EVALUATION OF GENETIC RISKS FROM IONIZING RADIATION. DETERMINATION OF CELLULAR RADIOSENSITIVITY AND RELATION TO REPAIR.
A novel complementary deoxyribonucleic acid (cDNA) expression vector has been constructed. Reconstruction experiments have shown that this system is classical vector systems described until now. Although the final goal, the cloning of the XP-A gene, has not yet been reached the cloning of the HPRT gene, from a cDNA library by correction of a human mutant cell line in a small scale experiment, shows that the episomal cDNA expression approach described here is by far the most efficient cloning by phenotypic correction system known today.
A novel complementary deoxyribonucleic acid (DNA) expression vector has been constructed. Reconstruction experiments have shown that this system is approximately 100 times more efficient than classical vector systems described until now. Although the final goal, the cloning of the xeroderma pigmentosum (XP) gene, has not yet been reached, the cloning of the hypoxanthine phosphoribosyltransferase (HPRT) gene from a complementary DNA (cDNA) library by correction of a human mutant cell line, in a small scale experiment, shows that the cDNA expression approach is by far the most efficient cloning by the phenotypic correction system known today.
THE PROJECT DESCRIBED WILL BE PERFORMED IN CLOSE COLLABORATION WITH 3 OTHER GROUPS. THE INTEREST OF THE DEPARTMENT OF MOLECULAR GENETICS WILL BE TWOFOLD:
1.- THE STUDY OF THE INFLUENCE OF KNOW E COLI AND YEAST REPAIR ENZYMES ON THE REPAIR ABILITIES OF MAMMALIAN (HUMAN) CELLS.
2.- THE CHARACTERIZATION OF RADIATION (UV) INDUCIBLE HUMAN GENES. IN CONTRAST TO THE PRECEDING CONTRACT PERIOD THE EMPHASIS WILL BE PUT ON THE SECOND PART OF THE PROJECT. THE FIRST PART OF THE PROPOSED PROGRAMM INITIATED 4 YEARS AGO, WILL BE COMPLETED DURING THE COMING CONTRACT PERIOD.
THE FOLLOWING EXPERIMENTS WILL BE CONTINUED OR INITIATED:
AD 1.- TRANSFECTION OF PLASMIDS HARBOURING THE UVRA, B AND C GENES MODIFIED BY MAMMALIAN REGULATORY ELEMENTS TO HUMAN OR RODENT REPAIR DEFICIENT CELL-LINES, CORRECTION STUDIES.
MICROINJECTION OF PURIFIED UVR PROTEINS INTO HUMAN XERODERMA PIGMENTOSUM CELL-LINES. UNSCHEDULED DNA SYNTHESIS (UDS) MEASUREMENTS. EXPRESSION OF UVR GENES IN SACCHAROMYCES CEREVISIAE. COMPLEMENTATION OF KNOWN YEAST RAD-MUTATIONS. CLONING OF YEAST RAD GENES. TRANSFECTION TO HUMAN CELLS VIA MAMMALIAN SHUTTLE VECTORS.
AD2.- IDENTIFICATION OF UV INDUCIBLE GENES IN HUMAN KERATINOCYTES BY DIFFERENTIAL SCREENING OF C-DNA CLONES. (DURING THE LAST CONTRACT PERIOD SEVERAL UV INDUCIBLE GENES HAVE ALREADY BEEN CLONED). STUDY OF THE INDUCTION KINETICS BY NORTHERN BLOTTING
STUDY OF THE INDUCIBILITY OF THESE GENES IN CELL-TYPES OTHER THAN KERATINOCYTES (E G FIBROBLASTS).
INDUCIBILITY BY AGENTS DIFFERENT THAN UV (TPA, MITOMYCIN, INTERFERON, ETC...)
CHARACTERIZATION OF COMPLETE C-DNA CLONES BY DNA SEQUENCING. COMPARISON WITH GENE DATA BANKS.
IDENTIFICATION OF THE CORRESPONDING GENES IN A GENOMIC COSMID LIBRARY.
INTRODUCTION OF COMPLETE C-DNAS INTO MAMMALIAN EXPRESSION VECTORS (NORMAL OR ANTI-ORIENTATION). EFFECT ON SURVIVAL AFTER UV-IRRADIATION.
IDENTIFICATION OF THE REGULATORY ELEMENTS OF THOSE GENES BY DELETION MAPPING OR SITE DIRECTED MUTAGENESIS.
EXPERIMENTS WILL BE PERFORMED IN ORDER TO DETERMINE WHETHER HOMOLOGOUS GENES ALSO EXIST IN YEAST. IF SUCH GENES ARE PRESENT IT WILL BE POSSIBLE TO CREATE YEAST MUTANTS IN THESE LOCI BY RECOMBINATIONAL INACTIVATION. SUCH EXPERIMENTS MIGHT BE NECESSARY IN ORDER TO DETERMINE THE ACTUAL FUNCTION OF THE HUMAN GENES.