Spontaneous and restriction enzyme-induced chromosomal recombination in mammalian cells.
We have derived Chinese hamster ovary (CHO) cell hybrids containing herpes simplex virus thymidine kinase (tk) heteroalleles for the study of spontaneous and restriction enzyme-induced interchromosomal recombination. These lines allowed us to make a direct comparison between spontaneous intrachromosomal and interchromosomal recombination using the same tk heteroalleles at the same genomic insertion site. We find that the frequency of interchromosomal recombination is less by a factor of at least 5000 than that of intrachromosomal recombination. Our results with mammalian cells differ markedly from results with Saccharomyces cerevisiae, with which similar studies typically give only a 10-to 30-fold difference. Next, to inquire into the fate of double-strand breaks at either of the two different Xho I linker insertion mutations, we electroporated PaeR7I enzyme, an isoschizomer of Xho I, into these hybrids. A priori, these breaks can be repaired either by recombination from the homology or by end-joining. Despite a predicted bias against recovering end-joining products in our system, all cells characterized by enzyme-induced resistance to hypoxanthine/aminopterin/thymidine were, in fact, due to nonhomologous recombination or end-joining. These results are in agreement with other studies that used extrachromosomal sequences to examine the relative efficiencies of end-joining and homologous recombination in mammalian cells, but are in sharp contrast to results of analogous studies in S. cerevisiae, wherein only products of homologous events are detected.
Godwin
A R
AR
Department of Genetics, Yale University School of Medicine, New Haven, CT 06510.
Bollag
R J
RJ
Christie
D M
DM
Liskay
R M
RM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D015183:Restriction Mapping; D013937:Thymidine Kinase / Q000235:genetics
D013937:Thymidine Kinase; C034510:CTCGAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific