Crossover Formation During Rice Meiosis Relies on Interaction of OsMSH4 and OsMSH5

MSH4 encodes a MutS protein that plays a specialized role in meiosis. In eukaryotic species, such as budding yeast, mice, Caenorhabditis elegans, and Arabidopsis, msh4 mutants display meiotic defects with a reduced number of chiasmata. Here, we characterized rice MSH4 by map-based cloning. In Osmsh4 mutants, the chiasma frequency was dramatically decreased to ∼10% of the wild type, but the synaptonemal complex was normally installed. The double mutant analysis showed that in the Osmsh4 Osmsh5 mutant, the reduction of chiasmata was greater than other zmm mutants. This was consistent with the absence of localization for OsZIP4 and OsMER3 in Osmsh4 and suggests an earlier role for OsMSH4 and OsMSH5 than other ZMM proteins where they may be required to stabilize progenitor Holliday junctions. Using yeast two-hybrid and pull-down assays, we verified the direct physical association between OsMSH4 and OsMSH5 and OsMSH5 and HEI10 in plants for the first time. The MSH4–MSH5 heterodimer has been demonstrated in mammals to stabilize the formation of progenitor and double Holliday junctions that may be resolved as crossovers (COs). We propose that OsMSH4 interacts with OsMSH5 to promote formation of the majority of COs in rice.     

Recruitment and diversification of an ecdysozoan family of neuropeptide hormones for black widow spider venom expression

Venoms have attracted enormous attention because of their potent physiological effects and dynamic evolution, including the convergent recruitment of homologous genes for venom expression. Here we provide novel evidence for the recruitment of genes from the Crustacean Hyperglycemic Hormone (CHH) and arthropod Ion Transport Peptide (ITP) superfamily for venom expression in black widow spiders. We characterized latrodectin peptides from venom gland cDNAs from the Western black widow spider (Latrodectus hesperus), the brown widow (Latrodectus geometricus) and cupboard spider (Steatoda grossa). Phylogenetic analyses of these sequences with homologs from other spider, scorpion and wasp venom cDNAs, as well as CHH/ITP neuropeptides, show latrodectins as derived members of the CHH/ITP superfamily. These analyses suggest that CHH/ITP homologs are more widespread in spider venoms, and were recruited for venom expression in two additional arthropod lineages. We also found that the latrodectin 2 gene and nearly all CHH/ITP genes include a phase 2 intron in the same position, supporting latrodectin's placement within the CHH/ITP superfamily. Evolutionary analyses of latrodectins suggest episodes of positive selection along some sequence lineages, and positive and purifying selection on specific codons, supporting its functional importance in widow venom. We consider how this improved understanding of latrodectin evolution informs functional hypotheses regarding its role in black widow venom as well as its potential convergent recruitment for venom expression across arthropods.     

Fine-Scale Heterogeneity in Crossover Rate in the garnet-scalloped Region of the Drosophila melanogaster X Chromosome

Homologous recombination affects myriad aspects of genome evolution, from standing levels of nucleotide diversity to the efficacy of natural selection. Rates of crossing over show marked variability at all scales surveyed, including species-, population-, and individual-level differences. Even within genomes, crossovers are nonrandomly distributed in a wide diversity of taxa. Although intra- and intergenomic heterogeneities in crossover distribution have been documented in Drosophila, the scale and degree of crossover rate heterogeneity remain unclear. In addition, the genetic features mediating this heterogeneity are unknown. Here we quantify fine-scale heterogeneity in crossover distribution in a 2.1-Mb region of the Drosophila melanogaster X chromosome by localizing crossover breakpoints in 2500 individuals, each containing a single crossover in this specific X chromosome region. We show 90-fold variation in rates of crossing over at a 5-kb scale, place this variation in the context of several aspects of genome evolution, and identify several genetic features associated with crossover rates. Our results shed new light on the scale and magnitude of crossover rate heterogeneity in D. melanogaster and highlight potential features mediating this heterogeneity.     

Effect of different salt ions on the propensity of aggregation and on the structure of Alzheimer's abeta(1-40) amyloid fibrils

The formation of amyloid fibrils and other polypeptide aggregates depends strongly on the physico-chemical environment. One such factor affecting aggregation is the presence and concentration of salt ions. We have examined the effects of salt ions on the aggregation propensity of Alzheimer's Abeta(1-40) peptide and on the structure of the dissolved and of the fibrillar peptide. All salts examined promote aggregation strongly. The most pronounced effect is seen within the cationic series, i.e. for MgCl2. Evaluation of different possible explanations suggests that Abeta(1-40) aggregation depends on direct interaction between ions and Abeta(1-40) peptide, and correlates with ion-induced changes of the surface tension. Salts have profound effects on the fibril structure. In the presence of salts, fibrils are associated with smaller diameters, narrower crossover distances and lower amide I maxima. Since Abeta(1-40) aggregation responds to salts in a manner unlike that for other polypeptides, such as glucagon, beta2-microglobulin or alpha-synuclein; these data argue that there is no fully uniform way in which salts affect aggregation of different polypeptide chains. These observations are important for understanding and predicting aggregation on the basis of simple physico-chemical properties.     

The crystal structure of lambda-Gam protein suggests a model for RecBCD inhibition

In Escherichia coli, RecBCD processes double-stranded DNA breaks during the initial stages of homologous recombination. RecBCD contains helicase and nuclease activities, and unwinds and digests the blunt-ended DNA until a specific eight-nucleotide sequence, Chi, is encountered. Chi modulates the nuclease activity of RecBCD and results in a resected DNA end, which is a substrate for RecA during subsequent steps in recombination. RecBCD also acts as a defence mechanism against bacteriophage infection by digesting linear viral DNA present during virus replication or resulting from the action of restriction endonucleases. To avoid this fate, bacteriophage lambda encodes the gene Gam whose product is an inhibitor of RecBCD. Gam has been shown to bind to RecBCD and inhibit its helicase and nuclease activities. We show that Gam inhibits RecBCD by preventing it from binding DNA. We have solved the crystal structure of Gam from two different crystal forms. Using the published crystal structure of RecBCD in complex with DNA we suggest models for the molecular mechanism of Gam-mediated inhibition of RecBCD. We also propose that Gam could be a mimetic of single-stranded, and perhaps also double-stranded, DNA.     

Recombination suppression in heterozygotes for a pericentric inversion induces the interchromosomal effect on crossovers in Arabidopsis

During meiosis, recombination ensures allelic exchanges through crossovers (COs) between the homologous chromosomes. Advances in our understanding of the rules of COs have come from studies of mutations including structural chromosomal rearrangements that, when heterozygous, are known to impair COs in various organisms. In this work, we investigate the effect of a large heterozygous pericentric inversion on male and female recombination in Arabidopsis. The inversion was discovered in the Atmcc1 mutant background and was characterized through genetic and next‐generation sequencing analysis. Reciprocal backcross populations, each consisting of over 400 individuals, obtained from the mutant and the wild type, both crossed with Landsberg erecta, were analyzed genome‐wide by 143 single‐nucleotide polymorphisms. The negative impact of inversion became evident in terms of CO loss in the rearranged chromosome in both male and female meiosis. No single‐CO event was detected within the inversion, consistent with a post‐meiotic selection operating against unbalanced gametes. Cytological analysis of chiasmata in F₁ plants confirmed that COs were reduced in male meiosis in the chromosome with inversion. Crossover suppression on the rearranged chromosome is associated with a significant increase of COs in the other chromosomes, thereby maintaining unchanged the number of COs per cell. The CO pattern observed in our study is consistent with the interchromosomal (IC) effect as first described in Drosophila. In contrast to male meiosis, in female meiosis no IC effect is visible. This may be related to the greater strength of interference that constrains the CO number in excess of the minimum value imposed by CO assurance in Arabidopsis female meiosis.     

SPO11.2 is essential for programmed double‐strand break formation during meiosis in bread wheat (Triticum aestivum L.)

Meiotic recombination is initiated by formation of DNA double‐strand breaks (DSBs). This involves a protein complex that includes in plants the two similar proteins, SPO11‐1 and SPO11‐2. We analysed the sequences of SPO11‐2 in hexaploid bread wheat (Triticum aestivum), as well as in its diploid and tetraploid progenitors. We investigated its role during meiosis using single, double and triple mutants. The three homoeologous SPO11‐2 copies of hexaploid wheat exhibit high nucleotide and amino acid similarities with those of the diploids, tetraploids and Arabidopsis. Interestingly, however, two nucleotides deleted in exon‐2 of the A copy lead to a premature stop codon and suggest that it encodes a non‐functional protein. Remarkably, the mutation was absent from the diploid A‐relative Triticum urartu, but present in the tetraploid Triticum dicoccoides and in different wheat cultivars indicating that the mutation occurred after the first polyploidy event and has since been conserved. We further show that triple mutants with all three copies (A, B, D) inactivated are sterile. Cytological analyses of these mutants show synapsis defects, accompanied by severe reductions in bivalent formation and numbers of DMC1 foci, thus confirming the essential role of TaSPO11‐2 in meiotic recombination in wheat. In accordance with its 2‐nucleotide deletion in exon‐2, double mutants for which only the A copy remained are also sterile. Notwithstanding, some DMC1 foci remain visible in this mutant, suggesting a residual activity of the A copy, albeit not sufficient to restore fertility.     

Longitudinal analysis of pre- and post-treatment measurements with equal baseline assumptions in randomized trials

For continuous variables of randomized controlled trials, recently, longitudinal analysis of pre- and posttreatment measurements as bivariate responses is one of analytical methods to compare two treatment groups. Under random allocation, means and variances of pretreatment measurements are expected to be equal between groups, but covariances and posttreatment variances are not. Under random allocation with unequal covariances and posttreatment variances, we compared asymptotic variances of the treatment effect estimators in three longitudinal models. The data-generating model has equal baseline means and variances, and unequal covariances and posttreatment variances. The model with equal baseline means and unequal variance–covariance matrices has a redundant parameter. In large sample sizes, these two models keep a nominal type I error rate and have high efficiency. The model with equal baseline means and equal variance–covariance matrices wrongly assumes equal covariances and posttreatment variances. Only under equal sample sizes, this model keeps a nominal type I error rate. This model has the same high efficiency with the data-generating model under equal sample sizes. In conclusion, longitudinal analysis with equal baseline means performed well in large sample sizes. We also compared asymptotic properties of longitudinal models with those of the analysis of covariance (ANCOVA) and t-test.     

Replication protein A (AtRPA1a) is required for class I crossover formation but is dispensable for meiotic DNA break repair

Replication protein A (RPA) is involved in many aspects of DNA metabolism including meiotic recombination. Many species possess a single RPA1 gene but Arabidopsis possesses five RPA1 paralogues. This feature has enabled us to gain further insight into the meiotic role of RPA1. Proteomic analysis implicated one of the AtRPA1 family (AtRPA1a) in meiosis. Immunofluorescence studies confirmed that AtRPA1a is associated with meiotic chromosomes from leptotene through to early pachytene. Analysis of an Atrpa1a mutant revealed that AtRPA1a is not essential at early stages in the recombination pathway. DNA double‐strand breaks are repaired in Atrpa1a, but the mutant is defective in the formation of crossovers, exhibiting a 60% reduction in chiasma frequency. Consistent with this, localization of recombination proteins AtRAD51 and AtMSH4 appears normal, whereas the numbers of AtMLH1 and AtMLH3 foci at pachytene are significantly reduced. This suggests that the defect in Atrpa1a is manifested at the stage of second‐end capture. Analysis of Atrpa1a/Atmsh4 and Atrpa1a/Atmlh3 double mutants indicates that loss of AtRPA1a predominantly affects the formation of class I, interference‐dependent crossovers.