The aim is the in-depth biochemical characterization of several Chinese hamster ovary (CHO) glycosylation mutants recently-isolated by this laboratory. The mutants were selected for resistance to plant lectins and exhibit properties typical of those that we have previously shown to arise from altered carbohydrate biosynthesis. Genetic studies have shown that each mutant type is novel and thus would be expected to express biochemical defects previously undescribed in CHO cells. The experimental approach is to define the structural carbohydrate change expressed by each mutant type and, based on this knowledge, to assay for the activity that might be responsible for the observed glycosylation phenotype. The mutants to be studied with highest priority include four dominant mutants (LEC14, LEC16, LEC17 and LEC 8) and four recessive mutants (Lec9, Lec19, Lec20 and Lec21). The biochemical bases of dominance are of interest to mechanisms by which cells may change their array of carbohydrates in a single step. The characterization of Lec9 mutants is important because they are the only mutant in our repertoire to exhibit markedly-reduced tumorigenicity in the nude mouse. Carbohydrates from the glycoproteins of animal viruses such as vesicular stomatitis virus or from the cell surface will be radiolabeled and structurally characterised by lectin-affinity and gel filtration chromatography with the aid of specific glycosidases. Additional structural information will be obtained from designated species by 1H-NMR spectroscopy at 500 MHz. Once the structural changes expressed by a mutant are known, assays for glycosyltransferases and other enzyme activities will be developed to pinpoint the enzymic basis of each defect.
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