BIOCHEMICAL GENETICS OF MAMMALIAN NUCLEOSIDE TRANSPORT

  • Ullman, Buddy, (PI)

Project: Research project

Description

Nucleoside transport plays an important role in cellular physiology. The
existence of mutant cell lines which have lost their capacity to transport
virtually all purine and pyrimidine nucleosides across the plasma membrane
suggests a common transport function for all nucleosides in mammalian
cells. The transport-deficient cells are resistant to nucleoside-mediated
growth inhibitory effects. We propose to develop a system to analyze nucleoside transport in
mammalian cells. Genetic studies will be performed on multiple human
nucleoside transport-defective cells to confirm the specificity of this
transport system. We have demonstrated genetically that the nucleoside
transport function has a profound regulatory effect on nucleoside efflux
and on the rates of de novo purine synthesis and purine salvage.
Complementation analysis by cell fusion techniques will determine the
number of genetic loci affecting transport function. A mapping system
based on human-mouse cell hybrids will be developed to ascertain the
chromosomal location of the human genes for nucleoside transport. Proteins from wild-type and mutant cells deficient in nucleoside transport
will be compared by two-dimensional electrophoresis and by photoaffinity
labeling in order to identify the relevant gene product(s). Competitive
binding assays with photoaffinity ligands and purine and pyrimidine
nucleosides will substantiate the biochemical identification of the
nucleoside transporter. The photoaffinity-labeled nucleoside transport
protein and an inhibitor binding assay will be used to monitor
solubilization and purification by affinity chromatography. For further
detailed characterization of the nucleoside transport functions in
wild-type and mutant cells, immunological reagents will be generated. (P)
StatusFinished
Effective start/end date9/30/858/31/86

Funding

  • National Institutes of Health

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Nucleosides
Molecular Biology
Pyrimidine Nucleosides
Purine Nucleosides
Genetic Loci
Cell Fusion
Hybrid Cells
Mutant Proteins
Affinity Chromatography
Genes
Electrophoresis
Ligands
Cell Line

ASJC

  • Medicine(all)