FIGL1 prevents aberrant chromosome associations and fragmentation and limits crossovers in polyploid wheat meiosis.

Meiotic crossovers (COs) generate genetic diversity and are crucial for viable gamete production. Plant COs are typically limited to 1-3 per chromosome pair, constraining the development of improved varieties, which in wheat is exacerbated by an extreme distal localisation bias. Advances in wheat genomics and related technologies provide new opportunities to investigate, and possibly modify, recombination in this important crop species. Here, we investigate the disruption of FIGL1 in tetraploid and hexaploid wheat as a potential strategy for modifying CO frequency/position. We analysed figl1 mutants and virus-induced gene silencing lines cytogenetically. Genetic mapping was performed in the hexaploid. FIGL1 prevents abnormal meiotic chromosome associations/fragmentation in both ploidies. It suppresses class II COs in the tetraploid such that CO/chiasma frequency increased 2.1-fold in a figl1 msh5 quadruple mutant compared with a msh5 double mutant. It does not appear to affect class I COs based on HEI10 foci counts in a hexaploid figl1 triple mutant. Genetic mapping in the triple mutant suggested no significant overall increase in total recombination across examined intervals but revealed large increases in specific individual intervals. Notably, the tetraploid figl1 double mutant was sterile but the hexaploid triple mutant was moderately fertile, indicating potential utility for wheat breeding.

Synthetically induced Arabidopsis thaliana autotetraploids provide insights into the analysis of meiotic mutants with altered crossover frequency.

Mutations affecting crossover (CO) frequency and distribution lead to the presence of univalents during meiosis, giving rise to aneuploid gametes and sterility. These mutations may have a different effect after chromosome doubling. The combination of altered ploidy and mutations could be potentially useful to gain new insights into the mechanisms and regulation of meiotic recombination; however, studies using autopolyploid meiotic mutants are scarce. Here, we have analyzed the cytogenetic consequences in colchicine-induced autotetraploids (colchiploids) from different Arabidopsis mutants with an altered CO frequency. We have found that there are three types of mutants: mutants in which chiasma frequency is doubled after chromosome duplication (zip4, mus81), as in the control; mutants in which polyploidy leads to a higher-than-expected increase in chiasma frequency (asy1, mer3, hei10, and mlh3); and mutants in which the rise in chiasma frequency produced by the presence of two extrachromosomal sets is less than doubled (msh5, fancm). In addition, the proportion of class I/class II COs varies after chromosome duplication in the control. The results obtained reveal the potential of colchiploid meiotic mutants for better understanding of the function of key proteins during plant meiosis. This is especially relevant considering that most crops are polyploids.

Partial cytological diploidization of neoautotetraploid meiosis by induced cross-over rate reduction.

Polyploids, which arise from whole-genome duplication events, have contributed to genome evolution throughout eukaryotes. Among plants, novel features of neopolyploids include traits that can be evolutionarily or agriculturally beneficial, such as increased abiotic stress tolerance. Thus, in addition to being interesting from an evolutionary perspective, genome duplication is also increasingly recognized as a promising crop improvement tool. However, newly formed (neo)polyploids commonly suffer from fertility problems, which have been attributed to abnormal associations among the multiple homologous chromosome copies during meiosis (multivalents). Here, we test the long-standing hypothesis that reducing meiotic cross-over number may be sufficient to limit multivalent formation, favoring diploid-like bivalent associations (cytological diploidization). To do so, we developed Arabidopsis thaliana lines with low cross-over rates by combining mutations for HEI10 and TAF4b. Double mutants showed a reduction of ~33% in cross-over numbers in diploids without compromising meiotic stability. Neopolyploids derived from the double mutant show a cross-over rate reduction of about 40% relative to wild-type neotetraploids, and groups of four homologs indeed formed fewer multivalents and more bivalents. However, we also show that the reduction in multivalents comes with the cost of a slightly increased frequency of univalents and that it does not rescue neopolyploid fertility. Thus, while our results do show that reducing cross-over rates can reduce multivalent frequency in neopolyploids, they also emphasize that there are additional factors affecting both meiotic stability and neopolyploid fertility that will need to be considered in solving the neopolyploid fertility challenge.

Identification, characterization, and rescue of CRISPR/Cas9 generated wheat SPO11-1 mutants.

Increasing crop yields through plant breeding is time consuming and laborious, with the generation of novel combinations of alleles being limited by chromosomal linkage blocks and linkage-drag. Meiotic recombination is essential to create novel genetic variation via the reshuffling of parental alleles. The exchange of genetic information between homologous chromosomes occurs at crossover (CO) sites but CO frequency is often low and unevenly distributed. This bias creates the problem of linkage-drag in recombination 'cold' regions, where undesirable variation remains linked to useful traits. In plants, programmed meiosis-specific DNA double-strand breaks, catalysed by the SPO11 complex, initiate the recombination pathway, although only ~5% result in the formation of COs. To study the role of SPO11-1 in wheat meiosis, and as a prelude to manipulation, we used CRISPR/Cas9 to generate edits in all three SPO11-1 homoeologues of hexaploid wheat. Characterization of progeny lines shows plants deficient in all six SPO11-1 copies fail to undergo chromosome synapsis, lack COs and are sterile. In contrast, lines carrying a single copy of any one of the three wild-type homoeologues are phenotypically indistinguishable from unedited plants both in terms of vegetative growth and fertility. However, cytogenetic analysis of the edited plants suggests that homoeologues differ in their ability to generate COs and in the dynamics of synapsis. In addition, we show that the transformation of wheat mutants carrying six edited copies of SPO11-1 with the TaSPO11-1B gene, restores synapsis, CO formation, and fertility and hence opens a route to modifying recombination in this agronomically important crop.

FANCM promotes class I interfering crossovers and suppresses class II non-interfering crossovers in wheat meiosis.

FANCM suppresses crossovers in plants by unwinding recombination intermediates. In wheat, crossovers are skewed toward the chromosome ends, thus limiting generation of novel allelic combinations. Here, we observe that FANCM maintains the obligate crossover in tetraploid and hexaploid wheat, thus ensuring that every chromosome pair exhibits at least one crossover, by localizing class I crossover protein HEI10 at pachytene. FANCM also suppresses class II crossovers that increased 2.6-fold in fancm msh5 quadruple mutants. These data are consistent with a role for FANCM in second-end capture of class I designated crossover sites, whilst FANCM is also required to promote formation of non-crossovers. In hexaploid wheat, genetic mapping reveals that crossovers increase by 31% in fancm compared to wild type, indicating that fancm could be an effective tool to accelerate breeding. Crossover rate differences in fancm correlate with wild type crossover distributions, suggesting that chromatin may influence the recombination landscape in similar ways in both wild type and fancm.

Cytogenetic Techniques for Analyzing Meiosis in Hexaploid Bread Wheat.

This chapter describes several cytogenetic procedures developed for investigating meiotic recombination in pollen mother cells (PMCs) of hexaploid wheat (Triticum aestivum) using standard fluorescence microscopy. Two basic methods are used to prepare slides for microscopy. In the cytological technique, wheat anthers are excised, fixed and used to prepare chromosome spreads which can be visualized following the application of a fluorescent DNA stain. In the immunocytological technique, fresh anthers are used to prepare chromosome spreads for analyzing the localization of meiotic proteins by applying specific antibodies followed by fluorescently tagged secondary antibodies. Both methods can be combined with the use of DNA probes to label specific chromosome regions such as telomeres, centromeres, and rDNA sequences in a procedure known as fluorescence in situ hybridisation (FISH). In addition, the cytological technique can be used in conjunction with S-phase incorporation of the DNA base analog, 5-bromo-2'-deoxyuridine (BrdU), and a modified immunolocalization procedure for a convenient meiotic time course assay. Although these protocols were developed for T. aestivum cv. Cadenza, they are directly applicable to other varieties and we have used them successfully for several other hexaploid cultivars and the tetraploid Triticum turgidum cv. Kronos.

The Role of DNA Topoisomerase Binding Protein 1 (TopBP1) in Genome Stability in Arabidopsis.

DNA topoisomerase II (TOPII) plays a very important role in DNA topology and in different biological processes such as DNA replication, transcription, repair, and chromosome condensation in higher eukaryotes. TOPII has been found to interact directly with a protein called topoisomerase II binding protein 1 (TopBP1) which also seems to have important roles in DNA replication and repair. In this study, we conducted different experiments to assess the roles of TopBP1 in DNA repair, mitosis, and meiosis, exploring the relationship between TOPII activity and TopBP1. We found that topbp1 mutant seedlings of Arabidopsis thaliana were hypersensitive to cisplatin treatment and the inhibition of TOPII with etoposide produced similar hypersensitivity levels. Furthermore, we recognised that there were no significant differences between the WT and topbp1 seedlings treated with cisplatin and etoposide together, suggesting that the hypersensitivity to cisplatin in the topbp1 mutant could be related to the functional interaction between TOPII and TopBP1. Somatic and meiotic anaphase bridges appeared in the topbp1 mutant at similar frequencies to those when TOPII was inhibited with merbarone, etoposide, or ICFR-187. The effects on meiosis of TOPII inhibition were produced at S phase/G2 stage, suggesting that catenanes could be produced at the onset of meiosis. Thus, if the processing of the catenanes is impaired, some anaphase bridges can be formed. Also, the appearance of anaphase bridges at first and second division is discussed.

The Role of Topoisomerase II in DNA Repair and Recombination in Arabidopsis thaliana.

DNA entanglements and supercoiling arise frequently during normal DNA metabolism. DNA topoisomerases are highly conserved enzymes that resolve the topological problems that these structures create. Topoisomerase II (TOPII) releases topological stress in DNA by removing DNA supercoils through breaking the two DNA strands, passing a DNA duplex through the break and religating the broken strands. TOPII performs key DNA metabolic roles essential for DNA replication, chromosome condensation, heterochromatin metabolism, telomere disentanglement, centromere decatenation, transmission of crossover (CO) interference, interlock resolution and chromosome segregation in several model organisms. In this study, we reveal the endogenous role of Arabidopsis thaliana TOPII in normal root growth and cell cycle, and mitotic DNA repair via homologous recombination. Additionally, we show that the protein is required for meiotic DSB repair progression, but not for CO formation. We propose that TOPII might promote mitotic HR DNA repair by relieving stress needed for HR strand invasion and D-loop formation.

Crossover-active regions of the wheat genome are distinguished by DMC1, the chromosome axis, H3K27me3, and signatures of adaptation.

The hexaploid bread wheat genome comprises over 16 gigabases of sequence across 21 chromosomes. Meiotic crossovers are highly polarized along the chromosomes, with elevation in the gene-dense distal regions and suppression in the Gypsy retrotransposon-dense centromere-proximal regions. We profiled the genomic landscapes of the meiotic recombinase DMC1 and the chromosome axis protein ASY1 in wheat and investigated their relationships with crossovers, chromatin state, and genetic diversity. DMC1 and ASY1 chromatin immunoprecipitation followed by sequencing (ChIP-seq) revealed strong co-enrichment in the distal, crossover-active regions of the wheat chromosomes. Distal ChIP-seq enrichment is consistent with spatiotemporally biased cytological immunolocalization of DMC1 and ASY1 close to the telomeres during meiotic prophase I. DMC1 and ASY1 ChIP-seq peaks show significant overlap with genes and transposable elements in the Mariner and Mutator superfamilies. However, DMC1 and ASY1 ChIP-seq peaks were detected along the length of each chromosome, including in low-crossover regions. At the fine scale, crossover elevation at DMC1 and ASY1 peaks and genes correlates with enrichment of the Polycomb histone modification H3K27me3. This indicates a role for facultative heterochromatin, coincident with high DMC1 and ASY1, in promoting crossovers in wheat and is reflected in distalized H3K27me3 enrichment observed via ChIP-seq and immunocytology. Genes with elevated crossover rates and high DMC1 and ASY1 ChIP-seq signals are overrepresented for defense-response and immunity annotations, have higher sequence polymorphism, and exhibit signatures of selection. Our findings are consistent with meiotic recombination promoting genetic diversity, shaping host-pathogen co-evolution, and accelerating adaptation by increasing the efficiency of selection.

Distal Bias of Meiotic Crossovers in Hexaploid Bread Wheat Reflects Spatio-Temporal Asymmetry of the Meiotic Program.

Meiotic recombination generates genetic variation and provides physical links between homologous chromosomes (crossovers) essential for accurate segregation. In cereals the distribution of crossovers, cytologically evident as chiasmata, is biased toward the distal regions of chromosomes. This creates a bottleneck for plant breeders in the development of varieties with improved agronomic traits, as genes situated in the interstitial and centromere proximal regions of chromosomes rarely recombine. Recent advances in wheat genomics and genome engineering combined with well-developed wheat cytogenetics offer new opportunities to manipulate recombination and unlock genetic variation. As a basis for these investigations we have carried out a detailed analysis of meiotic progression in hexaploid wheat (Triticum aestivum) using immunolocalization of chromosome axis, synaptonemal complex and recombination proteins. 5-Bromo-2'-deoxyuridine (BrdU) labeling was used to determine the chronology of key events in relation to DNA replication. Axis morphogenesis, synapsis and recombination initiation were found to be spatio-temporally coordinated, beginning in the gene-dense distal chromosomal regions and later occurring in the interstitial/proximal regions. Moreover, meiotic progression in the distal regions was coordinated with the conserved chromatin cycles that are a feature of meiosis. This mirroring of the chiasma bias was also evident in the distribution of the gene-associated histone marks, H3K4me3 and H3K27me3; the repeat-associated mark, H3K27me1; and H3K9me3. We believe that this study provides a cytogenetic framework for functional studies and ongoing initiatives to manipulate recombination in the wheat genome.

Meiotic chromosome axis remodelling is critical for meiotic recombination in Brassica rapa.

Meiosis generates genetic variation through homologous recombination (HR) that is harnessed during breeding. HR occurs in the context of meiotic chromosome axes and the synaptonemal complex. To study the role of axis remodelling in crossover (CO) formation in a crop species, we characterized mutants of the axis-associated protein ASY1 and the axis-remodelling protein PCH2 in Brassica rapa. asy1 plants form meiotic chromosome axes that fail to synapse. CO formation is almost abolished, and residual chiasmata are proportionally enriched in terminal chromosome regions, particularly in the nucleolar organizing region (NOR)-carrying chromosome arm. pch2 plants show impaired ASY1 loading and remodelling, consequently achieving only partial synapsis, which leads to reduced CO formation and loss of the obligatory CO. PCH2-independent chiasmata are proportionally enriched towards distal chromosome regions. Similarly, in Arabidopsis pch2, COs are increased towards telomeric regions at the expense of (peri-) centromeric COs compared with the wild type. Taken together, in B. rapa, axis formation and remodelling are critical for meiotic fidelity including synapsis and CO formation, and in asy1 and pch2 CO distributions are altered. While asy1 plants are sterile, pch2 plants are semi-sterile and thus PCH2 could be an interesting target for breeding programmes.

Varietal variation and chromosome behaviour during meiosis in Solanum tuberosum.

Naturally occurring autopolyploid species, such as the autotetraploid potato Solanum tuberosum, face a variety of challenges during meiosis. These include proper pairing, recombination and correct segregation of multiple homologous chromosomes, which can form complex multivalent configurations at metaphase I, and in turn alter allelic segregation ratios through double reduction. Here, we present a reference map of meiotic stages in diploid and tetraploid S. tuberosum using fluorescence in situ hybridisation (FISH) to differentiate individual meiotic chromosomes 1 and 2. A diploid-like behaviour at metaphase I involving bivalent configurations was predominant in all three tetraploid varieties. The crossover frequency per bivalent was significantly reduced in the tetraploids compared with a diploid variety, which likely indicates meiotic adaptation to the autotetraploid state. Nevertheless, bivalents were accompanied by a substantial frequency of multivalents, which varied by variety and by chromosome (7-48%). We identified possible sites of synaptic partner switching, leading to multivalent formation, and found potential defects in the polymerisation and/or maintenance of the synaptonemal complex in tetraploids. These findings demonstrate the rise of S. tuberosum as a model for autotetraploid meiotic recombination research and highlight constraints on meiotic chromosome configurations and chiasma frequencies as an important feature of an evolved autotetraploid meiosis.

TOPII and chromosome movement help remove interlocks between entangled chromosomes during meiosis.

During the zygotene stage of meiosis, normal progression of chromosome synapsis and homologous recombination frequently lead to the formation of structural interlocks between entangled chromosomes. The persistence of interlocks through to the first meiotic division can jeopardize normal synapsis and occasionally chromosome segregation. However, they are generally removed by pachytene. It has been postulated that interlock removal requires one or more active processes, possibly involving topoisomerase II (TOPII) and/or chromosome movement. However, experimental evidence has been lacking. Analysis of a hypomorphic topII mutant and a meiosis-specific topII RNAi knockdown of Arabidopsis thaliana using immunocytochemistry and structured illumination microscopy (SIM) has now enabled us to demonstrate a role for TOPII in interlock resolution. Furthermore, analysis using a nucleoporin nup136 mutant, which affects chromosome movement, reveals that although TOPII activity is required for the removal of some interlock structures, for others, chromosome movement is also necessary. Thus, our study demonstrates that at least two mechanisms are required to ensure interlock removal.

Development of a new fluorescent reporter:operator system: location of AraC regulated genes in Escherichia coli K-12.

BACKGROUND: In bacteria, many transcription activator and repressor proteins regulate multiple transcription units that are often distally distributed on the bacterial genome. To investigate the subcellular location of DNA bound proteins in the folded bacterial nucleoid, fluorescent reporters have been developed which can be targeted to specific DNA operator sites. Such Fluorescent Reporter-Operator System (FROS) probes consist of a fluorescent protein fused to a DNA binding protein, which binds to an array of DNA operator sites located within the genome. Here we have developed a new FROS probe using the Escherichia coli MalI transcription factor, fused to mCherry fluorescent protein. We have used this in combination with a LacI repressor::GFP protein based FROS probe to assess the cellular location of commonly regulated transcription units that are distal on the Escherichia coli genome. RESULTS: We developed a new DNA binding fluorescent reporter, consisting of the Escherichia coli MalI protein fused to the mCherry fluorescent protein. This was used in combination with a Lac repressor:green fluorescent protein fusion to examine the spatial positioning and possible co-localisation of target genes, regulated by the Escherichia coli AraC protein. We report that induction of gene expression with arabinose does not result in co-localisation of AraC-regulated transcription units. However, measurable repositioning was observed when gene expression was induced at the AraC-regulated promoter controlling expression of the araFGH genes, located close to the DNA replication terminus on the chromosome. Moreover, in dividing cells, arabinose-induced expression at the araFGH locus enhanced chromosome segregation after replication. CONCLUSION: Regions of the chromosome regulated by AraC do not colocalise, but transcription events can induce movement of chromosome loci in bacteria and our observations suggest a role for gene expression in chromosome segregation.

m6A potentiates Sxl alternative pre-mRNA splicing for robust Drosophila sex determination.

N6-methyladenosine (m6A) is the most common internal modification of eukaryotic messenger RNA (mRNA) and is decoded by YTH domain proteins. The mammalian mRNA m6A methylosome is a complex of nuclear proteins that includes METTL3 (methyltransferase-like 3), METTL14, WTAP (Wilms tumour 1-associated protein) and KIAA1429. Drosophila has corresponding homologues named Ime4 and KAR4 (Inducer of meiosis 4 and Karyogamy protein 4), and Female-lethal (2)d (Fl(2)d) and Virilizer (Vir). In Drosophila, fl(2)d and vir are required for sex-dependent regulation of alternative splicing of the sex determination factor Sex lethal (Sxl). However, the functions of m6A in introns in the regulation of alternative splicing remain uncertain. Here we show that m6A is absent in the mRNA of Drosophila lacking Ime4. In contrast to mouse and plant knockout models, Drosophila Ime4-null mutants remain viable, though flightless, and show a sex bias towards maleness. This is because m6A is required for female-specific alternative splicing of Sxl, which determines female physiognomy, but also translationally represses male-specific lethal 2 (msl-2) to prevent dosage compensation in females. We further show that the m6A reader protein YT521-B decodes m6A in the sex-specifically spliced intron of Sxl, as its absence phenocopies Ime4 mutants. Loss of m6A also affects alternative splicing of additional genes, predominantly in the 5' untranslated region, and has global effects on the expression of metabolic genes. The requirement of m6A and its reader YT521-B for female-specific Sxl alternative splicing reveals that this hitherto enigmatic mRNA modification constitutes an ancient and specific mechanism to adjust levels of gene expression.

The Papaver rhoeas S determinants confer self-incompatibility to Arabidopsis thaliana in planta.

Self-incompatibility (SI) is a major genetically controlled system used to prevent inbreeding in higher plants. S determinants regulate allele-specific rejection of "self" pollen by the pistil. SI is an important model system for cell-to-cell recognition and signaling and could be potentially useful for first-generation (F1) hybrid breeding. To date, the transfer of S determinants has used the complementation of orthologs to "restore" SI in close relatives. We expressed the Papaver rhoeas S determinants PrsS and PrpS in Arabidopsis thaliana. This enabled pistils to reject pollen expressing cognate PrpS. Moreover, plants coexpressing cognate PrpS and PrsS exhibit robust SI. This demonstrates that PrsS and PrpS are sufficient for a functional synthetic S locus in vivo. This transfer of novel S determinants into a highly divergent species (>140 million years apart) with no orthologs suggests their potential utility in crop production.

Analysis of the Relationships between DNA Double-Strand Breaks, Synaptonemal Complex and Crossovers Using the Atfas1-4 Mutant.

Chromatin Assembly Factor 1 (CAF-1) is a histone chaperone that assembles acetylated histones H3/H4 onto newly synthesized DNA, allowing the de novo assembly of nucleosomes during replication. CAF-1 is an evolutionary conserved heterotrimeric protein complex. In Arabidopsis, the three CAF-1 subunits are encoded by FAS1, FAS2 and MSI1. Atfas1-4 mutants have reduced fertility due to a decrease in the number of cells that enter meiosis. Interestingly, the number of DNA double-strand breaks (DSBs), measured by scoring the presence of γH2AX, AtRAD51 and AtDMC1 foci, is higher than in wild-type (WT) plants, and meiotic recombination genes such AtCOM1/SAE2, AtBRCA1, AtRAD51 and AtDMC1 are overexpressed. An increase in DSBs in this mutant does not have a significant effect in the mean chiasma frequency at metaphase I, nor a different number of AtMLH1 nor AtMUS81 foci per cell compared to WT at pachytene. Nevertheless, this mutant does show a higher gene conversion (GC) frequency. To examine how an increase in DSBs influences meiotic recombination and synaptonemal complex (SC) formation, we analyzed double mutants defective for AtFAS1 and different homologous recombination (HR) proteins. Most showed significant increases in both the mean number of synapsis initiation points (SIPs) and the total length of AtZYP1 stretches in comparison with the corresponding single mutants. These experiments also provide new insight into the relationships between the recombinases in Arabidopsis, suggesting a prominent role for AtDMC1 versus AtRAD51 in establishing interhomolog interactions. In Arabidopsis an increase in the number of DSBs does not translate to an increase in the number of crossovers (COs) but instead in a higher GC frequency. We discuss different mechanisms to explain these results including the possible existence of CO homeostasis in plants.

Juxtaposition of heterozygous and homozygous regions causes reciprocal crossover remodelling via interference during Arabidopsis meiosis.

During meiosis homologous chromosomes undergo crossover recombination. Sequence differences between homologs can locally inhibit crossovers. Despite this, nucleotide diversity and population-scaled recombination are positively correlated in eukaryote genomes. To investigate interactions between heterozygosity and recombination we crossed Arabidopsis lines carrying fluorescent crossover reporters to 32 diverse accessions and observed hybrids with significantly higher and lower crossovers than homozygotes. Using recombinant populations derived from these crosses we observed that heterozygous regions increase crossovers when juxtaposed with homozygous regions, which reciprocally decrease. Total crossovers measured by chiasmata were unchanged when heterozygosity was varied, consistent with homeostatic control. We tested the effects of heterozygosity in mutants where the balance of interfering and non-interfering crossover repair is altered. Crossover remodeling at homozygosity-heterozygosity junctions requires interference, and non-interfering repair is inefficient in heterozygous regions. As a consequence, heterozygous regions show stronger crossover interference. Our findings reveal how varying homolog polymorphism patterns can shape meiotic recombination.

Location and dynamics of an active promoter in Escherichia coli K-12.

In the present paper, we report that transcription affects the location of a DNA target in Escherichia coli K-12. A strain whose chromosome had been engineered to encode a lac repressor-GFP (green fluorescent protein) fusion was used as a host for a low copy number plasmid that carries an array of five lac operator sites. Individual cells of this strain exhibited a diffuse fluorescence signal, suggesting that the plasmid is distributed throughout the cell cytoplasm. However, a derivative of this plasmid carrying a cloned constitutive promoter is targeted to a location at the edge of the nucleoid towards the pole of the host cell. We conclude that transcription from the cloned promoter is driving the location of the plasmid and that specific locations in bacterial cells may favour gene expression.

Together yes, but not coupled: new insights into the roles of RAD51 and DMC1 in plant meiotic recombination.

The eukaryotic recombinases RAD51 and DMC1 are essential for DNA strand-exchange between homologous chromosomes during meiosis. RAD51 is also expressed during mitosis, and mediates homologous recombination (HR) between sister chromatids. It has been suggested that DMC1 might be involved in the switch from intersister chromatid recombination in somatic cells to interhomolog meiotic recombination. At meiosis, the Arabidopsis Atrad51 null mutant fails to synapse and has extensive chromosome fragmentation. The Atdmc1 null mutant is also asynaptic, but in this case chromosome fragmentation is absent. Thus in plants, AtDMC1 appears to be indispensable for interhomolog homologous recombination, whereas AtRAD51 seems to be more involved in intersister recombination. In this work, we have studied a new AtRAD51 knock-down mutant, Atrad51-2, which expresses only a small quantity of RAD51 protein. Atrad51-2 mutant plants are sterile and hypersensitive to DNA double-strand break induction, but their vegetative development is apparently normal. The meiotic phenotype of the mutant consists of partial synapsis, an elevated frequency of univalents, a low incidence of chromosome fragmentation and multivalent chromosome associations. Surprisingly, non-homologous chromosomes are involved in 51% of bivalents. The depletion of AtDMC1 in the Atrad51-2 background results in the loss of bivalents and in an increase of chromosome fragmentation. Our results suggest that a critical level of AtRAD51 is required to ensure the fidelity of HR during interchromosomal exchanges. Assuming the existence of asymmetrical DNA strand invasion during the initial steps of recombination, we have developed a working model in which the initial step of strand invasion is mediated by AtDMC1, with AtRAD51 required to check the fidelity of this process.