Molecular cloning and chromosomal localization of DNA sequences associated with a human DNA repair gene.

Rubin, Jaime S.; Prideaux, V.; Willard, H.F.; Dulhanty, Ann M.; Whitmore, G.F.; Bernstein, Alan

doi:10.1128/mcb.5.2.398

Cited by 46 publications

(13 citation statements)

References 32 publications

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“…Human chromosomes (9,21) and specific human DNA sequences (16,17,22) 6,1986 human chromosomes or DNA sequences that correct repair deficiencies in hamster cells have not yet been found to correct repair deficiencies in human cells (20).…”

Section: Resultsmentioning

confidence: 99%

Repair-deficient xeroderma pigmentosum cells made UV light resistant by fusion with X-ray-inactivated Chinese hamster cells.

Karentz¹,

Cleaver²

1986

Mol. Cell. Biol.

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Xeroderma pigmentosum (XP) is an inherited human disease caused by a malfunction in the dimer excision step of DNA repair. The disease is manifested as an extreme sensitivity to UV light resulting in the development of skin cancers and, in some cases, neurological disorders. At least nine complementation groups have been identified, indicating that the disease comprises a variety of mutations at several genetic loci (2, 5). The genetic basis of the physiological defect of XP has not yet been characterized. Neither genes nor gene products have been identified, and it is not known whether the deficiency is due to mutations in structural or regulatory aspects of DNA repair.Introduction of isolated DNA into XP cells by standard DNA transfection methods has thus far been unsuccessful in introducing genes that code for UV resistance, presenting a major obstacle to the cloning of genetic information that can complement the deficiency of XP (12,18). In contrast, the introduction of human DNA into repair-deficient hamster cells has resulted in the identification of DNA repair loci not represented in the XP complementation groups (4,16,21,22). We used a method of fusing X-irradiated Chinese hamster ovary (CHO) cells with XP cells so that fragments of CHO chromosomes, which appear to support DNA repair, were transferred into the human cells. This report presents the initial work of establishing stable UV-resistant, repaircompetent hybrid cell lines, which will then be used to clone hamster DNA sequences that correct the repair defect of XP cells. MATERIALS AND METHODSCell lines. Normal human fibroblasts (1508), simian virus 40-transformed normal human fibroblasts (A13-6F and GM637), simian virus 40-transformed XP group A fibroblasts (XP12RO), and Chinese hamster ovary (CHO) cells (AA8) were grown in Eagle minimal essential medium with 10% fetal calf serum.Cell fusion. The cell fusion procedure was similar to that described by Cirullo et al. (1) and was adopted because it * Corresponding author. predisposes hybrids to lose hamster chromosomes rather than human chromosomes. CHO cells were exposed to a lethal dose of X rays (50 Gy, 10 Gy/min), mixed with approximately equal numbers of unirradiated simian virus 40-transformed XP fibroblasts, and dispersed into 100-mm dishes. After 5 h at 37°C, cell fusion was mediated by exposure for 30 s to 40% polyethylene glycol 1000 and 10% dimethyl sulfoxide in minimal essential medium, followed by three rinses with 10% dimethyl sulfoxide in minimal essential medium. Complete culture medium was added after the final rinse, and cells were incubated overnight. To select for hybrids that contained functional CHO DNA repair genes and to eliminate unfused XP cells, we irradiated cells with 13 J of UV light per m2 (254 nm, 1.3 J/m2 per s) once a day for 3 consecutive days. At this dosage, XP12RO cell survival is 5 x 10-3, as determined by colony formation. Cultures were incubated for 2 to 3 weeks until colonies developed, and colonies on each plate were pooled to establish primary hybrid cell l...

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Section: Resultsmentioning

confidence: 99%

Repair-deficient xeroderma pigmentosum cells made UV light resistant by fusion with X-ray-inactivated Chinese hamster cells.

Karentz¹,

Cleaver²

1986

Mol. Cell. Biol.

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“…In mammalian cells, the situation is even more complex. Repair deficiency has been recognized in a series of human genetic diseases and also in a set of mouse and hamster cell mutants selected in v i t r~.~-~ As will be explained in more detail below, the human diseases and rodent cell mutants together demonstrate that up to 15 genes may be directly involved in the repair of the same photoproducts (pyrimidine dimers) that T4 can repair with a single gene. This contrast makes one wonder which are the more 'advanced' or ' efficient' organisms.…”

Section: Introductionmentioning

confidence: 98%

DNA repair in man: Regulation by a multigene family and association with human disease

Cleaver

Karentz

1987

BioEssays

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“…In this way a human gene, which corrects the hypersensitivity to u.v. and mitomycin C (MMC) and the incision defect of a CHO cell line, has been identified, partly characterized and assigned to chromosome 19 (Westerveld et al 1984;Rubin et al 1985).…”

Section: Conclusion and Future Prospectsmentioning

confidence: 99%

DNA Maintenance and its Relation to Human Pathology

Giannelli

1986

Journal of Cell Science

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Molecular cloning and chromosomal localization of DNA sequences associated with a human DNA repair gene.

Cited by 46 publications

References 32 publications

Repair-deficient xeroderma pigmentosum cells made UV light resistant by fusion with X-ray-inactivated Chinese hamster cells.

Repair-deficient xeroderma pigmentosum cells made UV light resistant by fusion with X-ray-inactivated Chinese hamster cells.

DNA repair in man: Regulation by a multigene family and association with human disease

DNA Maintenance and its Relation to Human Pathology

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