Evidence for intrachromosomal gene conversion in cultured mouse cells

Abstract

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.

Extrachromosomal and chromosomal gene conversion in mammalian cells.

“…The average gene conversion frequency (Table 3) was approximately fivefold higher than the average single-crossover frequency we have measured earlier (20). This result is consistent with those of Liskay et al (10,11), who found that gene conversion between defective tk genes occurred more frequently than reciprocal recombination. The gene conversion frequencies in Table 3 were 16-to 67-fold higher than the background reversion frequency of the neoX gene.…”

“…Recent experiments on chromosomal homologous recombination in a number of laboratories have relied on the use of either overlapping truncated fragments or mutant copies of the herpesvirus thymidine kinase (tk) (9)(10)(11) or neomycin resistance (neo) gene (16,20) to detect recombination events which restore one or more copies of the selected marker gene. Although these studies have measured recombination frequencies and characterized various reciprocal and nonreciprocal intrachromosomal recombination events, attempts to estimate the true frequencies of these events have been complicated by the ability of the recombination substrates to undergo both reciprocal and nonreciprocal recombination.…”

Genetic Effects of Dioxins in the Spot Test with Mice

“…A possible explanation comes from experiments using yeast, in which the probability that a heterozygous recessive gene becomes homozygous is two orders of magnitude higher for nonreciprocal recombination than for gene mutation (22,23). Also, observations in cultured mouse cells showed that the frequency of nonreciprocal recombination (gene conversion) between repetitive genes is several orders of magni- tude higher than the frequency of gene mutation (24). With reciprocal recombination, a single event is sufficient to result in the expression of all recessive mutations of a chromosomal segment, whereas with gene mutations several single events would be needed to achieve a similar effect.…”

Transfection of mouse fibroblast cells with a promoterless herpes simplex virus thymidine kinase gene: number of integrated gene copies and structure of single and amplified gene sequences.

“…Previous work has shown that mammalian cells in culture have a highly efficient machinery which promotes homologous recombination between exogeneous DNA molecules (10,(16)(17)(18). Recombination should therefore occur between the circular plasmid molecules added to the cells.…”