Glycoconjugates are a major class of molecules found on the surface of all cells including developing male germ cells. Glycans are biosynthesized by the coordinate action of a functional family of membrane-bound enzymes, termed glycosyltransferases. In spite of the diverse role that glycans serve in the proper functioning of a cell, there is a paucity of information on the regulation of glycosyltransferases genes. Using the beta4-galactosyltransferase-I (beta4GalT-I) gene as a """"""""model system"""""""" we have shown that during spermatogenesis there is a switch to a male germ cell-specific transcriptional start site that is used exclusively in round spermatids. Furthermore, we have identified a new 14-base pair regulatory motif (the TASS-1 motif) upstream of this start site that is essential for in vivo expression of beta4GalT-I in round spermatids. Coincident with the switch to the male germ cell-specific promoter there is also a switch to the use of one or two alternative, internal poly- adenylation sites. The net result of this switch is the generation of truncated male germ cell-specific beta4GalT-1 mRNAs in which approximately 1.7 kb of the lung (2.5 kb) 3'-untranslated region (3'-UTR) that distinguishes the beta4GalT-I somatic transcript, has been deleted. While the use of an internal polyadenylation site and the consequent truncation of the 3'-UTR is an emerging general paradigm for genes expressed in both somatic and male germ cells, the biological significance of this truncation is unknown. In this proposal we have four complementary goals. First, the TASS-1 will be characterized at the protein level, to determine if it is a novel member of a known family of transcription factors or defines a new family of transcription factors. Third, transgenic mouse lines in which the TASS-1 gene has been inactivated by homologous recombination will be analyzed to determine the consequences on male germ cell development. Fourth, we will test the hypothesis that the truncation of the consequences on male germ cell development. Fourth, we will tet the hypothesis that the truncation of the 3'-UTR results in male germ cell-restricted beta4GalT-1 transcripts with increased stability, relative to that of the somatic transcript. Assays using transfected 3'-UTR/reporter gene constructs and transgenic analysis will be used to examined mRNA stability both in vitro and in vivo. This proposal offers the potential to identify and characterize a new family of transcription factors essential for male germ cell development, and provide insight as to why germ cell transcripts contain truncated 3'-UTRs relative to their somatic cell counterparts. Consequently, it impacts only on male germ cell development but also on male infertility.
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