GLYCOSYLATION MUTANTS OF ANIMAL CELLS

Project: Research project

Project Details

Description

The overall objective of this research is to identify functional roles for developmentally regulated carbohydrate structures that are unique to mammalian cells. The long term experimental strategy has been to isolate Chinese hamster ovary (CHO) cell mutants with altered glycosylation patterns, and to use the mutants in both functional studies and to clone genes encoding glycosyltransferases by complementation. with the ultimate aim of exploring the consequences of manipulating glycosylation gene expression in the mouse. We have recently cloned two glycosyltransferase genes[N-acetylglucosaminyltransferase I (GlcNAc-T1) and an alpha(1,3)fucosyltransferase (alpha(l.3)Fuc-T)] by transfection of human genomic DNA into CHO cells and gene rescue in lambda vectors. Thus we are now in a position to pursue a molecular genetic approach to identify functions for mammalian carbohydrates. Our future studies will focus on four glycosyltransferases required for the synthesis of developmentally regulated carbohydrates: GlcNAc-Tl whose activity is essential for the production of all developmentally regulated, N-linked carbohydrates; GlcNAc-TIII which synthesizes the bisecting GlcNAc residue in N-linked carbohydrates; and alpha(1,3)Fuc-Tl and alpha(l.3)Fuc-T2, two distinct enzymes which synthesize fucosylated lactosamine units on different glycoconjugates. The structures made by the latter three enzymes are developmentally regulated in vivo and expression of each is characteristic of the dominant CHO glycosylation mutants LEC10 (GlcNAc-TIII), LEC11(alpha(l.3)Fuc-T1) and LEC12(alpha(l.3)Fuc-T2) respectively. The SPECIFIC AIMS of this proposal are: 1)To isolate cDNAs for the CHO transferases GlcNAc-TIII, alpha(l.3)Fuc-Tl and alpha(l.3)Fuc-T2 by cDNA expression cloning and to use the cDNAs to investigate the molecular genetic basis of the dominant CHO mutations LEC10, LEC11 and LEC12. 2)To use the cDNAs to generate specific probes for isolating the corresponding mouse glycosyltransferase genes in order to study their expression in the tissues of embryonic land adult mice. 3)To develop chimeric and transgenic mouse experimental systems to abrogate(GlcNAc-T1) or alter (alpha(1,3)Fuc-T) the expression of specific glycosyltransferases in vivo.
StatusFinished
Effective start/end date5/28/922/29/00

Funding

  • National Cancer Institute

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