Introduction of the yeast DNA repair gene PHR1 into normal and xeroderma pigmentosum human cells
The goal of the work described herein is to determine how UV light kills and mutates human cells. Specifically, the hypothesis to be tested states that the major cause of cell death is the cyclobutane dimer. The yeast (S. cerevisiae) enzyme photolyase provides an elegant means of dissecting the biological effects of the two lesions. Photolyase, the product of the PHR1 gene, catalyzes the visible light-dependent reversal of cyclobutane pyrimidine dimers. Introducing the gene for photolyase into human cells, which do not have a functional photoreactivation mechanism, should allow specific repair of cyclobutane pyrimidine dimers. To express the yeast DNA repair gene in human cells, the yeast PHR1 coding sequence was cloned into the mammalian expression vector pRSV4NEO-I. The resulting plasmid, pRSVPHR1, contains the coding sequence of the yeast gene, under control of transcription signals recognized by mammalian cells, and the dominant selectable gene neo. pRSVPHR1 was introduced into normal and XP SV40-transformed fibroblasts by the calcium phosphate coprecipitation technique, and G418-resistant clones were isolated. The level of PHR1 expression was determined by cytoplasmic RNA dot blots. Two clones, XP-3B and GM-20A, had high levels of expression.
- Research Organization:
- California Univ., San Francisco, CA (USA)
- OSTI ID:
- 6983412
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
LYASES
GENES
MUTATIONS
RADIOINDUCTION
XP CELLS
DNA REPAIR
CLONING
FIBROBLASTS
GENE REGULATION
MAN
PYRIMIDINE DIMERS
SACCHAROMYCES CEREVISIAE
ULTRAVIOLET RADIATION
ANIMAL CELLS
ANIMALS
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
CONNECTIVE TISSUE CELLS
ELECTROMAGNETIC RADIATION
ENZYMES
EUMYCOTA
FUNGI
MAMMALS
MICROORGANISMS
PLANTS
PRIMATES
RADIATIONS
RECOVERY
REPAIR
SACCHAROMYCES
SOMATIC CELLS
VERTEBRATES
YEASTS
560120* - Radiation Effects on Biochemicals
Cells
& Tissue Culture