The CHO UV-sensitive mutants UV24 and UV135 (complementation groups 3 and 5, respectively) are defective in nucleotide excision repair. After fusing each mutant with human lymphocytes, resistant hybrid clones showing genetic complementation were isolated by repeated exposure to UV radiation. Using a combination of isozyme markers, DNA probes, and cytogenetic methods to analyze the primary hybrids and their subclones, correction of the repair defect was shown to be correlated with the presence of a specific human chromosome in each case. Chromosome 2 corrected UV24, and the gene responsible was designated ERCC3. Line UV135 was corrected by human chromosome 13 and the gene designated ERCC5. The UV-sensitive mouse cell line, Q31, was shown not to complement UV135 and thus appears to be mutated in the same genetic locus (homologous to ERCC5) as UV135. Breakage of complementing chromosomes with retention of the genes correcting repair defects allowed the following provisional assignments: regional localization of ERCC5 to 13q14-q34, exclusion of ERCC3 from the region of chromosome 2 distal to p23, and relief of the ambiguity of ACP1 assignment (2p23 or 2p25) to 2p23 proximal to MDH1.
Mitomycin C (MMC)-resistant interspecific somatic cell hybrids made between human cells and the MMC-sensitive, Chinese hamster ovary (CHO) excision repair-deficient UV41 cells generally contained human chromosome 16, while other human chromosomes were randomly present. MMC-sensitive and -resistant subclones were isolated from resistant clones, and resistance generally segregated concordantly with human chromosome 16 markers. UV radiation survival analysis of subclones indicated that MMC and UV resistance were correlated. Therefore, the complementing gene, Excision Repair Cross Complementing 4 (ERCC4), was assigned to human chromosome 16. Complementation of UV41 by human cells derived from patients with xeroderma pigmentosum groups A, C, D and F excluded ERCC4 from involvement in those disease syndromes. Resistant hybrids containing only portions of chromosome 16 were identified by the lack of concordance of multiple chromosome 16 markers. When such hybrids were used as a source of probe for fluorescent in situ hybridization onto normal human metaphases, the only region of chromosome 16 identified as being consistently present was 16p13.1-p13.3. Genetic marker analysis of informative hybrids with mapped probes refined the position of ERCC4 to 16p13.13-p13.2 and allowed the following order of markers within the region to be established: pter--(PRM1, D16S215)-D16S213-D16S53-(D16S214,ERCC4) -D16S3-D16S96-cen.
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