High Frequency Of Recombination Research Articles

Bioinformatics analysis of the recent MERS-CoV with special reference to the virus-encoded Spike protein

Coronaviruses (CoVs) are characterized by high recombination frequencies, resulting in sudden outbreak of newly evolved viruses with different pathogenicity, tissue tropism and high genome sequence variability. Recently, an outbreak of CoVs was evolved in the Arabian Pennsylvania, which is known as Middle East Respiratory Syndrome Coronavirus (MERS-CoV). Full genome sequence analysis of MERS-CoV isolates and its comparison with other CoV full genome sequences revealed a low to medium sequence identity. Furthermore, it showed more sequence identity and phylogenetic relations with bat-derived CoVs and lower values with animal-derived CoVs, indicating low possibility of zoonotic origin and possible incrimination of bats in the spread of MERS-CoV. The higher neighbor homology was evident with BetaCoV and their associated SARS-CoV. The spike protein, which is a highly variable part of CoV genome and responsible for difference in tissue tropism and virus entry to the cell, showed more or less similar profile of the whole genome analysis. Furthermore, the highest identity was with those in bats with Asian origin of CoV and there was lower homology with isolates from other continents. With low human-to-human transmission and low homology with CoV of animal origin, bats are thought to be the source of MERS-CoV, especially those bearing the Asian isolates of CoV.https://maltcasino.fun/https://pinbahis.fun/https://onwin.direct/https://130betebet.com/https://detectivestripes.com/

DNA repair in plants studied by comet assay

Comet assay in plants.From the first description of the comet assay with isolated nuclei rather than whole cells it became evident that assay is well suited to be applied in plants (Koppen & Verschaeve, 1996). Disintegration of tissue by quick chopping with a razor blade, direct collection of released nuclei by patting and pipetting enables to process samples in time shorter than 2 minutes, the time prerequisite to study quick repair (Kozak et al, 2009).Plants are due to their sessile nature permanently exposed to environmental stresses (drought, salinity), ionizing (IR) and UV radiation and genotoxins, which directly or indirectly via generation of reactive oxidative species (ROS) damage their DNA. Radiomimetic Bleomycin functions as a catalyst to produce ROS leading to clusters of oxidized DNA lesions, single (SSB) and double (DSBs) strand breaks similarly as IR (Steighner & Povirk, 1990). Incurred SSBs and DSBs are easily distinguished and measured by comet assay when varying conditions of mainly used protocol with electrophoresis in 0.3 M NaOH, pH>13 solution (A/A assay). DSBs are detected under “neutral” conditions by N/N assay in regular electrophoretic buffer (Kozak et al, 2009; Olive & Banath, 2006), whilst SSBs are revealed by A/N assay, with alkali-unwinding step in 0.3 M NaOH prior electrophoresis (Angelis et al, 1999; Menke et al, 2001). Better resolution in DSBs and SSBs assays is observed when “neutral” conditions are set between pH 9-10, still well bellow DNA denaturing pH<11.6 (Bradley & Kohn, 1979).Repair of DSBs.DSBs are one of the most cytotoxic forms of DNA damage that must be repaired by recombination, predominantly via non-homologous joining of DNA ends (NHEJ) in higher eukaryotes. However, analysis of DSB repair kinetics of Arabidopsis NHEJ mutants atlig4 and atku80 with the N/N assay showed that repair of Bleomycin induced DSB is biphasic and rapid alternative repair pathways is active (Fig. 1). Surprisingly, kinetic measurements showed that rapid DSB repair was faster in the NHEJ mutant lines (t1/2 5.5 min.) than in wild-type Arabidopsis (t1/2 7.9 min.). Kozak et al. (Kozak et al, 2009) provided the first characterization of this alternate KU/LIG4-independent repair pathway that rapidly removes the majority of DSBs present in nuclear DNA and found its dependence on components of structural maintenance of chromosomes (SMC) complexes, namely SMC6b (MIM) of SMC5/6 complex, kleisin AtRAD21.1 and 3 of cohesin SMC1/3 complex (da Costa-Nunes et al, 2014) and structurally related SMCHD protein GMI1 (Bohmdorfer et al, 2011). Moreno-Romero et al. (Moreno-Romero et al, 2012) studied CK2 protein required for maintenance and control of genomic stability and used N/N assay to show that DSBs were more rapidly repaired in atck2 mutant than in control plants. Their results suggest that atck2 plants are more proficient in repairing DSBs and other lesions produced by IR or Bleomycin, despite their hypersensitivity to these agents.The moss Physcomitrella patens is unique besides the high frequency of homologous recombination for haploid state and filamentous growth during early stages of the vegetative growth. Sheared filaments enables to establish protonemal cultures with up to 50% dividing cells and makes Physcomitrella an excellent model plant to study DNA damage responses. Kamisugi et al. (Kamisugi et al, 2012) used N/N assay to study Physcomitrella mutants of pivotal DSB repair MRN complex (MRE11, RAD50, NBS1). Kinetics of DNA-DSB repair in wild-type and mutant plants revealed that Bleomycin-induced fragmentation of genomic DNA was quickly repaired at approximately equal rates (t1/2 3 min) in each genotype, although both, the ppmre11 and pprad50 mutants exhibited severely restricted growth and development and enhanced sensitivity to UV-B and Bleomycin-induced DNA damage. This implies that while extensive DNA repair can occur in the absence of a functional MRN complex; it is unsupervised in nature and results in the accumulation of deleterious mutations incompatible with normal growth and development, the similar phenomenon as observed by Moreno-Romero et al. (Moreno-Romero et al, 2012) in atck2 plants. When MRN complex and CK2, both assumed to be associated with error-free homologous recombination are disabled, then NHEJ error-prone repair prevails at a cost of induced mutations in continuous DNA. And indeed this was proved by sequencing of mutated APT locus in which we identified deletions of various lengths as could be expected as an outcome of NHEJ repair mechanism. Combination of comet and mutation assays explained contradictory observation of rapid DSB repair in mutants with phenotype sensitive to induction of DSBs. Evidently plants evolved this attitude of rapid reconstitution of genome integrity from the early evolution stages (mosses were one of the first plants to colonize land) to higher eukaryote seed plants, where during every plant cycle embryos undergo severe DNA damage during seed maturation (generated by ROS released upon desiccation) and efficient recovery during seed germination.Repair of SSBs.

Extensive recombination due to heteroduplexes generates large amounts of artificial gene fragments during PCR.

Artificial recombinants can be generated during PCR when more than two genetically distinct templates coexist in a single PCR reaction. These recombinant amplicons can lead to the false interpretation of genetic diversity and incorrect identification of biological phenotypes that do not exist in vivo. We investigated how recombination between 2 or 35 genetically distinct HIV-1 genomes was affected by different PCR conditions using the parallel allele-specific sequencing (PASS) assay and the next generation sequencing method. In a standard PCR condition, about 40% of amplicons in a PCR reaction were recombinants. The high recombination frequency could be significantly reduced if the number of amplicons in a PCR reaction was below a threshold of 1013–1014 using low thermal cycles, fewer input templates, and longer extension time. Heteroduplexes (each DNA strand from a distinct template) were present at a large proportion in the PCR products when more thermal cycles, more templates, and shorter extension time were used. Importantly, the majority of recombinants were identified in heteroduplexes, indicating that the recombinants were mainly generated through heteroduplexes. Since prematurely terminated extension fragments can form heteroduplexes by annealing to different templates during PCR amplification, recombination has a better chance to occur with samples containing different genomes when the number of amplicons accumulate over the threshold. New technologies are warranted to accurately characterize complex quasispecies gene populations.

Observation of high recombination occurrence of Porcine Reproductive and Respiratory Syndrome Virus in field condition

Recombination in Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is a well-documented phenomenon. A high recombination frequency has been reported in experimental conditions both in vitro and in vivo, and its role in driving viral evolution has been postulated by several authors. However field evidences are rare, mainly obtained from large-scale sampling and typically represented by single sequences rather than by groups of circulating “recombinant progenies”. The present work was aimed to investigate the gray area between experimental studies and large-scale epidemiological investigations. The study was performed on ORF5, ORF7 and concatenated sequences obtained in our laboratory or available in GenBank collected between 2009 and 2012 in northern Italy. Six independent recombinant strains out of 66 concatenated sequences (∼9%) were found, demonstrating a high recombination frequency respect to previous field studies but comparable to in vitro experiments. In silico analysis let speculate that this new strain displayed physicochemical features diverse enough to potentially alter its immunological properties. Taken altogether, the results of our study support previous experimental evidences that depict PRRSV to be extremely prone to recombination. The limited temporal and geographical spread of recombinant strains however states in favor of a limited fitness of the recombinant progeny compared to parental strains and the marginal role of this phenomenon in PRRSV evolution.

Bidirectional genomic exchange between Helicobacter pylori strains from a family in Coventry, United Kingdom

The human gastric pathogen Helicobacter pylori is characterised by a high mutation rate and frequent recombination during mixed infection, which result in extensive genetic diversity and rapid allelic diversification. Mixed infections are believed to be much more common in regions with a high H. pylori prevalence than in industrialised countries. To better understand the genomic flexibility of H. pylori in a low prevalence region, we used 454 sequencing technology to investigate whole genome sequences of H. pylori strains isolated from members of three generations of a family living in Coventry, UK. The genomes of four H. pylori strains isolated from a grandfather, two of his sons and one grandson were sequenced. Three of these genomes showed a high overall sequence similarity, suggesting a recent common ancestor. The genomes differed by 316–336 SNPs, and recombination events (imports) resulted in 170–251 clusters of polymorphisms (CNPs). Imports were particularly frequent in genes encoding Helicobacter outer membrane proteins, suggesting an adaptation of the strains to their individual host. The fourth strain differed substantially from these three highly related strains but still shared long fragments of identical sequence, which most likely reflect imports from the highly related family variants. The data show extensive bidirectional exchange of DNA between the strains isolated from the family members, illustrating both the convergence and divergence effect that recombination can lead to. Detailed analysis of the distribution of SNPs and imports permits to draw up a complex scenario of the transmission history involving infection with at least two, and probably more separate strains. This complexity and the resulting high frequency of recombination were unexpected for an industrialised country where the prevalence of H. pylori infection has strongly declined in recent decades.

Comprehensive identification of single nucleotide polymorphisms associated with beta-lactam resistance within pneumococcal mosaic genes.

Traditional genetic association studies are very difficult in bacteria, as the generally limited recombination leads to large linked haplotype blocks, confounding the identification of causative variants. Beta-lactam antibiotic resistance in Streptococcus pneumoniae arises readily as the bacteria can quickly incorporate DNA fragments encompassing variants that make the transformed strains resistant. However, the causative mutations themselves are embedded within larger recombined blocks, and previous studies have only analysed a limited number of isolates, leading to the description of “mosaic genes” as being responsible for resistance. By comparing a large number of genomes of beta-lactam susceptible and non-susceptible strains, the high frequency of recombination should break up these haplotype blocks and allow the use of genetic association approaches to identify individual causative variants. Here, we performed a genome-wide association study to identify single nucleotide polymorphisms (SNPs) and indels that could confer beta-lactam non-susceptibility using 3,085 Thai and 616 USA pneumococcal isolates as independent datasets for the variant discovery. The large sample sizes allowed us to narrow the source of beta-lactam non-susceptibility from long recombinant fragments down to much smaller loci comprised of discrete or linked SNPs. While some loci appear to be universal resistance determinants, contributing equally to non-susceptibility for at least two classes of beta-lactam antibiotics, some play a larger role in resistance to particular antibiotics. All of the identified loci have a highly non-uniform distribution in the populations. They are enriched not only in vaccine-targeted, but also non-vaccine-targeted lineages, which may raise clinical concerns. Identification of single nucleotide polymorphisms underlying resistance will be essential for future use of genome sequencing to predict antibiotic sensitivity in clinical microbiology.

A genomic survey of thirty soybean-infecting bean common mosaic virus (BCMV) isolates from China pointed BCMV as a potential threat to soybean production

Widely known as a severe pathogen of bean plants, the bean common mosaic virus (BCMV) has been reported to infect soybeans only sporadically and the involved strains were all found in China regions. To explore variations among soybean-infecting BCMV strains, hundreds of soybean mosaic leave samples were collected throughout China, with a total of 30 BCMV isolates detected and their genomes sequenced. These newly obtained genomes, together with 16 other BCMV genomes available in GenBank were examined from multiple aspects to characterize BCMV evolutionary processes. Phylogenetic analysis showed that both soybean-infecting BCMVs (group I) and peanut-infecting BCMVs (group II) are distantly related to other BCMVs, suggesting ancestral differentiation and host adaptation. Genetic variation analysis showed that P1, P3 and 6K2 genes and the beginning portion of CP gene showed higher levels of variation relative to other genes. Moreover, selection analyses further confirmed that a number of sites within the P1 and P3 genes have suffered positive selection. These obtained BCMV sequences also exhibit high recombination frequencies, indicating a more dynamic evolutionary history. Finally, 12 different soybean cultivars were challenged with two BCMV isolates (DXH015 and HZZB011), with most of the cultivars successfully infected. These findings suggest that BCMV is indeed a potential threat to soybean production.

Template Switching During Break-Induced Replication Is Promoted by the Mph1 Helicase in Saccharomyces cerevisiae

Chromosomal double-strand breaks (DSBs) that have only one end with homology to a donor duplex undergo repair by strand invasion followed by replication to the chromosome terminus (break-induced replication, BIR). Using a transformation-based assay system, it was previously shown that BIR could occur by several rounds of strand invasion, DNA synthesis, and dissociation. Here we describe a modification of the transformation-based assay to facilitate detection of switching between donor templates during BIR by genetic selection in diploid yeast. In addition to the expected recovery of template switch products, we found a high frequency of recombination between chromosome homologs during BIR, suggesting transfer of the DSB from the transforming linear DNA to the donor chromosome, initiating secondary recombination events. The frequency of BIR increased in the mph1Δ mutant, but the percentage of template switch events was significantly decreased, revealing an important role for Mph1 in promoting BIR-associated template switching. In addition, we show that the Mus81, Rad1, and Yen1 structure-selective nucleases act redundantly to facilitate BIR.

Mutagenic Organized Recombination Process by Homologous In Vivo Grouping (MORPHING) for Directed Enzyme Evolution

Approaches that depend on directed evolution require reliable methods to generate DNA diversity so that mutant libraries can focus on specific target regions. We took advantage of the high frequency of homologous DNA recombination in Saccharomyces cerevisiae to develop a strategy for domain mutagenesis aimed at introducing and in vivo recombining random mutations in defined segments of DNA. Mutagenic Organized Recombination Process by Homologous IN vivo Grouping (MORPHING) is a one-pot random mutagenic method for short protein regions that harnesses the in vivo recombination apparatus of yeast. Using this approach, libraries can be prepared with different mutational loads in DNA segments of less than 30 amino acids so that they can be assembled into the remaining unaltered DNA regions in vivo with high fidelity. As a proof of concept, we present two eukaryotic-ligninolytic enzyme case studies: i) the enhancement of the oxidative stability of a H2O2-sensitive versatile peroxidase by independent evolution of three distinct protein segments (Leu28-Gly57, Leu149-Ala174 and Ile199-Leu268); and ii) the heterologous functional expression of an unspecific peroxygenase by exclusive evolution of its native 43-residue signal sequence.

Molecular Characterization of Southern Rice Blacked‐Streaked Dwarf Virus (SRBSDV) from Vietnam

AbstractSouthern rice black‐streaked dwarf virus (SRBSDV) is a novel putative member of the genus Fijivirus, family Reoviridae. We report here the genomic sequences of a Vietnamese isolate (SRBSDV‐V). The total genome of SRBSDV‐V has 29 115 nucleotides (nt), nine nt shorter than SRBSDV‐GD or ‐HN, but similar in organization to these two Chinese isolates. Nucleotide diversities among SRBSDV isolates were much lower than those among the corresponding ORFs of the available RBSDV isolates and there was a lower purifying selection pressure on SRBSDV than RBSDV, providing first molecular evidence for the view that SRBSDV is of recent origin. In studies of all available SRBSDV sequences, there was no obvious correlation between geographic distances and phylogenetic distribution. A high frequency of genetic recombination was found among both Chinese and Vietnam SRBSDV isolates, suggesting that recombination may play an important role in the molecular variation and evolution of SRBSDV.

Genetic structure of Trypanosoma cruziin Colombia revealed by a High-throughput Nuclear Multilocus Sequence Typing (nMLST) approach

BackgroundChagas disease is a systemic pathology caused by Trypanosoma cruzi. This parasite reveals remarkable genetic variability, evinced in six Discrete Typing Units (DTUs) named from T. cruzi I to T. cruzi VI (TcI to TcVI). Recently newly identified genotypes have emerged such as TcBat in Brazil, Colombia and Panama associated to anthropogenic bats. The genotype with the broadest geographical distribution is TcI, which has recently been associated to severe cardiomyopathies in Argentina and Colombia. Therefore, new studies unraveling the genetic structure and natural history of this DTU must be pursued.ResultsWe conducted a spatial and temporal analysis on 50 biological clones of T. cruzi I (TcI) isolated from humans with different clinical phenotypes, triatomine bugs and mammal reservoirs across three endemic regions for Chagas disease in Colombia. These clones were submitted to a nuclear Multilocus Sequence Typing (nMLST) analysis in order to elucidate its genetic diversity and clustering. After analyzing 13 nuclear housekeeping genes and obtaining a 5821 bp length alignment, we detected two robust genotypes within TcI henceforth named TcIDOM (associated to human infections) and a second cluster associated to peridomestic and sylvatic populations. Additionaly, we detected putative events of recombination and an intriguing lack of linkage disequilibrium.ConclusionsThese findings reinforce the emergence of an enigmatic domestic T. cruzi genotype (TcIDOM), and demonstrates the high frequency of recombination at nuclear level across natural populations of T. cruzi. Therefore, the need to pursue studies focused on the diferential virulence profiles of TcI strains. The biological and epidemiological implications of these findings are herein discussed.

Analysis of homozygosity and genetic similarity between two succes-sive generations in a meiogynogenetic Japanese flounder family

The artificial induction of gynogenesis is a form of chromosome manipulation that has several applica-tions,including the rapid establishment of inbred lines or strains.The technique offers a high degree of homozy-gosity,sex-control,and accelerated elimination of recessive deleterious genes from a cultured population.We produced two meiotic gynogenetic Japanese flounder(Paralichthys olivaceus) family lines: meio-G1(the first generation) and meio-G2(the second generation).We evaluated the efficacy of artificial meiotic gynogenesis for producing pure inbred lines by quantifying the homozygosity and genetic similarity of meio-G1 and meio-G2 us-ing microsatellite markers The average homozygosity among 15 loci was 0.124 7 for meio-G1 and 0.221 5 for meio-G2,which was higher than that in the natural mating family(0.091 7).The average similarity indices among individuals within meio-G1 and meio-G2 were 0.891 7 and 0.923 8,which were higher than within the natural mating family(0.5603).The average similarity in meio-G2 was slightly higher than in meio-G1.The observed heterozygosity in meio-G1 and meio-G2 was 1 at the Poli9-8tuf,Poli18tuf,Poli107tuf loci,all of which were lo-cated at some distance from the centromere(i.e.,had high recombination frequencies).The loci(Poli33tuf,Po-li24MHFS) closest to the centromere(i.e.,with a low recombination frequency) were homozygous in meio-G1 and meio-G2.Our results suggest that artificially induced meiotic gynogenesis is an efficient method for inbreeding to purify the genome,increase genetic similarity,and fix maternal genetic traits in the Japanese flounder.The mei-ogynogenetic families cultured in our study may be used in further selective breeding research.

Abstract LB-71: Carcinogenic significance of integrated hepatitis b viral factors and its genotype in hepatocarcinogenesis.

Abstract Integration of Hepatitis B Virus (HBV) is one of the major causes underlying Hepatocellular carcinoma (HCC) development. Recent studies revealed that altered expression of multiple cellular genes due to multiple random integration of viral factors lead to development of HCC. The present study aims to identify the carcinogenic significance of integrated HBV genome and its genotype in HCC development. Host-Viral Junctions were determined in HCC (T) /adjacent tissues (NT) and cirrhotic (-HCC) tissues by sequencing of Alu PCR product followed by sequence blast. Host gene expression was checked by real time PCR. Competition between two coexisting genotypes was studied by replication assay using genotype specific PCR from media and viral core particle isolated from genotype specific plasmid transfected HepG2 cell line. Similarly, each viral genotype mediated DNA damage (γH2aX foci by confocal microscopy), ROS generation (DCFDA quantitation by Facs analysis and ER stress marker GRP78 luciferase assay), homologous recombination efficiency were also compared. HBV integration was observed in 66.7% of T (8/12), 33.3% (4/12) NT and 75% (6/8) cirrhotic tissue samples in 18, 4 and 8 different locations respectively. These viral integration analysis within or close to several host genes, such as genes involved in differentiation, signaling, stress response, cell cycle, telomere regulations (8/30, 26.7%) etc and most of them showed altered expression. Interestingly, C-terminal truncated HBX which lost its growth suppressive domain were observed more in T (5/8, 62.5%) than NT (2/4, 50%) tissue samples. Most importantly, upon genotype analysis of each five clones in serum and tissue showed genotype C preferentially integrated in coexistence of both C and D genotype as “free virus” in tissue but D genotype observed in circulation. Genotype specific PCR from HBV/C &amp; D cotransfected media showed that in presence of HBV/C, D replicates better than C but HBV/C with high recombination frequency generates more DNA damage and facilitate viral integration. Thus random integration of HBV in host chromosome causes alterations in oncogenic gene expression and these alterations presumably lead to clonal selection of hepatocyte that acquires a growth advantage and proceeds towards HCC. Genotype of HBV is an important determining factor for HBV integration and liver inury. Citation Format: Soma Banerjee, Somenath Datta, Shrabasti Roychoudhury, Debanjali Dasgupta, Amit Ghosh, Gaurav Roy, Simanti Datta, Abhijit Chowdhury. Carcinogenic significance of integrated hepatitis b viral factors and its genotype in hepatocarcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-71. doi:10.1158/1538-7445.AM2013-LB-71

Analysis of 454 sequencing error rate, error sources, and artifact recombination for detection of Low-frequency drug resistance mutations in HIV-1 DNA

Background454 sequencing technology is a promising approach for characterizing HIV-1 populations and for identifying low frequency mutations. The utility of 454 technology for determining allele frequencies and linkage associations in HIV infected individuals has not been extensively investigated. We evaluated the performance of 454 sequencing for characterizing HIV populations with defined allele frequencies.ResultsWe constructed two HIV-1 RT clones. Clone A was a wild type sequence. Clone B was identical to clone A except it contained 13 introduced drug resistant mutations. The clones were mixed at ratios ranging from 1% to 50% and were amplified by standard PCR conditions and by PCR conditions aimed at reducing PCR-based recombination. The products were sequenced using 454 pyrosequencing. Sequence analysis from standard PCR amplification revealed that 14% of all sequencing reads from a sample with a 50:50 mixture of wild type and mutant DNA were recombinants. The majority of the recombinants were the result of a single crossover event which can happen during PCR when the DNA polymerase terminates synthesis prematurely. The incompletely extended template then competes for primer sites in subsequent rounds of PCR. Although less often, a spectrum of other distinct crossover patterns was also detected. In addition, we observed point mutation errors ranging from 0.01% to 1.0% per base as well as indel (insertion and deletion) errors ranging from 0.02% to nearly 50%. The point errors (single nucleotide substitution errors) were mainly introduced during PCR while indels were the result of pyrosequencing. We then used new PCR conditions designed to reduce PCR-based recombination. Using these new conditions, the frequency of recombination was reduced 27-fold. The new conditions had no effect on point mutation errors. We found that 454 pyrosequencing was capable of identifying minority HIV-1 mutations at frequencies down to 0.1% at some nucleotide positions.ConclusionStandard PCR amplification results in a high frequency of PCR-introduced recombination precluding its use for linkage analysis of HIV populations using 454 pyrosequencing. We designed a new PCR protocol that resulted in a much lower recombination frequency and provided a powerful technique for linkage analysis and haplotype determination in HIV-1 populations. Our analyses of 454 sequencing results also demonstrated that at some specific HIV-1 drug resistant sites, mutations can reliably be detected at frequencies down to 0.1%.

Genotoxin Induced Mutagenesis in the Model PlantPhyscomitrella patens

The moss Physcomitrella patens is unique for the high frequency of homologous recombination, haploid state, and filamentous growth during early stages of the vegetative growth, which makes it an excellent model plant to study DNA damage responses. We used single cell gel electrophoresis (comet) assay to determine kinetics of response to Bleomycin induced DNA oxidative damage and single and double strand breaks in wild type and mutant lig4 Physcomitrella lines. Moreover, APT gene when inactivated by induced mutations was used as selectable marker to ascertain mutational background at nucleotide level by sequencing of the APT locus. We show that extensive repair of DSBs occurs also in the absence of the functional LIG4, whereas repair of SSBs is seriously compromised. From analysis of induced mutations we conclude that their accumulation rather than remaining lesions in DNA and blocking progression through cell cycle is incompatible with normal plant growth and development and leads to sensitive phenotype.

Changes in accumulation of heteroplasmic mitochondrial DNA and frequency of recombination via short repeats during plant lifetime in Phaseolus vulgaris.

Recombination via short repeats in plant mitochondrial genomes results in sublimons--DNA molecules with a copy number much lower compared to the main mitochondrial genome. Coexistence of stoichiometrically different mitotypes, called heteroplasmy, plays an important evolutionary role, since sublimons occasionally replace the main genome resulting in a new plant phenotype. It is not clear, how frequency of recombination and sublimon production is regulated and how it is related to changes in the quantity of the main genome and sublimons. We analyzed the accumulation of two recombining main genome sequences and two resulting sublimons in apical meristems, undifferentiated tissues and leaves of different age of Phaseolus vulgaris. Copy numbers of the main genome sequences varied greatly depending on tissue type and organ age while accumulation of sublimons remained much more stable. Although the overall accumulation of plant mtDNA decreased with the leaf age, the quantity of sublimons increased relative to the main genome indicating a higher frequency of recombination via the short 314 bp repeat. Recombination was symmetrical in young developing leaves while in senescent tissues it shifted towards asymmetric events resulting in overrepresentation of one product. We propose that during plant lifetime replication and recombination frequencies change oppositely sustaining heteroplasmic compositions of the genome, which are favorable for inheritance and maintenance of complex plant mtDNA.

Genomic disorders on chromosome 22

Chromosome 22, the first human chromosome to be completely sequenced, is prone to genomic alterations. Copy-number variants (CNVs) are common because of an enrichment of low-copy repeat sequences that precipitate a high frequency of nonallelic homologous misalignments and unequal recombination during meiosis. Among these is one of the most common multiple anomaly syndromes in humans and the most common microdeletion syndrome, velocardiofacial syndrome (VCFS), also known as 22q11.2 deletion syndrome and DiGeorge syndrome. This review will focus on the recent literature dealing with both the molecular and clinical aspects of chromosome 22 genomic variations. Although the literature covering this area is expansive, the majority is descriptive or analytical of the problems presented by these genomic disorders, and there is little evidence of translational research including treatment outcomes. With the increased use of microarray analysis in both research and clinical practice, variations in CNVs are becoming elucidated. Genomic analysis continues to characterize genes and gene effect. Research on the COMT gene continues to yield interesting findings, including a possible sex-mediated effect because of its regulatory role with estrogen. There is a small amount of treatment outcome data relevant to neuropsychiatric disorders in VCFS, but based on small samples and short-term follow-up. Although hundreds of studies in the past year have focused on genomic disorders of chromosome 22, little progress has been made in the implementation of translational research, even for more common disorders including VCFS.

Mapping Bacterial Functional Networks and Pathways in &lt;em&gt;Escherichia Coli&lt;/em&gt; using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10. Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping bacterial functional networks and pathways in Escherichia Coli using synthetic genetic arrays.

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)(1). While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships(2). Large-scale GI maps have been reported for eukaryotic organisms like yeast(3-7), but GI information remains sparse for prokaryotes(8), which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods(9, 10). Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale(9), using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection(11)) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity(9, 12, 13)) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system(14). GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected(9). Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process(2). Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex(2) as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred(9).

Low Species Barriers in Halophilic Archaea and the Formation of Recombinant Hybrids

Speciation of sexually reproducing organisms requires reproductive barriers. Prokaryotes reproduce asexually butoften exchange DNA by lateral gene transfer mechanisms and recombination [1], yet distinct lineages are still observed. Thus, barriers to gene flow such as geographic isolation, genetic incompatibility or a physiological inability to transfer DNA represent potential underlying mechanisms behind preferred exchange groups observed in prokaryotes [2-6]. In Bacteria, experimental evidence showed that sequence divergence impedes homologous recombination between bacterial species [7-11]. Here we study interspecies gene exchange in halophilic archaea that possess a parasexual mechanism of genetic exchange that is functional between species [12, 13]. In this process, cells fuse forming a diploid state containing the full genetic repertoire of both parental cells, which facilitates genetic exchange and recombination. Later, cells separate, occasionally resulting in hybrids of the parental strains [14]. We show high recombination frequencies between Haloferax volcanii and Haloferax mediterranei, two species that have an average nucleotide sequence identity of 86.6%. Whole genome sequencing of Haloferax interspecies hybrids revealed the exchange of chromosomal fragments ranging from 310Kb to 530Kb. These results show that recombination barriers may be more permissive in halophilic archaea than they are in bacteria.