Analysis of the meiotic recombination frequency in transgenic tomato hybrids expressing recA and NLS-recA-licBM3 genes

To study and induce meiotic recombination in plants, we generated and analyzed transgenic tomato hybrids F1-RecA and F1-NLS-recA-LicBM3 expressing, respectively, the recA gene of Escherichia coli and the NLS-recA-licBM3 gene. It was found that the recA and NLS-recA-licBM3 genes are inherited through the maternal and paternal lineages, they have no selective influence on the pollen and are contained in tomato F1-RecA and F1-NLS-RecA-LicBM3 hybrids outside the second chromosome in the hemizygous state. The comparative analysis of the meiotic recombination frequency (rf) in the progenies of the transgenic and nontransgenic hybrids showed that only the expression of the recA gene of E. coli in cells of the F1-RecA plants produced a 1.2-1.5-fold increase in the frequency of recombination between some linked marker genes of the second chromosome of tomato.     

Transgenic tomato plants expressing <Emphasis Type="Italic">recA</Emphasis> and <Emphasis Type="Italic">NLS-recA-licBM3</Emphasis> genes as a model for studying meiotic recombination

Homologous DNA recombination in eukaryotes is necessary to maintain genome stability and integrity and for correct chromosome segregation and formation of new haplotypes in meiosis. At the same time, genetic determination and nonrandomness of meiotic recombination restrict the introgression of genes and generation of unique genotypes. As one of the approaches to study and induce meiotic recombination in plants, it is recommended to use the recA gene of Escherichia coli. It is shown that the recA and NLS-recAlicBM3 genes have maternal inheritance and are expressed in the progeny of transgenic tomato plants. Plants expressing recA or NLS-recA-licBM3 and containing one T-DNA insertion do not differ in pollen fertility from original nontransgenic forms and can therefore be used for comparative studies of the effect of bacterial recombinases on meiotic recombination between linked genes.     

Saccharomyces cerevisiae checkpoint genes MEC1, RAD17 and RAD24 are required for normal meiotic recombination partner choice.

Checkpoint gene function prevents meiotic progression when recombination is blocked by mutations in the recA homologue DMC1. Bypass of dmc1 arrest by mutation of the DNA damage checkpoint genes MEC1, RAD17, or RAD24 results in a dramatic loss of spore viability, suggesting that these genes play an important role in monitoring the progression of recombination. We show here that the role of mitotic checkpoint genes in meiosis is not limited to maintaining arrest in abnormal meioses; mec1-1, rad24, and rad17 single mutants have additional meiotic defects. All three mutants display Zip1 polycomplexes in two- to threefold more nuclei than observed in wild-type controls, suggesting that synapsis may be aberrant. Additionally, all three mutants exhibit elevated levels of ectopic recombination in a novel physical assay. rad17 mutants also alter the fraction of recombination events that are accompanied by an exchange of flanking markers. Crossovers are associated with up to 90% of recombination events for one pair of alleles in rad17, as compared with 65% in wild type. Meiotic progression is not required to allow ectopic recombination in rad17 mutants, as it still occurs at elevated levels in ndt80 mutants that arrest in prophase regardless of checkpoint signaling. These observations support the suggestion that MEC1, RAD17, and RAD24, in addition to their proposed monitoring function, act to promote normal meiotic recombination.     

Meiosis,recombination and chromosomes: a review of gene isolation and fluorescent in situ hybridization data in plants

Evidence is now increasing that many functions and processes of meiotic genes are similar in yeast and higher eukaryotes. However, there are significant differences and, most notably, yeast has considerably higher recombination frequencies than higher eukaryotes, different cross-over interference and possibly more than one pathway for recombination, one late and one early. Other significant events are the timing of double-strand breaks (induced by Spo11) that could be either cause or consequence of homologous chromosome synapsis and SC formation depending on the organisms, yeast plants and mammals versus Drosophila melanogaster and Caenorhabditis elegans. Many plant homologues and heterologues to meiotic genes of yeast and other organisms have now been isolated, in particular in Arabidopsis thaliana, showing that overall recombination genes are very conserved while synaptonemal complex and cohesion proteins are not. In addition to the importance of unravelling the meiotic processes by gene discovery, this review discusses the significance of chromatin packaging, genome organization, and distribution of specific repeated DNA sequences for homologous chromosome cognition and pairing, and the distribution of recombination events along the chromosomes.     

Recombination and UV resistance of Escherichia coli with the cloned recA and recBCD genes of Serratia marcescens and Proteus mirabilis: evidence for an advantage of intraspecies combination of P. mirabilis RecA protein and RecBCD enzyme.

In Escherichia coli, constituents of the main recombination pathway are provided by the genes recA (RecA protein) and recBCD (RecBCD enzyme). Recombination in conjugation experiments and repair of UV damage of E. coli mutants deleted for recA, for recBCD or for recA plus recBCD were restored, although to different degrees, by the cloned recA and recBCD genes from Serratia marcescens or Proteus mirabilis. When both recombination enzymes were from the same species, repair and recombination efficiencies had the order E. coli greater than S. marcescens greater than P. mirabilis. However, the P. mirabilis recA plus recBCD genes resulted in higher levels of repair and recombination than those obtained with one component from P. mirabilis (recA or recBCD) and the other from E. coli or S. marcescens. The data provide evidence for the similarity of RecABCD pathways of recombination among enteric bacteria and suggest an in vivo advantage of an intraspecies combination of P. mirabilis RecA protein and RecBCD enzyme over interspecies combinations. This could point to a cooperation between these basic recombination enzymes. The molecular processes which could be involved are discussed.     

An expanded inventory of conserved meiotic genes provides evidence for sex in Trichomonas vaginalis

Meiosis is a defining feature of eukaryotes but its phylogenetic distribution has not been broadly determined, especially among eukaryotic microorganisms (i.e. protists)-which represent the majority of eukaryotic 'supergroups'. We surveyed genomes of animals, fungi, plants and protists for meiotic genes, focusing on the evolutionarily divergent parasitic protist Trichomonas vaginalis. We identified homologs of 29 components of the meiotic recombination machinery, as well as the synaptonemal and meiotic sister chromatid cohesion complexes. T. vaginalis has orthologs of 27 of 29 meiotic genes, including eight of nine genes that encode meiosis-specific proteins in model organisms. Although meiosis has not been observed in T. vaginalis, our findings suggest it is either currently sexual or a recent asexual, consistent with observed, albeit unusual, sexual cycles in their distant parabasalid relatives, the hypermastigotes. T. vaginalis may use meiotic gene homologs to mediate homologous recombination and genetic exchange. Overall, this expanded inventory of meiotic genes forms a useful "meiosis detection toolkit". Our analyses indicate that these meiotic genes arose, or were already present, early in eukaryotic evolution; thus, the eukaryotic cenancestor contained most or all components of this set and was likely capable of performing meiotic recombination using near-universal meiotic machinery.     

Genetic control of recombination partner preference in yeast meiosis. Isolation and characterization of mutants elevated for meiotic unequal sister-chromatid recombination.

Meiotic exchange occurs preferentially between homologous chromatids, in contrast to mitotic recombination, which occurs primarily between sister chromatids. To identify functions that direct meiotic recombination events to homologues, we screened for mutants exhibiting an increase in meiotic unequal sister-chromatid recombination (SCR). The msc (meiotic sister-chromatid recombination) mutants were quantified in spo13 meiosis with respect to meiotic unequal SCR frequency, disome segregation pattern, sporulation frequency, and spore viability. Analysis of the msc mutants according to these criteria defines three classes. Mutants with a class I phenotype identified new alleles of the meiosis-specific genes RED1 and MEK1, the DNA damage checkpoint genes RAD24 and MEC3, and a previously unknown gene, MSC6. The genes RED1, MEK1, RAD24, RAD17, and MEC1 are required for meiotic prophase arrest induced by a dmc1 mutation, which defines a meiotic recombination checkpoint. Meiotic unequal SCR was also elevated in a rad17 mutant. Our observation that meiotic unequal SCR is elevated in meiotic recombination checkpoint mutants suggests that, in addition to their proposed monitoring function, these checkpoint genes function to direct meiotic recombination events to homologues. The mutants in class II, including a dmc1 mutant, confer a dominant meiotic lethal phenotype in diploid SPO13 meiosis in our strain background, and they identify alleles of UBR1, INP52, BUD3, PET122, ELA1, and MSC1-MSC3. These results suggest that DMC1 functions to bias the repair of meiosis-specific double-strand breaks to homologues. We hypothesize that the genes identified by the class II mutants function in or are regulators of the DMC1-promoted interhomologue recombination pathway. Class III mutants may be elevated for rates of both SCR and homologue exchange.     

Mutations in mating-type genes of the heterothallic fungus Podospora anserina lead to self-fertility

It has been established that meiotic recombination and chromosome segregation are inhibited when meiotic DNA replication is blocked. Here we demonstrate that early meiotic gene (EMG) expression is also inhibited by a block in replication. Since early meiotic genes are required to promote meiotic recombination and DNA division, the low expression of these genes may contribute to the block in meiotic progression. We have identified three Hur- (HU reduced recombination) mutants that fail to couple meiotic recombination and gene expression with replication. One of these mutations is in RPD3, a gene required to maintain meiotic gene repression in mitotic cells. Complete deletions of RPD3 and the repression adapter SIN3 permitted recombination and early meiotic gene expression when replication was inhibited with hydroxyurea (HU). Biochemical analysis showed that the Rpd3p-Sin3p-Ume6p repression complex does exist in meiotic cells. These observations suggest that repression of early meiotic genes by SIN3 and RPD3 is critical for the normal response to inhibited replication. A second response to inhibited replication has also been discovered. HU-inhibited replication reduced the accumulation of phospho-Ume6p in meiotic cells. Phosphorylation of Ume6p normally promotes interaction with the meiotic activator Ime1p, thereby activating EMG expression. Thus, inhibited replication may also reduce the Ume6p-dependent activation of EMGs. Taken together, our data suggest that both active repression and reduced activation combine to inhibit EMG expression when replication is inhibited.     

Brca2 is involved in meiosis in Arabidopsis thaliana as suggested by its interaction with Dmc1

Two BRCA2-like sequences are present in the Arabidopsis genome. Both genes are expressed in flower buds and encode nearly identical proteins, which contain four BRC motifs. In a yeast two-hybrid assay, the Arabidopsis Brca2 proteins interact with Rad51 and Dmc1. RNAi constructs aimed at silencing the BRCA2 genes at meiosis triggered a reproducible sterility phenotype, which was associated with dramatic meiosis alterations. We obtained the same phenotype upon introduction of RNAi constructs aimed at silencing the RAD51 gene at meiosis in dmc1 mutant plants. The meiotic figures we observed strongly suggest that homologous recombination is highly disturbed in these meiotic cells, leaving aberrant recombination events to repair the meiotic double-strand breaks. The 'brca2' meiotic phenotype was eliminated in spo11 mutant plants. Our experiments point to an essential role of Brca2 at meiosis in Arabidopsis. We also propose a role for Rad51 in the dmc1 context.     

Pathways to meiotic recombination in Arabidopsis thaliana

Meiosis is a central feature of sexual reproduction. Studies in plants have made and continue to make an important contribution to fundamental research aimed at the understanding of this complex process. Moreover, homologous recombination during meiosis provides the basis for plant breeders to create new varieties of crops. The increasing global demand for food, combined with the challenges from climate change, will require sustained efforts in crop improvement. An understanding of the factors that control meiotic recombination has the potential to make an important contribution to this challenge by providing the breeder with the means to make fuller use of the genetic variability that is available within crop species. Cytogenetic studies in plants have provided considerable insights into chromosome organization and behaviour during meiosis. More recently, studies, predominantly in Arabidopsis thaliana, are providing important insights into the genes and proteins that are required for crossover formation during plant meiosis. As a result, substantial progress in the understanding of the molecular mechanisms that underpin meiosis in plants has begun to emerge. This article summarizes current progress in the understanding of meiotic recombination and its control in Arabidopsis. We also assess the relationship between meiotic recombination in Arabidopsis and other eukaryotes, highlighting areas of close similarity and apparent differences.     

The recA-recBCD dependent recombination pathways of Serratia marcescens and Proteus mirabilis in Escherichia coli: functions of hybrid enzymes and hybrid pathways.

The physical maps of cloned recBCD gene regions of Serratia marcescens and Proteus mirabilis were correlated to genes located in this region. The genes thyA, recC, recB, recD and argA were organized as in Escherichia coli. The 3 rec genes code for the 3 different subunits of the RecBCD enzyme and produced enzymes promoting recombination and repair of UV damage in E coli. The recBCD-dependent stimulation of recombination at specific nucleotide sequences called Chi (Chi-activation) was determined in lambda red-gam-crosses. Chi-activation by the different RecBCD enzymes decreased in the order E coli greater than S marcescens greater than P mirabilis. In E coli cloned subunits genes from S marcescens and P mirabilis led to the formation of functional hybrid enzymes consisting of subunits from 2 or even 3 species. The origin of the RecC subunit present in the hybrid enzymes affected the degree of Chi-activation. Further, changes in Chi-activation occurred when the RecD subunit in the enzyme from E coli was replaced by RecD proteins from S marcescens or P mirabilis. This suggested that the RecD subunit determines not only whether or not Chi-activation is possible but also to which extent it occurs. Finally we have reconstituted recombination pathways of S marcescens and P mirabilis by combining the cloned recA and recBCD genes from these species in E coli deleted for recA and recBCD. Both pathways can efficiently promote recombination and repair. Studies are summarized which showed that levels of repair and recombination promoted by the recA-recBCD genes are mostly higher when the recA and recBCD genes came from the same species than from 2 different species (hybrid RecBCD recombination pathway). The data are interpreted to provide evidence that in vivo the RecA protein co-operates with the RecBCD enzyme in recombination and repair of UV damage.     

抑制植物减数分裂重组的分子机理

减数分裂重组不仅保证了真核生物有性生殖过程中染色体数量的稳定,还通过父母亲本间遗传物质的互换在后代中产生遗传变异。因此,减数分裂重组是遗传多样性形成的重要途径,也是生物多样性和物种进化的主要动力。在绝大多数真核生物中,不管染色体数目的多少或基因组的大小,减数分裂重组的形成都受到严格的调控,但抑制减数分裂重组的分子机理目前仍不清楚。近年来,通过正向遗传学筛选鉴定出多个减数分裂重组抑制基因,揭示了抑制基因的功能和调控途径。本文基于拟南芥中减数分裂重组抑制基因的研究现状,综述了植物减数分裂重组抑制基因研究取得的突破性进展,并结合基因功能与其调控网络阐述了抑制植物减数分裂重组的分子机理。     

Phage λ Has an Analog of Escherichia Coli Reco, Recr and Recf Genes

The RecF pathway catalyzes generalized recombination in Escherichia coli that is mutant for recBC, sbcB and sbcC. This pathway operating on conjugational recombination requires the recA, recF, recJ, recN, recO, recQ, recR, ruvA, ruvB and ruvC genes. In contrast, lambda mutant for its own recombination genes, int, red alpha and red beta, requires only the recA and recJ genes to recombine efficiently in recBC sbcB sbcC cells. Deletion of an open reading frame in the ninR region of lambda results in an additional requirement for recO, recR and recF in order to recombine in recBC sbcB sbcC mutant cells. This function, designated orf for recO-, recR- and recF-like function, is largely RecF pathway specific.     

Deletions Generated by the Transposon Tn10 in the srl recA Region of the ESCHERICHIA COLI K-12 Chromosome

A negative regulatory gene for the srl operon (srlR) was recognized by the characteristics of an insertion mutation generated by the transposon Tn10 determining tetracycline resistance. This finding is discussed in light of previous hypotheses on the regulation of the srl genes, which mediate metabolism of glucitol (i.e., sorbitol). Mapping showed that the order of genes in this region is: srlR srlD srlC recA alaS. Using two different methods, five mutations of both srl and recA were detected. The phenotype conferred by these mutations, UV sensitivity and extreme recombination deficiency, is characteristic of standard recA point mutants. Three of the mutations were deletions that also removed the genes for tetracycline resistance of the nearby transposon. A fourth mutation ended at a distance from Tn10 sufficient to allow separation of the two by recombination following P1 transduction; our tests did not allow us to conclude whether this mutation was an inversion or a deletion. The fifth mutation was a deletion that seemed to end immediately adjacent to the boundary of Tn10, proximal to recA. Mechanisms for the generation of these srl recA mutations are discussed.     

Complementation of bacteriophage induction and recombination defects in Escherichia coli RecA(-) mutants by expression of the cloned T4 bacteriophage uvsX gene

Previous workers reported that the T4 bacteriophage UvsX protein could promote neither RecA-LexA-mediated DNA repair nor induction of lysogenized bacteriophage, only recombination. Reexamination of these phenotypes demonstrated that, in contrast to these prior studies, when this gene was cloned into a medium but not a low-copy-number vector, it stimulated both a high frequency of spontaneous induction and mitomycin C-stimulated bacteriophage induction in a strain containing a recA13 mutation, but not a recA1 defect. The gene when cloned into a low- or medium- copy-number vector also promoted a low frequency of recombination of two duplicated genes in Escherichia coli in a strain with a complete recA gene deletion. These results suggest that a narrow concentration range of T4 UvsX protein is required to promote both high-frequency spontaneous and mitomycin C-stimulated bacteriophage induction in a recA13 gene mutant, but it facilitates recombination of duplicated genes at only a very low frequency in E. coli RecA(-) mutants with a complete recA deletion. These results also suggest that the different UvsX phenotypes are affected differentially by the concentration of UvsX protein present.     

A Simple Model for the Influence of Meiotic Conversion Tracts on GC Content

A strong correlation between GC content and recombination rate is observed in many eukaryotes, which is thought to be due to conversion events linked to the repair of meiotic double-strand breaks. In several organisms, the length of conversion tracts has been shown to decrease exponentially with increasing distance from the sites of meiotic double-strand breaks. I show here that this behavior leads to a simple analytical model for the evolution and the equilibrium state of the GC content of sequences devoid of meiotic double-strand break sites. In the yeast Saccharomyces cerevisiae, meiotic double-strand breaks are practically excluded from protein-coding sequences. A good fit was observed between the predictions of the model and the variations of the average GC content of the third codon position (GC3) of S. cerevisiae genes. Moreover, recombination parameters that can be extracted by fitting the data to the model coincide with experimentally determined values. These results thus indicate that meiotic recombination plays an important part in determining the fluctuations of GC content in yeast coding sequences. The model also accounted for the different patterns of GC variations observed in the genes of Candida species that exhibit a variety of sexual lifestyles, and hence a wide range of meiotic recombination rates. Finally, the variations of the average GC3 content of human and chicken coding sequences could also be fitted by the model. These results suggest the existence of a widespread pattern of GC variation in eukaryotic genes due to meiotic recombination, which would imply the generality of two features of meiotic recombination: its association with GC-biased gene conversion and the quasi-exclusion of meiotic double-strand breaks from coding sequences. Moreover, the model points out to specific constraints on protein fragments encoded by exon terminal sequences, which are the most affected by the GC bias.     

The Drosophila Meiotic Recombination Gene Mei-9 Encodes a Homologue of the Yeast Excision Repair Protein Rad1

Meiotic recombination and DNA repair are mediated by overlapping sets of genes. In the yeast Saccharomyces cerevisiae, many genes required to repair DNA double-strand breaks are also required for meiotic recombination. In contrast, mutations in genes required for nucleotide excision repair (NER) have no detectable effects on meiotic recombination in S. cerevisiae. The Drosophila melanogaster mei-9 gene is unique among known recombination genes in that it is required for both meiotic recombination and NER. We have analyzed the mei-9 gene at the molecular level and found that it encodes a homologue of the S. cerevisiae excision repair protein Rad1, the probable homologue of mammalian XPF/ERCC4. Hence, the predominant process of meiotic recombination in Drosophila proceeds through a pathway that is at least partially distinct from that of S. cerevisiae, in that it requires an NER protein. The biochemical properties of the Rad1 protein allow us to explain the observation that mei-9 mutants suppress reciprocal exchange without suppressing the frequency of gene conversion.     

The Genetic Dependence of Recombination in Recd Mutants of Escherichia Coli

RecBCD enzyme has multiple activities including helicase, exonuclease and endonuclease activities. Mutations in the genes recB or recC, encoding two subunits of the enzyme, reduce the frequency of many types of recombinational events. Mutations in recD, encoding the third subunit, do not reduce recombination even though most of the activities of the RecBCD enzyme are severely reduced. In this study, the genetic dependence of different types of recombination in recD mutants has been investigated. The effects of mutations in genes in the RecBCD pathway (recA and recC) as well as the genes specific for the RecF pathway (recF, recJ, recN, recO, recQ, ruv and lexA) were tested on conjugational, transductional and plasmid recombination, and on UV survival. recD mutants were hyper-recombinogenic for all the monitored recombination events, especially those involving plasmids, and all recombination events in recD strains required recA and recC. In addition, unlike recD+ strains, chromosomal recombination events and the repair of UV damage to DNA in recD strains were dependent on one RecF pathway gene, recJ. Only a subset of the tested recombination events were affected by ruv, recN, recQ, recO and lexA mutations.