GLYCOSYLTRANSFERASE GENE EXPRESSION IN TRANSFORMED CELLS

Project: Research project

Description

Most cell surface proteins of mammalian cells are posttranslationally
modified to express branched oligosaccharides. One of the characteristic
changes in cellular pathology during neoplastic transformation is the
increase in the branching of ASN-linked oligosaccharides. Furthermore,
these changes in branching patterns have been correlated with metastatic
potential of malignant cells. For example, a 2-fold increase in
[GlcNAcbeta(1 ,6)Man] sequences occurs when fibroblasts are transformed by
tumor viruses or by the ras oncogene alone. The addition of these branches
is performed by the sequential action of specific glycosyltransferases.
The regulation and expression of the specific glycosyltransferases. the
regulation and expression of the genes encoding these enzymes in both
normal and malignant cells are not well characterized. A better
understanding of the control of these genes is necessary to understand, in
detail, their role in regulating oncologic pathology and the consequences
of the cell surface changes which they cause. The goals of this project are to identify and isolate by molecular cloning
the genes and cDNAs encoding N-acetylglucosaminyltransferases I and V to be
used as probes for the analysis of the expression of these genes. We
describe preliminary data to show that we can generate, identify and
isolate revertants of mutant, enzyme-deficient cell lines using DNA-
mediated transformation with human genomic DNA combined with screening by
differential lectin binding. We are now cloning the human DNA sequences
from these revertants to determine whether they contain the human
glycosyltransferase gene. We also will use a complementary approach to
directly clone the cDNAs for these genes using eucaryotic expression
vectors and a transient, panning selection scheme which will be much
faster. After these sequences have been cloned, characterized and
sequenced, they will be used as probes to study the expression of these
genes during Neoplastic transformation and during the in vitro
differentiation of HL60 cells. These probe will also be used to determine
the molecular basis of the lesions in the mutant cells lines.
StatusFinished
Effective start/end date7/1/906/30/94

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $126,634.00

Fingerprint

Glycosyltransferases
Gene Expression
alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase
Oligosaccharides
Genes
DNA
Complementary DNA
Pathology
Cell Line
ras Genes
HL-60 Cells
Enzymes
Lectins
Organism Cloning
Membrane Proteins
Clone Cells
Fibroblasts
Viruses

ASJC

  • Medicine(all)