The goal of this application is to examine the role of the ALG7 gene in mammalian salivary gland development. Conserved in evolution and essential for viability, ALG7 functions by initiating the dolichol pathway of protein N-glycosylation and is the key gene regulating protein N-glycosylation. Significant changes in ALG7 expression accompany developmental programs of diverse eukaryotic systems, ranging from the yeast Saccharomyces cerevisiae through zebrafish to hamster salivary glands. To date, ALG7 has been shown to play a regulatory role in yeast development, where attenuating ALG7 expression 4-fold has deleterious effects on cell proliferation. The regulatory role of ALG7 extends through various animal systems, including mammalian tissue culture cells. Since postnatal development of hamster submandibular gland (SMG) entails downregulation of ALG7 activity, the Principal Investigator proposes to examine this gene's expression and consequences of its misregulation in the developing murine SMG using transgenic and other genetically perturbed mice. The hypothesis on which this application is based is that changes in ALG7 expression are critical for the normal development of SMGs, and that misregulation of this gene's activity will result in developmental defects.
The specific aims are: 1) to determine the temporal and spatial expression of ALG7 at different stages of embryonic and postnatal development of SMGs using in vivo and in vitro detection assays; 2) to interfere with ALG7 expression at distinct stages of SMG postnatal development in tissue explants and mice using tunicamycin and antisense oligonucleotides and RNAs; 3) to construct transgenic mouse lines with perturbed ALG7 expression by engineering different 3' untranslated region knockouts; and 4) to examine the effects of deregulated ALG7 expression in SMGs from transgenic and exogenously-perturbed mice, as well as SMG explants, on cell proliferation, a function shown to be altered in yeast and mammalian cells in culture.
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