TY - JOUR
T1 - Spontaneous and restriction enzyme-induced chromosomal recombination in mammalian cells
AU - Godwin, Alan R.
AU - Bollag, Roni J.
AU - Christie, Donna Marie
AU - Liskay, R. Michael
PY - 1994/12/20
Y1 - 1994/12/20
N2 - 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 PaeR71 enzyme, an isoschizomer of Xho I, into these hybrids. A priori, these breaks can be repaired either by recombination from the homolog 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/aminopter- in/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.
AB - 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 PaeR71 enzyme, an isoschizomer of Xho I, into these hybrids. A priori, these breaks can be repaired either by recombination from the homolog 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/aminopter- in/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.
KW - double- strand break
KW - interchromosomal recombination
KW - intrachromosomal recombination
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U2 - 10.1073/pnas.91.26.12554
DO - 10.1073/pnas.91.26.12554
M3 - Article
C2 - 7809076
AN - SCOPUS:0028577296
SN - 0027-8424
VL - 91
SP - 12554
EP - 12558
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 26
ER -