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

ASJC

  • Genetics
  • Molecular Biology

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