated in this study from a spermatocyte cDNA library. Making a cDNA library from only one kind of germinal cells of testis is difficult due to failure of purity of cell separation and contamination by other cells but it is encouraging that rat and mouse

homologues of the gene were separately obtained from two cDNA libraries each made using spermatocyte cells. Two of the mice ESTs (BF319562, BF018459) which matched to TSGAIO are derived from the later stage of spermatid (round spermatid).

III. 13.3 Copy num ber of TSGAIO

The human genomic BAG library which was screened consisted of 125,000 BAG colonies, each with an average size of 160,000-200,000 bp. This gives an

estimated ten fold coverage of the genome. The screen yielded three positive colonies. This indicates that the gene is likely to be single copy. In addition, the mouse genomic library which was used in this experiment consisted of 128,000 PAC clones with an average insert size 150,000bp equivalent to approximately six fold genome coverage and the hybridisation results (8 colonies) is also consistent with the single copy hypothesis.