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DNA repair genes of mammalian cells
Abstract:
In the Chinese hamster ovary (CHO) cell line, various mutations affecting DNA repair have been obtained. Mutants that belong to 5 genetic complementation groups for ultraviolet (UV) sensitivity and resemble the cells from individuals having the cancer-prone genetic disorder xeroderma pigmentosum (XP) were previously identified. Each mutant is defective in the incision step of nucleotide excision repair and hypersensitive to bulky DNA lesions. These UV mutants can be divided into two subgroups; only Groups 2 and 4 are extremely sensitive to mitomycin C and other DNA cross-linking agents. The clear-cut phenotypes of the CHO mutants have allowed us to construct hybrid cells by fusion with human lymphocytes and thereby identify which human chromosomes carry genes that correct the CHO mutations. The first two mutations analyzed, UV20 (excision-repair deficient; UV Group 2) and EM9, which has a very high frequency of sister chromatid exchange (SCE), are both corrected by chromosome 19. Efforts are underway to isolate complementing repair genes by DNA-mediated gene transfer. The human gene that corrects mutant EM9 and the hamster gene that corrects UV135 (UV Group 5) have been introduced by cotransfer of genomic DNA and the dominant selectable marker gpt (guanine phosphoribosyltransferase) gene. In each case, the DNA repair function was co-selected based on resistance to 5-chlorodeoxyuridine (CldUrd) or repeated UV irradiation, respectively. The presence of a functional human repair gene in the EM9 transformants is shown by the presence of common human DNA sequences on some fragments produced by restriction enzyme cleavage. In UV135, transfer of a repair gene is indicated by a colony distribution containing "jackpots" and by instability of the resistant phenotype.
Insights
Chinese hamster ovary (CHO) cells with DNA repair defects were studied. Human chromosome 19 corrects mutations in UV-sensitive CHO cells, identifying genes involved in DNA repair and xeroderma pigmentosum (XP).
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Chinese hamster ovary (CHO) cell lines with mutations affecting DNA repair were previously identified.
- These mutants exhibit sensitivity to ultraviolet (UV) radiation and resemble xeroderma pigmentosum (XP) cells.
- Mutants are defective in nucleotide excision repair and sensitive to bulky DNA lesions and cross-linking agents.
Purpose of the Study:
- To identify human chromosomes carrying genes that correct DNA repair defects in CHO mutants.
- To investigate the genetic basis of DNA repair and its relation to human genetic disorders like XP.
- To isolate and characterize DNA repair genes through gene transfer techniques.
Main Methods:
- Fusion of mutant CHO cells with human lymphocytes to create hybrid cells.
- Genetic complementation analysis to identify correcting human chromosomes.
- DNA-mediated gene transfer using selectable markers (gpt) and resistance assays (CldUrd, UV irradiation).
Main Results:
- Human chromosome 19 was found to correct two CHO mutations: UV20 (excision-repair deficient) and EM9 (high sister chromatid exchange frequency).
- DNA repair genes were successfully introduced into EM9 and UV135 (UV Group 5) mutant cells via gene transfer.
- Presence of functional human repair genes in transformants was confirmed by DNA sequence analysis and phenotypic changes.
Conclusions:
- Human chromosome 19 harbors genes crucial for correcting specific DNA repair deficiencies in CHO cells.
- This study provides a framework for identifying and isolating human DNA repair genes.
- The findings contribute to understanding the molecular mechanisms underlying DNA repair and related genetic disorders.