Although glycoproteins with N-linked glycans are ubiquitous and involved in diverse physiological processes, little is known about regulation in the N-glycosylation pathway. The yeast Saccharomyces cerevisiae provides a good experimental system in which molecular, genetic and biochemical approaches can be used to study various levels of controls of N-linked glycoprotein biosynthesis. In yeast N-linked glycoproteins are the major components of the cell wall, this structure is responsible for morphology during growth and undergoes changes in composition upon mating and sporulation. This proposal focusses on the expression of ALG7, the first gene of the N-linked glycosylation pathway (the ALG pathway) in yeast. The ALG7 gene encodes UDP-G1cNAc: Dol-P transferase, it is essential and structurally complex. ALG7 expression appears to be regulated through production of multiple transcripts differing in the length of their 3' noncoding region. This 3' untranslated region contains information critical to the regulation of ALG7 transferase levels. The amount of ALG7 enzyme, in turn, affects cell morphology and viability. The goal of this project is to define relationships between molecular controls of ALG7 expression and cell growth and development. Two approaches will be used. First I will study the mechanisms of multiple transcript production, factors involved in the formation of mature 3' ends in ALG7 mRNAs, and transcriptional activation of the gene. The biological roles of the controlling elements will be correlated with ALG7 enzyme levels, with cell morphology, and with cellular functions involving glycoproteins. The second approach will examine the roles of diversity in ALG7 expression in cell growth, mating and sporulation. Thus, changes in cell wall composition and in cell shape accompanying development will be related to ALG7 enzyme levels and specific transcripts and controlling factors. Since two other genes in the ALG pathway also produce multiple mRNAs, studies with ALG7 may point to common features of expression in the early steps of N-glycosylation in yeast, and in eukaryotes generally.
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