Clastogenicity of PvuII and EcoRI in electroporated CHO cells assayed by metaphase chromosomal aberrations and by micronuclei using the cytokinesis-block technique.
The clastogenicity of two restriction endonucleases with almost equal cutting frequencies: PvuII, generating blunt-ended DNA double-strand breaks (dsb) and EcoRI, generating cohesive-ended dsb, has been measured after treatment of electroporated CHO cells with these enzymes. Chromosome damage was assessed by the micronucleus cytokinesis-block technique, and for certain electroporation voltages by analysis of metaphase preparations. As has been found in previous studies, PvuII was found to be more effective in causing chromosomal damage than EcoRI, indicating the greater importance of blunt-ended dsb in chromosome aberration induction. These findings also validate the use of the micronucleus cytokinesis-block technique for evaluating chromosomal damage from restriction endonucleases. The results show a biphasic induction of micronuclei in binucleate cells as a function of time after treatment. This pattern is interpreted as indicating variable sensitivity of cells to restriction endonucleases at different stages of the cell cycle. The micronucleus data show that late collection times (40-48 h after treatment) give higher frequencies than short times. Both micronucleus and metaphase aberration data indicate that voltages in excess of 260 V are more efficient in porating cells than lower voltages and, as a result, lower restriction endonuclease concentrations could be used.
Moses
S A
SA
Department of Biology and Preclinical Medicine, University of St Andrews, Fife, UK.
Christie
A F
AF
Bryant
P E
PE
D000818:Animals; D002460:Cell Line; D002869:Chromosome Aberrations; D006224:Cricetinae; D003412:Cricetulus; D015246:Deoxyribonuclease EcoRI / Q000494:pharmacology*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004558:Electric Stimulation; D005260:Female; D008677:Metaphase / Q000187:drug effects; D015162:Micronucleus Tests; D009152:Mutagenicity Tests; D009153:Mutagens; D010053:Ovary
D009153:Mutagens; D015246:Deoxyribonuclease EcoRI; C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific