Ectopic mitotic recombination in Drosophila probed with bacterial beta-galactosidase gene-based reporter transgenes.

Plasmids were constructed to investigate homologous mitotic recombination in Drosophila cells. Heteroalleles containing truncated but overlapping segments of the bacterial beta-galactosidase gene (lacZ) were positioned either on separate plasmids or as direct repeats on the same chromosome. Recombination reconstituted a functional lacZgene leading to expression of LacZ+activity detectable by histochemical staining. High extrachromosomal recombination (ECR) frequencies between unlinked heteroalleles were observed upon transient co-transfection into Drosophila melanogaster Schneider line 2 (S2) cells. Stably transfected cells containing the lacZ heteroalleles linked on a chromosome exhibited intrachromosomal recombination (ICR) frequencies two orders of magnitude lower than ECR frequencies. Recombination was inducible by exposing the cells to ethyl methanesulphonate or mitomycin C. Recombination products were characterized by multiplex PCR analysis and unequal sister chromatid recombination was found as the predominant mechanism reconstituting the lacZ gene. To investigate recombination in vivo imaginal disc cells from transgenic larvae carrying the reporter gene on the X chromosome were isolated and stained for LacZ+ activity. The presence of a few LacZ+ clones indicated that mitotic recombination events occurred at frequencies two orders of magnitude lower than the corresponding event in cultured cells and late during larval development.     

Influence of genetic background and heterozygosity on meiotic recombination in Arabidopsis thaliana.

Plant breeding relies on genetic variability generated by meiotic recombination. Control of recombination frequencies is not yet possible, but would significantly extend the options for plant-breeding strategies. A prerequisite would be variability of recombination frequencies. In this study, 15 transgenic kanamycin (KR) and hygromycin (HR) resistance gene insertions mapping to the five Arabidopsis thaliana chromosomes were used as genetic markers. Recombination frequencies were determined from the frequencies of resistance phenotypes within populations segregating for linked KR and HR markers. Recombination frequencies of marker pairs were compared among these four ecotypes, among F1s in both reciprocal forms derived from these ecotypes, and between F1s and their parent lines. On average, the recombination frequencies in F1 crosses were substantially higher (up to 2-fold) than in the homozygous parental ecotypes. A strong negative correlation between genetic similarities of ecotypes and recombination frequencies was detected for two adjacent marker pairs located on the long arm of chromosome 3, but not for marker pairs in other genomic regions. Our results suggest that heterozygosity influences recombination in plant breeding, and cannot be ignored in genetic mapping of genomes.     

Interchromatid and interhomolog recombination in Arabidopsis thaliana

Intermolecular recombination events were monitored in Arabidopsis thaliana lines using specially designed recombination traps consisting of tandem disrupted beta-glucuronidase or luciferase reporter genes in direct repeat orientation. Recombination frequencies (RFs) varied between the different lines, indicating possible position effects influencing intermolecular recombination processes. The RFs between sister chromatids and between homologous chromosomes were measured in plants either hemizygous or homozygous for a transgene locus. The RFs in homozygous plants exceeded those of hemizygous plants by a factor of >2, implying that in somatic plant cells both sister chromatid recombination and recombination between homologous chromosomes exist for recombinational DNA repair. In addition, different DNA-damaging agents stimulated recombination in homozygous and hemizygous plants to different extents in a manner dependent on the type of DNA damage and on the genomic region. The genetic and molecular analysis of recombination events showed that most of the somatic recombination events result from gene conversion, although a pop-out event has also been characterized.     

Induction of intrachromosomal homologous recombination in whole plants

The influence of different factors on frequencies of intrachromosomal homologous recombination in whole Arabidopsis thaliana and tobacco plants was analyzed using a disrupted β-glucuronidase marker gene. Recombination frequencies were enhanced severalfold by DNA damaging agents like UV-light or MMS (methyl methanesulfonate). Applying 3-methoxybenzamide (3-MB), an inhibitor of poly(ADP)ribose polymerase (PARP), an enzyme that is postulated to be involved in DNA repair, enhanced homologous recombination frequencies strongly. These findings indicate that homologous recombination is involved in DNA repair and can (at least partially) compensate for other DNA repair pathways. Indications that recombination in plants can be induced by environmental stress factors that are not likely to be involved in DNA metabolism were also found; Arabidopsis plants growing in a medium containing 0.1 M NaCl exhibited elevated recombination frequencies. The possible general effects of ‘environmental’ challenges on genome flexibility are discussed.     

Determination of the minimal length DNA homologous region required for plasmid integration into the Bacillus subtilis chromosome via homologous recombination]

With a view to determine a minimal sequence length of homology necessary for RecE-dependent homologous recombination in Bacillus subtilis cells, we developed a system, based on interaction between plasmid replicon and bacterial chromosome. Recombination frequencies were measured between ts plasmid pE194 derivatives carrying chromosomal beta-glucuronidase gene (bglS) fragments of various length, and a bacterial chromosome. The homologous recombination events resulted in bglS gene disruption. Approx. 70 bp of homology were found to be necessary for detectable homologous recombination. Homologous recombination was not detected when homology was equal 25 bp. These data indicate that homology requirement for recombination in B. subtilis differs from that in Escherichia coli.     

Genetic recombination of bacterial plasmid DNA. Physical and genetic analysis of the products of plasmid recombination in Escherichia coli

Derivatives of plasmid pBR322 DNA containing tet mutations were constructed by inserting XhoI linkers at various sites in the tetracycline resistance gene. Monomer plasmids containing either the tet-10 allele located at nucleotide position 23 or the tet-14 allele located at nucleotide position 1267 were used to construct a circular dimer containing one copy of each allele and a circular trimer containing one copy of the tet-10 allele and two copies of the tet-14 allele. Genetic recombination of these plasmid DNAs to produce a functional tetracycline resistance gene could be detected as the production of tetracycline-resistant progeny during the growth of transformants or using a restriction mapping assay which detected the rearrangement of the mutant alleles. The structure of individual tetracycline-resistant recombination products was determined by restriction mapping. This analysis suggested that as many as 70% of the plasmid recombination events in Escherichia coli AB1157 could have involved gene conversion events. The formation of these recombination products was most easily predicted by a model involving figure 8 recombination intermediates and the formation of symmetric regions of heteroduplex. Recombination in JC10287 delta(srlR-recA)304 occurred at 5% of the wild-type frequency and appeared to occur by a similar mechanism. Recombination in JC9604 recA56 recB21 recC22 sbcA23 occurred at 20 times the wild-type frequency and appeared to involve multiple independent recombination events.     

Somatic and Meiotic Chromosomal Recombination between Inverted Duplications in Transgenic Tobacco Plants

Homologous recombination has been extensively studied in bacteria, yeast, and more recently in animal cells, but little is known about this process in plants. We present here an analysis of meiotic and somatic chromosomal recombination between closely linked inverted duplications located on a single chromosomal region in tobacco. Transgenic tobacco lines were constructed by Agrobacterium transformation with plasmid vectors containing a functional hygromycin phosphotransferase (hyg) selectable marker flanked by a pair of defective neomycin phosphotransferase (neo) genes positioned as inverted repeats. As each neo gene is mutated in a different site, recombination between the two defective genes can be detected following selection for kanamycin-resistant plant cells. The recombination substrates were designed to allow investigation into the nature of molecular events underlying homologous recombination by restriction endonuclease analysis. Chromosomal recombination was studied in mitotically dividing cells (cultured leaf mesophyll cells) and after meiosis (germinated seedlings). Spontaneous somatic recombinants were recovered at frequencies between ~3 x 10-5 to 10-6 events per cell. Low dose [gamma] irradiation of somatic cells resulted in a threefold maximum increase in the recovery of recombinants. Recombinants were also detected at low frequency when transgenic T3 seeds were germinated under kanamycin selection. DNA gel blot analyses demonstrated that homologous recombination occurred mainly as gene conversion unassociated with reciprocal exchange, although a variety of other events including gene coconversion were also observed.     

Increase of homologous recombination frequency in vascular tissue of Arabidopsis plants exposed to salt stress

Here we analyzed the influence of salt stress on plant genome stability. Homologous recombination events were detected in transgenic Arabidopsis plants that carried in their genome a beta-glucuronidase recombination marker. Recombination events were scored as blue sectors using a stereo microscope. Exposure to 50 mM salt resulted in a 3.0-fold increase in recombination frequency. To analyze the organ and tissue specificity of recombination events, we examined cross-sections of leaves, stems and roots. We found that nearly 30% of recombination events in plants grown under normal conditions and nearly 50% of events in plants grown on salt were undetected by the conventional method. Most of the recombination events represented a cluster/group of cells (12 on average), although events with single cells were also detected. Recombination events were very frequent in leaf mesophyll cells. On average, individual recombination events located on leaves contained more cells than events located on roots or stems. Analysis of recombination events in cross-sectioned tissue of salt-treated plants revealed a shift in the distribution of recombination events towards the vascular tissue. We discuss the significance of the finding for plant stress physiology.     

Intrachromosomal recombination mediated by papovavirus large T antigens.

To investigate the mechanism by which the large T antigen (T-Ag) of polyomavirus and simian virus 40 can promote recombination in mammalian cells, we analyzed homologous recombination events occurring between two defective copies of the polyomavirus middle T (pmt) oncogene lying in close proximity on the same chromosome in a rat cell line. Reconstitution of a functional pmt gene by spontaneous recombination occurred at a rate of about 2 x 10(-7) per cell generation. Introduction of the polyomavirus large T (plt) oncogene into the cell line by DNA transfection promoted recombination very efficiently, with rates in the range of 10(-1) to 10(-2) per cell generation. Recombination was independent of any amplification of viral sequences and could even be promoted by the large T-Ag from simian virus 40, which cannot activate polyomavirus DNA replication. To explain the role of large T-Ag, we propose a novel mechanism of nonconservative recombination involving slipped-strand mispairing between the two viral repeats followed by gap repair synthesis.     

Unequal homologous recombination between tandemly arranged sequences stably incorporated into cultured rat cells.

Cultured rat cells deficient in endogenous thymidine kinase activity (tk) were stably transformed with a recombination-indicator DNA substrate constructed in vitro by rearrangement of the herpes simplex virus tk gene sequences into a partially redundant permutation of the functional gene. The recombination-indicator DNA did not express tk, but was designed to allow formation of a functional tk gene via homologous recombination. A clonal cell line (519) was isolated that harbored several permuted herpes simplex virus tk genes. 519 cells spontaneously produced progeny that survived in medium containing hypoxanthine, aminopterin, and thymidine. Acquisition of resistance to hypoxanthine, aminopterin, and thymidine was accompanied by the rearrangement of the defective tk gene to functional configuration. The rearrangement apparently occurred by unequal exchange between one permuted tk gene and a replicated copy of itself. Recombination was between 500-base-pair tracts of DNA sequence homology that were separated by 3.4 kilobases. Exchanges occurred spontaneously at a frequency of approximately 5 X 10(-6) events per cell per generation. Recombination also mediated reversion to the tk- phenotype; however, the predominant mechanism by which cells escaped death in the presence of drugs rendered toxic by thymidine kinase was not recombination, but rather inactivation of the intact tk gene.     

Use of a Chromosomal Inverted Repeat to Demonstrate That the Rad51 and Rad52 Genes of Saccharomyces Cerevisiae Have Different Roles in Mitotic Recombination

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 had functions in addition to those of the Rad51/Rad52 protein complex.     

Homeologous recombination in Solanum lycopersicoides introgression lines of cultivated tomato

A library of "introgression lines" containing Solanum lycopersicoides chromosome segments in the genetic background of cultivated tomato (Lycopersicon esculentum) was used to study factors affecting homeologous recombination. Recombination rates were estimated in progeny of 43 heterozygous introgressions and whole-chromosome substitution lines, together representing 11 of the 12 tomato chromosomes. Recombination within homeologous segments was reduced to as little as 0-10% of expected frequencies. Relative recombination rates were positively correlated with the length of introgressed segments on the tomato map. The highest recombination (up to 40-50% of normal) was observed in long introgressions or substitution lines. Double-introgression lines containing two homeologous segments on opposite chromosome arms were synthesized to increase their combined length. Recombination was higher in the double than in the single segment lines, despite a preference for crossovers in the region of homology between segments. A greater increase in homeologous recombination was obtained by crossing the S. lycopersicoides introgression lines to L. pennellii--a phylogenetically intermediate species--or to L. esculentum lines containing single L. pennellii segments on the same chromosome. Recombination rates were highest in regions of overlap between S. lycopersicoides and L. pennellii segments. The potential application of these results to breeding with introgression lines is discussed.     

Mutations affecting meiosis in Podospora anserina. II. Effect of mei2 mutants on recombination

Summary Three meiosis-deficient mutants of gene mei2 (mei2-1, mei-2-2 and mei2-3) are blocked during the prophase I of meiosis, before normal pachytene. The mutant mei-2-2 is leaky and there is a partial complementation in crosses mei2-2xmei-2-1 and mei2-2xmei2-3. It has thus been possible to analyse descendants of these crosses. This analysis shows an important alteration in recombination frequencies on at least three different linkage groups. Recombination frequencies appear to be increased near the centromere and decreased in other regions of the chromosomes. This coincides with a decrease in chiasma interference. Intergenic recombination is increased in a locus located very near to the chromosome II centromere. Moreover, the relative proportion of crossovers among the recombination events is stronger than in the control. Though it is impossible at present to formulate a precise hypothesis for the action of the mei2 gene at the molecular level, it is proposed that it might well control a stage of the DNA repair or synthesis.     

Genetic approaches for studying transgene inheritance and genetic recombination in three successive generations of transformed tobacco

Transgene integration into plant genomes is a complex process accompanied by molecular rearrangements. Classic methods that are normally used to study transgenic population genetics are generally inadequate for assessing such integration. Two major characteristics of transgenic populations are that a transgenic genome may harbor many copies of the transgene and that molecular rearrangements can create an unstable transgenic locus. In this work, we examined the segregation of T1, T2 and T3 transgenic tobacco progenies. Since transfer DNA (T-DNA) contains the NptII selectable marker gene that confers resistance to kanamycin, we used this characteristic in developing a method to estimate the number of functional inserts integrated into the genome. This approach was based on calculation of the theoretical segregation ratios in successive generations. Mendelian ratios of 3:1, 15:1 and 63:1 were confirmed for five transformation events whereas six transformation events yielded non-segregating progenies, a finding that raised questions about causal factors. A second approach based on a maximum likelihood method was performed to estimate recombination frequencies between linked inserts. Recombination estimates varied among transformation events and over generations. Some transgenic loci were unstable and evolved continuously to segregate independently in the T3 generation. Recombination and amplification of the transgene and filler DNA yielded additional transformed genotypes.     

Poliovirus RNA recombination: mechanistic studies in the absence of selection.

Direct and quantitative detection of recombinant RNA molecules by polymerase chain reaction (PCR) provides a novel method for studying recombination in RNA viruses without selection for viable progeny. The parental poliovirus strains used in this study contained polymorphic marker loci approximately 600 bases apart; both exhibited wild-type growth characteristics. We established conditions under which the amount of PCR product was linearly proportional to the amount of input template, and the reproducibility was high. Recombinant progeny were predominantly homologous and arose at frequencies up to 2 x 10(-3). Recombination events increased in frequency throughout replication, indicating that there is no viral RNA sequestration or inhibition of recombination late in infection as proposed in earlier genetic studies. Previous studies have demonstrated that poliovirus recombination occurs by a copy-choice mechanism in which the viral polymerase switches templates during negative-strand synthesis. Varying the relative amount of input parental virus markedly altered reciprocal recombination frequencies. This, in conjunction with the kinetics data, indicated that acceptor template concentration is a determinant of template switching frequency. Since positive strands greatly outnumber negative strands throughout poliovirus infection, this would explain the bias toward recombination during negative-strand synthesis.     

A high-throughput system for genome-wide measurement of genetic recombination in Arabidopsis thaliana based on transgenic markers

A high-throughput system for the measurement of recombination frequencies in the genetic model plant, Arabidopsis thaliana, is described. It is based on 21 mono-transgenic isogenic lines harboring antibiotic resistance genes on all five chromosomes. Recombination between pairs of gene insertions in repulsion phase that confer resistance against kanamycin (kan) and hygromycin (hyg) is determined by a phenotypic assay of progeny (DART: Double Antibiotic Resistance Technique). DART allows testing for the influence of numerous environmental and genetic factors, including candidate genes, on recombination frequencies in specific genomic regions as well as the entire genome. Its usefulness is demonstrated by investigating the effects of UV treatment, different temperature and phosphorus supply regimes, and sex on recombination frequencies for all five chromosomes of A. thaliana. Electronic Publication     

The M26 hotspot of Schizosaccharomyces pombe stimulates meiotic ectopic recombination and chromosomal rearrangements.

Homologous recombination is increased during meiosis between DNA sequences at the same chromosomal position (allelic recombination) and at different chromosomal positions (ectopic recombination). Recombination hotspots are important elements in controlling meiotic allelic recombination. We have used artificially dispersed copies of the ade6 gene in Schizosaccharomyces pombe to study hotspot activity in meiotic ectopic recombination. Ectopic recombination was reduced 10-1000-fold relative to allelic recombination, and was similar to the low frequency of ectopic recombination between naturally repeated sequences in S. pombe. The M26 hotspot was active in ectopic recombination in some, but not all, integration sites, with the same pattern of activity and inactivity in ectopic and allelic recombination. Crossing over in ectopic recombination, resulting in chromosomal rearrangements, was associated with 35-60% of recombination events and was stimulated 12-fold by M26. These results suggest overlap in the mechanisms of ectopic and allelic recombination and indicate that hotspots can stimulate chromosomal rearrangements.     

The Impact of Recombination Hotspots on Genome Evolution of a Fungal Plant Pathogen

Recombination has an impact on genome evolution by maintaining chromosomal integrity, affecting the efficacy of selection, and increasing genetic variability in populations. Recombination rates are a key determinant of the coevolutionary dynamics between hosts and their pathogens. Historic recombination events created devastating new pathogens, but the impact of ongoing recombination in sexual pathogens is poorly understood. Many fungal pathogens of plants undergo regular sexual cycles, and sex is considered to be a major factor contributing to virulence. We generated a recombination map at kilobase-scale resolution for the haploid plant pathogenic fungus Zymoseptoria tritici. To account for intraspecific variation in recombination rates, we constructed genetic maps from two independent crosses. We localized a total of 10,287 crossover events in 441 progeny and found that recombination rates were highly heterogeneous within and among chromosomes. Recombination rates on large chromosomes were inversely correlated with chromosome length. Short accessory chromosomes often lacked evidence for crossovers between parental chromosomes. Recombination was concentrated in narrow hotspots that were preferentially located close to telomeres. Hotspots were only partially conserved between the two crosses, suggesting that hotspots are short-lived and may vary according to genomic background. Genes located in hotspot regions were enriched in genes encoding secreted proteins. Population resequencing showed that chromosomal regions with high recombination rates were strongly correlated with regions of low linkage disequilibrium. Hence, genes in pathogen recombination hotspots are likely to evolve faster in natural populations and may represent a greater threat to the host.     

Unequal SCE is a rare event in homologous recombination between duplicated neo gene fragments in CHO cells

The frequency of sister-chromatid exchange (SCE) was studied in Chinese hamster ovary (CHO) cell lines with stable insertions of the vector pIII-14gpt which contains 2 truncated neomycin resistance (neo) gene fragments. Recombination between regions of homology in the 2 fragments can restore a functional neo gene and make the cell resistant to the antibiotic G418, a neomycin analogue. Unequal SCE would be one of several possible mechanisms for this event. The observed spontaneous rate of formation of G418-resistant subclones was approximately 6.4 x 10(-6) per cell per generation, as compared to the estimated spontaneous frequency of 3 SCE per cell per generation. Given this SCE frequency, the probability of an SCE occurring in a target site of about 1600 bp (the distance separating the homologous regions in the neo fragments) would be about 8 x 10(-7) per cell per generation, or approximately one tenth of the estimated rate of recombination. Treatment of the cells with methyl methanesulfonate (MMS, 50 x 10(-6) M) induced about 80-90 SCE per cell, corresponding to a probability of 2 x 10(-5) SCE per 1600-bp target per cell. In the same cell culture, MMS treatment induced 4-8 x 10(-4) recombination events per cell giving rise to G418 resistance. Cells treated with HN2 (up to 4 x 10(-6) M) showed a significant increase in SCEs, but no change in the frequency of G418-resistant revertants. These results suggest that the 2 pathways leading to SCE and recombination respectively are uncoupled, and only a small fraction of the recombination events, if any, are due to unequal SCE in this system.