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Random transgene integration is a powerful tool for developing new genome-wide screening approaches. These techniques have already been used for functional gene annotation by transposon-insertion sequencing, for identif ication of transcription factor binding sites and regulatory sequences, and for dissecting chromatin position effects. Precise localization of transgenes and accurate artifact f iltration are essential for this type of method. To date, many mapping assays have been developed, including Inverse-PCR, TLA, LAM-PCR, and splinkerette PCR. However, none of them is able to ensure localization of both transgene's f lanking regions simultaneously, which would be necessary for some applications. Here we proposed a cheap and simple NGS-based approach that overcomes this limitation. The developed assay requires using intentionally designed vectors that lack recognition sites of one or a set of restriction enzymes used for DNA fragmentation. By looping and sequencing these DNA fragments, we obtain special data that allows us to link the two f lanking regions of the transposon. This can be useful for precise insertion mapping and for screening approaches in the f ield of chromosome engineering, where chromosomal recombination events between transgenes occur in a cell population. To demonstrate the method's feasibility, we applied it for mapping SB transposon integration in the human HAP1 cell line. Our technique allowed us to eff iciently localize genomic transposon integrations, which was conf irmed via PCR analysis. For practical application of this approach, we proposed a set of recommendations and a normalization strategy. The developed method can be used for multiplex transgene localization and detection of rearrangements between them.Полногеномные скрининговые методы, основанные на случайной интеграции экзогенных генетических конструкций, – новейший класс инструментов, открывающий возможности для изучения широкого спектра геномных процессов. Данный подход уже был применен к функциональному аннотированию генов млекопитающих, скринингу приспособленности бактерий, определению сайтов связывания факторов транскрипции, идентификации регуляторных генетических элементов и исследованию хромосомного эффекта положения. Все эти эксперименты требуют точной локализации трансгенов в геноме. Существующие на сегодняшний день методы картирования, такие как Inverse-PCR, TLA, splinkerette PCR и LAM-PCR, не позволяют одновременно определять оба участка генома, фланкирующие одну интеграцию трансгена, что ограничивает применимость подходов, в том числе связанных с хромосомной инженерией. В настоящей работе мы предлагаем метод, с помощью которого можно преодолеть это ограничение. Разработанная технология основана на фрагментации геномной ДНК, не затрагивающей интеграции трансгена. Это достигается путем исключения из последовательности вектора сайтов узнавания одного или нескольких ферментов рестрикции. Затем, как и в Inverse-PCR, были закольцованы молекулы лигированием в разбавленной смеси и секвенированы. Полученные данные дают возможность с высокой точностью идентифицировать перестройки и отделить их от артефактов лигирования, и, кроме того, отследить события транслокаций между интеграциями трансгенов. Это может быть использовано в экспериментах по изучению индуцируемых хромосомных перестроек. Для доказательства применимости метода мы с его помощью картировали интеграции транспозона Sleeping Beauty в клетки человека линии Hap1. Картированные интеграции были валидированы с помощью ПЦР-анализа. В статье приведен ряд рекомендаций для практического использования этого метода в экспериментах по множественной локализации интеграций трансгенных конструкций.

As promising biomarkers and therapy targets, microRNAs (miRNAs) are involved in various physiological and tumorigenic processes. Genetic variants in miRNA‐binding sites can lead to dysfunction of miRNAs and contribute to disease. However, systematic investigation of the miRNA‐related single nucleotide polymorphisms (SNPs) for pancreatic cancer (PC) risk remains elusive. We performed integrative bioinformatics analyses to select 31 SNPs located in miRNA‐target binding sites using the miRNASNP v2.0, a solid database providing miRNA‐related SNPs for genetic research, and investigated their associations with risk of PC in two large case‐control studies totally including 1847 cases and 5713 controls. We observed that the SNP rs3802266 is significantly associated with increased risk of PC (odds ratio (OR) = 1.21, 95% confidence intervals (CI) = 1.11‐1.31, P = 1.29E‐05). Following luciferase reporter gene assays show that rs3802266‐G creates a stronger binding site for miR‐181a‐2‐3p in 3′ untranslated region (3′UTR) of the gene ZHX2. Expression quantitative trait loci (eQTL) analysis suggests that ZHX2 expression is lower in individuals carrying rs3802266‐G with increased PC risk. In conclusion, our findings highlight the involvement of miRNA‐binding SNPs in PC susceptibility and provide new clues for PC carcinogenesis.

Conjugal transfer is a major driving force of genetic exchange in eubacteria, and the system in IncP1-type broad-host-range plasmids transfers DNA even to eukaryotes and archaea in a process known as trans-kingdom conjugation (TKC). Although conjugation factors encoded on plasmids have been extensively analyzed, those on the donor chromosome have not. To identify the potential conjugation factor(s), a genome-wide survey on a comprehensive collection of Escherichia coli gene knockout mutants (Keio collection) as donors to Saccharomyces cerevisiae recipients was performed using a conjugal transfer system mediated by the type IV secretion system (T4SS) of the IncP1α plasmid. Out of 3,884 mutants, three mutants (ΔfrmR, ΔsufA, and ΔiscA) were isolated, which showed an increase by one order of magnitude in both E. coli–E. coli and E. coli–yeast conjugations without an increase in the mRNA accumulation level for the conjugation related genes examined. The double-knockout mutants for these genes (ΔfrmRΔsufA and ΔiscAΔfrmR) did not show synergistic effects on the conjugation efficiency, suggesting that these factors affect a common step in the conjugation machinery. The three mutants demonstrated increased conjugation efficiency in IncP1β-type but not in IncN- and IncW-type broad-host-range plasmid transfers, and the homologous gene knockout mutants against the three genes in Agrobacterium tumefaciens also showed increased TKC efficiency. These results suggest the existence of a specific regulatory system in IncP1 plasmids that enables the control of conjugation efficiency in different hosts, which could be utilized for the development of donor strains as gene introduction tools into bacteria, eukaryotes, and archaea.

The latent HIV‐1 reservoir is a major barrier to viral eradication. However, our understanding of how HIV‐1 establishes latency is incomplete. Here, by performing a genome‐wide CRISPR‐Cas9 knockout library screen, we identify phosphatidylethanolamine‐binding protein 1 (PEBP1), also known as Raf kinase inhibitor protein (RKIP), as a novel gene inducing HIV latency. Depletion of PEBP1 leads to the reactivation of HIV‐1 in multiple models of latency. Mechanistically, PEBP1 de‐phosphorylates Raf1/ERK/IκB and IKK/IκB signaling pathways to sequestrate NF‐κB in the cytoplasm, which transcriptionally inactivates HIV‐1 to induce latency. Importantly, the induction of PEBP1 expression by the green tea compound epigallocatechin‐3‐gallate (EGCG) prevents latency reversal by inhibiting nuclear translocation of NF‐κB, thereby suppressing HIV‐1 transcription in primary CD4+ T cells isolated from patients receiving antiretroviral therapy (ART). These results suggest a critical role for PEBP1 in the regulation of upstream NF‐κB signaling pathways governing HIV transcription. Targeting of this pathway could be an option to control HIV reservoirs in patients in the future., The latent HIV‐1 reservoir prevents efficient viral eradication. The phosphatase PEBP1 plays a critical role in the regulation of NF‐κB‐dependent pathways governing HIV‐1 transcription, and its targeting could control HIV reservoirs in patients.

Small GTPases function as molecular switches to active or inactive signaling cascades via binding or hydrolyzing GTP. A type of plant specific small GTPases, the ROPs are known to be involved in plant growth, development and immunity. We determined whether ROPs are conserved in Solanaceous species and whether they are involved in plant growth, development and resistance against Phytophthora capsisi. In genome-wide screening, a total of 66 ROPs in six Solanaceous species (SolROPs) were identified, including 16 ROPs in Solanum tuberosum L. (potato), 9 in Solanum lycopersicum L. (tomato), 5 in Solanum melongena L. (eggplant), 9 in Capsicum annuum L. (pepper), 13 in Nicotiana benthamiana Domin and 14 in Nicotiana tabacum L. (tobacco). Phylogenetic analysis revealed that 11 AtROPs and 66 SolROPs fall into five distinct clades (I-V) and hence a novel and systematic gene nomenclature was proposed. In addition, a comprehensive expression analysis was performed by making use of an online database. This revealed that ROP genes are differentially expressed during plant growth and development. Moreover, gene expression of SlROP-II.1 in S. lycopersicum could be significantly induced by P. capsici. Subsequently, SlROP-II.1 and its homologues in N. benthamiana and C. annuum (NbROP-II.1 and CaROP-II.1) were selected for functional analysis using virus-induced gene silencing. Infection assays with P. capsici on silenced plants revealed that SlROP-II.1, NbROP-II.1 and CaROP-II.1 play a role in P. capsici resistance, suggesting conserved function of ROP-II clade across different Solanaceous species. In addition, NbROP-II.1 is also involved in regulating plant growth and development. This study signified the diversity of Solanaceous ROPs and their potential roles in plant growth, development and immunity.

The conjugal transfer is a major driving force in the spread of antibiotic resistance genes. Nevertheless, an effective approach has not yet been developed to target conjugal transfer to prevent the acquisition of antibiotic resistance by this mechanism. This study aimed to identify potential targets for plasmid transfer blockade by isolating mutants defective in the completion of the acquisition of antibiotic resistance via conjugal transfer. We performed genome-wide screening by combining an IncP1α-type broad host range plasmid conjugation system with a comprehensive collection of Escherichia coli gene knockout mutants (Keio collection; 3884 mutants). We followed a six-step screening procedure to identify the mutants showing conjugation deficiency precisely. No mutants defective in the conjugal transfer were isolated, strongly suggesting that E. coli cannot escape from being a recipient organism for P1α plasmid transfer. However, several mutants with low viability were identified, as well as mutants defective in establishing resistance to chloramphenicol, which was used for transconjugant selection. These results suggest that developing drugs capable of inhibiting the establishment of antibiotic resistance is a better approach than attempting to prevent the conjugal transfer to block the spread of antibiotic resistance genes. Our screening system based on the IncP1α-type plasmid transfer can be extended to isolation of target genes for other drugs. This study could be the foundation for further research to understand its underlying molecular mechanism through functional analysis of the identified genes.

Background The parasite Leishmania panamensis is the main cause of leishmaniasis in Panama. The disease is largely uncontrolled, with a rising incidence and no appropriate control measures. While microsatellites are considered some of the best genetic markers to study population genetics and molecular epidemiology in these and other parasites, none has been developed for L. panamensis. Findings Here we have developed and tested a new panel of microsatellites for this species, based on high-throughput genome-wide screening. The new set of microsatellites is composed of seventeen loci, mainly spanning trinucleotide or longer motifs. We have evaluated the sensitivity and specificity of the panel based on a sample of 27 isolates obtained from cutaneous leishmaniasis patients from central Panama and also several reference species from both L. (Leishmania) and L. (Viannia) subgenera. The genetic equilibrium was assessed both intra- and inter-loci, while the reproductive mode was evaluated using several tests. The new SSR panel shows high polymorphism and sensitivity, as well as good specificity. The preliminary data described here for L. panamensis suggest extensive departure from Hardy-Weinberg proportions, significant linkage disequilibrium and strong deficit of heterozygotes. Several recombination tests involving multilocus linkage disequilibrium and a phylogenetic approach allowed rejection of frequent recombination in our dataset. Conclusions The genome-wide strategy described here proved to be useful to identify and test new polymorphic SSR loci in Leishmania. The new panel of polymorphic microsatellites is a valuable contribution to the existing molecular markers for the study of genetic structure and other aspects of this important species. Electronic supplementary material The online version of this article (doi:10.1186/s13071-015-1153-2) contains supplementary material, which is available to authorized users.

Background RNA interference (RNAi) leads to sequence specific knock-down of gene expression and has emerged as an important tool to analyse gene functions, pathway analysis and gene therapy. Although RNAi is a conserved cellular process involving common elements and factors, species-specific differences have been observed among different eukaryotes. Identification of components for RNAi pathway is pursued intensively and successful genome-wide screens have been performed for components of RNAi pathways in various organisms. Functional comparative genomics analysis offers evolutionary insight that forms basis of discoveries of novel RNAi-factors within related organisms. Keeping in view the academic and commercial utility of insect derived cell-line from Spodoptera frugiperda, we pursued the identification and functional analysis of components of RNAi-machinery of Sf21 cell-line using genome-wide application. Results The genome and transcriptome of Sf21 was assembled and annotated. In silico application of comparative genome analysis among insects allowed us to identify several RNAi factors in Sf21 line. The candidate RNAi factors from assembled genome were validated by knockdown analysis of candidate factors using the siRNA screens on the Sf21-gfp reporter cell-line. Forty two (42) potential factors were identified using the cell based assay. These include core RNAi elements including Dicer-2, Argonaute-1, Drosha, Aubergine and auxiliary modules like chromatin factors, RNA helicases, RNA processing module, signalling allied proteins and others. Phylogenetic analyses and domain architecture revealed that Spodoptera frugiperda homologs retained identity with Lepidoptera (Bombyx mori) or Coleoptera (Tribolium castaneum) sustaining an evolutionary conserved scaffold in post-transcriptional gene silencing paradigm within insects. Conclusion The database of RNAi-factors generated by whole genome association survey offers comprehensive outlook about conservation as well as specific differences of the proteins of RNAi machinery. Understanding the interior involved in different phases of gene silencing also offers impending tool for RNAi-based applications. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-775) contains supplementary material, which is available to authorized users.

The PBAT software package (v2.5) provides a unique set of tools for complex family-based association analysis at a genome-wide level. PBAT can handle nuclear families with missing parental genotypes, extended pedigrees with missing genotypic information, analysis of single nucleotide polymorphisms (SNPs), haplotype analysis, quantitative traits, multivariate/longitudinal data and time to onset phenotypes. The data analysis can be adjusted for covariates and gene/environment interactions. Haplotype-based features include sliding windows and the reconstruction of the haplotypes of the probands. PBAT's screening tools allow the user successfully to handle the multiple comparisons problem at a genome-wide level, even for 100,000 SNPs and more. Moreover, PBAT is computationally fast. A genome scan of 300,000 SNPs in 2,000 trios takes 4 central processing unit (CPU)-days. PBAT is available for Linux, Sun Solaris and Windows XP. ispartof: Human Genomics vol:2 issue:1 pages:67-69 ispartof: location:England status: published

The chromosomal translocation t(12;21) resulting in the ETV6/RUNX1 fusion gene is the most frequent structural cytogenetic abnormality among patients with childhood acute lymphoblastic leukaemia (ALL). We investigated 62 ETV6/RUNX1‐positive childhood ALL patients by single nucleotide polymorphism array to explore acquired copy number alterations (CNAs) at diagnosis. The mean number of CNAs was 2·82 (range 0–14). Concordance with available G‐band karyotyping and comparative genomic hybridization was 93%. Based on three major protein‐protein complexes disrupted by these CNAs, patients could be categorized into four distinct subgroups, defined by different underlying biological mechanisms relevant to the aetiology of childhood ALL. When recurrent CNAs were evaluated by an oncogenetic tree analysis classifying their sequential order, the most common genetic aberrations (deletions of 6q, 9p, 13q and X, and gains of 10 and 21) seemed independent of each other. Finally, we identified the most common regions with recurrent gains and losses, which comprise microRNA clusters with known oncogenic or tumour‐suppressive roles. The present study sheds further light on the genetic diversity of ETV6/RUNX1‐positive childhood ALL, which may be important for understanding poor responses among this otherwise highly curable subset of ALL and lead to novel targeted treatment strategies.

Item does not contain fulltext Array-based whole genome investigation or molecular karyotyping enables the genome-wide detection of submicroscopic imbalances. Proof-of-principle experiments have demonstrated that molecular karyotyping outperforms conventional karyotyping with regard to detection of chromosomal imbalances. This article identifies areas for which the technology seems matured and areas that require more investigations. Molecular karyotyping should be part of the genetic diagnostic work-up of patients with developmental disorders. For the implementation of the technique for other constitutional indications and in prenatal diagnosis, more research is appropriate. Also, the article aims to provide best practice guidelines for the application of array comparative genomic hybridisation to ensure both technical and clinical quality criteria that will optimise and standardise results and reports in diagnostic laboratories. In short, both the specificity and the sensitivity of the arrays should be evaluated in every laboratory offering the diagnostic test. Internal and external quality control programmes are urgently needed to evaluate and standardise the test results between laboratories.

To study genetic loci influencing obesity in nuclear families with type 2 diabetes, we performed a genome-wide screen with 325 microsatellite markers that had an average spacing of 11 cM and a mean heterozygosity of ~75% covering all 22 autosomes. Genotype data were obtained from 562 individuals from 178 families from the Breda Study Cohort. These families were determined to have at least two members with type 2 diabetes. As a measure of obesity, the BMI of each diabetes patient was determined. The genotypes were analyzed using variance components (VCs) analysis implemented in GENEHUNTER 2 to determine quantitative trait loci influencing BMI. The VC analysis revealed two genomic regions showing VC logarithm of odds (LOD) scores >1.0 on chromosome 1 and chromosome 11. The regions of interest on both chromosomes were further investigated by fine-mapping with additional markers, resulting in a VC LOD score of 1.5 on chromosome 1q and a VC LOD of 2.4 on chromosome 11q. The locus on chromosome 1 has been implicated previously in diabetes. The locus on chromosome 11 has been implicated previously in diabetes and obesity. Our study to determine linkage for BMI confirms the presence of quantitative trait loci influencing obesity in subjects with type 2 diabetes on chromosomes 1q31-q42 and 11q14-q24.

To study genetic loci influencing obesity in nuclear families with type 2 diabetes, we performed a genome-wide screen with 325 microsatellite markers that had an average spacing of 11 cM and a mean heterozygosity of ~75% covering all 22 autosomes. Genotype data were obtained from 562 individuals from 178 families from the Breda Study Cohort. These families were determined to have at least two members with type 2 diabetes. As a measure of obesity, the BMI of each diabetes patient was determined. The genotypes were analyzed using variance components (VCs) analysis implemented in GENEHUNTER 2 to determine quantitative trait loci influencing BMI. The VC analysis revealed two genomic regions showing VC logarithm of odds (LOD) scores >1.0 on chromosome 1 and chromosome 11. The regions of interest on both chromosomes were further investigated by fine-mapping with additional markers, resulting in a VC LOD score of 1.5 on chromosome 1q and a VC LOD of 2.4 on chromosome 11q. The locus on chromosome 1 has been implicated previously in diabetes. The locus on chromosome 11 has been implicated previously in diabetes and obesity. Our study to determine linkage for BMI confirms the presence of quantitative trait loci influencing obesity in subjects with type 2 diabetes on chromosomes 1q31-q42 and 11q14-q24.