Lennon, Kelley L.2023-03-242023-03-2419981998(OCoLC)41953618b22455772https://hdl.handle.net/2144/45804Thesis (D.Sc.D.)--Boston University, Henry M. Goldman School of Dental Medicine, 1998 (Oral Biology).Includes bibliographical references: (leaves 110-176).Protein N-glycosylation is metabolic process that is obligatory for viability in all eukaryotes. It results in the modification of appropriate asparagine residues of polypeptides with oligosaccharide structures, influencing their properties and bioactivities. N-glycosylation is initiated, via the dolichol pathway, with the synthesis of the lipid-linked oligosaccharide precursor that is later transferred to protein. This synthesis occurs in the endoplasmic reticulum and involves the expression of a set of evolutionarily conserved ALG genes. Contrary to the previous dogma, recent studies suggested that the early dolichol pathway genes are highly regulated, early growth response genes (Kukuruzinska and Lennon, 1994). RNA blotting assays presented here indicate that all the ALG genes, as well as two oligosaccharyltransferase (OT) subunits, are coordinately regulated with growth early in G1, at the GJG1 transition point. In contrast, at a second point in the cell cycle critical to proliferation, late in G1, the genes of the dolichol pathway and OT are differentially regulated. Specifically, ALG7 expression is selectively downregulated in response to cell cycle arrest. This proliferation­ dependent expression of the first dolichol pathway gene, ALG7, is also demonstrated in a higher vertebrate system, zebrafish, via immunohistochemistry and Western blotting. The mechanisms involved in the regulation of ALG7 expression in yeast were assessed with RNA blotting assays. Evidence is provided that this gene is controlled on transcriptional and post-transcriptional levels. Moreover, data from in vitro transcription/translation studies of the mammalian ALG7 gene suggest the possibility of additional control of this gene's expression, on the level of translation. Finally, the functional significance of the early dolichol pathway genes, and ALG7 in particular, is demonstrated through the use of disparate methodologies that include RNA blotting, flow cytometry, fluorescence and light microscopy. The results suggest that the early ALG genes have developmental relevance, since unwarranted changes in their expression cause defects in proliferation, differentiation and cytoskeletal organization. Moreover, these studies reveal potential downstream genetic targets for the ALG genes, and protein N-­glycosylation in general, that include G1 cyclins and proteins involved in organization of the actin cytoskeleton.en-USThis work is being made available in OpenBU by permission of its author, and is available for research purposes only. All rights are reserved to the author.DolicholGenetics, biochemicalGlycosylationThesis/Dissertation