In order to investigate structure/function relationships of animal cell carbohydrates, as series of glycosylation mutants of Chinese hamster ovary (CHO) cells have been isolated. All of the mutants exhibit altered carbohydrate biosynthesis and express new carbohydrate structures at the cell surface. Twenty-five distinct phenotypes have been identified and classified into seven dominant types and fourteen recessive complementation groups. They have been used by many laboratories to study the roles of carbohydrate moieties in the biosynthesis, compartmentalization and functions of endogeneous or introduced glycoproteins. Recent studies in this laboratory have shown that the altered carbohydrates synthesized by Lec9 CHO mutants correlate directly with their reduced tumorigenicity in nude mice. The CHO glycosylation mutants provide an avenue to the isolation of glycosylation genes and the determination of mechanisms that control their expression. It is proposed to isolate new glycosylation mutants and revertants using lectins as selective agents so that all the genes involved in carbohydrate biosynthesis are identified. Studies on molecular mechanisms that regulate glycosylation gene expression will focus on the three dominant mutants, LEC10, LEC11 and LEC12. Their phenotypes appear to be the result of gene activation since each of them expresses a novel glycosyltransferase activity not detected in parental CHO. The abilities of reagents that cause mutations and epigenetic changes such as hypomethylation to alter the rates of appearance of these mutants and their revertants will be examined. Co-transfection of genomic DNA with a plasmid carrying a dominant selectable marker will be used to isolate the genes and their expression will be compared in parental and mutant cells. These studies should reveal mechanisms that regulate glycosylation gene expression in CHO cells and provide probes to investigate the changes in carbohydrate synthesis that occur during development, differentiation and oncogenic transformation.
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