Information transfer between duplicated chromosomal sequences in mammalian cells involves contiguous regions of DNA.

Abstract

We have investigated the nature of Information transfer that appears to occur nonreciprocally between duplicated chromosomal sequences in cultured mouse L cells. We have studied gene conversion between two different defective thymidine kinase genes derived from two closely related strains of type 1 herpes simplex virus and that share a silent restriction site polymorphism. Our results demonstrate that this silent site can be coconverted along with the selected mutant sites. The findings are consistent with a mechanism of gene conversion that involves contiguous blocks of DNA differing in length, position, or both. An additional firding is that the products of coconversion events involving the silent site are unequally recovered although the rates of conversion observed at four different selected sites are similar.Gene conversion, a nonreciprocal form of information transfer, was originally described in fungi some 30 years ago. In 1970, Edelman and Gally suggested that a mechanism similar to gene conversion could be active in maintaining sequence homogeneity in mammalian multigene families (1). Since then, numerous DNA sequence comparisons ofrelated genes have reinforced the idea that genetic information could be transferred between related mammalian genes, at least when viewed on an evolutionary time scale. In one early study (2), the process responsible for such transfer of information was proposed to be gene conversion.We have previously described studies designed to systematically examine homologous recombination events occurring between a pair of genes lying in close proximity on the same chromosome in cultured mammalian cells (3,4 involve regions of DNA as in fungi, or might the process act only on the mutation itself? Assuming that regions are involved, is information transferred as a contiguous block or in a discontinuous manner so that transfer is "patchy"? Are these tracts of conversion fixed or can different regions of information be transferred?In brief, the conversion events studied appear to involve transfer of a contiguous block of information. In addition, the findings indicate that conversion tracts between duplicated elements sharing 1.2 kilobase pairs (kbp) of homologous sequence can involve as much as 358 bp of information, and that these tracts are of variable length, position, or both. Finally, another outcome of these studies is that products of coconversion events are unequally recovered, although the rates of conversion per se are similar within the duplication. MATERIALS AND METHODSCell Culture and Transformation. Mouse L cells lacking thymidine kinase (TK) (LTK-cells) were cultured and transformed with DNA as previously described (3, 4).Plasmid Construction. The plasmid vector is a derivative of pSV2-neo (6) with restriction site alterations as previously described (4). Xho I linker insertion mutations of the herpes simplex virus (HSV) type 1 (strain F) TK gene were a generous gift from David Zipser and Jesse Kwoh. The four "recipient" Xho I mutant TK genes used in these studies...

Dependence of intrachromosomal recombination in mammalian cells on uninterrupted homology.

“…We argue that the reduction in recombination was not due to such a sequence-specific effect but rather was due to a general heterology effect that resulted in an insufficient recombination target size, as follows. (i) Previously, we observed near equal recombination rates at five different sites within the HSV F tk gene (7,9,10), thereby suggesting that there are no recombination hotspots within the tk gene. (ii) The rate of recombination seen with sequences contained in construct pF-F in this study is in agreement with the rate measured in a previous study (7) involving a similarly sized fragment of the strain F tk gene mapping immediately adjacent to, but not overlapping, the interval of the tk gene studied here.…”

“…Fluctuation analyses were done as described previously (9). Ten independent subclones were used in the analysis of each cell line.…”

High-frequency germ line gene conversion in transgenic mice.

“…However, this would be possible only if the hybrid contained the unmutated (donor) version of the recipient frameshift mutation, which depends on the location of the initial crossover following microinjection. Regardless, data from other systems indicate that gene conversion would still be the predominant mechanism responsible for lacZ correction in these two lines (23,25,26,30).…”

“…CELL. BIOL. on May 10, 2018 by guest http://mcb.asm.org/ Downloaded from selecting for gene conversion has been used successfully in mammalian tissue culture systems (28,30). Male mice carrying this construct should transcribe the recipient gene specifically in haploid germ cells but will not produce functional lacZ unless a spontaneous mutation or recombination event corrects the frameshift mutation.…”

“…Similar results were obtained at the HIS4 locus (22,23,25), leading to a proposal that a mechanism exists to suppress intrachromosomal reciprocal exchanges (23). Studies of recombination in mammalian cells also show that gene conversion is five times more prevalent than reciprocal recombination for intrachromosomal gene corrections (30,31,50). Since the percentage of blue spermatids in these lines is similar to that in the unrearranged lines, it is likely that the predominant recombination mechanism for lacZ correction was gene conversion.…”