A regulatory role for the cohesin loader NIPBL in nonhomologous end joining during immunoglobulin class switch recombination.

DNA double strand breaks (DSBs) are mainly repaired via homologous recombination (HR) or nonhomologous end joining (NHEJ). These breaks pose severe threats to genome integrity but can also be necessary intermediates of normal cellular processes such as immunoglobulin class switch recombination (CSR). During CSR, DSBs are produced in the G1 phase of the cell cycle and are repaired by the classical NHEJ machinery. By studying B lymphocytes derived from patients with Cornelia de Lange Syndrome, we observed a strong correlation between heterozygous loss-of-function mutations in the gene encoding the cohesin loading protein NIPBL and a shift toward the use of an alternative, microhomology-based end joining during CSR. Furthermore, the early recruitment of 53BP1 to DSBs was reduced in the NIPBL-deficient patient cells. Association of NIPBL deficiency and impaired NHEJ was also observed in a plasmid-based end-joining assay and a yeast model system. Our results suggest that NIPBL plays an important and evolutionarily conserved role in NHEJ, in addition to its canonical function in sister chromatid cohesion and its recently suggested function in HR.

New species emergence via recombination among isolates of the Brazilian tomato infecting Begomovirus complex

Partial nucleotide sequences of five tomato infecting Begomovirus isolates were determined from DNA-A fragments, corresponding to the 5' region of the replication associated protein gene, the intergenic region and the 5' region of the coat protein gene. Isolate DFM shared 95% identity with Tomato mottle leaf curl virus (TMoLCV), isolates 34, PA-05, and Ta4 were 88% identical to Tomato yellow vein streak virus and isolate DF-BR3 shared 77% identity with TMoLCV. Recombination analysis indicated that isolate DF-BR3 was a chimaera, and it provided evidence that there is a complex and actively recombining population of tomato infecting begomoviruses in Brazil.

Detection of metabolite induction in fungal co-cultures on solid media by high-throughput differential ultra-high pressure liquid chromatography-time-of-flight mass spectrometry fingerprinting.

Access to new biological sources is a key element of natural product research. A particularly large number of biologically active molecules have been found to originate from microorganisms. Very recently, the use of fungal co-culture to activate the silent genes involved in metabolite biosynthesis was found to be a successful method for the induction of new compounds. However, the detection and identification of the induced metabolites in the confrontation zone where fungi interact remain very challenging. To tackle this issue, a high-throughput UHPLC-TOF-MS-based metabolomic approach has been developed for the screening of fungal co-cultures in solid media at the petri dish level. The metabolites that were overexpressed because of fungal interactions were highlighted by comparing the LC-MS data obtained from the co-cultures and their corresponding mono-cultures. This comparison was achieved by subjecting automatically generated peak lists to statistical treatments. This strategy has been applied to more than 600 co-culture experiments that mainly involved fungal strains from the Fusarium genera, although experiments were also completed with a selection of several other filamentous fungi. This strategy was found to provide satisfactory repeatability and was used to detect the biomarkers of fungal induction in a large panel of filamentous fungi. This study demonstrates that co-culture results in consistent induction of potentially new metabolites.

Genetic variability of rice recurrent selection populations as affected by male sterility or manual recombination

The objective of this work was to determine the effect of male sterility or manual recombination on genetic variability of rice recurrent selection populations. The populations CNA-IRAT 4, with a gene for male sterility, and CNA 12, which was manually recombined, were evaluated. Genetic variability among selection cycles was estimated using14 simple sequence repeat (SSR) markers. A total of 926 plants were analyzed, including ten genitors and 180 individuals from each of the evaluated cycles (1, 2 and 5) of the population CNA-IRAT 4, and 16 genitors and 180 individuals from each of the cycles (1 and 2) of CNA 12. The analysis allowed the identification of alleles not present among the genitors for both populations, in all cycles, especially for the CNA-IRAT 4 population. These alleles resulted from unwanted fertilization with genotypes that were not originally part of the populations. The parameters of Wright's F-statistic (F IS and F IT) indicated that the manual recombination expands the genetic variability of the CNA 12 population, whereas male sterility reduces the one of CNA-IRAT 4.

Recombination is suppressed and variability reduced in a nascent Y chromosome.

Several hypotheses have been elaborated to account for the evolutionary decay commonly observed in full-fledged Y chromosomes. Enhanced drift, background selection and selective sweeps, which are expected to result from reduced recombination, may all share responsibilities in the initial decay of proto-Y chromosomes, but little empirical information has been gathered so far. Here we take advantage of three markers that amplify on both of the morphologically undifferentiated sex chromosomes of the European tree frog (Hyla arborea) to show that recombination is suppressed in males (the heterogametic sex) but not in females. Accordingly, genetic variability is reduced on the Y, but in a way that can be accounted for by merely the number of chromosome copies per breeding pair, without the need to invoke background selection or selective sweeps.

The effect of recombination in Aeromonas

Although several approaches have been attempted, the estimation of recombination frequencies in natural populations ofbacteria remains challenging. Previous studies have demonstrated awide variety of situations among bacterial species, ranging from theclonal diversification of Salmonella or Escherichia coli, which aremainly due to mutation, to the frequent recombination found inNeisseria gonorrhoeae or Helicobacter pylori. Most of the populationstudies done with bacterial species suggest that recombination occursin nature but that it is infrequent compared to mutation. Consequently,bacterial populations consist largely of independent clonal lineages.Our research suggests little or null influence of recombination in thegenetic structure of "Aeromonas hydrophila Species Complex", despite the presence of some strains with recombinant gene fragments.

Classification and fingerprinting of different berries based on biochemical profiling and antioxidant capacity

The objective of this work was to evaluate the biochemical composition of six berry types belonging to Fragaria, Rubus, Vaccinium and Ribes genus. Fruit samples were collected in triplicate (50 fruit each) from 18 different species or cultivars of the mentioned genera, during three years (2008 to 2010). Content of individual sugars, organic acids, flavonols, and phenolic acids were determined by high performance liquid chromatography (HPLC) analysis, while total phenolics (TPC) and total antioxidant capacity (TAC), by using spectrophotometry. Principal component analysis (PCA) and hierarchical cluster analysis (CA) were performed to evaluate the differences in fruit biochemical profile. The highest contents of bioactive components were found in Ribes nigrum and in Fragaria vesca, Rubus plicatus, and Vaccinium myrtillus. PCA and CA were able to partially discriminate between berries on the basis of their biochemical composition. Individual and total sugars, myricetin, ellagic acid, TPC and TAC showed the highest impact on biochemical composition of the berry fruits. CA separated blackberry, raspberry, and blueberry as isolate groups, while classification of strawberry, black and red currant in a specific group has not occurred. There is a large variability both between and within the different types of berries. Metabolite fingerprinting of the evaluated berries showed unique biochemical profiles and specific combination of bioactive compound contents.

Incidence of Garlic common latent virus in Argentina, and phylogenetic and recombination analyses of isolates

The objective of this work was to estimate the incidence and prevalence of Garlic common latent virus (GarCLV) in the main production regions of garlic (Allium sativum) in Argentina, and to perform phylogenetic and recombination analyses in isolates from these regions. Leaf samples (3,050) were taken from four garlic commercial types, in 13 departments of the four main garlic-producing provinces of Argentina, in a 1,175-ha sampling area. Virus infection was evaluated with DAS-Elisa test using specific antiserum, and the phylogenetic and recombination analyses were done with capsid protein (CP) nucleotide sequence of seven GarCLV isolates from the provinces. The incidence of GarCLV in the evaluated provinces varied between 6.7 and 22% of the samples, whereas the prevalence varied between 52.6 and 70%. In the analysis of garlic commercial types, Morado showed the highest incidence of the virus, in the province of San Juan, whereas Rosado Paraguayo had the lowest incidence, in the province of Cordoba. Nucleotide identity in the CP sequences ranged between 80.3 and 97.6%. The phylogenetic analysis shows the presence of two main groups of GarCLV and of a possible third group that would include only a German isolate. The recombination analysis between isolates from different parts of the world evidences the presence of recombinant isolates from Poland and Australia.

Evidence of recombination in putative ancient asexuals.

Ancient asexuals have been considered to be a contradiction of the basic tenets of evolutionary theory. Barred from rearranging genetic variation by recombination, their reduced number of gene arrangements is thought to hamper their response to changing environments. For the same reason, it should be difficult for them to avoid the build-up of deleterious mutations. Several groups of taxonomically diverse organisms are thought to be ancient asexuals, although clear evidence for or against the existence of recombination events is scarce. Several methods have recently been developed for predicting recombination events by analyzing aligned sequences of a given region of DNA that all originate from one species. The methods are based on phylogenetic, substitution, and compatibility analyses. Here we present the results of analyses of sequence data from different loci studied in several groups of evolutionarily distant species that are considered to be ancient asexuals, using seven different types of analysis. The groups of organisms were the arbuscular mycorrhizal fungi (Glomales), Darwinula stevensoni (Darwinuloidea crustacean ostracods) and the bdelloid rotifers (Bdelloidea), which are thought to have been asexual for the last 400, 25-100, and 35-40 Myr, respectively. The seven different analytical methods evaluated the evolutionary relationships among haplotypes, and these methods had previously been shown to be reliable for predicting the occurrence of recombination events. Despite the different degree of genetic variation among the different groups of organisms, at least some evidence for recombination was found in all species groups. In particular, predictions of recombination events in the arbuscular mycorrhizal fungi were frequent. Predictions of recombination were also found for sequence data that have previously been used to infer the absence of recombination in bdelloid rotifers. Although our results have to be taken with some caution because they could signal very ancient recombination events or possibly other genetic variation of nonrecombinant origin, they suggest that some cryptic recombination events may exist in these organisms.

Cooperation of breast cancer proteins PALB2 and piccolo BRCA2 in stimulating homologous recombination.

Inherited mutations in human PALB2 are associated with a predisposition to breast and pancreatic cancers. PALB2's tumor-suppressing effect is thought to be based on its ability to facilitate BRCA2's function in homologous recombination. However, the biochemical properties of PALB2 are unknown. Here we show that human PALB2 binds DNA, preferentially D-loop structures, and directly interacts with the RAD51 recombinase to stimulate strand invasion, a vital step of homologous recombination. This stimulation occurs through reinforcing biochemical mechanisms, as PALB2 alleviates inhibition by RPA and stabilizes the RAD51 filament. Moreover, PALB2 can function synergistically with a BRCA2 chimera (termed piccolo, or piBRCA2) to further promote strand invasion. Finally, we show that PALB2-deficient cells are sensitive to PARP inhibitors. Our studies provide the first biochemical insights into PALB2's function with piBRCA2 as a mediator of homologous recombination in DNA double-strand break repair.

Analysis of meiotic recombination in 22q11.2, a region that frequently undergoes deletions and duplications

Background: The 22q11.2 deletion syndrome is the most frequent genomic disorder with an estimated frequency of 1/4000 live births. The majority of patients (90%) have the same deletion of 3 Mb (Typically Deleted Region, TDR) that results from aberrant recombination at meiosis between region specific low-copy repeats (LCRs). Methods: As a first step towards the characterization of recombination rates and breakpoints within the 22q11.2 region we have constructed a high resolution recombination breakpoint map based on pedigree analysis and a population-based historical recombination map based on LD analysis. Results: Our pedigree map allows the location of recombination breakpoints with a high resolution (potential recombination hotspots), and this approach has led to the identification of 5 breakpoint segments of 50 kb or less (8.6 kb the smallest), that coincide with historical hotspots. It has been suggested that aberrant recombination leading to deletion (and duplication) is caused by low rates of Allelic Homologous Recombination (AHR) within the affected region. However, recombination rate estimates for 22q11.2 region show that neither average recombination rates in the 22q11.2 region or within LCR22-2 (the LCR implicated in most deletions and duplications), are significantly below chromosome 22 averages. Furthermore, LCR22-2, the repeat most frequently implicated in rearrangements, is also the LCR22 with the highest levels of AHR. In addition, we find recombination events in the 22q11.2 region to cluster within families. Within this context, the same chromosome recombines twice in one family; first by AHR and in the next generation by NAHR resulting in an individual affected with the del22q11.2 syndrome. Conclusion: We show in the context of a first high resolution pedigree map of the 22q11.2 region that NAHR within LCR22 leading to duplications and deletions cannot be explained exclusively under a hypothesis of low AHR rates. In addition, we find that AHR recombination events cluster within families. If normal and aberrant recombination are mechanistically related, the fact that LCR22s undergo frequent AHR and that we find familial differences in recombination rates within the 22q11.2 region would have obvious health-related implications.

Regulation of recombination at yeast nuclear pores controls repair and triplet repeat stability.

Secondary structure-forming DNA sequences such as CAG repeats interfere with replication and repair, provoking fork stalling, chromosome fragility, and recombination. In budding yeast, we found that expanded CAG repeats are more likely than unexpanded repeats to localize to the nuclear periphery. This positioning is transient, occurs in late S phase, requires replication, and is associated with decreased subnuclear mobility of the locus. In contrast to persistent double-stranded breaks, expanded CAG repeats at the nuclear envelope associate with pores but not with the inner nuclear membrane protein Mps3. Relocation requires Nup84 and the Slx5/8 SUMO-dependent ubiquitin ligase but not Rad51, Mec1, or Tel1. Importantly, the presence of the Nup84 pore subcomplex and Slx5/8 suppresses CAG repeat fragility and instability. Repeat instability in nup84, slx5, or slx8 mutant cells arises through aberrant homologous recombination and is distinct from instability arising from the loss of ligase 4-dependent end-joining. Genetic and physical analysis of Rad52 sumoylation and binding at the CAG tract suggests that Slx5/8 targets sumoylated Rad52 for degradation at the pore to facilitate recovery from acute replication stress by promoting replication fork restart. We thereby confirmed that the relocation of damage to nuclear pores plays an important role in a naturally occurring repair process.

Recombination dynamics in ZnO nanowires: Surfaces states versus mode quality factor

In this work, we investigate the influence of finite size on the recombinations dynamics of ZnO nanowires. We demonstrate that diameter as well as lenght of nanowires determine the lifetime of the neutral donor bound excitons. Our findings suggest that while the length is mainly responsible for different mode quality factors of the cavity-like nanowires, the diameter determines the influence of surface states as alternative recombinations channels for the optical modes trapped in the nanocavity. In addition, comparing nanowires grown using different catalyst we show that the surfaces states strongly depend on each precursor characteristics.

Sex-Chromosome Homomorphy in Palearctic Tree Frogs Results from Both Turnovers and X-Y Recombination.

Contrasting with birds and mammals, poikilothermic vertebrates often have homomorphic sex chromosomes, possibly resulting from high rates of sex-chromosome turnovers and/or occasional X-Y recombination. Strong support for the latter mechanism was provided by four species of European tree frogs, which inherited from a common ancestor (∼5 Ma) the same pair of homomorphic sex chromosomes (linkage group 1, LG1), harboring the candidate sex-determining gene Dmrt1. Here, we test sex linkage of LG1 across six additional species of the Eurasian Hyla radiation with divergence times ranging from 6 to 40 Ma. LG1 turns out to be sex linked in six of nine resolved cases. Mapping the patterns of sex linkage to the Hyla phylogeny reveals several transitions in sex-determination systems within the last 10 My, including one switch in heterogamety. Phylogenetic trees of DNA sequences along LG1 are consistent with occasional X-Y recombination in all species where LG1 is sex linked. These patterns argue against one of the main potential causes for turnovers, namely the accumulation of deleterious mutations on nonrecombining chromosomes. Sibship analyses show that LG1 recombination is strongly reduced in males from most species investigated, including some in which it is autosomal. Intrinsically low male recombination might facilitate the evolution of male heterogamety, and the presence of important genes from the sex-determination cascade might predispose LG1 to become a sex chromosome.