Inducible expression and cytogenetic effects of the EcoRI restriction endonuclease in Chinese hamster ovary cells.
The cytogenetic endpoints sister chromatid exchange (SCE) and chromosome aberrations are widely used as indicators of DNA damage induced by mutagenic carcinogens. Chromosome aberrations appear to result directly from DNA double-strand breaks, but the lesion(s) giving rise to SCE formation remains unknown. Most compounds that induce SCEs induce a spectrum of lesions in DNA. To investigate the role of double-strand breakage in SCE formation, we constructed a plasmid that gives rise to one specific lesion, a staggered-end ("cohesive") DNA double-strand break. This plasmid, designated pMENs, contains a selectable marker, neo, which is a bacterial gene for neomycin resistance, and the coding sequence for the bacterial restriction endonuclease EcoRI attached to the mouse metallothionein gene promoter. EcoRI recognizes G decreases AATTC sequences in DNA and makes DNA double-strand breaks with four nucleotides overhanging as staggered ends. Cells transfected with pMENS were resistant to the antibiotic G418 and contained an integrated copy of the EcoRI gene, detectable by DNA filter hybridization. The addition of the heavy metal CdSO4 resulted in the intracellular production of EcoRI, as measured by an anti-EcoRI antibody. Cytogenetic analysis after the addition of CdSO4 indicated a dramatic increase in the frequency of chromosome aberrations but very little effect on SCE frequency. Although there was some intercellular heterogeneity, these results confirm that DNA double-strand breaks do result in chromosome aberrations but that these breaks are not sufficient to give rise to SCE formation.
Morgan
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WF
Laboratory of Radiobiology and Environmental Health, University of California, San Francisco 94143.
Fero
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ML
Land
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MC
Winegar
R A
RA
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002869:Chromosome Aberrations; D006224:Cricetinae; D004249:DNA Damage; D015246:Deoxyribonuclease EcoRI / Q000235:genetics*; D004790:Enzyme Induction; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D008668:Metallothionein / Q000235:genetics; D011994:Recombinant Proteins; D012854:Sister Chromatid Exchange
D011994:Recombinant Proteins; D008668:Metallothionein; D015246:Deoxyribonuclease EcoRI