The effects of insertions on mammalian intrachromosomal recombination.

Genetics ◽  
1994 ◽  
Vol 136 (2) ◽  
pp. 607-617
Author(s):  
A R Godwin ◽  
R M Liskay
Keyword(s):  
Gene Conversion ◽  
Direct Repeat ◽  
Insertion Mutation ◽  
Inverted Repeats ◽  
Crossing Over ◽  
Intrachromosomal Recombination ◽  
Mouse Cells ◽  
Simplex Virus ◽  
Insertion Mutations ◽  
Gene Conversions

Abstract We examined the effects of insertion mutations on intrachromosomal recombination. A series of mouse L cell lines carrying mutant herpes simplex virus thymidine kinase (tk) heteroalleles was generated; these lines differed in the nature of their insertion mutations. In direct repeat lines with different large insertions in each gene, there was a 20-fold drop in gene conversion rate and only a five-fold drop in crossover rate relative to the analogous rates in lines with small insertions in each gene. Surprisingly, in direct repeat lines carrying the same large insertion in each gene, there was a larger drop in both types of recombination. When intrachromosomal recombination between inverted repeat tk genes with different large insertions was examined, we found that the rate of gene conversion dropped five-fold relative to small insertions, while the rate of crossing over was unaffected. The differential effects on conversion and crossing over imply that gene conversion is more sensitive to insertion mutation size. Finally, the fraction of gene conversions associated with a crossover increased from 2% for inverted repeats with small insertions to 18% for inverted repeats with large insertions. One interpretation of this finding is that during intrachromosomal recombination in mouse cells long conversion tracts are more often associated with crossing over.

Conservative intrachromosomal recombination between inverted repeats in mouse cells: association between reciprocal exchange and gene conversion.

Genetics ◽  
1988 ◽  
Vol 119 (1) ◽  
pp. 161-169
Author(s):  
R J Bollag ◽  
R M Liskay
Keyword(s):  
Gene Conversion ◽  
Mammalian Cells ◽  
Inverted Repeats ◽  
Intrachromosomal Recombination ◽  
Reciprocal Exchange ◽  
Sequence Inversion ◽  
Mouse Cells ◽  
Simplex Virus ◽  
The Relationship ◽  
Recombination Gene

Abstract Recombination in mammalian cells is thought to involve both reciprocal and nonreciprocal modes of exchange, although rigorous proof is lacking due to the inability to recover all products of an exchange. To investigate further the relationship between these modes of exchange, we have analyzed intrachromosomal recombination between duplicated herpes simplex virus thymidine kinase (HSV tk) mutant alleles arranged as inverted repeats in cultured mouse L cells. In crosses between inverted repeats, a single intrachromatid reciprocal exchange leads to inversion of the sequence between the crossover sites and recovery of both genes involved in the event. The majority of recombinant products do not display such inversion and are thus consistent with a nonreciprocal mode of recombination (gene conversion). The remaining products display the sequence inversion predicted for intrachromatid reciprocal exchange. In light of the fact that intrachromatid exchanges occur, the rarity of intrachromatid double reciprocal exchanges strengthens the interpretation that the majority of events in this and previous investigations involve gene conversion. Furthermore, in accord with prediction, one-third of the reciprocal recombinants (inversions) display associated gene conversion. This association suggests that reciprocal and nonreciprocal modes of exchange are mechanistically related in mammalian cells. Finally, the occurrence of inversion recombinants suggests that intrachromosomal recombination can be a conservative (nondestructive) process.

Direct-repeat analysis of chromatid interactions during intrachromosomal recombination in mouse cells

1991 ◽  
Vol 11 (10) ◽  
pp. 4839-4845
Author(s):  
R J Bollag ◽  
R M Liskay
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Direct Repeat ◽  
Direct Repeats ◽  
Heteroduplex Dna ◽  
Repeat Analysis ◽  
Intrachromosomal Recombination ◽  
Sister Chromatids ◽  
Mouse Cells ◽  
Simplex Virus

Homologous intrachromosomal recombination between linked genes can involve interactions that are either intramolecular (intrachromatid) or intermolecular (sister chromatid). To assess the relative proportions of chromatid interactions, we report studies of intrachromosomal recombination in mouse L cells containing herpes simplex virus thymidine kinase genes in two alternative configurations of direct repeats. By comparing products of reciprocal exchanges between these two configurations, we conclude that the majority of interactions that give rise to crossover products involve unequally paired sister chromatids after DNA replication. Analyses of an additional class of crossover products that involve discontinuous associated gene conversion suggest that these recombination events involve a heteroduplex DNA intermediate.

scholarly journals Direct-repeat analysis of chromatid interactions during intrachromosomal recombination in mouse cells.

1991 ◽  
Vol 11 (10) ◽  
pp. 4839-4845 ◽  
Author(s):  
R J Bollag ◽  
R M Liskay
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Direct Repeat ◽  
Direct Repeats ◽  
Heteroduplex Dna ◽  
Repeat Analysis ◽  
Intrachromosomal Recombination ◽  
Sister Chromatids ◽  
Mouse Cells ◽  
Simplex Virus

Homologous intrachromosomal recombination between linked genes can involve interactions that are either intramolecular (intrachromatid) or intermolecular (sister chromatid). To assess the relative proportions of chromatid interactions, we report studies of intrachromosomal recombination in mouse L cells containing herpes simplex virus thymidine kinase genes in two alternative configurations of direct repeats. By comparing products of reciprocal exchanges between these two configurations, we conclude that the majority of interactions that give rise to crossover products involve unequally paired sister chromatids after DNA replication. Analyses of an additional class of crossover products that involve discontinuous associated gene conversion suggest that these recombination events involve a heteroduplex DNA intermediate.

Homologous recombination involving a large heterology in Escherichia coli.

Genetics ◽  
1988 ◽  
Vol 119 (4) ◽  
pp. 759-769
Author(s):  
K Yamamoto ◽  
N Takahashi ◽  
H Yoshikura ◽  
I Kobayashi
Keyword(s):  
Escherichia Coli ◽  
Gene Conversion ◽  
Kanamycin Resistance ◽  
Coli Strain ◽  
The Other ◽  
Reca Gene ◽  
Inverted Repeats ◽  
Crossing Over ◽  
Wild Type ◽  
Parental Allele

Abstract Recombination between two different deletion alleles of a gene (neo) for neomycin and kanamycin resistance was studied in an Escherichia coli sbcA- recB-C- strain. The two homologous regions were in an inverted orientation on the same plasmid molecule. Kanamycin-resistant plasmids were selected and analyzed. The rate of recombination to form kanamycin-resistant plasmids was decreased by mutations in the recE, recF and recJ genes, but was not decreased by a mutation in the recA gene. It was found that these plasmids often possessed one wild-type kanamycin-resistant allele (neo+) while the other neo allele was still in its original (deletion) form. Among kanamycin-resistant plasmids with one wild-type and one parental allele it was often found that the region between the inverted repeats had been flipped (turned around) with respect to sites outside the inverted repeats. These results were interpreted as follows. Gene conversion, analogous to gene conversion in eukaryotic meiosis, is responsible for a unidirectional transfer of information from one neo deletion allele to the other. The flipping of the region between the inverted repeats is interpreted as analogous to the crossing over associated with gene conversion in eukaryotic meiosis. In contrast with a rec+ strain, these products cannot be explained by two rounds of reciprocal crossing over involving a dimeric form as an intermediate. In the accompanying paper we present evidence that gene conversion by double-strand gap repair takes place in the same E. coli strain.

scholarly journals Suppression of Intrachromosomal Gene Conversion in Mammalian Cells by Small Degrees of Sequence Divergence

Genetics ◽  
1999 ◽  
Vol 151 (4) ◽  
pp. 1559-1568 ◽  
Author(s):  
Tamas Lukacsovich ◽  
Alan S Waldman
Keyword(s):  
Herpes Simplex ◽  
Gene Conversion ◽  
Mammalian Cells ◽  
Sequence Divergence ◽  
Insertion Mutation ◽  
Single Nucleotide ◽  
Heteroduplex Dna ◽  
Simplex Virus ◽  
Hsv Thymidine Kinase ◽  
Low Rate

Abstract Pairs of closely linked defective herpes simplex virus (HSV) thymidine kinase (tk) gene sequences exhibiting various nucleotide heterologies were introduced into the genome of mouse Ltk– cells. Recombination events were recovered by selecting for the correction of a 16-bp insertion mutation in one of the tk sequences. We had previously shown that when two tk sequences shared a region of 232 bp of homology, interruption of the homology by two single nucleotide heterologies placed 19 bp apart reduced recombination nearly 20-fold. We now report that either one of the nucleotide heterologies alone reduces recombination only about 2.5-fold, indicating that the original pair of single nucleotide heterologies acted synergistically to inhibit recombination. We tested a variety of pairs of single nucleotide heterologies and determined that they reduced recombination from 7- to 175-fold. Substrates potentially leading to G-G or C-C mispairs in presumptive heteroduplex DNA (hDNA) intermediates displayed a particularly low rate of recombination. Additional experiments suggested that increased sequence divergence causes a shortening of gene conversion tracts. Collectively, our results suggest that suppression of recombination between diverged sequences is mediated via processing of a mispaired hDNA intermediate.

scholarly journals Long Inverted Repeats Are an At-Risk Motif for Recombination in Mammalian Cells

Genetics ◽  
1999 ◽  
Vol 153 (4) ◽  
pp. 1873-1883 ◽  
Author(s):  
Alan S Waldman ◽  
Hiep Tran ◽  
Edie C Goldsmith ◽  
Michael A Resnick
Keyword(s):  
At Risk ◽  
Homologous Recombination ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Direct Repeat ◽  
Fold Increase ◽  
Inverted Repeats ◽  
Sequence Motifs ◽  
Common Motif ◽  
Simplex Virus

Abstract Certain DNA sequence motifs and structures can promote genomic instability. We have explored instability induced in mouse cells by long inverted repeats (LIRs). A cassette was constructed containing a herpes simplex virus thymidine kinase (tk) gene into which was inserted an LIR composed of two inverted copies of a 1.1-kb yeast URA3 gene sequence separated by a 200-bp spacer sequence. The tk gene was introduced into the genome of mouse Ltk− fibroblasts either by itself or in conjunction with a closely linked tk gene that was disrupted by an 8-bp XhoI linker insertion; rates of intrachromosomal homologous recombination between the markers were determined. Recombination between the two tk alleles was stimulated 5-fold by the LIR, as compared to a long direct repeat (LDR) insert, resulting in nearly 10−5 events per cell per generation. Of the tk+ segregants recovered from LIR-containing cell lines, 14% arose from gene conversions that eliminated the LIR, as compared to 3% of the tk+ segregants from LDR cell lines, corresponding to a >20-fold increase in deletions at the LIR hotspot. Thus, an LIR, which is a common motif in mammalian genomes, is at risk for the stimulation of homologous recombination and possibly other genetic rearrangements.

Dependence of intrachromosomal recombination in mammalian cells on uninterrupted homology

1988 ◽  
Vol 8 (12) ◽  
pp. 5350-5357
Author(s):  
A S Waldman ◽  
R M Liskay
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Base Pair ◽  
Mammalian Cells ◽  
Insertion Mutation ◽  
Single Nucleotide ◽  
Intrachromosomal Recombination ◽  
Simplex Virus

Recombination between a 360-base-pair (bp) segment of a wild-type thymidine kinase gene (tk) from each of three different strains (F, MP, and 101) of herpes simplex virus type one and a complete herpes simplex virus type 1 (strain F) tk gene containing an 8-bp insertion mutation was studied. The pairs of tk sequences resided as closely linked repeats within the genome of mouse LTK- cells. The frequency of recombination between sequences exhibiting 232 bp of uninterrupted homology and containing no mismatches other than the insertion mutation was comparable to the frequency of recombination between two sequences exhibiting four additional nucleotide mismatches distributed in such a way to preserve the 232-bp stretch of contiguous homology. In contrast, the placement of only two single-nucleotide mismatches (in addition to the insertion mutation) in such a manner to reduce the longest uninterrupted homology to 134 bp resulted in a 20-fold reduction in recombination. We conclude that the rate of intrachromosomal recombination in mammalian cells is determined by the amount of uninterrupted homology available and not by the total number of mismatches within a given interval of DNA. Furthermore, efficient recombination appears to require between 134 and 232 bp of uninterrupted homology; single-nucleotide heterologies are most likely sufficient to disrupt the minimal efficient recombination target. We also observed that if recombination was allowed to initiate within sequences exhibiting perfect homology, the event could propagate through and terminate within adjacent sequences exhibiting 19% base pair mismatch. We interpret this to mean that heterology exerts most of its impact on early rather than late steps of intrachromosomal recombination in mammalian cells.

Use of a chromosomal inverted repeat to demonstrate that the RAD51 and RAD52 genes of Saccharomyces cerevisiae have different roles in mitotic recombination.

Genetics ◽  
1994 ◽  
Vol 138 (3) ◽  
pp. 587-595 ◽  
Author(s):  
A J Rattray ◽  
L S Symington
Keyword(s):  
Gene Conversion ◽  
Type Strain ◽  
Wild Type Strain ◽  
Inverted Repeats ◽  
Crossing Over ◽  
Wild Type ◽  
Double Mutant Strain ◽  
In The Wild ◽  
Rad52 Protein ◽  
Recombination Assay

Abstract An intrachromosomal recombination assay that monitors events between alleles of the ade2 gene oriented as inverted repeats was developed. Recombination to adenine prototrophy occurred at a rate of 9.3 x 10(-5)/cell/generation. Of the total recombinants, 50% occurred by gene conversion without crossing over, 35% by crossover and 15% by crossover associated with conversion. The rate of recombination was reduced 3,000-fold in a rad52 mutant, but the distribution of residual recombination events remained similar to that seen in the wild type strain. In rad51 mutants the rate of recombination was reduced only 4-fold. In this case, gene conversion events unassociated with a crossover were reduced 18-fold, whereas crossover events were reduced only 2.5-fold. A rad51 rad52 double mutant strain showed the same reduction in the rate of recombination as the rad52 mutant, but the distribution of events resembled that seen in rad51. From these observations it is concluded that (i) RAD52 is required for high levels of both gene conversions and reciprocal crossovers, (ii) that RAD51 is not required for intrachromosomal crossovers, and (iii) that RAD51 and RAD52 have different functions, or that RAD52 has functions in addition to those of the Rad51/Rad52 protein complex.

scholarly journals MEI4, a yeast gene required for meiotic recombination.

Genetics ◽  
1989 ◽  
Vol 123 (4) ◽  
pp. 675-682 ◽  
Author(s):  
T M Menees ◽  
G S Roeder
Keyword(s):  
Gene Conversion ◽  
Meiotic Recombination ◽  
Genomic Library ◽  
Insertion Mutation ◽  
Crossing Over ◽  
Yeast Gene ◽  
Meiotic Gene ◽  
Recombination Pathway ◽  
Meiosis I

Abstract Mutants at the MEI4 locus were detected in a search for mutants defective in meiotic gene conversion. mei4 mutants exhibit decreased sporulation and produce inviable spores. The spore inviability phenotype is rescued by a spo13 mutation, which causes cells to bypass the meiosis I division. The MEI4 gene has been cloned from a yeast genomic library by complementation of the recombination defect and has been mapped to chromosome V near gln3. Strains carrying a deletion/insertion mutation of the MEI4 gene display no meiotically induced gene conversion but normal mitotic conversion frequencies. Both meiotic interchromosomal and intrachromosomal crossing over are completely abolished in mei4 strains. The mei4 mutation is able to rescue the spore-inviability phenotype of spo13 and 52 strains (i.e., mei4 spo13 rad52 mutants produce viable spores), indicating that MEI4 acts before RAD52 in the meiotic recombination pathway.

scholarly journals Different types of recombination events are controlled by the RAD1 and RAD52 genes of Saccharomyces cerevisiae.

Genetics ◽  
1988 ◽  
Vol 120 (2) ◽  
pp. 367-377
Author(s):  
H L Klein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Conversion ◽  
Recombination Event ◽  
Repeated Sequences ◽  
Direct Repeats ◽  
Intrachromosomal Recombination ◽  
Sister Chromatids ◽  
Different Types ◽  
Gene Conversions

Abstract Intrachromosomal recombination within heteroallelic duplications located on chromosomes III and XV of Saccharomyces cerevisiae has been examined. Both possible orientations of alleles have been used in each duplication. Three recombinant classes, gene conversions, pop-outs and triplications, were recovered. Some of the recombinant classes were not anticipated from the particular allele orientation of the duplication. Recovery of these unexpected recombinants requires the RAD1 gene. These studies show that RAD1 has a role in recombination between repeated sequences, and that the recombination event is a gene conversion associated with a crossover. These events appear to involve very localized conversion of a heteroduplex region and are distinct from RAD52 mediated gene conversion events. Evidence is also presented to suggest that most recombination events between direct repeats are intrachromatid, not between sister chromatids.

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