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

R. M. Liskay, J. L. Stachelek
1986 Proceedings of the National Academy of Sciences of the United States of America  
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
more » ... istent 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). We observed that events consistent with both nonreciprocal and reciprocal exchange could occur in the same parent line containing a single gene duplication (4). Events consistent with nonreciprocal information transfer, or gene conversion, were found to make up a majority (80-85%) of the total recombination events (4). In light of these findings, we have undertaken a detailed inquiry into the mechanism of gene conversion between artificially created gene duplications in mammalian cells. Studies such as these should be of interest both in terms of understanding how repeated sequences interact and in terms ofproviding information on homologous recombination between chromosomal sequences. In fungi, gene conversion can act in a single event on two or more markers-that is, the markers can be "coconverted." Coconversion of separate markers suggests that conversion events in fungi involve regions of DNA more extensive than simply a single site within a gene (for a review see ref. 5). In this report, we present experiments designed to answer several related questions about the nature of information transfer between duplicated Herpes virus thymidine kinase gene sequences in mammalian cells. Do the events 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 METHODS Cell 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 were inserted on a 2.4-kbp fragment into the unique BamHI site of the pSV2-neo plasmid after attachment of synthetic BamHI linkers (New England Biolabs). The 1.2-kbp "donor" fragment was originally isolated from the wild-type TK gene from strain 101 of HSV type 1 by a HincII/Sma I double digestion, and was inserted into the HindIII site of our pSV2-neo plasmid derivative (4) by using synthetic HindIII linkers (New England Biolabs). This fragment encodes most of the coding region but lacks the normal HSV TK promotor region and polyadenylylation signals.
doi:10.1073/pnas.83.6.1802 pmid:3006074 pmcid:PMC323172 fatcat:ow6gs6c2kngsrkrjyigi4subhe