It is clear that correct attachment of sialic acids is essential to a diverse array of physiologically significant events. Quantitatively and qualitatively correct sialylation requires precise orchestration in the expression of the cognate sialyltransferases. Recent efforts by a number of laboratories have resulted in the molecular cloning of a number of sialyltransferases. However, the molecular pathways that dictate the expression of these sialyltransferases remain mostly unknown. Past and current effort from this laboratory have been devoted to elucidating the gene structure of one of these sialyltransferases, the beta-galactoside alpha2,6-sialyltransferase (SIAT1), and the regulation of its expression. The experiments proposed here are a continuation of these studies. The eventual goal is an in-depth understanding of the regulatory pathways controlling SIAT1 expression. The initial aims will be to establish, by means of cell transfection assays, genetic sequences required for tissue- and cellular-specificity in SIAT1 expression. Putative regulatory sequences will be tested in vivo by mean of transgene fusion constructs containing reporter sequences. Finally, experiments are proposed that will utilize the recently established technology to generate mutant strains of mice using homologous recombination in embryonic stem cells. This will allow unprecedented in vivo studies addressing issues concerning not only regulation, but also functional contributions of sialyltransferase expression in different tissues. Experiments are also proposed to initiate a similar analysis on the beta- galactoside alpha2,3-sialyltransferases (SIAT4). Three distinct enzymes encoded on separate genes are competent in the elaboration of the SIAT4 sialyl- linkage. As with the proposed studies on SIAT1, these initial studies will address the molecular basis and regulation of expression of the SIAT4 genes. Later, if time permits, experiments will be directed at understanding the significance of multiple SIAT4 genes, and functional differences, if any, of the SIAT4s.
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