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In this report we present an experimental scheme that facilitates the study of homologous recombination between closely linked genes in cultured mammalian cells. Two different Xho I linker insertion mutants of the herpes simplex virus type 1 thymidine kinase (HTK) gene were introduced into mouse LTK- cells as direct repeats on a plasmid carrying a dominant selectable marker. Following stabilization of these sequences in the recipient cell, selection for TK+ was applied to detect recombinational events between different TK- genes. TK+ segregants were observed at a frequency of 10(-4)-10(-5) in lines harboring both mutant genes. Control lines carrying only one type of mutant HTK gene yielded TK+ cells at frequencies of 10(-7) or less. Physical analysis of the TK+ segregants has revealed the presence of an apparently normal HTK gene that is resistant to Xho l endonuclease digestion in each TK+ line examined. Analyses of the TK gene pairs before and after recombination suggest that at least 50% of the recombinants are the result of nonreciprocal exchanges of genetic information, or gene conversion events.

Thymidine kinase-deficient mouse L cells have been transformed with plasmid DNAs carrying 8-base-pair Xho I linker insertion mutations in the coding region of the herpes simplex virus type 1 thymidine kinase gene. When the mutant plasmids are introduced individually into LTK-cells, transformation efficiencies are greatly reduced relative to the wild type. However, when two mutant plasmids are cotransferred into the same LTK-recipients, significantly higher frequencies of transformation are observed (30-300 times). Here we demonstrate the usefulness of linker insertions for the study of homologous recombination in detecting the existence of normal thymidine kinase gene sequences (i.e., sequences lacking the insertions after recombination are substantiated by DNADNA hybridization). In addition, the frequencies of recombination in the various "crosses" are consistent with the known positions of the mutations.Until recently, genetic and molecular experiments on homologous recombination in cultured mammalian cells have been limited to viral systems such as simian virus 40 and adenovirus (1)(2)(3)(4)(5). In these studies the presence of viral recombination function is not precluded, although the design of certain experiments (3-5) makes this problem seem unlikely. The development of DNA-mediated gene transfer (6, 7) and recombinant DNA techniques provides a means for the study of homologous recombination between defined selectable DNA sequences in mammalian cells free of potential viral-encoded recombination functions. While our studies were in progress, several reports appeared presenting findings that are consistent with homologous recombination after introduction of different plasmid DNA molecules into mouse cells (8)(9)(10). One of these studies used microinjection of physically marked sequences (8). Two other investigations applied selective systems (9, 10), similar to the one described here, in which mutant genes introduced into mammalian cells were shown to recombine to yield wild-type genes. In addition, when truncated H2 mouse genes were introduced into mouse cells, complete H2 gene products were observed at low frequency (11). The authors state that these findings are consistent, at least, with homologous recombination between incoming and resident H2 sequences. However, in this latter study, evidence for recombination at the DNA level is at present lacking.In this communication, we present evidence for homologous recombination between various mutant genes of herpes simplex virus type 1 thymidine kinase (HTK). These mutants result from insertion of 8-base-pair (bp) synthetic oligonucleotide linkers that specify a unique recognition site for the restriction endonuclease Xho I. These mutations represent subtle alterations of the HTK coding region and yet provide an easy means for distinguishing between mutant and wild-type genes. As expected, when defective HTK genes bearing frameshift mutations are introduced into thymidine kinase (TK)-deficient mouse L cells, transformation frequencies are considera...

The hexokinase A (HKA) and hexokinase B (HKB) genes of Saccharomyces cerevisiae have been cloned from a library of yeast genomic DNA. Using an in vitro glucose phosphorylation assay, the HKB gene was located on a plasmid carrying a 13.6 kb fragment of yeast DNA. After subcloning the relevant restriction fragments, the nucleotide sequence of the HKB gene was determined. Using this information, we were able to locate the HKA gene on a plasmid carrying this gene, which we then sequenced. Approximately 43% of the amino acid sequence of HKB was determined directly from 24 tryptic peptides. The results are in complete agreement with those derived from the DNA sequence and are consistent with the results of x-ray crystallography. Comparison of the amino acid sequences of HKA and HKB show that 378 out of 485 residues are identical. The 5' flanking region of the A gene contains nucleotide sequences expected for genes that are expressed at relatively high levels in yeast. The 24 base pair hyphenated palindrome at the 3' end of the HKB gene may be a site for termination of transcription of this gene.

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...