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Sample records for gene targeting reveals

  1. Targeted capture and resequencing of 1040 genes reveal environmentally driven functional variation in grey wolves.

    PubMed

    Schweizer, Rena M; Robinson, Jacqueline; Harrigan, Ryan; Silva, Pedro; Galverni, Marco; Musiani, Marco; Green, Richard E; Novembre, John; Wayne, Robert K

    2016-01-01

    In an era of ever-increasing amounts of whole-genome sequence data for individuals and populations, the utility of traditional single nucleotide polymorphisms (SNPs) array-based genome scans is uncertain. We previously performed a SNP array-based genome scan to identify candidate genes under selection in six distinct grey wolf (Canis lupus) ecotypes. Using this information, we designed a targeted capture array for 1040 genes, including all exons and flanking regions, as well as 5000 1-kb nongenic neutral regions, and resequenced these regions in 107 wolves. Selection tests revealed striking patterns of variation within candidate genes relative to noncandidate regions and identified potentially functional variants related to local adaptation. We found 27% and 47% of candidate genes from the previous SNP array study had functional changes that were outliers in sweed and bayenv analyses, respectively. This result verifies the use of genomewide SNP surveys to tag genes that contain functional variants between populations. We highlight nonsynonymous variants in APOB, LIPG and USH2A that occur in functional domains of these proteins, and that demonstrate high correlation with precipitation seasonality and vegetation. We find Arctic and High Arctic wolf ecotypes have higher numbers of genes under selection, which highlight their conservation value and heightened threat due to climate change. This study demonstrates that combining genomewide genotyping arrays with large-scale resequencing and environmental data provides a powerful approach to discern candidate functional variants in natural populations. PMID:26562361

  2. Targeted next-generation sequencing reveals multiple deleterious variants in OPLL-associated genes.

    PubMed

    Chen, Xin; Guo, Jun; Cai, Tao; Zhang, Fengshan; Pan, Shengfa; Zhang, Li; Wang, Shaobo; Zhou, Feifei; Diao, Yinze; Zhao, Yanbin; Chen, Zhen; Liu, Xiaoguang; Chen, Zhongqiang; Liu, Zhongjun; Sun, Yu; Du, Jie

    2016-01-01

    Ossification of the posterior longitudinal ligament of the spine (OPLL), which is characterized by ectopic bone formation in the spinal ligaments, can cause spinal-cord compression. To date, at least 11 susceptibility genes have been genetically linked to OPLL. In order to identify potential deleterious alleles in these OPLL-associated genes, we designed a capture array encompassing all coding regions of the target genes for next-generation sequencing (NGS) in a cohort of 55 unrelated patients with OPLL. By bioinformatics analyses, we successfully identified three novel and five extremely rare variants (MAF < 0.005). These variants were predicted to be deleterious by commonly used various algorithms, thereby resulting in missense mutations in four OPLL-associated genes (i.e., COL6A1, COL11A2, FGFR1, and BMP2). Furthermore, potential effects of the patient with p.Q89E of BMP2 were confirmed by a markedly increased BMP2 level in peripheral blood samples. Notably, seven of the variants were found to be associated with the patients with continuous subtype changes by cervical spinal radiological analyses. Taken together, our findings revealed for the first time that deleterious coding variants of the four OPLL-associated genes are potentially pathogenic in the patients with OPLL. PMID:27246988

  3. Targeted next-generation sequencing reveals multiple deleterious variants in OPLL-associated genes

    PubMed Central

    Chen, Xin; Guo, Jun; Cai, Tao; Zhang, Fengshan; Pan, Shengfa; Zhang, Li; Wang, Shaobo; Zhou, Feifei; Diao, Yinze; Zhao, Yanbin; Chen, Zhen; Liu, Xiaoguang; Chen, Zhongqiang; Liu, Zhongjun; Sun, Yu; Du, Jie

    2016-01-01

    Ossification of the posterior longitudinal ligament of the spine (OPLL), which is characterized by ectopic bone formation in the spinal ligaments, can cause spinal-cord compression. To date, at least 11 susceptibility genes have been genetically linked to OPLL. In order to identify potential deleterious alleles in these OPLL-associated genes, we designed a capture array encompassing all coding regions of the target genes for next-generation sequencing (NGS) in a cohort of 55 unrelated patients with OPLL. By bioinformatics analyses, we successfully identified three novel and five extremely rare variants (MAF < 0.005). These variants were predicted to be deleterious by commonly used various algorithms, thereby resulting in missense mutations in four OPLL-associated genes (i.e., COL6A1, COL11A2, FGFR1, and BMP2). Furthermore, potential effects of the patient with p.Q89E of BMP2 were confirmed by a markedly increased BMP2 level in peripheral blood samples. Notably, seven of the variants were found to be associated with the patients with continuous subtype changes by cervical spinal radiological analyses. Taken together, our findings revealed for the first time that deleterious coding variants of the four OPLL-associated genes are potentially pathogenic in the patients with OPLL. PMID:27246988

  4. Genomewide analysis of Drosophila GAGA factor target genes reveals context-dependent DNA binding

    PubMed Central

    van Steensel, Bas; Delrow, Jeffrey; Bussemaker, Harmen J.

    2003-01-01

    The association of sequence-specific DNA-binding factors with their cognate target sequences in vivo depends on the local molecular context, yet this context is poorly understood. To address this issue, we have performed genomewide mapping of in vivo target genes of Drosophila GAGA factor (GAF). The resulting list of ≈250 target genes indicates that GAF regulates many cellular pathways. We applied unbiased motif-based regression analysis to identify the sequence context that determines GAF binding. Our results confirm that GAF selectively associates with (GA)n repeat elements in vivo. GAF binding occurs in upstream regulatory regions, but less in downstream regions. Surprisingly, GAF binds abundantly to introns but is virtually absent from exons, even though the density of (GA)n is roughly the same. Intron binding occurs equally frequently in last introns compared with first introns, suggesting that GAF may not only regulate transcription initiation, but possibly also elongation. We provide evidence for cooperative binding of GAF to closely spaced (GA)n elements and explain the lack of GAF binding to exons by the absence of such closely spaced GA repeats. Our approach for revealing determinants of context-dependent DNA binding will be applicable to many other transcription factors. PMID:12601174

  5. Transcriptome Analysis of Targeted Mouse Mutations Reveals the Topography of Local Changes in Gene Expression

    PubMed Central

    Adkisson, Michael; Nava, A. J.; Kirov, Julia V.; Cipollone, Andreanna; Willis, Brandon; Rapp, Jared; de Jong, Pieter J.; Lloyd, Kent C.

    2016-01-01

    The unintended consequences of gene targeting in mouse models have not been thoroughly studied and a more systematic analysis is needed to understand the frequency and characteristics of off-target effects. Using RNA-seq, we evaluated targeted and neighboring gene expression in tissues from 44 homozygous mutants compared with C57BL/6N control mice. Two allele types were evaluated: 15 targeted trap mutations (TRAP); and 29 deletion alleles (DEL), usually a deletion between the translational start and the 3’ UTR. Both targeting strategies insert a bacterial beta-galactosidase reporter (LacZ) and a neomycin resistance selection cassette. Evaluating transcription of genes in +/- 500 kb of flanking DNA around the targeted gene, we found up-regulated genes more frequently around DEL compared with TRAP alleles, however the frequency of alleles with local down-regulated genes flanking DEL and TRAP targets was similar. Down-regulated genes around both DEL and TRAP targets were found at a higher frequency than expected from a genome-wide survey. However, only around DEL targets were up-regulated genes found with a significantly higher frequency compared with genome-wide sampling. Transcriptome analysis confirms targeting in 97% of DEL alleles, but in only 47% of TRAP alleles probably due to non-functional splice variants, and some splicing around the gene trap. Local effects on gene expression are likely due to a number of factors including compensatory regulation, loss or disruption of intragenic regulatory elements, the exogenous promoter in the neo selection cassette, removal of insulating DNA in the DEL mutants, and local silencing due to disruption of normal chromatin organization or presence of exogenous DNA. An understanding of local position effects is important for understanding and interpreting any phenotype attributed to targeted gene mutations, or to spontaneous indels. PMID:26839965

  6. Targeted and genome-scale methylomics reveals gene body signatures in human cell lines

    PubMed Central

    Ball, Madeleine Price; Li, Jin Billy; Gao, Yuan; Lee, Je-Hyuk; LeProust, Emily; Park, In-Hyun; Xie, Bin; Daley, George Q.; Church, George M.

    2012-01-01

    Cytosine methylation, an epigenetic modification of DNA, is a target of growing interest for developing high throughput profiling technologies. Here we introduce two new, complementary techniques for cytosine methylation profiling utilizing next generation sequencing technology: bisulfite padlock probes (BSPPs) and methyl sensitive cut counting (MSCC). In the first method, we designed a set of ~10,000 BSPPs distributed over the ENCODE pilot project regions to take advantage of existing expression and chromatin immunoprecipitation data. We observed a pattern of low promoter methylation coupled with high gene body methylation in highly expressed genes. Using the second method, MSCC, we gathered genome-scale data for 1.4 million HpaII sites and confirmed that gene body methylation in highly expressed genes is a consistent phenomenon over the entire genome. Our observations highlight the usefulness of techniques which are not inherently or intentionally biased in favor of only profiling particular subsets like CpG islands or promoter regions. PMID:19329998

  7. RNA Seq profiling reveals a novel expression pattern of TGF-β target genes in human blood eosinophils.

    PubMed

    Shen, Zhong-Jian; Hu, Jie; Esnault, Stephane; Dozmorov, Igor; Malter, James S

    2015-09-01

    Despite major advances in our understanding of TGF-β signaling in multiple cell types, little is known about the direct target genes of this pathway in human eosinophils. These cells constitute the major inflammatory component present in the sputum and lung of active asthmatics and their numbers correlate well with disease severity. During the transition from acute to chronic asthma, TGF-β levels rise several fold in the lung which drives fibroblasts to produce extracellular matrix (ECM) and participate in airway and parenchymal remodeling. In this report, we use purified blood eosinophils from healthy donors and analyze baseline and TGF-β responsive genes by RNA Seq, and demonstrate that eosinophils (PBE) express 7981 protein-coding genes of which 178 genes are up-regulated and 199 genes are down-regulated by TGF-β. While 18 target genes have been previously associated with asthma and eosinophilic disorders, the vast majority have been implicated in cell death and survival, differentiation, and cellular function. Ingenuity pathway analysis revealed that 126 canonical pathways are activated by TGF-β including iNOS, TREM1, p53, IL-8 and IL-10 signaling. As TGF-β is an important cytokine for eosinophil function and survival, and pulmonary inflammation and fibrosis, our results represent a significant step toward the identification of novel TGF-β responsive genes and provide a potential therapeutic opportunity by selectively targeting relevant genes and pathways. PMID:26112417

  8. Target genes of Dpp/BMP signaling pathway revealed by transcriptome profiling in the early D.melanogaster embryo.

    PubMed

    Dominguez, Calixto; Zuñiga, Alejandro; Hanna, Patricia; Hodar, Christian; Gonzalez, Mauricio; Cambiazo, Verónica

    2016-10-10

    In the early Drosophila melanogaster embryo, the gene regulatory network controlled by Dpp signaling is involved in the subdivision of dorsal ectoderm into the presumptive dorsal epidermis and amnioserosa. In this work, we aimed to identify new Dpp downstream targets involved in dorsal ectoderm patterning. We used oligonucleotide D. melanogaster microarrays to identify the set of genes that are differential expressed between wild type embryos and embryos that overexpress Dpp (nos-Gal4>UAS-dpp) during early stages of embryo development. By using this approach, we identified 358 genes whose relative abundance significantly increased in response to Dpp overexpression. Among them, we found the entire set of known Dpp target genes that function in dorsal ectoderm patterning (zen, doc, hnt, pnr, ush, tup, and others) in addition to several up-regulated genes of unknown functions. Spatial expression pattern of up-regulated genes in response to Dpp overexpression as well as their opposing transcriptional responses to Dpp loss- and gain-of-function indicated that they are new candidate target genes of Dpp signaling pathway. We further analyse one of the candidate genes, CG13653, which is expressed at the dorsal-most cells of the embryo during a restricted period of time. CG13653 orthologs were not detected in basal lineages of Dipterans, which unlike D. melanogaster develop two extra-embryonic membranes, amnion and serosa. We characterized the enhancer region of CG13653 and revealed that CG13653 is directly regulated by Dpp signaling pathway. PMID:27397649

  9. Comparative Analysis of Gene Expression Data Reveals Novel Targets of Senescence-Associated microRNAs

    PubMed Central

    Napolitano, Marco; Comegna, Marika; Succoio, Mariangela; Leggiero, Eleonora; Pastore, Lucio; Faraonio, Raffaella; Cimino, Filiberto; Passaro, Fabiana

    2014-01-01

    In the last decades, cellular senescence is viewed as a complex mechanism involved in different processes, ranging from tumor suppression to induction of age-related degenerative alterations. Senescence-inducing stimuli are myriad and, recently, we and others have demonstrated the role exerted by microRNAs in the induction and maintenance of senescence, by the identification of a subset of Senescence-Associated microRNAs (SAmiRs) up-regulated during replicative or stress-induced senescence and able to induce a premature senescent phenotype when over-expressed in human primary cells. With the intent to find novel direct targets of two specific SAmiRs, SAmiR-494 and -486-5p, and cellular pathways which they are involved in, we performed a comparative analysis of gene expression profiles available in literature to select genes down-regulated upon replicative senescence of human primary fibroblasts. Among them, we searched for SAmiR’s candidate targets by analyzing with different target prediction algorithms their 3’UTR for the presence of SAmiR-binding sites. The expression profiles of selected candidates have been validated on replicative and stress-induced senescence and the targeting of the 3’UTRs was assessed by luciferase assay. Results allowed us to identify Cell Division Cycle Associated 2 (CDCA2) and Inhibitor of DNA binding/differentiation type 4 (ID4) as novel targets of SAmiR-494 and SAmiR-486-5p, respectively. Furthermore, we demonstrated that the over-expression of CDCA2 in human primary fibroblasts was able to partially counteract etoposide-induced senescence by mitigating the activation of DNA Damage Response. PMID:24905922

  10. High-throughput analysis of promoter occupancy reveals new targets for Arx, a gene mutated in mental retardation and interneuronopathies.

    PubMed

    Quillé, Marie-Lise; Carat, Solenne; Quéméner-Redon, Sylvia; Hirchaud, Edouard; Baron, Daniel; Benech, Caroline; Guihot, Jeanne; Placet, Morgane; Mignen, Olivier; Férec, Claude; Houlgatte, Rémi; Friocourt, Gaëlle

    2011-01-01

    Genetic investigations of X-linked intellectual disabilities have implicated the ARX (Aristaless-related homeobox) gene in a wide spectrum of disorders extending from phenotypes characterised by severe neuronal migration defects such as lissencephaly, to mild or moderate forms of mental retardation without apparent brain abnormalities but with associated features of dystonia and epilepsy. Analysis of Arx spatio-temporal localisation profile in mouse revealed expression in telencephalic structures, mainly restricted to populations of GABAergic neurons at all stages of development. Furthermore, studies of the effects of ARX loss of function in humans and animal models revealed varying defects, suggesting multiple roles of this gene during brain development. However, to date, little is known about how ARX functions as a transcription factor and the nature of its targets. To better understand its role, we combined chromatin immunoprecipitation and mRNA expression with microarray analysis and identified a total of 1006 gene promoters bound by Arx in transfected neuroblastoma (N2a) cells and in mouse embryonic brain. Approximately 24% of Arx-bound genes were found to show expression changes following Arx overexpression or knock-down. Several of the Arx target genes we identified are known to be important for a variety of functions in brain development and some of them suggest new functions for Arx. Overall, these results identified multiple new candidate targets for Arx and should help to better understand the pathophysiological mechanisms of intellectual disability and epilepsy associated with ARX mutations. PMID:21966449

  11. Targeted Gene Disruption Reveals an Adhesin Indispensable for Pathogenicity of Blastomyces dermatitidis

    PubMed Central

    Tristan Brandhorst, T.; Wüthrich, Marcel; Warner, Thomas; Klein, Bruce

    1999-01-01

    Systemic fungal infections are becoming more common and difficult to treat, yet the pathogenesis of these infectious diseases remains poorly understood. In many cases, pathogenicity can be attributed to the ability of the fungi to adhere to target tissues, but the lack of tractable genetic systems has limited progress in understanding and interfering with the offending fungal products. In Blastomyces dermatitidis, the agent of blastomycosis, a respiratory and disseminated mycosis of people and animals worldwide, expression of the putative adhesin encoded by the WI-1 gene was investigated as a possible virulence factor. DNA-mediated gene transfer was used to disrupt the WI-1 locus by allelic replacement, resulting in impaired binding and entry of yeasts into macrophages, loss of adherence to lung tissue, and abolishment of virulence in mice; each of these properties was fully restored after reconstitution of WI-1 by means of gene transfer. These findings establish the pivotal role of WI-1 in adherence and virulence of B. dermatitidis yeasts. To our knowledge, they offer the first example of a genetically proven virulence determinant among systemic dimorphic fungi, and underscore the value of reverse genetics for studies of pathogenesis in these organisms. PMID:10209038

  12. Transcriptome analysis of a barley breeding program examines gene expression diversity and reveals target genes for malting quality improvement

    PubMed Central

    2010-01-01

    Background Advanced cycle breeding utilizes crosses among elite lines and is a successful method to develop new inbreds. However, it results in a reduction in genetic diversity within the breeding population. The development of malting barley varieties requires the adherence to a narrow malting quality profile and thus the use of advanced cycle breeding strategies. Although attention has been focused on diversity in gene expression and its association with genetic diversity, there are no studies performed in a single breeding program examining the implications that consecutive cycles of breeding have on gene expression variation and identifying the variability still available for future improvement. Results Fifteen lines representing the historically important six-rowed malting barley breeding program of the University of Minnesota were genotyped with 1,524 SNPs, phenotypically examined for six malting quality traits, and analyzed for transcript accumulation during germination using the Barley1 GeneChip array. Significant correlation was detected between genetic and transcript-level variation. We observed a reduction in both genetic and gene expression diversity through the breeding process, although the expression of many genes have not been fixed. A high number of quality-related genes whose expression was fixed during the breeding process was identified, indicating that much of the diversity reduction was associated with the improvement of the complex phenotype "malting quality", the main goal of the University of Minnesota breeding program. We also identified 49 differentially expressed genes between the most recent lines of the program that were correlated with one or more of the six primary malting quality traits. These genes constitute potential targets for the improvement of malting quality within the breeding program. Conclusions The present study shows the repercussion of advanced cycle breeding on gene expression diversity within an important barley

  13. Targeted next-generation sequencing of candidate genes reveals novel mutations in patients with dilated cardiomyopathy

    PubMed Central

    ZHAO, YUE; FENG, YUE; ZHANG, YUN-MEI; DING, XIAO-XUE; SONG, YU-ZHU; ZHANG, A-MEI; LIU, LI; ZHANG, HONG; DING, JIA-HUAN; XIA, XUE-SHAN

    2015-01-01

    Dilated cardiomyopathy (DCM) is a major cause of sudden cardiac death and heart failure, and it is characterized by genetic and clinical heterogeneity, even for some patients with a very poor clinical prognosis; in the majority of cases, DCM necessitates a heart transplant. Genetic mutations have long been considered to be associated with this disease. At present, mutations in over 50 genes related to DCM have been documented. This study was carried out to elucidate the characteristics of gene mutations in patients with DCM. The candidate genes that may cause DCM include MYBPC3, MYH6, MYH7, LMNA, TNNT2, TNNI3, MYPN, MYL3, TPM1, SCN5A, DES, ACTC1 and RBM20. Using next-generation sequencing (NGS) and subsequent mutation confirmation with traditional capillary Sanger sequencing analysis, possible causative non-synonymous mutations were identified in ~57% (12/21) of patients with DCM. As a result, 7 novel mutations (MYPN, p.E630K; TNNT2, p.G180A; MYH6, p.R1047C; TNNC1, p.D3V; DES, p.R386H; MYBPC3, p.C1124F; and MYL3, p.D126G), 3 variants of uncertain significance (RBM20, p.R1182H; MYH6, p.T1253M; and VCL, p.M209L), and 2 known mutations (MYH7, p.A26V and MYBPC3, p.R160W) were revealed to be associated with DCM. The mutations were most frequently found in the sarcomere (MYH6, MYBPC3, MYH7, TNNC1, TNNT2 and MYL3) and cytoskeletal (MYPN, DES and VCL) genes. As genetic testing is a useful tool in the clinical management of disease, testing for pathogenic mutations is beneficial to the treatment of patients with DCM and may assist in predicting disease risk for their family members before the onset of symptoms. PMID:26458567

  14. RNA-Seq reveals common and unique PXR- and CAR-target gene signatures in the mouse liver transcriptome.

    PubMed

    Cui, Julia Yue; Klaassen, Curtis D

    2016-09-01

    The pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are well-known xenobiotic-sensing nuclear receptors with overlapping functions. However, there lacks a quantitative characterization to distinguish between the PXR and CAR target genes and signaling pathways in the liver. The present study performed a transcriptomic comparison of the PXR- and CAR-targets using RNA-Seq in livers of adult wild-type mice that were treated with the prototypical PXR ligand PCN (200mg/kg, i.p. once daily for 4days in corn oil) or the prototypical CAR ligand TCPOBOP (3mg/kg, i.p., once daily for 4days in corn oil). At the given doses, TCPOBOP differentially regulated many more genes (2125) than PCN (212), and 147 of the same genes were differentially regulated by both chemicals. As expected, the top pathways differentially regulated by both PCN and TCPOBOP were involved in xenobiotic metabolism, and they also up-regulated genes involved in retinoid metabolism, but down-regulated genes involved in inflammation and iron homeostasis. Regarding unique pathways, PXR activation appeared to overlap with the aryl hydrocarbon receptor signaling, whereas CAR activation appeared to overlap with the farnesoid X receptor signaling, acute-phase response, and mitochondrial dysfunction. The mRNAs of differentially regulated drug-processing genes (DPGs) partitioned into three patterns, namely TCPOBOP-induced, PCN-induced, as well as TCPOBOP-suppressed gene clusters. The cumulative mRNAs of the differentially regulated DPGs, phase-I and -II enzymes, as well as efflux transporters were all up-regulated by both PCN and TCPOBOPOP, whereas the cumulative mRNAs of the uptake transporters were down-regulated only by TCPOBOP. The absolute mRNA abundance in control and receptor-activated conditions was examined in each DPG category to predict the contribution of specific DPG genes in the PXR/CAR-mediated pharmacokinetic responses. The preferable differential regulation by TCPOBOP in the

  15. Cloning genes responsive to a hepatocarcinogenic peroxisome proliferator chemical reveals novel targets of regulation.

    PubMed

    Corton, J C; Moreno, E S; Merritt, A; Bocos, C; Cattley, R C

    1998-12-11

    To better understand the molecular basis of the hepatocyte proliferation and induction of hepatocellular adenomas by exposure to peroxisome proliferator chemicals (PPC), a systematic search for genes modulated by a PPC (WY-14643) in rat liver was carried out using the differential display technique. The fragments fell into two classes based on the time of initial and maximal induction by WY-14643. The class I genes (clones 5 and 30) were induced 3 h after a gavage exposure to WY-14643 with maximal expression at 24 h. The class II genes (clones 13 and 16) were induced after 24 h with maximal expression at 78 weeks. Expression of the class II genes was also increased after other treatments that cause cell proliferation. Clone 30 was identified as CYP4A2, previously shown to be regulated by PPC. Clone 13 was homologous to the mouse protein H gene, a component of the heterogeneous nuclear ribonucleoprotein particle important in mRNA splicing. Clone 16 was identified as cyclophilin-A, the receptor for the immunosuppressant drug cyclosporin A. The sequence of clone 5 was unique. These data demonstrate that WY-14643 increases the levels of a number of novel genes that are coordinately regulated with increases in chronic cell proliferation and fatty acid metabolism. PMID:10381131

  16. Transgenic banana plants expressing Xanthomonas wilt resistance genes revealed a stable non-target bacterial colonization structure.

    PubMed

    Nimusiima, Jean; Köberl, Martina; Tumuhairwe, John Baptist; Kubiriba, Jerome; Staver, Charles; Berg, Gabriele

    2015-01-01

    Africa is among the continents where the battle over genetically modified crops is currently being played out. The impact of GM in Africa could potentially be very positive. In Uganda, researchers have developed transgenic banana lines resistant to banana Xanthomonas wilt. The transgenic lines expressing hrap and pflp can provide a timely solution to the pandemic. However, the impact of the transgenes expression on non-target microorganisms has not yet been investigated. To study this effect, transgenic and control lines were grown under field conditions and their associated microbiome was investigated by 16S rRNA gene profiling combining amplicon sequencing and molecular fingerprinting. Three years after sucker planting, no statistically significant differences between transgenic lines and their non-modified predecessors were detected for their associated bacterial communities. The overall gammaproteobacterial rhizosphere microbiome was highly dominated by Xanthomonadales, while Pseudomonadales and Enterobacteriales were accumulated in the pseudostem. Shannon indices revealed much higher diversity in the rhizosphere than in the pseudostem endosphere. However, the expression of the transgenes did not result in changes in the diversity of Gammaproteobacteria, the closest relatives of the target pathogen. In this field experiment, the expression of the resistance genes appears to have no consequences for non-target rhizobacteria and endophytes. PMID:26657016

  17. Transgenic banana plants expressing Xanthomonas wilt resistance genes revealed a stable non-target bacterial colonization structure

    PubMed Central

    Nimusiima, Jean; Köberl, Martina; Tumuhairwe, John Baptist; Kubiriba, Jerome; Staver, Charles; Berg, Gabriele

    2015-01-01

    Africa is among the continents where the battle over genetically modified crops is currently being played out. The impact of GM in Africa could potentially be very positive. In Uganda, researchers have developed transgenic banana lines resistant to banana Xanthomonas wilt. The transgenic lines expressing hrap and pflp can provide a timely solution to the pandemic. However, the impact of the transgenes expression on non-target microorganisms has not yet been investigated. To study this effect, transgenic and control lines were grown under field conditions and their associated microbiome was investigated by 16S rRNA gene profiling combining amplicon sequencing and molecular fingerprinting. Three years after sucker planting, no statistically significant differences between transgenic lines and their non-modified predecessors were detected for their associated bacterial communities. The overall gammaproteobacterial rhizosphere microbiome was highly dominated by Xanthomonadales, while Pseudomonadales and Enterobacteriales were accumulated in the pseudostem. Shannon indices revealed much higher diversity in the rhizosphere than in the pseudostem endosphere. However, the expression of the transgenes did not result in changes in the diversity of Gammaproteobacteria, the closest relatives of the target pathogen. In this field experiment, the expression of the resistance genes appears to have no consequences for non-target rhizobacteria and endophytes. PMID:26657016

  18. DNA Methylation and Gene Expression Profiling of Ewing Sarcoma Primary Tumors Reveal Genes That Are Potential Targets of Epigenetic Inactivation

    PubMed Central

    Patel, Nikul; Black, Jennifer; Chen, Xi; Marcondes, A. Mario; Grady, William M.; Lawlor, Elizabeth R.; Borinstein, Scott C.

    2012-01-01

    The role of aberrant DNA methylation in Ewing sarcoma is not completely understood. The methylation status of 503 genes in 52 formalin-fixed paraffin-embedded EWS tumors and 3 EWS cell lines was compared to human mesenchymal stem cell primary cultures (hMSCs) using bead chip methylation analysis. Relative expression of methylated genes was assessed in 5-Aza-2-deoxycytidine-(5-AZA)-treated EWS cell lines and in a cohort of primary EWS samples and hMSCs by gene expression and quantitative RT-PCR. 129 genes demonstrated statistically significant hypermethylation in EWS tumors compared to hMSCs. Thirty-six genes were profoundly methylated in EWS and unmethylated in hMSCs. 5-AZA treatment of EWS cell lines resulted in upregulation of expression of hundreds of genes including 162 that were increased by at least 2-fold. The expression of 19 of 36 candidate hypermethylated genes was increased following 5-AZA. Analysis of gene expression from an independent cohort of tumors confirmed decreased expression of six of nineteen hypermethylated genes (AXL, COL1A1, CYP1B1, LYN, SERPINE1,) and VCAN. Comparing gene expression and DNA methylation analyses proved to be an effective way to identify genes epigenetically regulated in EWS. Further investigation is ongoing to elucidate the role of these epigenetic alterations in EWS pathogenesis. PMID:23024594

  19. High-resolution chemical dissection of a model eukaryote reveals targets, pathways and gene functions.

    PubMed

    Hoepfner, Dominic; Helliwell, Stephen B; Sadlish, Heather; Schuierer, Sven; Filipuzzi, Ireos; Brachat, Sophie; Bhullar, Bhupinder; Plikat, Uwe; Abraham, Yann; Altorfer, Marc; Aust, Thomas; Baeriswyl, Lukas; Cerino, Raffaele; Chang, Lena; Estoppey, David; Eichenberger, Juerg; Frederiksen, Mathias; Hartmann, Nicole; Hohendahl, Annika; Knapp, Britta; Krastel, Philipp; Melin, Nicolas; Nigsch, Florian; Oakeley, Edward J; Petitjean, Virginie; Petersen, Frank; Riedl, Ralph; Schmitt, Esther K; Staedtler, Frank; Studer, Christian; Tallarico, John A; Wetzel, Stefan; Fishman, Mark C; Porter, Jeffrey A; Movva, N Rao

    2014-01-01

    Due to evolutionary conservation of biology, experimental knowledge captured from genetic studies in eukaryotic model organisms provides insight into human cellular pathways and ultimately physiology. Yeast chemogenomic profiling is a powerful approach for annotating cellular responses to small molecules. Using an optimized platform, we provide the relative sensitivities of the heterozygous and homozygous deletion collections for nearly 1800 biologically active compounds. The data quality enables unique insights into pathways that are sensitive and resistant to a given perturbation, as demonstrated with both known and novel compounds. We present examples of novel compounds that inhibit the therapeutically relevant fatty acid synthase and desaturase (Fas1p and Ole1p), and demonstrate how the individual profiles facilitate hypothesis-driven experiments to delineate compound mechanism of action. Importantly, the scale and diversity of tested compounds yields a dataset where the number of modulated pathways approaches saturation. This resource can be used to map novel biological connections, and also identify functions for unannotated genes. We validated hypotheses generated by global two-way hierarchical clustering of profiles for (i) novel compounds with a similar mechanism of action acting upon microtubules or vacuolar ATPases, and (ii) an un-annotated ORF, YIL060w, that plays a role in respiration in the mitochondria. Finally, we identify and characterize background mutations in the widely used yeast deletion collection which should improve the interpretation of past and future screens throughout the community. This comprehensive resource of cellular responses enables the expansion of our understanding of eukaryotic pathway biology. PMID:24360837

  20. Captured metagenomics: large-scale targeting of genes based on ‘sequence capture’ reveals functional diversity in soils

    PubMed Central

    Manoharan, Lokeshwaran; Kushwaha, Sandeep K.; Hedlund, Katarina; Ahrén, Dag

    2015-01-01

    Microbial enzyme diversity is a key to understand many ecosystem processes. Whole metagenome sequencing (WMG) obtains information on functional genes, but it is costly and inefficient due to large amount of sequencing that is required. In this study, we have applied a captured metagenomics technique for functional genes in soil microorganisms, as an alternative to WMG. Large-scale targeting of functional genes, coding for enzymes related to organic matter degradation, was applied to two agricultural soil communities through captured metagenomics. Captured metagenomics uses custom-designed, hybridization-based oligonucleotide probes that enrich functional genes of interest in metagenomic libraries where only probe-bound DNA fragments are sequenced. The captured metagenomes were highly enriched with targeted genes while maintaining their target diversity and their taxonomic distribution correlated well with the traditional ribosomal sequencing. The captured metagenomes were highly enriched with genes related to organic matter degradation; at least five times more than similar, publicly available soil WMG projects. This target enrichment technique also preserves the functional representation of the soils, thereby facilitating comparative metagenomics projects. Here, we present the first study that applies the captured metagenomics approach in large scale, and this novel method allows deep investigations of central ecosystem processes by studying functional gene abundances. PMID:26490729

  1. Code-Assisted Discovery of TAL Effector Targets in Bacterial Leaf Streak of Rice Reveals Contrast with Bacterial Blight and a Novel Susceptibility Gene

    PubMed Central

    Cernadas, Raul A.; Doyle, Erin L.; Niño-Liu, David O.; Wilkins, Katherine E.; Bancroft, Timothy; Wang, Li; Schmidt, Clarice L.; Caldo, Rico; Yang, Bing; White, Frank F.; Nettleton, Dan; Wise, Roger P.; Bogdanove, Adam J.

    2014-01-01

    Bacterial leaf streak of rice, caused by Xanthomonas oryzae pv. oryzicola (Xoc) is an increasingly important yield constraint in this staple crop. A mesophyll colonizer, Xoc differs from X. oryzae pv. oryzae (Xoo), which invades xylem to cause bacterial blight of rice. Both produce multiple distinct TAL effectors, type III-delivered proteins that transactivate effector-specific host genes. A TAL effector finds its target(s) via a partially degenerate code whereby the modular effector amino acid sequence identifies nucleotide sequences to which the protein binds. Virulence contributions of some Xoo TAL effectors have been shown, and their relevant targets, susceptibility (S) genes, identified, but the role of TAL effectors in leaf streak is uncharacterized. We used host transcript profiling to compare leaf streak to blight and to probe functions of Xoc TAL effectors. We found that Xoc and Xoo induce almost completely different host transcriptional changes. Roughly one in three genes upregulated by the pathogens is preceded by a candidate TAL effector binding element. Experimental analysis of the 44 such genes predicted to be Xoc TAL effector targets verified nearly half, and identified most others as false predictions. None of the Xoc targets is a known bacterial blight S gene. Mutational analysis revealed that Tal2g, which activates two genes, contributes to lesion expansion and bacterial exudation. Use of designer TAL effectors discriminated a sulfate transporter gene as the S gene. Across all targets, basal expression tended to be higher than genome-average, and induction moderate. Finally, machine learning applied to real vs. falsely predicted targets yielded a classifier that recalled 92% of the real targets with 88% precision, providing a tool for better target prediction in the future. Our study expands the number of known TAL effector targets, identifies a new class of S gene, and improves our ability to predict functional targeting. PMID:24586171

  2. Integrated Expression Profiling and Genome-Wide Analysis of ChREBP Targets Reveals the Dual Role for ChREBP in Glucose-Regulated Gene Expression

    PubMed Central

    Lee, Yong Seok; Kim, Ha-Jung; Han, Jung-Youn; Im, Seung-Soon; Chong, Hansook Kim; Kwon, Je-Keun; Cho, Yun-Ho; Kim, Woo Kyung; Osborne, Timothy F.; Horton, Jay D.; Jun, Hee-Sook; Ahn, Yong-Ho; Ahn, Sung-Min; Cha, Ji-Young

    2011-01-01

    The carbohydrate response element binding protein (ChREBP), a basic helix-loop-helix/leucine zipper transcription factor, plays a critical role in the control of lipogenesis in the liver. To identify the direct targets of ChREBP on a genome-wide scale and provide more insight into the mechanism by which ChREBP regulates glucose-responsive gene expression, we performed chromatin immunoprecipitation-sequencing and gene expression analysis. We identified 1153 ChREBP binding sites and 783 target genes using the chromatin from HepG2, a human hepatocellular carcinoma cell line. A motif search revealed a refined consensus sequence (CABGTG-nnCnG-nGnSTG) to better represent critical elements of a functional ChREBP binding sequence. Gene ontology analysis shows that ChREBP target genes are particularly associated with lipid, fatty acid and steroid metabolism. In addition, other functional gene clusters related to transport, development and cell motility are significantly enriched. Gene set enrichment analysis reveals that ChREBP target genes are highly correlated with genes regulated by high glucose, providing a functional relevance to the genome-wide binding study. Furthermore, we have demonstrated that ChREBP may function as a transcriptional repressor as well as an activator. PMID:21811631

  3. Homozygosity mapping and targeted genomic sequencing reveal the gene responsible for cerebellar hypoplasia and quadrupedal locomotion in a consanguineous kindred

    PubMed Central

    Gulsuner, Suleyman; Tekinay, Ayse Begum; Doerschner, Katja; Boyaci, Huseyin; Bilguvar, Kaya; Unal, Hilal; Ors, Aslihan; Onat, O. Emre; Atalar, Ergin; Basak, A. Nazli; Topaloglu, Haluk; Kansu, Tulay; Tan, Meliha; Tan, Uner; Gunel, Murat; Ozcelik, Tayfun

    2011-01-01

    The biological basis for the development of the cerebro-cerebellar structures required for posture and gait in humans is poorly understood. We investigated a large consanguineous family from Turkey exhibiting an extremely rare phenotype associated with quadrupedal locomotion, mental retardation, and cerebro-cerebellar hypoplasia, linked to a 7.1-Mb region of homozygosity on chromosome 17p13.1–13.3. Diffusion weighted imaging and fiber tractography of the patients' brains revealed morphological abnormalities in the cerebellum and corpus callosum, in particular atrophy of superior, middle, and inferior peduncles of the cerebellum. Structural magnetic resonance imaging showed additional morphometric abnormalities in several cortical areas, including the corpus callosum, precentral gyrus, and Brodmann areas BA6, BA44, and BA45. Targeted sequencing of the entire homozygous region in three affected individuals and two obligate carriers uncovered a private missense mutation, WDR81 p.P856L, which cosegregated with the condition in the extended family. The mutation lies in a highly conserved region of WDR81, flanked by an N-terminal BEACH domain and C-terminal WD40 beta-propeller domains. WDR81 is predicted to be a transmembrane protein. It is highly expressed in the cerebellum and corpus callosum, in particular in the Purkinje cell layer of the cerebellum. WDR81 represents the third gene, after VLDLR and CA8, implicated in quadrupedal locomotion in humans. PMID:21885617

  4. Integrated gene co-expression network analysis in the growth phase of Mycobacterium tuberculosis reveals new potential drug targets.

    PubMed

    Puniya, Bhanwar Lal; Kulshreshtha, Deepika; Verma, Srikant Prasad; Kumar, Sanjiv; Ramachandran, Srinivasan

    2013-11-01

    We have carried out weighted gene co-expression network analysis of Mycobacterium tuberculosis to gain insights into gene expression architecture during log phase growth. The differentially expressed genes between at least one pair of 11 different M. tuberculosis strains as source of biological variability were used for co-expression network analysis. This data included genes with highest coefficient of variation in expression. Five distinct modules were identified using topological overlap based clustering. All the modules together showed significant enrichment in biological processes: fatty acid biosynthesis, cell membrane, intracellular membrane bound organelle, DNA replication, Quinone biosynthesis, cell shape and peptidoglycan biosynthesis, ribosome and structural constituents of ribosome and transposition. We then extracted the co-expressed connections which were supported either by transcriptional regulatory network or STRING database or high edge weight of topological overlap. The genes trpC, nadC, pitA, Rv3404c, atpA, pknA, Rv0996, purB, Rv2106 and Rv0796 emerged as top hub genes. After overlaying this network on the iNJ661 metabolic network, the reactions catalyzed by 15 highly connected metabolic genes were knocked down in silico and evaluated by Flux Balance Analysis. The results showed that in 12 out of 15 cases, in 11 more than 50% of reactions catalyzed by genes connected through co-expressed connections also had altered fluxes. The modules 'Turquoise', 'Blue' and 'Red' also showed enrichment in essential genes. We could map 152 of the previously known or proposed drug targets in these modules and identified 15 new potential drug targets based on their high degree of co-expressed connections and strong correlation with module eigengenes. PMID:24056838

  5. Neurospora crassa transcriptomics reveals oxidative stress and plasma membrane homeostasis biology genes as key targets in response to chitosan

    PubMed Central

    Lopez-Moya, Federico; Kowbel, David; Nueda, Ma José; Palma-Guerrero, Javier; Glass, N. Louise; Lopez-Llorca, Luis Vicente

    2016-01-01

    Chitosan is a natural polymer with antimicrobial activity. Chitosan causes plasma membrane permeabilization and induction of intracellular reactive oxygen species (ROS) in Neurospora crassa. We have determined the transcriptional profile of N. crassa to chitosan and identified the main gene targets involved in the cellular response to this compound. Global network analyses showed membrane, transport and oxidoreductase activity as key nodes affected by chitosan. Activation of oxidative metabolism indicates the importance of ROS and cell energy together with plasma membrane homeostasis in N. crassa response to chitosan. Deletion strain analysis of chitosan susceptibility pointed, NCU03639 encoding a class 3 lipase, involved in plasma membrane repair by lipid replacement and NCU04537 a MFS monosaccharide transporter related with assimilation of simple sugars, as main gene targets of chitosan. NCU10521, a glutathione S-transferase-4 involved in the generation of reducing power for scavenging intracellular ROS is also a determinant chitosan gene target. Ca2+ increased tolerance to chitosan in N. crassa. Growth of NCU10610 (fig 1 domain) and SYT1 (a synaptotagmin) deletion strains was significantly increased by Ca2+ in presence of chitosan. Both genes play a determinant role in N. crassa membrane homeostasis. Our results are of paramount importance for developing chitosan as antifungal. PMID:26694141

  6. Neurospora crassa transcriptomics reveals oxidative stress and plasma membrane homeostasis biology genes as key targets in response to chitosan.

    PubMed

    Lopez-Moya, Federico; Kowbel, David; Nueda, Maria José; Palma-Guerrero, Javier; Glass, N Louise; Lopez-Llorca, Luis Vicente

    2016-02-01

    Chitosan is a natural polymer with antimicrobial activity. Chitosan causes plasma membrane permeabilization and induction of intracellular reactive oxygen species (ROS) in Neurospora crassa. We have determined the transcriptional profile of N. crassa to chitosan and identified the main gene targets involved in the cellular response to this compound. Global network analyses showed membrane, transport and oxidoreductase activity as key nodes affected by chitosan. Activation of oxidative metabolism indicates the importance of ROS and cell energy together with plasma membrane homeostasis in N. crassa response to chitosan. Deletion strain analysis of chitosan susceptibility pointed NCU03639 encoding a class 3 lipase, involved in plasma membrane repair by lipid replacement, and NCU04537 a MFS monosaccharide transporter related to assimilation of simple sugars, as main gene targets of chitosan. NCU10521, a glutathione S-transferase-4 involved in the generation of reducing power for scavenging intracellular ROS is also a determinant chitosan gene target. Ca(2+) increased tolerance to chitosan in N. crassa. Growth of NCU10610 (fig 1 domain) and SYT1 (a synaptotagmin) deletion strains was significantly increased by Ca(2+) in the presence of chitosan. Both genes play a determinant role in N. crassa membrane homeostasis. Our results are of paramount importance for developing chitosan as an antifungal. PMID:26694141

  7. Global Identification of SMAD2 Target Genes Reveals a Role for Multiple Co-regulatory Factors in Zebrafish Early Gastrulas*

    PubMed Central

    Liu, Zhaoting; Lin, Xiwen; Cai, Zhaoping; Zhang, Zhuqiang; Han, Chunsheng; Jia, Shunji; Meng, Anming; Wang, Qiang

    2011-01-01

    Nodal and Smad2/3 signals play pivotal roles in mesendoderm induction and axis determination during late blastulation and early gastrulation in vertebrate embryos. However, Smad2/3 direct target genes during those critical developmental stages have not been systematically identified. Here, through ChIP-chip assay, we show that the promoter/enhancer regions of 679 genes are bound by Smad2 in the zebrafish early gastrulas. Expression analyses confirm that a significant proportion of Smad2 targets are indeed subjected to Nodal/Smad2 regulation at the onset of gastrulation. The co-existence of DNA-binding sites of other transcription factors in the Smad2-bound regions allows the identification of well known Smad2-binding partners, such as FoxH1 and Lef1/β-catenin, as well as many previously unknown Smad2 partners, including Oct1 and Gata6, during embryogenesis. We demonstrate that Oct1 physically associates with and enhances the transcription and mesendodermal induction activity of Smad2, whereas Gata6 exerts an inhibitory role in Smad2 signaling and mesendodermal induction. Thus, our study systemically uncovers a large number of Smad2 targets in early gastrulas and suggests cooperative roles of Smad2 and other transcription factors in controlling target gene transcription, which will be valuable for studying regulatory cascades during germ layer formation and patterning of vertebrate embryos. PMID:21669877

  8. The low-abundance transcriptome reveals novel biomarkers, specific intracellular pathways and targetable genes associated with advanced gastric cancer.

    PubMed

    Bizama, Carolina; Benavente, Felipe; Salvatierra, Edgardo; Gutiérrez-Moraga, Ana; Espinoza, Jaime A; Fernández, Elmer A; Roa, Iván; Mazzolini, Guillermo; Sagredo, Eduardo A; Gidekel, Manuel; Podhajcer, Osvaldo L

    2014-02-15

    Studies on the low-abundance transcriptome are of paramount importance for identifying the intimate mechanisms of tumor progression that can lead to novel therapies. The aim of the present study was to identify novel markers and targetable genes and pathways in advanced human gastric cancer through analyses of the low-abundance transcriptome. The procedure involved an initial subtractive hybridization step, followed by global gene expression analysis using microarrays. We observed profound differences, both at the single gene and gene ontology levels, between the low-abundance transcriptome and the whole transcriptome. Analysis of the low-abundance transcriptome led to the identification and validation by tissue microarrays of novel biomarkers, such as LAMA3 and TTN; moreover, we identified cancer type-specific intracellular pathways and targetable genes, such as IRS2, IL17, IFNγ, VEGF-C, WISP1, FZD5 and CTBP1 that were not detectable by whole transcriptome analyses. We also demonstrated that knocking down the expression of CTBP1 sensitized gastric cancer cells to mainstay chemotherapeutic drugs. We conclude that the analysis of the low-abundance transcriptome provides useful insights into the molecular basis and treatment of cancer. PMID:23907728

  9. Phenotypic profiling of the human genome reveals gene products involved in plasma membrane targeting of SRC kinases

    PubMed Central

    Ritzerfeld, Julia; Remmele, Steffen; Wang, Tao; Temmerman, Koen; Brügger, Britta; Wegehingel, Sabine; Tournaviti, Stella; Strating, Jeroen R.P.M.; Wieland, Felix T.; Neumann, Beate; Ellenberg, Jan; Lawerenz, Chris; Hesser, Jürgen; Erfle, Holger; Pepperkok, Rainer; Nickel, Walter

    2011-01-01

    SRC proteins are non-receptor tyrosine kinases that play key roles in regulating signal transduction by a diverse set of cell surface receptors. They contain N-terminal SH4 domains that are modified by fatty acylation and are functioning as membrane anchors. Acylated SH4 domains are both necessary and sufficient to mediate specific targeting of SRC kinases to the inner leaflet of plasma membranes. Intracellular transport of SRC kinases to the plasma membrane depends on microdomains into which SRC kinases partition upon palmitoylation. In the present study, we established a live-cell imaging screening system to identify gene products involved in plasma membrane targeting of SRC kinases. Based on siRNA arrays and a human model cell line expressing two kinds of SH4 reporter molecules, we conducted a genome-wide analysis of SH4-dependent protein targeting using an automated microscopy platform. We identified and validated 54 gene products whose down-regulation causes intracellular retention of SH4 reporter molecules. To detect and quantify this phenotype, we developed a software-based image analysis tool. Among the identified gene products, we found factors involved in lipid metabolism, intracellular transport, and cellular signaling processes. Furthermore, we identified proteins that are either associated with SRC kinases or are related to various known functions of SRC kinases such as other kinases and phosphatases potentially involved in SRC-mediated signal transduction. Finally, we identified gene products whose function is less defined or entirely unknown. Our findings provide a major resource for future studies unraveling the molecular mechanisms that underlie proper targeting of SRC kinases to the inner leaflet of plasma membranes. PMID:21795383

  10. Transcriptome analysis of PPARγ target genes reveals the involvement of lysyl oxidase in human placental cytotrophoblast invasion.

    PubMed

    Segond, Nadine; Degrelle, Séverine A; Berndt, Sarah; Clouqueur, Elodie; Rouault, Christine; Saubamea, Bruno; Dessen, Philippe; Fong, Keith S K; Csiszar, Katalin; Badet, Josette; Evain-Brion, Danièle; Fournier, Thierry

    2013-01-01

    Human placental development is characterized by invasion of extravillous cytotrophoblasts (EVCTs) into the uterine wall during the first trimester of pregnancy. Peroxisome proliferator-activated receptor γ (PPARγ) plays a major role in placental development, and activation of PPARγ by its agonists results in inhibition of EVCT invasion in vitro. To identify PPARγ target genes, microarray analysis was performed using GeneChip technology on EVCT primary cultures obtained from first-trimester human placentas. Gene expression was compared in EVCTs treated with the PPARγ agonist rosiglitazone versus control. A total of 139 differentially regulated genes were identified, and changes in the expression of the following 8 genes were confirmed by reverse transcription-quantitative polymerase chain reaction: a disintegrin and metalloproteinase domain12 (ADAM12), connexin 43 (CX43), deleted in liver cancer 1 (DLC1), dipeptidyl peptidase 4 (DPP4), heme oxygenase 1 (HMOX-1), lysyl oxidase (LOX), plasminogen activator inhibitor 1 (PAI-1) and PPARγ. Among the upregulated genes, lysyl oxidase (LOX) was further analyzed. In the LOX family, only LOX, LOXL1 and LOXL2 mRNA expression was significantly upregulated in rosiglitazone-treated EVCTs. RNA and protein expression of the subfamily members LOX, LOXL1 and LOXL2 were analyzed by absolute RT-qPCR and western blotting, and localized by immunohistochemistry and immunofluorescence-confocal microscopy. LOX protein was immunodetected in the EVCT cytoplasm, while LOXL1 was found in the nucleus and nucleolus. No signal was detected for LOXL2 protein. Specific inhibition of LOX activity by β-aminopropionitrile in cell invasion assays led to an increase in EVCT invasiveness. These results suggest that LOX, LOXL1 and LOXL2 are downstream PPARγ targets and that LOX activity is a negative regulator of trophoblastic cell invasion. PMID:24265769

  11. Targeted Sequencing Reveals Large-Scale Sequence Polymorphism in Maize Candidate Genes for Biomass Production and Composition

    PubMed Central

    Ulpinnis, Chris; Scholz, Uwe; Altmann, Thomas

    2015-01-01

    A major goal of maize genomic research is to identify sequence polymorphisms responsible for phenotypic variation in traits of economic importance. Large-scale detection of sequence variation is critical for linking genes, or genomic regions, to phenotypes. However, due to its size and complexity, it remains expensive to generate whole genome sequences of sufficient coverage for divergent maize lines, even with access to next generation sequencing (NGS) technology. Because methods involving reduction of genome complexity, such as genotyping-by-sequencing (GBS), assess only a limited fraction of sequence variation, targeted sequencing of selected genomic loci offers an attractive alternative. We therefore designed a sequence capture assay to target 29 Mb genomic regions and surveyed a total of 4,648 genes possibly affecting biomass production in 21 diverse inbred maize lines (7 flints, 14 dents). Captured and enriched genomic DNA was sequenced using the 454 NGS platform to 19.6-fold average depth coverage, and a broad evaluation of read alignment and variant calling methods was performed to select optimal procedures for variant discovery. Sequence alignment with the B73 reference and de novo assembly identified 383,145 putative single nucleotide polymorphisms (SNPs), of which 42,685 were non-synonymous alterations and 7,139 caused frameshifts. Presence/absence variation (PAV) of genes was also detected. We found that substantial sequence variation exists among genomic regions targeted in this study, which was particularly evident within coding regions. This diversification has the potential to broaden functional diversity and generate phenotypic variation that may lead to new adaptations and the modification of important agronomic traits. Further, annotated SNPs identified here will serve as useful genetic tools and as candidates in searches for phenotype-altering DNA variation. In summary, we demonstrated that sequencing of captured DNA is a powerful approach for

  12. Novel Genes Involved in Endosomal Traffic in Yeast Revealed by Suppression of a Targeting-defective Plasma Membrane ATPase Mutant

    PubMed Central

    Luo, Wen-jie; Chang, Amy

    1997-01-01

    A novel genetic selection was used to identify genes regulating traffic in the yeast endosomal system. We took advantage of a temperature-sensitive mutant in PMA1, encoding the plasma membrane ATPase, in which newly synthesized Pma1 is mislocalized to the vacuole via the endosome. Diversion of mutant Pma1 from vacuolar delivery and rerouting to the plasma membrane is a major mechanism of suppression of pma1ts. 16 independent suppressor of pma1 (sop) mutants were isolated. Identification of the corresponding genes reveals eight that are identical with VPS genes required for delivery of newly synthesized vacuolar proteins. A second group of SOP genes participates in vacuolar delivery of mutant Pma1 but is not essential for delivery of the vacuolar protease carboxypeptidase Y. Because the biosynthetic pathway to the vacuole intersects with the endocytic pathway, internalization of a bulk membrane endocytic marker FM 4-64 was assayed in the sop mutants. By this means, defective endosome-to-vacuole trafficking was revealed in a subset of sop mutants. Another subset of sop mutants displays perturbed trafficking between endosome and Golgi: impaired pro-α factor processing in these strains was found to be due to defective recycling of the trans-Golgi protease Kex2. One of these strains defective in Kex2 trafficking carries a mutation in SOP2, encoding a homologue of mammalian synaptojanin (implicated in synaptic vesicle endocytosis and recycling). Thus, cell surface delivery of mutant Pma1 can occur as a consequence of disturbances at several different sites in the endosomal system. PMID:9265642

  13. Targeted high-throughput growth hormone 1 gene sequencing reveals high within-breed genetic diversity in South African goats.

    PubMed

    Ncube, K T; Mdladla, K; Dzomba, E F; Muchadeyi, F C

    2016-06-01

    This study assessed the genetic diversity in the growth hormone 1 gene (GH1) within and between South African goat breeds. Polymerase chain reaction-targeted gene amplification together with Illumina MiSeq next-generation sequencing (NGS) was used to generate the full length (2.54 kb) of the growth hormone 1 gene and screen for SNPs in the South African Boer (SAB) (n = 17), Tankwa (n = 15) and South African village (n = 35) goat populations. A range of 27-58 SNPs per population were observed. Mutations resulting in amino acid changes were observed at exons 2 and 5. Higher within-breed diversity of 97.37% was observed within the population category consisting of SA village ecotypes and the Tankwa goats. Highest pairwise FST values ranging from 0.148 to 0.356 were observed between the SAB and both the South African village and Tankwa feral goat populations. Phylogenetic analysis indicated nine genetic clusters, which reflected close relationships between the South African populations and the other international breeds with the exception of the Italian Sarda breeds. Results imply greater potential for within-population selection programs, particularly with SA village goats. PMID:26919178

  14. Targeted disruption of the LAMA3 gene in mice reveals abnormalities in survival and late stage differentiation of epithelial cells.

    PubMed

    Ryan, M C; Lee, K; Miyashita, Y; Carter, W G

    1999-06-14

    Laminin 5 regulates anchorage and motility of epithelial cells through integrins alpha6beta4 and alpha3beta1, respectively. We used targeted disruption of the LAMA3 gene, which encodes the alpha3 subunit of laminin 5 and other isoforms, to examine developmental functions that are regulated by adhesion to the basement membrane (BM). In homozygous null animals, profound epithelial abnormalities were detected that resulted in neonatal lethality, consistent with removal of all alpha3-laminin isoforms from epithelial BMs. Alterations in three different cellular functions were identified. First, using a novel tissue adhesion assay, we found that the mutant BM could not induce stable adhesion by integrin alpha6beta4, consistent with the presence of junctional blisters and abnormal hemidesmosomes. In the absence of laminin 5 function, we were able to detect a new ligand for integrin alpha3beta1 in the epidermal BM, suggesting that basal keratinocytes can utilize integrin alpha3beta1 to interact with an alternative ligand. Second, we identified a survival defect in mutant epithelial cells that could be rescued by exogenous laminin 5, collagen, or an antibody against integrin alpha6beta4, suggesting that signaling through beta1 or beta4 integrins is sufficient for survival. Third, we detected abnormalities in ameloblast differentiation in developing mutant incisors indicating that events downstream of adhesion are affected in mutant animals. These results indicate that laminin 5 has an important role in regulating tissue organization, gene expression, and survival of epithelium. PMID:10366601

  15. Gene Expression Analysis of CL-20-induced Reversible Neurotoxicity Reveals GABAA Receptors as Potential Target in the Earthworm Eisenia fetida

    PubMed Central

    Gong, Ping; Guan, Xin; Pirooznia, Mehdi; Liang, Chun; Perkins, Edward J.

    2012-01-01

    The earthworm Eisenia fetida is one of the most used species in standardized soil ecotoxicity tests. Endpoints such as survival, growth and reproduction are eco-toxicologically relevant but provide little mechanistic insight into toxicity pathways, especially at the molecular level. Here we applied a toxicogenomic approach to investigate the mode of action underlying the reversible neurotoxicity of hexanitrohexaazaisowurtzitane (CL-20), a cyclic nitroamine explosives compound. We developed an E. fetida-specific shotgun microarray targeting 15119 unique E. fetida transcripts. Using this array we profiled gene expression in E. fetida in response to exposure to CL-20. Eighteen earthworms were exposed for 6 days to 0.2 μg/cm2 of CL-20 on filter paper, half of which were allowed to recover in a clean environment for 7 days. Nine vehicle control earthworms were sacrificed at day 6 and 13, separately. Electrophysiological measurements indicated that the conduction velocity of earthworm medial giant nerve fiber decreased significantly after 6-day exposure to CL-20, but was restored after 7 days of recovery. Total RNA was isolated from the four treatment groups including 6-day control, 6-day exposed, 13-day control and 13-day exposed (i.e. 6-day exposure followed by 7-day recovery), and was hybridized to the 15K shot-gun oligo array. Statistical and bioinformatic analyses suggest that CL-20 initiated neurotoxicity by non-competitively blocking the ligand-gated GABAA receptor ion channel, leading to altered expression of genes involved in GABAergic, cholinergic, and Agrin-MuSK pathways. In the recovery phase, expression of affected genes returned to normality, possibly as a result of autophagy and CL-20 dissociation/metabolism. This study provides significant insights into potential mechanisms of CL-20-induced neurotoxicity and the recovery of earthworms from transient neurotoxicity stress. PMID:22191394

  16. Genome wide transcriptional profiling in breast cancer cells reveals distinct changes in hormone receptor target genes and chromatin modifying enzymes after proteasome inhibition

    PubMed Central

    Kinyamu, H. Karimi; Collins, Jennifer B.; Grissom, Sherry F.; Hebbar, Pratibha B.; Archer, Trevor K.

    2010-01-01

    Steroid hormone receptors, like glucocorticoid (GR) and estrogen receptors (ER), are master regulators of genes that control many biological processes implicated in health and disease. Gene expression is dependent on receptor levels which are tightly regulated by the ubiquitin-proteasome system. Previous studies have shown that proteasome inhibition increases GR, but decreases ER-mediated gene expression. At the gene expression level this divergent role of the proteasome in receptor-dependent transcriptional regulation is not well understood. We have used a genomic approach to examine the impact of proteasome activity on GR and ER-mediated gene expression in MCF-7 breast cancer cells treated with dexamethasone (DEX) or 17β-estradiol (E2), the proteasome inhibitor MG132 (MG) or MG132 and either hormone (MD or ME2) for 24h. Transcript profiling reveals that inhibiting proteasome activity modulates gene expression by GR and ER in a similar manner in that several GR and ER target genes are up-regulated and down-regulated after proteasome inhibition. In addition, proteasome inhibition modulates receptor-dependent genes involved in the etiology of a number of human pathological states, including multiple myeloma, leukemia, breast/prostate cancer, HIV/AIDS and neurodegenerative disorders. Importantly, our analysis reveals that a number of transcripts encoding histone and DNA modifying enzymes, prominently histone/DNA methyltransferases and demethylases, are altered after proteasome inhibition. As proteasome inhibitors are currently in clinical trials as therapy for multiple myeloma, HIV/AIDs and leukemia, the possibility that some of the target molecules are hormone regulated and by chromatin modifying enzymes is intriguing in this era of epigenetic therapy. PMID:18381591

  17. Targeted ultra-deep sequencing reveals recurrent and mutually exclusive mutations of cancer genes in blastic plasmacytoid dendritic cell neoplasm

    PubMed Central

    Pfarr, Nicole; Andrulis, Mindaugas; Jöhrens, Korinna; Klauschen, Frederick; Siebolts, Udo; Wolf, Thomas; Koch, Philipp-Sebastian; Schulz, Miriam; Hartschuh, Wolfgang; Goerdt, Sergij; Lennerz, Jochen K.; Wickenhauser, Claudia; Klapper, Wolfram

    2014-01-01

    Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare haematopoietic malignancy characterized by dismal prognosis and overall poor therapeutic response. Since the biology of BPDCN is barely understood, our study aims to shed light on the genetic make-up of these highly malignant tumors. Using targeted high-coverage massive parallel sequencing, we investigated 50 common cancer genes in 33 BPDCN samples. We detected point mutations in NRAS (27.3% of cases), ATM (21.2%), MET, KRAS, IDH2, KIT (9.1% each), APC and RB1 (6.1% each), as well as in VHL, BRAF, MLH1, TP53 and RET (3% each). Moreover, NRAS, KRAS and ATM mutations were found to be mutually exclusive and we observed recurrent mutations in NRAS, IDH2, APC and ATM. CDKN2A deletions were detected in 27.3% of the cases followed by deletions of RB1 (9.1%), PTEN and TP53 (3% each). The mutual exclusive distribution of some mutations may point to different subgroups of BPDCN whose biological significance remains to be explored. PMID:25115387

  18. Targeted ultra-deep sequencing reveals recurrent and mutually exclusive mutations of cancer genes in blastic plasmacytoid dendritic cell neoplasm.

    PubMed

    Stenzinger, Albrecht; Endris, Volker; Pfarr, Nicole; Andrulis, Mindaugas; Jöhrens, Korinna; Klauschen, Frederick; Siebolts, Udo; Wolf, Thomas; Koch, Philipp-Sebastian; Schulz, Miriam; Hartschuh, Wolfgang; Goerdt, Sergij; Lennerz, Jochen K; Wickenhauser, Claudia; Klapper, Wolfram; Anagnostopoulos, Ioannis; Weichert, Wilko

    2014-08-15

    Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare haematopoietic malignancy characterized by dismal prognosis and overall poor therapeutic response. Since the biology of BPDCN is barely understood, our study aims to shed light on the genetic make-up of these highly malignant tumors. Using targeted high-coverage massive parallel sequencing, we investigated 50 common cancer genes in 33 BPDCN samples. We detected point mutations in NRAS (27.3% of cases), ATM (21.2%), MET, KRAS, IDH2, KIT (9.1% each), APC and RB1 (6.1%), as well as in VHL, BRAF, MLH1, TP53 and RET1 (3% each). Moreover, NRAS-, KRAS- and ATM-mutations were found to be mutually exclusive and we observed recurrent mutations in NRAS, IDH2, APC and ATM. CDKN2A deletions were detected in 27.3% of the cases followed by deletions of RB1 (9.1%), PTEN and TP53 (3% each). The mutual exclusive distribution of some mutations may point to different subgroups of BPDCN whose biological significance remains to be explored. PMID:25115387

  19. Time-Course Small RNA Profiling Reveals Rice miRNAs and Their Target Genes in Response to Rice Stripe Virus Infection.

    PubMed

    Lian, Sen; Cho, Won Kyong; Kim, Sang-Min; Choi, Hoseong; Kim, Kook-Hyung

    2016-01-01

    It has been known that many microRNAs (miRNAs) are involved in the regulation for the plant development and defense mechanism by regulating the expression of the target gene. Several previous studies has demonstrated functional roles of miRNAs in antiviral defense mechanisms. In this study, we employed high-throughput sequencing technology to identify rice miRNAs upon rice stripe virus (RSV) infection at three different time points. Six libraries from mock and RSV-infected samples were subjected for small RNA sequencing. Bioinformatic analyses revealed 374 known miRNAs and 19 novel miRNAs. Expression of most identified miRNAs was not dramatically changed at 3 days post infection (dpi) and 7 dpi by RSV infection. However, many numbers of miRNAs were up-regulated in mock and RSV-infected samples at 15 dpi by RSV infection. Moreover, expression profiles of identified miRNAs revealed that only few numbers of miRNAs were strongly regulated by RSV infection. In addition, 15 resistance genes were targets of six miRNAs suggesting that those identified miRNAs and 15 NBS-LRR resistance genes might be involved in RSV infection. Taken together, our results provide novel insight into the dynamic expression profiles of rice miRNAs upon RSV infection and clues for the understanding of the regulatory roles of miRNAs via time-course. PMID:27626631

  20. Camelid Ig V genes reveal significant human homology not seen in therapeutic target genes, providing for a powerful therapeutic antibody platform

    PubMed Central

    Klarenbeek, Alex; Mazouari, Khalil El; Desmyter, Aline; Blanchetot, Christophe; Hultberg, Anna; de Jonge, Natalie; Roovers, Rob C; Cambillau, Christian; Spinelli, Sylvia; Del-Favero, Jurgen; Verrips, Theo; de Haard, Hans J; Achour, Ikbel

    2015-01-01

    Camelid immunoglobulin variable (IGV) regions were found homologous to their human counterparts; however, the germline V repertoires of camelid heavy and light chains are still incomplete and their therapeutic potential is only beginning to be appreciated. We therefore leveraged the publicly available HTG and WGS databases of Lama pacos and Camelus ferus to retrieve the germline repertoire of V genes using human IGV genes as reference. In addition, we amplified IGKV and IGLV genes to uncover the V germline repertoire of Lama glama and sequenced BAC clones covering part of the Lama pacos IGK and IGL loci. Our in silico analysis showed that camelid counterparts of all human IGKV and IGLV families and most IGHV families could be identified, based on canonical structure and sequence homology. Interestingly, this sequence homology seemed largely restricted to the Ig V genes and was far less apparent in other genes: 6 therapeutically relevant target genes differed significantly from their human orthologs. This contributed to efficient immunization of llamas with the human proteins CD70, MET, interleukin (IL)-1β and IL-6, resulting in large panels of functional antibodies. The in silico predicted human-homologous canonical folds of camelid-derived antibodies were confirmed by X-ray crystallography solving the structure of 2 selected camelid anti-CD70 and anti-MET antibodies. These antibodies showed identical fold combinations as found in the corresponding human germline V families, yielding binding site structures closely similar to those occurring in human antibodies. In conclusion, our results indicate that active immunization of camelids can be a powerful therapeutic antibody platform. PMID:26018625

  1. Camelid Ig V genes reveal significant human homology not seen in therapeutic target genes, providing for a powerful therapeutic antibody platform.

    PubMed

    Klarenbeek, Alex; El Mazouari, Khalil; Desmyter, Aline; Blanchetot, Christophe; Hultberg, Anna; de Jonge, Natalie; Roovers, Rob C; Cambillau, Christian; Spinelli, Sylvia; Del-Favero, Jurgen; Verrips, Theo; de Haard, Hans J; Achour, Ikbel

    2015-01-01

    Camelid immunoglobulin variable (IGV) regions were found homologous to their human counterparts; however, the germline V repertoires of camelid heavy and light chains are still incomplete and their therapeutic potential is only beginning to be appreciated. We therefore leveraged the publicly available HTG and WGS databases of Lama pacos and Camelus ferus to retrieve the germline repertoire of V genes using human IGV genes as reference. In addition, we amplified IGKV and IGLV genes to uncover the V germline repertoire of Lama glama and sequenced BAC clones covering part of the Lama pacos IGK and IGL loci. Our in silico analysis showed that camelid counterparts of all human IGKV and IGLV families and most IGHV families could be identified, based on canonical structure and sequence homology. Interestingly, this sequence homology seemed largely restricted to the Ig V genes and was far less apparent in other genes: 6 therapeutically relevant target genes differed significantly from their human orthologs. This contributed to efficient immunization of llamas with the human proteins CD70, MET, interleukin (IL)-1β and IL-6, resulting in large panels of functional antibodies. The in silico predicted human-homologous canonical folds of camelid-derived antibodies were confirmed by X-ray crystallography solving the structure of 2 selected camelid anti-CD70 and anti-MET antibodies. These antibodies showed identical fold combinations as found in the corresponding human germline V families, yielding binding site structures closely similar to those occurring in human antibodies. In conclusion, our results indicate that active immunization of camelids can be a powerful therapeutic antibody platform. PMID:26018625

  2. Analysis of Post-Traumatic Brain Injury Gene Expression Signature Reveals Tubulins, Nfe2l2, Nfkb, Cd44, and S100a4 as Treatment Targets

    PubMed Central

    Lipponen, Anssi; Paananen, Jussi; Puhakka, Noora; Pitkänen, Asla

    2016-01-01

    We aimed to define the chronically altered gene expression signature of traumatic brain injury (TBI-sig) to discover novel treatments to reverse pathologic gene expression or reinforce the expression of recovery-related genes. Genome-wide RNA-sequencing was performed at 3 months post-TBI induced by lateral fluid-percussion injury in rats. We found 4964 regulated genes in the perilesional cortex and 1966 in the thalamus (FDR < 0.05). TBI-sig was used for a LINCS analysis which identified 11 compounds that showed a strong connectivity with the TBI-sig in neuronal cell lines. Of these, celecoxib and sirolimus were recently reported to have a disease-modifying effect in in vivo animal models of epilepsy. Other compounds revealed by the analysis were BRD-K91844626, BRD-A11009626, NO-ASA, BRD-K55260239, SDZ-NKT-343, STK-661558, BRD-K75971499, ionomycin, and desmethylclomipramine. Network analysis of overlapping genes revealed the effects on tubulins (Tubb2a, Tubb3, Tubb4b), Nfe2l2, S100a4, Cd44, and Nfkb2, all of which are linked to TBI-relevant outcomes, including epileptogenesis and tissue repair. Desmethylclomipramine modulated most of the gene targets considered favorable for TBI outcome. Our data demonstrate long-lasting transcriptomics changes after TBI. LINCS analysis predicted that these changes could be modulated by various compounds, some of which are already in clinical use but never tested in TBI. PMID:27530814

  3. Analysis of Post-Traumatic Brain Injury Gene Expression Signature Reveals Tubulins, Nfe2l2, Nfkb, Cd44, and S100a4 as Treatment Targets.

    PubMed

    Lipponen, Anssi; Paananen, Jussi; Puhakka, Noora; Pitkänen, Asla

    2016-01-01

    We aimed to define the chronically altered gene expression signature of traumatic brain injury (TBI-sig) to discover novel treatments to reverse pathologic gene expression or reinforce the expression of recovery-related genes. Genome-wide RNA-sequencing was performed at 3 months post-TBI induced by lateral fluid-percussion injury in rats. We found 4964 regulated genes in the perilesional cortex and 1966 in the thalamus (FDR < 0.05). TBI-sig was used for a LINCS analysis which identified 11 compounds that showed a strong connectivity with the TBI-sig in neuronal cell lines. Of these, celecoxib and sirolimus were recently reported to have a disease-modifying effect in in vivo animal models of epilepsy. Other compounds revealed by the analysis were BRD-K91844626, BRD-A11009626, NO-ASA, BRD-K55260239, SDZ-NKT-343, STK-661558, BRD-K75971499, ionomycin, and desmethylclomipramine. Network analysis of overlapping genes revealed the effects on tubulins (Tubb2a, Tubb3, Tubb4b), Nfe2l2, S100a4, Cd44, and Nfkb2, all of which are linked to TBI-relevant outcomes, including epileptogenesis and tissue repair. Desmethylclomipramine modulated most of the gene targets considered favorable for TBI outcome. Our data demonstrate long-lasting transcriptomics changes after TBI. LINCS analysis predicted that these changes could be modulated by various compounds, some of which are already in clinical use but never tested in TBI. PMID:27530814

  4. RNA-seq Profiling Reveals Novel Target Genes of LexA in the Cyanobacterium Synechocystis sp. PCC 6803

    PubMed Central

    Kizawa, Ayumi; Kawahara, Akihito; Takimura, Yasushi; Nishiyama, Yoshitaka; Hihara, Yukako

    2016-01-01

    LexA is a well-established transcriptional repressor of SOS genes induced by DNA damage in Escherichia coli and other bacterial species. However, LexA in the cyanobacterium Synechocystis sp. PCC 6803 has been suggested not to be involved in SOS response. In this study, we performed RNA-seq analysis of the wild-type strain and the lexA-disrupted mutant to obtain the comprehensive view of LexA-regulated genes in Synechocystis. Disruption of lexA positively or negatively affected expression of genes related to various cellular functions such as phototactic motility, accumulation of the major compatible solute glucosylglycerol and subunits of bidirectional hydrogenase, photosystem I, and phycobilisome complexes. We also observed increase in the expression level of genes related to iron and manganese uptake in the mutant at the later stage of cultivation. However, none of the genes related to DNA metabolism were affected by disruption of lexA. DNA gel mobility shift assay using the recombinant LexA protein suggested that LexA binds to the upstream region of pilA7, pilA9, ggpS, and slr1670 to directly regulate their expression, but changes in the expression level of photosystem I genes by disruption of lexA is likely a secondary effect. PMID:26925056

  5. Comparative Transcriptome Analysis Reveals Significant Differences in MicroRNA Expression and Their Target Genes between Adipose and Muscular Tissues in Cattle

    PubMed Central

    Sun, Jiajie; Zhang, Bowen; Lan, Xianyong; Zhang, Chunlei; Lei, Chuzhao; Chen, Hong

    2014-01-01

    The posttranscriptional gene regulation mediated by microRNAs (miRNAs) plays an important role in various species. However, to date limited miRNAs have been reported between fat and muscle tissues in beef cattle. In this paper, 412 known and 22 novel miRNAs in backfat as well as 334 known and 10 novel miRNAs in longissimus thoracis were identified in the Chinese Qinchuan beef cattle. Bta-miR-199a-3p, -154c, -320a and -432 were expressed at higher levels in backfat tissue, while bta-miR-1, -133a, -206, and -378 were also significantly enriched in muscle tissue. Functional analysis revealed that fat-enriched miRNAs targeted PRKAA1/2, PPARA and PPARG genes to modulate lipid and fatty acid metabolism, and muscle-enriched miRNAs targeted CSRP3 gene to present function involved in skeletal and muscular system development. The results obtained may help in the design of new selection strategies to improve beef quality. PMID:25006962

  6. Gene Network Analysis of Metallo Beta Lactamase Family Proteins Indicates the Role of Gene Partners in Antibiotic Resistance and Reveals Important Drug Targets.

    PubMed

    Parimelzaghan, Anitha; Anbarasu, Anand; Ramaiah, Sudha

    2016-06-01

    Metallo Beta (β) Lactamases (MBL) are metal dependent bacterial enzymes that hydrolyze the β-lactam antibiotics. In recent years, MBL have received considerable attention because it inactivates most of the β-lactam antibiotics. Increase in dissemination of MBL encoding antibiotic resistance genes in pathogenic bacteria often results in unsuccessful treatments. Gene interaction network of MBL provides a complete understanding on the molecular basis of MBL mediated antibiotic resistance. In our present study, we have constructed the MBL network of 37 proteins with 751 functional partners from pathogenic bacterial spp. We found 12 highly interconnecting clusters. Among the 37 MBL proteins considered in the present study, 22 MBL proteins are from B3 subclass, 14 are from B1 subclass and only one is from B2 subclass. Global topological parameters are used to calculate and compare the probability of interactions in MBL proteins. Our results indicate that the proteins associated within the network have a strong influence in antibiotic resistance mechanism. Interestingly, several drug targets are identified from the constructed network. We believe that our results would be helpful for researchers exploring MBL-mediated antibiotic resistant mechanisms. J. Cell. Biochem. 117: 1330-1339, 2016. © 2015 Wiley Periodicals, Inc. PMID:26517410

  7. Targeted gene disruption reveals a role for natural secretory IgM in the maturation of the primary immune response.

    PubMed

    Ehrenstein, M R; O'Keefe, T L; Davies, S L; Neuberger, M S

    1998-08-18

    Accelerated development of the secondary immune response may be attributable in part to the rapid delivery of antigen to lymphoid follicles by circulating antibody elicited on primary immunization. Here we provide evidence indicating that the nonspecific IgM present in naive mice (natural antibody) plays a role in the acceleration of the primary response. Targeted deletion of the Ig microseconds polyadenylation site by use of Cre recombinase allowed the creation of mice that, although harboring a normal number of B cells expressing surface IgM, completely lacked serum IgM while retaining the other Ig isotypes. These mice retained a broadly normal B lymphocyte distribution (although containing a somewhat expanded peritoneal B1a subset) but exhibited substantial delays in mounting affinity-matured IgG responses to T cell-dependent antigens. The T cell-independent response, however, was augmented. The data indicate that the IgM present before antigen challenge (as well, possibly, as that elicited immediately after immunization) accelerates maturation of the primary response, presumably by complexing with the antigen and facilitating lymphocyte activation and/or antigen trapping. PMID:9707605

  8. A Targeted Glycan-Related Gene Screen Reveals Heparan Sulfate Proteoglycan Sulfation Regulates WNT and BMP Trans-Synaptic Signaling

    PubMed Central

    Dani, Neil; Nahm, Minyeop; Lee, Seungbok; Broadie, Kendal

    2012-01-01

    A Drosophila transgenic RNAi screen targeting the glycan genome, including all N/O/GAG-glycan biosynthesis/modification enzymes and glycan-binding lectins, was conducted to discover novel glycan functions in synaptogenesis. As proof-of-product, we characterized functionally paired heparan sulfate (HS) 6-O-sulfotransferase (hs6st) and sulfatase (sulf1), which bidirectionally control HS proteoglycan (HSPG) sulfation. RNAi knockdown of hs6st and sulf1 causes opposite effects on functional synapse development, with decreased (hs6st) and increased (sulf1) neurotransmission strength confirmed in null mutants. HSPG co-receptors for WNT and BMP intercellular signaling, Dally-like Protein and Syndecan, are differentially misregulated in the synaptomatrix of these mutants. Consistently, hs6st and sulf1 nulls differentially elevate both WNT (Wingless; Wg) and BMP (Glass Bottom Boat; Gbb) ligand abundance in the synaptomatrix. Anterograde Wg signaling via Wg receptor dFrizzled2 C-terminus nuclear import and retrograde Gbb signaling via synaptic MAD phosphorylation and nuclear import are differentially activated in hs6st and sulf1 mutants. Consequently, transcriptional control of presynaptic glutamate release machinery and postsynaptic glutamate receptors is bidirectionally altered in hs6st and sulf1 mutants, explaining the bidirectional change in synaptic functional strength. Genetic correction of the altered WNT/BMP signaling restores normal synaptic development in both mutant conditions, proving that altered trans-synaptic signaling causes functional differentiation defects. PMID:23144627

  9. Network analysis reveals a stress-affected common gene module among seven stress-related diseases/systems which provides potential targets for mechanism research

    PubMed Central

    Guo, Liyuan; Du, Yang; Wang, Jing

    2015-01-01

    Chronic stress (CS) was reported to associate with many complex diseases and stress-related diseases show strong comorbidity; however, molecular analyses have not been performed to date to evaluate common stress-induced biological processes across these diseases. We utilized networks constructed by genes from seven genetic databases of stress-related diseases or systems to explore the common mechanisms. Genes were connected based on the interaction information of proteins they encode. A common sub-network constructed by 561 overlapping genes and 8863 overlapping edges among seven networks was identified and it provides a common gene module among seven stress-related diseases/systems. This module is significantly overlapped with network that constructed by genes from the CS gene database. 36 genes with high connectivity (hub genes) were identified from seven networks as potential key genes in those diseases/systems, 33 of hub genes were included in the common module. Genes in the common module were enriched in 190 interactive gene ontology (GO) functional clusters which provide potential disease mechanism. In conclusion, by analyzing gene networks we revealed a stress-affected common gene module among seven stress-related diseases/systems which provides insight into the process of stress induction of disease and suggests potential gene and pathway candidates for further research. PMID:26245528

  10. First applications of a targeted exome sequencing approach in fetuses with ultrasound abnormalities reveals an important fraction of cases with associated gene defects

    PubMed Central

    Pangalos, Constantinos; Hagnefelt, Birgitta; Lilakos, Konstantinos

    2016-01-01

    Background. Fetal malformations and other structural abnormalities are relatively frequent findings in the course of routine prenatal ultrasonographic examination. Due to their considerable genetic and clinical heterogeneity, the underlying genetic cause is often elusive and the resulting inability to provide a precise diagnosis precludes proper reproductive and fetal risk assessment. We report the development and first applications of an expanded exome sequencing-based test, coupled to a bioinformatics-driven prioritization algorithm, targeting gene disorders presenting with abnormal prenatal ultrasound findings. Methods. We applied the testing strategy to14 euploid fetuses, from 11 on-going pregnancies and three products of abortion, all with various abnormalities or malformations detected through prenatal ultrasound examination. Whole exome sequencing (WES) was followed by variant prioritization, utilizing a custom analysis pipeline (Fetalis algorithm), targeting 758 genes associated with genetic disorders which may present with abnormal fetal ultrasound findings. Results. A definitive or highly-likely diagnosis was made in 6 of 14 cases (43%), of which 3 were abortuses (Ellis-van Creveld syndrome, Ehlers-Danlos syndrome and Nemaline myopathy 2) and 3 involved on-going pregnancies (Citrullinemia, Noonan syndrome, PROKR2-related Kallmann syndrome). In the remaining eight on-going pregnancy cases (57%), a ZIC1 variant of unknown clinical significance was detected in one case, while in seven cases testing did not reveal any pathogenic variant(s). Pregnancies were followed-up to birth, resulting in one neonate harboring the PROKR2 mutation, presenting with isolated minor structural cardiac abnormalities, and in seven apparently healthy neonates. Discussion. The expanded targeted exome sequencing-based approach described herein (Fetalis), provides strong evidence suggesting a definite and beneficial increase in our diagnostic capabilities in prenatal diagnosis of

  11. First applications of a targeted exome sequencing approach in fetuses with ultrasound abnormalities reveals an important fraction of cases with associated gene defects.

    PubMed

    Pangalos, Constantinos; Hagnefelt, Birgitta; Lilakos, Konstantinos; Konialis, Christopher

    2016-01-01

    Background. Fetal malformations and other structural abnormalities are relatively frequent findings in the course of routine prenatal ultrasonographic examination. Due to their considerable genetic and clinical heterogeneity, the underlying genetic cause is often elusive and the resulting inability to provide a precise diagnosis precludes proper reproductive and fetal risk assessment. We report the development and first applications of an expanded exome sequencing-based test, coupled to a bioinformatics-driven prioritization algorithm, targeting gene disorders presenting with abnormal prenatal ultrasound findings. Methods. We applied the testing strategy to14 euploid fetuses, from 11 on-going pregnancies and three products of abortion, all with various abnormalities or malformations detected through prenatal ultrasound examination. Whole exome sequencing (WES) was followed by variant prioritization, utilizing a custom analysis pipeline (Fetalis algorithm), targeting 758 genes associated with genetic disorders which may present with abnormal fetal ultrasound findings. Results. A definitive or highly-likely diagnosis was made in 6 of 14 cases (43%), of which 3 were abortuses (Ellis-van Creveld syndrome, Ehlers-Danlos syndrome and Nemaline myopathy 2) and 3 involved on-going pregnancies (Citrullinemia, Noonan syndrome, PROKR2-related Kallmann syndrome). In the remaining eight on-going pregnancy cases (57%), a ZIC1 variant of unknown clinical significance was detected in one case, while in seven cases testing did not reveal any pathogenic variant(s). Pregnancies were followed-up to birth, resulting in one neonate harboring the PROKR2 mutation, presenting with isolated minor structural cardiac abnormalities, and in seven apparently healthy neonates. Discussion. The expanded targeted exome sequencing-based approach described herein (Fetalis), provides strong evidence suggesting a definite and beneficial increase in our diagnostic capabilities in prenatal diagnosis of

  12. Receptor-Targeted Nipah Virus Glycoproteins Improve Cell-Type Selective Gene Delivery and Reveal a Preference for Membrane-Proximal Cell Attachment

    PubMed Central

    Bender, Ruben R.; Muth, Anke; Schneider, Irene C.; Friedel, Thorsten; Hartmann, Jessica; Plückthun, Andreas; Maisner, Andrea; Buchholz, Christian J.

    2016-01-01

    Receptor-targeted lentiviral vectors (LVs) can be an effective tool for selective transfer of genes into distinct cell types of choice. Moreover, they can be used to determine the molecular properties that cell surface proteins must fulfill to act as receptors for viral glycoproteins. Here we show that LVs pseudotyped with receptor-targeted Nipah virus (NiV) glycoproteins effectively enter into cells when they use cell surface proteins as receptors that bring them closely enough to the cell membrane (less than 100 Å distance). Then, they were flexible in receptor usage as demonstrated by successful targeting of EpCAM, CD20, and CD8, and as selective as LVs pseudotyped with receptor-targeted measles virus (MV) glycoproteins, the current standard for cell-type specific gene delivery. Remarkably, NiV-LVs could be produced at up to two orders of magnitude higher titers compared to their MV-based counterparts and were at least 10,000-fold less effectively neutralized than MV glycoprotein pseudotyped LVs by pooled human intravenous immunoglobulin. An important finding for NiV-LVs targeted to Her2/neu was an about 100-fold higher gene transfer activity when particles were targeted to membrane-proximal regions as compared to particles binding to a more membrane-distal epitope. Likewise, the low gene transfer activity mediated by NiV-LV particles bound to the membrane distal domains of CD117 or the glutamate receptor subunit 4 (GluA4) was substantially enhanced by reducing receptor size to below 100 Å. Overall, the data suggest that the NiV glycoproteins are optimally suited for cell-type specific gene delivery with LVs and, in addition, for the first time define which parts of a cell surface protein should be targeted to achieve optimal gene transfer rates with receptor-targeted LVs. PMID:27281338

  13. Receptor-Targeted Nipah Virus Glycoproteins Improve Cell-Type Selective Gene Delivery and Reveal a Preference for Membrane-Proximal Cell Attachment.

    PubMed

    Bender, Ruben R; Muth, Anke; Schneider, Irene C; Friedel, Thorsten; Hartmann, Jessica; Plückthun, Andreas; Maisner, Andrea; Buchholz, Christian J

    2016-06-01

    Receptor-targeted lentiviral vectors (LVs) can be an effective tool for selective transfer of genes into distinct cell types of choice. Moreover, they can be used to determine the molecular properties that cell surface proteins must fulfill to act as receptors for viral glycoproteins. Here we show that LVs pseudotyped with receptor-targeted Nipah virus (NiV) glycoproteins effectively enter into cells when they use cell surface proteins as receptors that bring them closely enough to the cell membrane (less than 100 Å distance). Then, they were flexible in receptor usage as demonstrated by successful targeting of EpCAM, CD20, and CD8, and as selective as LVs pseudotyped with receptor-targeted measles virus (MV) glycoproteins, the current standard for cell-type specific gene delivery. Remarkably, NiV-LVs could be produced at up to two orders of magnitude higher titers compared to their MV-based counterparts and were at least 10,000-fold less effectively neutralized than MV glycoprotein pseudotyped LVs by pooled human intravenous immunoglobulin. An important finding for NiV-LVs targeted to Her2/neu was an about 100-fold higher gene transfer activity when particles were targeted to membrane-proximal regions as compared to particles binding to a more membrane-distal epitope. Likewise, the low gene transfer activity mediated by NiV-LV particles bound to the membrane distal domains of CD117 or the glutamate receptor subunit 4 (GluA4) was substantially enhanced by reducing receptor size to below 100 Å. Overall, the data suggest that the NiV glycoproteins are optimally suited for cell-type specific gene delivery with LVs and, in addition, for the first time define which parts of a cell surface protein should be targeted to achieve optimal gene transfer rates with receptor-targeted LVs. PMID:27281338

  14. Gene targeting with retroviral vectors

    SciTech Connect

    Ellis, J.; Bernstein, A. )

    1989-04-01

    The authors have designed and constructed integration-defective retroviral vectors to explore their potential for gene targeting in mammalian cells. Two nonoverlapping deletion mutants of the bacterial neomycin resistance (neo) gene were used to detect homologous recombination events between viral and chromosomal sequences. Stable neo gene correction events were selected at a frequency of approximately 1 G418/sup r/ cell per 3 x 10/sup 6/ infected cells. Analysis of the functional neo gene in independent targeted cell clones indicated that unintegrated retroviral linear DNA recombined with the target by gene conversion for variable distances into regions of nonhomology. In addition, transient neo gene correction events which were associated with the complete loss of the chromosomal target sequences were observed. These results demonstrated that retroviral vectors can recombine with homologous chromosomal sequences in rodent and human cells.

  15. Genome-wide CNV analysis in 221 unrelated patients and targeted high-throughput sequencing reveal novel causative candidate genes for colorectal adenomatous polyposis.

    PubMed

    Horpaopan, Sukanya; Spier, Isabel; Zink, Alexander M; Altmüller, Janine; Holzapfel, Stefanie; Laner, Andreas; Vogt, Stefanie; Uhlhaas, Siegfried; Heilmann, Stefanie; Stienen, Dietlinde; Pasternack, Sandra M; Keppler, Kathleen; Adam, Ronja; Kayser, Katrin; Moebus, Susanne; Draaken, Markus; Degenhardt, Franziska; Engels, Hartmut; Hofmann, Andrea; Nöthen, Markus M; Steinke, Verena; Perez-Bouza, Alberto; Herms, Stefan; Holinski-Feder, Elke; Fröhlich, Holger; Thiele, Holger; Hoffmann, Per; Aretz, Stefan

    2015-03-15

    To uncover novel causative genes in patients with unexplained adenomatous polyposis, a model disease for colorectal cancer, we performed a genome-wide analysis of germline copy number variants (CNV) in a large, well characterized APC and MUTYH mutation negative patient cohort followed by a targeted next generation sequencing (NGS) approach. Genomic DNA from 221 unrelated German patients was genotyped on high-resolution SNP arrays. Putative CNVs were filtered according to stringent criteria, compared with those of 531 population-based German controls, and validated by qPCR. Candidate genes were prioritized using in silico, expression, and segregation analyses, data mining and enrichment analyses of genes and pathways. In 27% of the 221 unrelated patients, a total of 77 protein coding genes displayed rare, nonrecurrent, germline CNVs. The set included 26 candidates with molecular and cellular functions related to tumorigenesis. Targeted high-throughput sequencing found truncating point mutations in 12% (10/77) of the prioritized genes. No clear evidence was found for autosomal recessive subtypes. Six patients had potentially causative mutations in more than one of the 26 genes. Combined with data from recent studies of early-onset colorectal and breast cancer, recurrent potential loss-of-function alterations were detected in CNTN6, FOCAD (KIAA1797), HSPH1, KIF26B, MCM3AP, YBEY and in three genes from the ARHGAP family. In the canonical Wnt pathway oncogene CTNNB1 (β-catenin), two potential gain-of-function mutations were found. In conclusion, the present study identified a group of rarely affected genes which are likely to predispose to colorectal adenoma formation and confirmed previously published candidates for tumor predisposition as etiologically relevant. PMID:25219767

  16. Comparative analysis of the Dicer-like gene family reveals loss of miR162 target site in SmDCL1 from Salvia miltiorrhiza

    PubMed Central

    Shao, Fenjuan; Qiu, Deyou; Lu, Shanfa

    2015-01-01

    DCL1, the core component for miRNA biogenesis, is itself regulated by miR162 in Arabidopsis. MiRNA-mediated feedback regulation of AtDCL1 is important to maintain the proper level of DCL1 transcripts. However, it is unknown whether the miRNA-mediated regulation of DCL1 is conserved among plants. We analyzed the SmDCL gene family in Salvia miltiorrhiza, an emerging model plant for Traditional Chinese Medicine (TCM) studies, using a comprehensive approach integrating genome-wide prediction, molecular cloning, gene expression profiling, and posttranscriptional regulation analysis. A total of five SmDCLs were identified. Comparative analysis of SmDCLs and AtDCLs showed an apparent enlargement of SmDCL introns in S. miltiorrhiza. The absence of miR162 in S. miltiorrhiza and the loss of miR162 target site in SmDCL1 were unexpectedly found. Further analysis showed that the miR162 target site was not present in DCL1 from ancient plants and was gained during plant evolution. The gained miR162 target site might be lost in a few modern plants through nucleotide mutations. Our results provide evidence for the gain and loss of miR162 and its target sites in Dicer-like genes during evolution. The data is useful for understanding the evolution of miRNA-mediated feedback regulation of DCLs in plants. PMID:25970825

  17. Comparative analysis of the Dicer-like gene family reveals loss of miR162 target site in SmDCL1 from Salvia miltiorrhiza.

    PubMed

    Shao, Fenjuan; Qiu, Deyou; Lu, Shanfa

    2015-01-01

    DCL1, the core component for miRNA biogenesis, is itself regulated by miR162 in Arabidopsis. MiRNA-mediated feedback regulation of AtDCL1 is important to maintain the proper level of DCL1 transcripts. However, it is unknown whether the miRNA-mediated regulation of DCL1 is conserved among plants. We analyzed the SmDCL gene family in Salvia miltiorrhiza, an emerging model plant for Traditional Chinese Medicine (TCM) studies, using a comprehensive approach integrating genome-wide prediction, molecular cloning, gene expression profiling, and posttranscriptional regulation analysis. A total of five SmDCLs were identified. Comparative analysis of SmDCLs and AtDCLs showed an apparent enlargement of SmDCL introns in S. miltiorrhiza. The absence of miR162 in S. miltiorrhiza and the loss of miR162 target site in SmDCL1 were unexpectedly found. Further analysis showed that the miR162 target site was not present in DCL1 from ancient plants and was gained during plant evolution. The gained miR162 target site might be lost in a few modern plants through nucleotide mutations. Our results provide evidence for the gain and loss of miR162 and its target sites in Dicer-like genes during evolution. The data is useful for understanding the evolution of miRNA-mediated feedback regulation of DCLs in plants. PMID:25970825

  18. Mutational analysis of genes coding for cell surface proteins in colorectal cancer cell lines reveal novel altered pathways, druggable mutations and mutated epitopes for targeted therapy

    PubMed Central

    Correa, Bruna R.; Bettoni, Fabiana; Koyama, Fernanda C.; Navarro, Fabio C.P.; Perez, Rodrigo O.; Mariadason, John; Sieber, Oliver M.; Strausberg, Robert L.; Simpson, Andrew J.G.; Jardim, Denis L.F.; Reis, Luiz Fernando L.; Parmigiani, Raphael B.; Galante, Pedro A.F.; Camargo, Anamaria A.

    2014-01-01

    We carried out a mutational analysis of 3,594 genes coding for cell surface proteins (Surfaceome) in 23 colorectal cancer cell lines, searching for new altered pathways, druggable mutations and mutated epitopes for targeted therapy in colorectal cancer. A total of 3,944 somatic non-synonymous substitutions and 595 InDels, occurring in 2,061 (57%) Surfaceome genes were catalogued. We identified 48 genes not previously described as mutated in colorectal tumors in the TCGA database, including genes that are mutated and expressed in >10% of the cell lines (SEMA4C, FGFRL1, PKD1, FAM38A, WDR81, TMEM136, SLC36A1, SLC26A6, IGFLR1). Analysis of these genes uncovered important roles for FGF and SEMA4 signaling in colorectal cancer with possible therapeutic implications. We also found that cell lines express on average 11 druggable mutations, including frequent mutations (>20%) in the receptor tyrosine kinases AXL and EPHA2, which have not been previously considered as potential targets for colorectal cancer. Finally, we identified 82 cell surface mutated epitopes, however expression of only 30% of these epitopes was detected in our cell lines. Notwithstanding, 92% of these epitopes were expressed in cell lines with the mutator phenotype, opening new venues for the use of “general” immune checkpoint drugs in this subset of patients. PMID:25193853

  19. Visualized Gene Network Reveals the Novel Target Transcripts Sox2 and Pax6 of Neuronal Development in Trans-Placental Exposure to Bisphenol A

    PubMed Central

    Yang, Chung-Wei; Chou, Wei-Chun; Chen, Kuan-Hsueh; Cheng, An-Lin; Mao, I-Fang; Chao, How-Ran; Chuang, Chun-Yu

    2014-01-01

    Background Bisphenol A (BPA) is a ubiquitous endocrine disrupting chemical in our daily life, and its health effect in response to prenatal exposure is still controversial. Early-life BPA exposure may impact brain development and contribute to childhood neurological disorders. The aim of the present study was to investigate molecular target genes of neuronal development in trans-placental exposure to BPA. Methodology A meta-analysis of three public microarray datasets was performed to screen for differentially expressed genes (DEGs) in exposure to BPA. The candidate genes of neuronal development were identified from gene ontology analysis in a reconstructed neuronal sub-network, and their gene expressions were determined using real-time PCR in 20 umbilical cord blood samples dichotomized into high and low BPA level groups upon the median 16.8 nM. Principal Findings Among 36 neuronal transcripts sorted from DAVID ontology clusters of 457 DEGs using the analysis of Bioconductor limma package, we found two neuronal genes, sex determining region Y-box 2 (Sox2) and paired box 6 (Pax6), had preferentially down-regulated expression (Bonferroni correction p-value <10−4 and log2-transformed fold change ≤−1.2) in response to BPA exposure. Fetal cord blood samples had the obviously attenuated gene expression of Sox2 and Pax6 in high BPA group referred to low BPA group. Visualized gene network of Cytoscape analysis showed that Sox2 and Pax6 which were contributed to neural precursor cell proliferation and neuronal differentiation might be down-regulated through sonic hedgehog (Shh), vascular endothelial growth factor A (VEGFA) and Notch signaling. Conclusions These results indicated that trans-placental BPA exposure down-regulated gene expression of Sox2 and Pax6 potentially underlying the adverse effect on childhood neuronal development. PMID:25051057

  20. Direct Comparison of a Natural Loss-Of-Function Single Nucleotide Polymorphism with a Targeted Deletion in the Ncf1 Gene Reveals Different Phenotypes

    PubMed Central

    Sareila, Outi; Hagert, Cecilia; Rantakari, Pia; Poutanen, Matti; Holmdahl, Rikard

    2015-01-01

    The genetic targeting of mouse models has given insight into complex processes. However, phenotypes of genetically targeted mice are susceptible to artifacts due to gene manipulation, which may lead to misinterpretation of the observations. To directly address these issues, we have compared the immunological phenotypes of Ncf1 knockout mice with Ncf1m1J mice possessing a naturally occurring intronic loss-of-function SNP in their Ncf1 gene. Neutrophil cytosolic factor 1 (NCF1) is the key regulatory component of the phagocytic NADPH oxidase 2 (NOX2) complex. Defects in NCF1 lead to lower production of reactive oxygen species (ROS) associated with autoimmune diseases in humans. In mice, collagen induced arthritis (CIA) and psoriatic arthritis are autoimmune disorders known to be regulated by Ncf1, and they were utilized in the present study to compare the Ncf1 knockout with Ncf1m1J mice. Targeted Ncf1 knockout mice were generated on a pure C57BL/6N genetic background, and thereafter crossed with B10.Q.Ncf1m1J mice. The targeting silenced the Ncf1 gene as intended, and both the B6N;B10.Q.Ncf1m1J mice as well as the knockout littermates had reduced ROS production compared to wild type mice. Both also exhibited enhanced STAT1 (signal transducer and activator of transcription 1) protein expression as an indicator of pronounced interferon signature reported recently for Ncf1 deficient mice. Surprisingly, female Ncf1 knockout mice were protected from CIA whereas the Ncf1m1J females developed severe disease. Ovariectomization retrieved the susceptibility of Ncf1 knockout females pointing to a sex hormone regulated protection against CIA in these mice. The data partly explains the discrepancy of the phenotypes reported earlier utilizing the Ncf1m1J mice or Ncf1 knockout mice. These observations indicate that even a targeted knockout mutation may lead to a different biological outcome in comparison to the natural loss-of-function mutation of the same gene. PMID:26528554

  1. Genetic Labeling Reveals Novel Cellular Targets of Schizophrenia Susceptibility Gene: Distribution of GABA and Non-GABA ErbB4-Positive Cells in Adult Mouse Brain

    PubMed Central

    Bean, Jonathan C.; Lin, Thiri W.; Sathyamurthy, Anupama; Liu, Fang; Yin, Dong-Min; Xiong, Wen-Cheng

    2014-01-01

    Neuregulin 1 (NRG1) and its receptor ErbB4 are schizophrenia risk genes. NRG1-ErbB4 signaling plays a critical role in neural development and regulates neurotransmission and synaptic plasticity. Nevertheless, its cellular targets remain controversial. ErbB4 was thought to express in excitatory neurons, although recent studies disputed this view. Using mice that express a fluorescent protein under the promoter of the ErbB4 gene, we determined in what cells ErbB4 is expressed and their identity. ErbB4 was widely expressed in the mouse brain, being highest in amygdala and cortex. Almost all ErbB4-positive cells were GABAergic in cortex, hippocampus, basal ganglia, and most of amygdala in neonatal and adult mice, suggesting GABAergic transmission as a major target of NRG1-ErbB4 signaling in these regions. Non-GABAergic, ErbB4-positive cells were present in thalamus, hypothalamus, midbrain, and hindbrain. In particular, ErbB4 is expressed in serotoninergic neurons of raphe nuclei but not in norepinephrinergic neurons of the locus ceruleus. In hypothalamus, ErbB4 is present in neurons that express oxytocin. Finally, ErbB4 is expressed in a group of cells in the subcortical areas that are positive for S100 calcium binding protein β. These results identify novel cellular targets of NRG1-ErbB4 signaling. PMID:25274830

  2. Global gene expression profiling in mouse plasma cell tumor precursor and bystander cells reveals potential intervention targets for plasma cell neoplasia.

    PubMed

    LeGrand, Jason; Park, Eun Sung; Wang, Hongyang; Gupta, Shalu; Owens, James D; Nelson, Patrick J; DuBois, Wendy; Bair, Thomas; Janz, Siegfried; Mushinski, J Frederic

    2012-01-26

    Tumor progression usually proceeds through several sequential stages, any of which could be targets for interrupting the progression process if one understood these steps at the molecular level. We extracted nascent plasma cell tumor (PCT) cells from within inflammatory oil granulomas (OG) isolated from IP pristane-injected BALB/c.iMyc(Eμ) mice at 5 different time points during tumor progression. We used laser capture microdissection to collect incipient PCT cells and analyzed their global gene expression on Affymetrix Mouse Genome 430A microarrays. Two independent studies were performed with different sets of mice. Analysis of the expression data used ANOVA and Bayesian estimation of temporal regulation. Genetic pathway analysis was performed using MetaCore (GeneGo) and IPA (Ingenuity). The gene expression profiles of PCT samples and those of undissected OG samples from adjacent sections showed that different genes and pathways were mobilized in the tumor cells during tumor progression, compared with their stroma. Our analysis implicated several genetic pathways in PCT progression, including biphasic (up- and then down-regulation) of the Spp1/osteopontin-dependent network and up-regulation of mRNA translation/protein synthesis. The latter led to a biologic validation study that showed that the AMPK-activating diabetes drug, metformin, was a potent specific PCT inhibitor in vitro. PMID:22147894

  3. Integrative analysis and expression profiling of secondary cell wall genes in C4 biofuel model Setaria italica reveals targets for lignocellulose bioengineering

    PubMed Central

    Muthamilarasan, Mehanathan; Khan, Yusuf; Jaishankar, Jananee; Shweta, Shweta; Lata, Charu; Prasad, Manoj

    2015-01-01

    Several underutilized grasses have excellent potential for use as bioenergy feedstock due to their lignocellulosic biomass. Genomic tools have enabled identification of lignocellulose biosynthesis genes in several sequenced plants. However, the non-availability of whole genome sequence of bioenergy grasses hinders the study on bioenergy genomics and their genomics-assisted crop improvement. Foxtail millet (Setaria italica L.; Si) is a model crop for studying systems biology of bioenergy grasses. In the present study, a systematic approach has been used for identification of gene families involved in cellulose (CesA/Csl), callose (Gsl) and monolignol biosynthesis (PAL, C4H, 4CL, HCT, C3H, CCoAOMT, F5H, COMT, CCR, CAD) and construction of physical map of foxtail millet. Sequence alignment and phylogenetic analysis of identified proteins showed that monolignol biosynthesis proteins were highly diverse, whereas CesA/Csl and Gsl proteins were homologous to rice and Arabidopsis. Comparative mapping of foxtail millet lignocellulose biosynthesis genes with other C4 panicoid genomes revealed maximum homology with switchgrass, followed by sorghum and maize. Expression profiling of candidate lignocellulose genes in response to different abiotic stresses and hormone treatments showed their differential expression pattern, with significant higher expression of SiGsl12, SiPAL2, SiHCT1, SiF5H2, and SiCAD6 genes. Further, due to the evolutionary conservation of grass genomes, the insights gained from the present study could be extrapolated for identifying genes involved in lignocellulose biosynthesis in other biofuel species for further characterization. PMID:26583030

  4. Identification of Hypoxia-Inducible Target Genes of Aspergillus fumigatus by Transcriptome Analysis Reveals Cellular Respiration as an Important Contributor to Hypoxic Survival

    PubMed Central

    Kroll, Kristin; Pähtz, Vera; Hillmann, Falk; Vaknin, Yakir; Schmidt-Heck, Wolfgang; Roth, Martin; Jacobsen, Ilse D.; Osherov, Nir; Brakhage, Axel A.

    2014-01-01

    Aspergillus fumigatus is an opportunistic, airborne pathogen that causes invasive aspergillosis in immunocompromised patients. During the infection process, A. fumigatus is challenged by hypoxic microenvironments occurring in inflammatory, necrotic tissue. To gain further insights into the adaptation mechanism, A. fumigatus was cultivated in an oxygen-controlled chemostat under hypoxic and normoxic conditions. Transcriptome analysis revealed a significant increase in transcripts associated with cell wall polysaccharide metabolism, amino acid and metal ion transport, nitrogen metabolism, and glycolysis. A concomitant reduction in transcript levels was observed with cellular trafficking and G-protein-coupled signaling. To learn more about the functional roles of hypoxia-induced transcripts, we deleted A. fumigatus genes putatively involved in reactive nitrogen species detoxification (fhpA), NAD+ regeneration (frdA and osmA), nitrogen metabolism (niaD and niiA), and respiration (rcfB). We show that the nitric oxygen (NO)-detoxifying flavohemoprotein gene fhpA is strongly induced by hypoxia independent of the nitrogen source but is dispensable for hypoxic survival. By deleting the nitrate reductase gene niaD, the nitrite reductase gene niiA, and the two fumarate reductase genes frdA and osmA, we found that alternative electron acceptors, such as nitrate and fumarate, do not have a significant impact on growth of A. fumigatus during hypoxia, but functional mitochondrial respiratory chain complexes are essential under these conditions. Inhibition studies indicated that primarily complexes III and IV play a crucial role in the hypoxic growth of A. fumigatus. PMID:25084861

  5. Genome-Wide Mapping Targets of the Metazoan Chromatin Remodeling Factor NURF Reveals Nucleosome Remodeling at Enhancers, Core Promoters and Gene Insulators

    PubMed Central

    Kwon, So Yeon; Grisan, Valentina; Jang, Boyun; Herbert, John; Badenhorst, Paul

    2016-01-01

    NURF is a conserved higher eukaryotic ISWI-containing chromatin remodeling complex that catalyzes ATP-dependent nucleosome sliding. By sliding nucleosomes, NURF is able to alter chromatin dynamics to control transcription and genome organization. Previous biochemical and genetic analysis of the specificity-subunit of Drosophila NURF (Nurf301/Enhancer of Bithorax (E(bx)) has defined NURF as a critical regulator of homeotic, heat-shock and steroid-responsive gene transcription. It has been speculated that NURF controls pathway specific transcription by co-operating with sequence-specific transcription factors to remodel chromatin at dedicated enhancers. However, conclusive in vivo demonstration of this is lacking and precise regulatory elements targeted by NURF are poorly defined. To address this, we have generated a comprehensive map of in vivo NURF activity, using MNase-sequencing to determine at base pair resolution NURF target nucleosomes, and ChIP-sequencing to define sites of NURF recruitment. Our data show that, besides anticipated roles at enhancers, NURF interacts physically and functionally with the TRF2/DREF basal transcription factor to organize nucleosomes downstream of active promoters. Moreover, we detect NURF remodeling and recruitment at distal insulator sites, where NURF functionally interacts with and co-localizes with DREF and insulator proteins including CP190 to establish nucleosome-depleted domains. This insulator function of NURF is most apparent at subclasses of insulators that mark the boundaries of chromatin domains, where multiple insulator proteins co-associate. By visualizing the complete repertoire of in vivo NURF chromatin targets, our data provide new insights into how chromatin remodeling can control genome organization and regulatory interactions. PMID:27046080

  6. Genome-Wide Mapping Targets of the Metazoan Chromatin Remodeling Factor NURF Reveals Nucleosome Remodeling at Enhancers, Core Promoters and Gene Insulators.

    PubMed

    Kwon, So Yeon; Grisan, Valentina; Jang, Boyun; Herbert, John; Badenhorst, Paul

    2016-04-01

    NURF is a conserved higher eukaryotic ISWI-containing chromatin remodeling complex that catalyzes ATP-dependent nucleosome sliding. By sliding nucleosomes, NURF is able to alter chromatin dynamics to control transcription and genome organization. Previous biochemical and genetic analysis of the specificity-subunit of Drosophila NURF (Nurf301/Enhancer of Bithorax (E(bx)) has defined NURF as a critical regulator of homeotic, heat-shock and steroid-responsive gene transcription. It has been speculated that NURF controls pathway specific transcription by co-operating with sequence-specific transcription factors to remodel chromatin at dedicated enhancers. However, conclusive in vivo demonstration of this is lacking and precise regulatory elements targeted by NURF are poorly defined. To address this, we have generated a comprehensive map of in vivo NURF activity, using MNase-sequencing to determine at base pair resolution NURF target nucleosomes, and ChIP-sequencing to define sites of NURF recruitment. Our data show that, besides anticipated roles at enhancers, NURF interacts physically and functionally with the TRF2/DREF basal transcription factor to organize nucleosomes downstream of active promoters. Moreover, we detect NURF remodeling and recruitment at distal insulator sites, where NURF functionally interacts with and co-localizes with DREF and insulator proteins including CP190 to establish nucleosome-depleted domains. This insulator function of NURF is most apparent at subclasses of insulators that mark the boundaries of chromatin domains, where multiple insulator proteins co-associate. By visualizing the complete repertoire of in vivo NURF chromatin targets, our data provide new insights into how chromatin remodeling can control genome organization and regulatory interactions. PMID:27046080

  7. Search for Basonuclin Target Genes

    PubMed Central

    Wang, Junwen; Zhang, Shengliang; Schultz, Richard M.; Tseng, Hung

    2006-01-01

    Basonuclin (Bnc 1) is a transcription factor that has an unusual ability to interact with promoters of both RNA polymerases I and II. The action of basonuclin is mediated through three pairs of evolutionarily conserved zinc fingers, which produce three DNase I footprints on the promoters of rDNA and the basonuclin gene. Using these DNase footprints, we built a computational model for the basonuclin DNA-binding module, which was used to identify in silico potential RNA polymerase II target genes in the human and mouse promoter databases. The target genes of basonuclin show that it regulates the expression of proteins involved in chromatin structure, transcription/DNA-binding, ion-channels, adhesion/cell-cell junction, signal transduction and intracellular transport. Our results suggest that basonuclin, like MYC, may coordinate transcriptional activities among the three RNA polymerases. But basonuclin regulates a distinctive set of pathways, which differ from that regulated by MYC. PMID:16919236

  8. Role of group V phospholipase A2 in zymosan-induced eicosanoid generation and vascular permeability revealed by targeted gene disruption*

    PubMed Central

    Satake, Yoshiyuki; Diaz, Bruno L.; Balestrieri, Barbara; Lam, Bing K.; Kanaoka, Yoshihide; Grusby, Michael J.; Arm, Jonathan P.

    2005-01-01

    SUMMARY Conclusions regarding the contribution of low molecular weight secretory phospholipase A2 (sPLA2) enzymes in eicosanoid generation have relied on data obtained from transfected cells or the use of inhibitors that fail to discriminate between individual members of the large family of mammalian sPLA2 enzymes. To elucidate the role of group V sPLA2, we used targeted gene disruption to generate mice lacking this enzyme. Zymosan-induced generation of leukotriene C4 and prostaglandin E2 was attenuated ~50% in peritoneal macrophages from group V sPLA2-null mice compared to macrophages from wild-type littermates. Furthermore, the early phase of plasma exudation in response to intraperitoneal injection of zymosan and the accompanying in vivo generation of cysteinyl leukotrienes were markedly attenuated in group V sPLA2-null mice compared to wild-type controls. These data provide clear evidence of a role for group V sPLA2 in regulating eicosanoid generation in response to an acute innate stimulus of the immune response both in vitro and in vivo, suggesting a role for this enzyme in innate immunity. PMID:14761945

  9. Genome-wide profiling of histone H3 lysine 27 and lysine 4 trimethylation in multiple myeloma reveals the importance of Polycomb gene targeting and highlights EZH2 as a potential therapeutic target

    PubMed Central

    Párraga, Alba Atienza; Enroth, Stefan; Singh, Umashankar; Ungerstedt, Johanna; Österborg, Anders; Brown, Peter J.; Ma, Anqi; Jin, Jian; Nilsson, Kenneth; Öberg, Fredrik; Kalushkova, Antonia; Jernberg-Wiklund, Helena

    2016-01-01

    Multiple myeloma (MM) is a malignancy of the antibody-producing plasma cells. MM is a highly heterogeneous disease, which has hampered the identification of a common underlying mechanism for disease establishment as well as the development of targeted therapy. Here we present the first genome-wide profiling of histone H3 lysine 27 and lysine 4 trimethylation in MM patient samples, defining a common set of active H3K4me3-enriched genes and silent genes marked by H3K27me3 (H3K27me3 alone or bivalent) unique to primary MM cells, when compared to normal bone marrow plasma cells. Using this epigenome profile, we found increased silencing of H3K27me3 targets in MM patients at advanced stages of the disease, and the expression pattern of H3K27me3-marked genes correlated with poor patient survival. We also demonstrated that pharmacological inhibition of EZH2 had anti-myeloma effects in both MM cell lines and CD138+ MM patient cells. In addition, EZH2 inhibition decreased the global H3K27 methylation and induced apoptosis. Taken together, these data suggest an important role for the Polycomb repressive complex 2 (PRC2) in MM, and highlights the PRC2 component EZH2 as a potential therapeutic target in MM. PMID:26755663

  10. Genome-Wide Investigation Using sRNA-Seq, Degradome-Seq and Transcriptome-Seq Reveals Regulatory Networks of microRNAs and Their Target Genes in Soybean during Soybean mosaic virus Infection

    PubMed Central

    Yu, Kangfu; Wang, Aiming

    2016-01-01

    MicroRNAs (miRNAs) play key roles in a variety of cellular processes through regulation of their target gene expression. Accumulated experimental evidence has demonstrated that infections by viruses are associated with the altered expression profile of miRNAs and their mRNA targets in the host. However, the regulatory network of miRNA-mRNA interactions during viral infection remains largely unknown. In this study, we performed small RNA (sRNA)-seq, degradome-seq and as well as a genome-wide transcriptome analysis to profile the global gene and miRNA expression in soybean following infections by three different Soybean mosaic virus (SMV) isolates, L (G2 strain), LRB (G2 strain) and G7 (G7 strain). sRNA-seq analyses revealed a total of 253 soybean miRNAs with a two-fold or greater change in abundance compared with the mock-inoculated control. 125 transcripts were identified as the potential cleavage targets of 105 miRNAs and validated by degradome-seq analyses. Genome-wide transcriptome analysis showed that total 2679 genes are differentially expressed in response to SMV infection including 71 genes predicted as involved in defense response. Finally, complex miRNA-mRNA regulatory networks were derived using the RNAseq, small RNAseq and degradome data. This work represents a comprehensive, global approach to examining virus-host interactions. Genes responsive to SMV infection are identified as are their potential miRNA regulators. Additionally, regulatory changes of the miRNAs themselves are described and the regulatory relationships were supported with degradome data. Taken together these data provide new insights into molecular SMV-soybean interactions and offer candidate miRNAs and their targets for further elucidation of the SMV infection process. PMID:26963095

  11. Targeted gene flow for conservation.

    PubMed

    Kelly, Ella; Phillips, Ben L

    2016-04-01

    Anthropogenic threats often impose strong selection on affected populations, causing rapid evolutionary responses. Unfortunately, these adaptive responses are rarely harnessed for conservation. We suggest that conservation managers pay close attention to adaptive processes and geographic variation, with an eye to using them for conservation goals. Translocating pre-adapted individuals into recipient populations is currently considered a potentially important management tool in the face of climate change. Targeted gene flow, which involves moving individuals with favorable traits to areas where these traits would have a conservation benefit, could have a much broader application in conservation. Across a species' range there may be long-standing geographic variation in traits or variation may have rapidly developed in response to a threatening process. Targeted gene flow could be used to promote natural resistance to threats to increase species resilience. We suggest that targeted gene flow is a currently underappreciated strategy in conservation that has applications ranging from the management of invasive species and their impacts to controlling the impact and virulence of pathogens. PMID:26332195

  12. Targeted ablation of the Pde6h gene in mice reveals cross-species differences in cone and rod phototransduction protein isoform inventory.

    PubMed

    Brennenstuhl, Christina; Tanimoto, Naoyuki; Burkard, Markus; Wagner, Rebecca; Bolz, Sylvia; Trifunovic, Dragana; Kabagema-Bilan, Clement; Paquet-Durand, Francois; Beck, Susanne C; Huber, Gesine; Seeliger, Mathias W; Ruth, Peter; Wissinger, Bernd; Lukowski, Robert

    2015-04-17

    Phosphodiesterase-6 (PDE6) is a multisubunit enzyme that plays a key role in the visual transduction cascade in rod and cone photoreceptors. Each type of photoreceptor utilizes discrete catalytic and inhibitory PDE6 subunits to fulfill its physiological tasks, i.e. the degradation of cyclic guanosine-3',5'-monophosphate at specifically tuned rates and kinetics. Recently, the human PDE6H gene was identified as a novel locus for autosomal recessive (incomplete) color blindness. However, the three different classes of cones were not affected to the same extent. Short wave cone function was more preserved than middle and long wave cone function indicating that some basic regulation of the PDE6 multisubunit enzyme was maintained albeit by a unknown mechanism. To study normal and disease-related functions of cone Pde6h in vivo, we generated Pde6h knock-out (Pde6h(-/-)) mice. Expression of PDE6H in murine eyes was restricted to both outer segments and synaptic terminals of short and long/middle cone photoreceptors, whereas Pde6h(-/-) retinae remained PDE6H-negative. Combined in vivo assessment of retinal morphology with histomorphological analyses revealed a normal overall integrity of the retinal organization and an unaltered distribution of the different cone photoreceptor subtypes upon Pde6h ablation. In contrast to human patients, our electroretinographic examinations of Pde6h(-/-) mice suggest no defects in cone/rod-driven retinal signaling and therefore preserved visual functions. To this end, we were able to demonstrate the presence of rod PDE6G in cones indicating functional substitution of PDE6. The disparities between human and murine phenotypes caused by mutant Pde6h/PDE6H suggest species-to-species differences in the vulnerability of biochemical and neurosensory pathways of the visual signal transduction system. PMID:25739440

  13. Targeted deletion of the genes encoding NTH1 and NEIL1 DNA N-glycosylases reveals the existence of novel carcinogenic oxidative damage to DNA☆

    PubMed Central

    Chan, Michael K.; Ocampo-Hafalla, Maria T.; Vartanian, Vladimir; Jaruga, Pawel; Kirkali, Güldal; Koenig, Karen L.; Brown, Stuart; Lloyd, R. Stephen; Dizdaroglu, Miral; Teebor, George W.

    2016-01-01

    We have generated a strain of mice lacking two DNA N-glycosylases of base excision repair (BER), NTH1 and NEIL1, homologs of bacterial Nth (endonuclease three) and Nei (endonuclease eight). Although these enzymes remove several oxidized bases from DNA, they do not remove the well-known carcinogenic oxidation product of guanine: 7,8-dihydro-8-oxoguanine (8-OH-Gua), which is removed by another DNA N-glycosylase, OGG1. The Nth1−/−Neil1−/− mice developed pulmonary and hepatocellular tumors in much higher incidence than either of the single knockouts, Nth1−/− and Neil1−/−. The pulmonary tumors contained, exclusively, activating GGT→GAT transitions in codon 12 of K-ras of their DNA. Such transitions contrast sharply with the activating GGT→GTT transversions in codon 12 of K-ras of the pathologically similar pulmonary tumors, which arose in mice lacking OGG1 and a second DNA N-glycosylase, MUTY. To characterize the biochemical phenotype of the knockout mice, the content of oxidative DNA base damage was analyzed from three tissues isolated from control, single and double knockout mice. The content of 8-OH-Gua was indistinguishable among all genotypes. In contrast, the content of 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) derived from adenine and guanine, respectively, were increased in some but not all tissues of Neil1−/− and Neil1−/−Nth1−/− mice. The high incidence of tumors in our Nth1−/−Neil1−/− mice together with the nature of the activating mutation in the K-ras gene of their pulmonary tumors, reveal for the first time, the existence of mutagenic and carcinogenic oxidative damage to DNA which is not 8-OH-Gua. PMID:19346169

  14. Targeted Ablation of the Pde6h Gene in Mice Reveals Cross-species Differences in Cone and Rod Phototransduction Protein Isoform Inventory*

    PubMed Central

    Brennenstuhl, Christina; Tanimoto, Naoyuki; Burkard, Markus; Wagner, Rebecca; Bolz, Sylvia; Trifunovic, Dragana; Kabagema-Bilan, Clement; Paquet-Durand, Francois; Beck, Susanne C.; Huber, Gesine; Seeliger, Mathias W.; Ruth, Peter; Wissinger, Bernd; Lukowski, Robert

    2015-01-01

    Phosphodiesterase-6 (PDE6) is a multisubunit enzyme that plays a key role in the visual transduction cascade in rod and cone photoreceptors. Each type of photoreceptor utilizes discrete catalytic and inhibitory PDE6 subunits to fulfill its physiological tasks, i.e. the degradation of cyclic guanosine-3′,5′-monophosphate at specifically tuned rates and kinetics. Recently, the human PDE6H gene was identified as a novel locus for autosomal recessive (incomplete) color blindness. However, the three different classes of cones were not affected to the same extent. Short wave cone function was more preserved than middle and long wave cone function indicating that some basic regulation of the PDE6 multisubunit enzyme was maintained albeit by a unknown mechanism. To study normal and disease-related functions of cone Pde6h in vivo, we generated Pde6h knock-out (Pde6h−/−) mice. Expression of PDE6H in murine eyes was restricted to both outer segments and synaptic terminals of short and long/middle cone photoreceptors, whereas Pde6h−/− retinae remained PDE6H-negative. Combined in vivo assessment of retinal morphology with histomorphological analyses revealed a normal overall integrity of the retinal organization and an unaltered distribution of the different cone photoreceptor subtypes upon Pde6h ablation. In contrast to human patients, our electroretinographic examinations of Pde6h−/− mice suggest no defects in cone/rod-driven retinal signaling and therefore preserved visual functions. To this end, we were able to demonstrate the presence of rod PDE6G in cones indicating functional substitution of PDE6. The disparities between human and murine phenotypes caused by mutant Pde6h/PDE6H suggest species-to-species differences in the vulnerability of biochemical and neurosensory pathways of the visual signal transduction system. PMID:25739440

  15. Comparison of the gene expression profiles from normal and Fgfrl1 deficient mouse kidneys reveals downstream targets of Fgfrl1 signaling.

    PubMed

    Gerber, Simon D; Amann, Ruth; Wyder, Stefan; Trueb, Beat

    2012-01-01

    Fgfrl1 (fibroblast growth factor receptor-like 1) is a transmembrane receptor that is essential for the development of the metanephric kidney. It is expressed in all nascent nephrogenic structures and in the ureteric bud. Fgfrl1 null mice fail to develop the metanephric kidneys. Mutant kidney rudiments show a dramatic reduction of ureteric branching and a lack of mesenchymal-to-epithelial transition. Here, we compared the expression profiles of wildtype and Fgfrl1 mutant kidneys to identify genes that act downstream of Fgfrl1 signaling during the early steps of nephron formation. We detected 56 differentially expressed transcripts with 2-fold or greater reduction, among them many genes involved in Fgf, Wnt, Bmp, Notch, and Six/Eya/Dach signaling. We validated the microarray data by qPCR and whole-mount in situ hybridization and showed the expression pattern of candidate genes in normal kidneys. Some of these genes might play an important role during early nephron formation. Our study should help to define the minimal set of genes that is required to form a functional nephron. PMID:22432025

  16. Comparison of the Gene Expression Profiles from Normal and Fgfrl1 Deficient Mouse Kidneys Reveals Downstream Targets of Fgfrl1 Signaling

    PubMed Central

    Gerber, Simon D.; Amann, Ruth; Wyder, Stefan; Trueb, Beat

    2012-01-01

    Fgfrl1 (fibroblast growth factor receptor-like 1) is a transmembrane receptor that is essential for the development of the metanephric kidney. It is expressed in all nascent nephrogenic structures and in the ureteric bud. Fgfrl1 null mice fail to develop the metanephric kidneys. Mutant kidney rudiments show a dramatic reduction of ureteric branching and a lack of mesenchymal-to-epithelial transition. Here, we compared the expression profiles of wildtype and Fgfrl1 mutant kidneys to identify genes that act downstream of Fgfrl1 signaling during the early steps of nephron formation. We detected 56 differentially expressed transcripts with 2-fold or greater reduction, among them many genes involved in Fgf, Wnt, Bmp, Notch, and Six/Eya/Dach signaling. We validated the microarray data by qPCR and whole-mount in situ hybridization and showed the expression pattern of candidate genes in normal kidneys. Some of these genes might play an important role during early nephron formation. Our study should help to define the minimal set of genes that is required to form a functional nephron. PMID:22432025

  17. Code-assisted discovery of TAL effector targets in bacterial leaf streak of rice reveals contrast with bacterial blight and a novel susceptibility gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transcription activator-like (TAL) effectors found in Xanthomonas spp. promote bacterial growth and plant susceptibility by binding specific DNA sequences or, effector-binding elements (EBEs), and inducing host gene expression. In this study, we have found substantially different transcriptional pro...

  18. Genome-Wide Search Reveals the Existence of a Limited Number of Thyroid Hormone Receptor Alpha Target Genes in Cerebellar Neurons

    PubMed Central

    Chatonnet, Fabrice; Guyot, Romain; Picou, Frédéric; Bondesson, Maria; Flamant, Frederic

    2012-01-01

    Thyroid hormone (T3) has a major influence on cerebellum post-natal development. The major phenotypic landmark of exposure to low levels of T3 during development (hypothyroidism) in the cerebellum is the retarded inward migration of the most numerous cell type, granular neurons. In order to identify the direct genetic regulation exerted by T3 on cerebellar neurons and their precursors, we used microarray RNA hybridization to perform a time course analysis of T3 induced gene expression in primary cultures of cerebellar neuronal cell. These experiments suggest that we identified a small set of genes which are directly regulated, both in vivo and in vitro, during cerebellum post-natal development. These modest changes suggest that T3 does not acts directly on granular neurons and mainly indirectly influences the cellular interactions taking place during development. PMID:22586439

  19. Target-specific identification and characterization of the putative gene cluster for brasilinolide biosynthesis revealing the mechanistic insights and combinatorial synthetic utility of 2-deoxy-l-fucose biosynthetic enzymes.

    PubMed

    Chiu, Hsien-Tai; Weng, Chien-Pao; Lin, Yu-Chin; Chen, Kuan-Hung

    2016-02-14

    Brasilinolides exhibiting potent immunosuppressive and antifungal activities with remarkably low toxicity are structurally characterized by an unusual modified 2-deoxy-l-fucose (2dF) attached to a type I polyketide (PK-I) macrolactone. From the pathogenic producer Nocardia terpenica (Nocardia brasiliensis IFM-0406), a 210 kb genomic fragment was identified by target-specific degenerate primers and subsequently sequenced, revealing a giant nbr gene cluster harboring genes (nbrCDEF) required for TDP-2dF biosynthesis and those for PK-I biosynthesis, modification and regulation. The results showed that the genetic and domain arrangements of nbr PK-I synthases agreed colinearly with the PK-I structures of brasilinolides. Subsequent heterologous expression of nbrCDEF in Escherichia coli accomplished in vitro reconstitution of TDP-2dF biosynthesis. The catalytic functions and mechanisms of NbrCDEF enzymes were further characterized by systematic mix-and-match experiments. The enzymes were revealed to display remarkable substrate and partner promiscuity, leading to the establishment of in vitro hybrid deoxysugar biosynthetic pathways throughout an in situ one-pot (iSOP) method. This study represents the first demonstration of TDP-2dF biosynthesis at the enzyme and molecular levels, and provides new hope for expanding the structural diversity of brasilinolides by combinatorial biosynthesis. PMID:26754528

  20. Oncogenicity of L-type amino-acid transporter 1 (LAT1) revealed by targeted gene disruption in chicken DT40 cells: LAT1 is a promising molecular target for human cancer therapy

    SciTech Connect

    Ohkawa, Mayumi; Ohno, Yoshiya; Masuko, Kazue; Takeuchi, Akiko; Suda, Kentaro; Kubo, Akihiro; Kawahara, Rieko; Okazaki, Shogo; Tanaka, Toshiyuki; Saya, Hideyuki; Seki, Masayuki; Enomoto, Takemi; Yagi, Hideki; Hashimoto, Yoshiyuki; Masuko, Takashi

    2011-03-25

    Highlights: {yields} We established LAT1 amino-acid transporter-disrupted DT40 cells. {yields} LAT1-disrupted cells showed slow growth and lost the oncogenicity. {yields} siRNA and mAb inhibited human tumor growth in vitro and in vivo. {yields} LAT1 is a promising target molecule for cancer therapy. -- Abstract: L-type amino-acid transporter 1 (LAT1) is the first identified light chain of CD98 molecule, disulfide-linked to a heavy chain of CD98. Following cDNA cloning of chicken full-length LAT1, we have constructed targeting vectors for the disruption of chicken LAT1 gene from genomic DNA of chicken LAT1 consisting of 5.4 kb. We established five homozygous LAT1-disrupted (LAT1{sup -/-}) cell clones, derived from a heterozygous LAT1{sup +/-} clone of DT40 chicken B cell line. Reactivity of anti-chicken CD98hc monoclonal antibody (mAb) with LAT1{sup -/-} DT40 cells was markedly decreased compared with that of wild-type DT40 cells. All LAT1{sup -/-} cells were deficient in L-type amino-acid transporting activity, although alternative-splice variant but not full-length mRNA of LAT1 was detected in these cells. LAT1{sup -/-} DT40 clones showed outstandingly slow growth in liquid culture and decreased colony-formation capacity in soft agar compared with wild-type DT40 cells. Cell-cycle analyses indicated that LAT1{sup -/-} DT40 clones have prolonged cell-cycle phases compared with wild-type or LAT1{sup +/-} DT40 cells. Knockdown of human LAT1 by small interfering RNAs resulted in marked in vitro cell-growth inhibition of human cancer cells, and in vivo tumor growth of HeLa cells in athymic mice was significantly inhibited by anti-human LAT1 mAb. All these results indicate essential roles of LAT1 in the cell proliferation and occurrence of malignant phenotypes and that LAT1 is a promising candidate as a molecular target of human cancer therapy.

  1. The drug target genes show higher evolutionary conservation than non-target genes

    PubMed Central

    Liu, Panpan; Luan, Meiwei; Zhu, Hongjie; Liu, Guiyou; Zhang, Mingming; Lv, Hongchao; Duan, Lian; Shang, Zhenwei; Li, Jin; Jiang, Yongshuai; Zhang, Ruijie

    2016-01-01

    Although evidence indicates that drug target genes share some common evolutionary features, there have been few studies analyzing evolutionary features of drug targets from an overall level. Therefore, we conducted an analysis which aimed to investigate the evolutionary characteristics of drug target genes. We compared the evolutionary conservation between human drug target genes and non-target genes by combining both the evolutionary features and network topological properties in human protein-protein interaction network. The evolution rate, conservation score and the percentage of orthologous genes of 21 species were included in our study. Meanwhile, four topological features including the average shortest path length, betweenness centrality, clustering coefficient and degree were considered for comparison analysis. Then we got four results as following: compared with non-drug target genes, 1) drug target genes had lower evolutionary rates; 2) drug target genes had higher conservation scores; 3) drug target genes had higher percentages of orthologous genes and 4) drug target genes had a tighter network structure including higher degrees, betweenness centrality, clustering coefficients and lower average shortest path lengths. These results demonstrate that drug target genes are more evolutionarily conserved than non-drug target genes. We hope that our study will provide valuable information for other researchers who are interested in evolutionary conservation of drug targets. PMID:26716901

  2. Targeted Gene Therapies: Tools, Applications, Optimization

    PubMed Central

    Humbert, Olivier; Davis, Luther; Maizels, Nancy

    2012-01-01

    Many devastating human diseases are caused by mutations in a single gene that prevent a somatic cell from carrying out its essential functions, or by genetic changes acquired as a result of infectious disease or in the course of cell transformation. Targeted gene therapies have emerged as potential strategies for treatment of such diseases. These therapies depend upon rare-cutting endonucleases to cleave at specific sites in or near disease genes. Targeted gene correction provides a template for homology-directed repair, enabling the cell's own repair pathways to erase the mutation and replace it with the correct sequence. Targeted gene disruption ablates the disease gene, disabling its function. Gene targeting can also promote other kinds of genome engineering, including mutation, insertion, or gene deletion. Targeted gene therapies present significant advantages compared to approaches to gene therapy that depend upon delivery of stably expressing transgenes. Recent progress has been fueled by advances in nuclease discovery and design, and by new strategies that maximize efficiency of targeting and minimize off-target damage. Future progress will build on deeper mechanistic understanding of critical factors and pathways. PMID:22530743

  3. Nemertean toxin genes revealed through transcriptome sequencing.

    PubMed

    Whelan, Nathan V; Kocot, Kevin M; Santos, Scott R; Halanych, Kenneth M

    2014-12-01

    Nemerteans are one of few animal groups that have evolved the ability to utilize toxins for both defense and subduing prey, but little is known about specific nemertean toxins. In particular, no study has identified specific toxin genes even though peptide toxins are known from some nemertean species. Information about toxin genes is needed to better understand evolution of toxins across animals and possibly provide novel targets for pharmaceutical and industrial applications. We sequenced and annotated transcriptomes of two free-living and one commensal nemertean and annotated an additional six publicly available nemertean transcriptomes to identify putative toxin genes. Approximately 63-74% of predicted open reading frames in each transcriptome were annotated with gene names, and all species had similar percentages of transcripts annotated with each higher-level GO term. Every nemertean analyzed possessed genes with high sequence similarities to known animal toxins including those from stonefish, cephalopods, and sea anemones. One toxin-like gene found in all nemerteans analyzed had high sequence similarity to Plancitoxin-1, a DNase II hepatotoxin that may function well at low pH, which suggests that the acidic body walls of some nemerteans could work to enhance the efficacy of protein toxins. The highest number of toxin-like genes found in any one species was seven and the lowest was three. The diversity of toxin-like nemertean genes found here is greater than previously documented, and these animals are likely an ideal system for exploring toxin evolution and industrial applications of toxins. PMID:25432940

  4. Nemertean Toxin Genes Revealed through Transcriptome Sequencing

    PubMed Central

    Whelan, Nathan V.; Kocot, Kevin M.; Santos, Scott R.; Halanych, Kenneth M.

    2014-01-01

    Nemerteans are one of few animal groups that have evolved the ability to utilize toxins for both defense and subduing prey, but little is known about specific nemertean toxins. In particular, no study has identified specific toxin genes even though peptide toxins are known from some nemertean species. Information about toxin genes is needed to better understand evolution of toxins across animals and possibly provide novel targets for pharmaceutical and industrial applications. We sequenced and annotated transcriptomes of two free-living and one commensal nemertean and annotated an additional six publicly available nemertean transcriptomes to identify putative toxin genes. Approximately 63–74% of predicted open reading frames in each transcriptome were annotated with gene names, and all species had similar percentages of transcripts annotated with each higher-level GO term. Every nemertean analyzed possessed genes with high sequence similarities to known animal toxins including those from stonefish, cephalopods, and sea anemones. One toxin-like gene found in all nemerteans analyzed had high sequence similarity to Plancitoxin-1, a DNase II hepatotoxin that may function well at low pH, which suggests that the acidic body walls of some nemerteans could work to enhance the efficacy of protein toxins. The highest number of toxin-like genes found in any one species was seven and the lowest was three. The diversity of toxin-like nemertean genes found here is greater than previously documented, and these animals are likely an ideal system for exploring toxin evolution and industrial applications of toxins. PMID:25432940

  5. Problem-Solving Test: Targeted Gene Disruption

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2008-01-01

    Mutational inactivation of a specific gene is the most powerful technique to analyze the biological function of the gene. This approach has been used for a long time in viruses, bacteria, yeast, and fruit fly, but looked quite hopeless in more complex organisms. Targeted inactivation of specific genes (also known as knock-out mutation) in mice is…

  6. Targeted polymeric nanoparticles for cancer gene therapy

    PubMed Central

    Kim, Jayoung; Wilson, David R.; Zamboni, Camila G.; Green, Jordan J.

    2015-01-01

    In this article, advances in designing polymeric nanoparticles for targeted cancer gene therapy are reviewed. Characterization and evaluation of biomaterials, targeting ligands, and transcriptional elements are each discussed. Advances in biomaterials have driven improvements to nanoparticle stability and tissue targeting, conjugation of ligands to the surface of polymeric nanoparticles enable binding to specific cancer cells, and the design of transcriptional elements has enabled selective DNA expression specific to the cancer cells. Together, these features have improved the performance of polymeric nanoparticles as targeted non-viral gene delivery vectors to treat cancer. As polymeric nanoparticles can be designed to be biodegradable, non-toxic, and to have reduced immunogenicity and tumorigenicity compared to viral platforms, they have significant potential for clinical use. Results of polymeric gene therapy in clinical trials and future directions for the engineering of nanoparticle systems for targeted cancer gene therapy are also presented. PMID:26061296

  7. TCGA bladder cancer study reveals potential drug targets

    Cancer.gov

    Investigators with TCGA have identified new potential therapeutic targets for a major form of bladder cancer, including important genes and pathways that are disrupted in the disease. They also discovered that, at the molecular level, some subtypes of bla

  8. Seed Targeted Gene Confinement Strategies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The genetic improvement of plants using biotechnology is now centrally important to agriculture, food security, and the biofuels industry. It is also important to the continued health of the environment as the need for food (on existing arable land) and renewable energy becomes critical. New genes c...

  9. Gene Targeting in Mice: a Review

    PubMed Central

    Bouabe, Hicham; Okkenhaug, Klaus

    2015-01-01

    Summary The ability to introduce DNA sequences (e.g. genes) of interest into the germline genome has rendered the mouse a powerful and indispensable experimental model in fundamental and medical research. The DNA sequences can be integrated into the genome randomly or into a specific locus by homologous recombination, in order to: (i) delete or insert mutations into genes of interest to determine their function, (ii) introduce human genes into the genome of mice to generate animal models enabling study of human-specific genes and diseases, e.g. mice susceptible to infections by human-specific pathogens of interest, (iii) introduce individual genes or genomes of pathogens (such as viruses) in order to examine the contributions of such genes to the pathogenesis of the parent pathogens, (iv) and last but not least introduce reporter genes that allow monitoring in vivo or ex vivo the expression of genes of interest. Furthermore, the use of recombination systems, such as Cre/loxP or FRT/FLP, enables conditional induction or suppression of gene expression of interest in a restricted period of mouse’s lifetime, in a particular cell type, or in a specific tissue. In this review, we will give an updated summary of the gene targeting technology and discuss some important considerations in the design of gene-targeted mice. PMID:23996268

  10. Gene Therapy and Targeted Toxins for Glioma

    PubMed Central

    Castro, Maria G.; Candolfi, Marianela; Kroeger, Kurt; King, Gwendalyn D.; Curtin, James F.; Yagiz, Kader; Mineharu, Yohei; Assi, Hikmat; Wibowo, Mia; Muhammad, AKM Ghulam; Foulad, David; Puntel, Mariana; Lowenstein, Pedro R.

    2011-01-01

    The most common primary brain tumor in adults is glioblastoma. These tumors are highly invasive and aggressive with a mean survival time of nine to twelve months from diagnosis to death. Current treatment modalities are unable to significantly prolong survival in patients diagnosed with glioblastoma. As such, glioma is an attractive target for developing novel therapeutic approaches utilizing gene therapy. This review will examine the available preclinical models for glioma including xenographs, syngeneic and genetic models. Several promising therapeutic targets are currently being pursued in pre-clinical investigations. These targets will be reviewed by mechanism of action, i.e., conditional cytotoxic, targeted toxins, oncolytic viruses, tumor suppressors/oncogenes, and immune stimulatory approaches. Preclinical gene therapy paradigms aim to determine which strategies will provide rapid tumor regression and long-term protection from recurrence. While a wide range of potential targets are being investigated preclinically, only the most efficacious are further transitioned into clinical trial paradigms. Clinical trials reported to date are summarized including results from conditionally cytotoxic, targeted toxins, oncolytic viruses and oncogene targeting approaches. Clinical trial results have not been as robust as preclinical models predicted; this could be due to the limitations of the GBM models employed. Once this is addressed, and we develop effective gene therapies in models that better replicate the clinical scenario, gene therapy will provide a powerful approach to treat and manage brain tumors. PMID:21453286

  11. Gene Therapy and Targeted Toxins for Glioma

    PubMed Central

    King, Gwendalyn D.; Curtin, James F.; Candolfi, Marianela; Kroeger, Kurt; Lowenstein, Pedro R.; Castro, Maria G.

    2006-01-01

    The most common primary brain tumor in adults is glioblastoma. These tumors are highly invasive and aggressive with a mean survival time of nine to twelve months from diagnosis to death. Current treatment modalities are unable to significantly prolong survival in patients diagnosed with glioblastoma. As such, glioma is an attractive target for developing novel therapeutic approaches utilizing gene therapy. This review will examine the available preclinical models for glioma including xenographs, syngeneic and genetic models. Several promising therapeutic targets are currently being pursued in pre-clinical investigations. These targets will be reviewed by mechanism of action, i.e., conditional cytotoxic, targeted toxins, oncolytic viruses, tumor suppressors/oncogenes, and immune stimulatory approaches. Preclinical gene therapy paradigms aim to determine which strategies will provide rapid tumor regression and long-term protection from recurrence. While a wide range of potential targets are being investigated preclinically, only the most efficacious are further transitioned into clinical trial paradigms. Clinical trials reported to date are summarized including results from conditionally cytotoxic, targeted toxins, oncolytic viruses and oncogene targeting approaches. Clinical trial results have not been as robust as preclinical models predicted, this could be due to the limitations of the GBM models employed. Once this is addressed, and we develop effective gene therapies in models that better replicate the clinical scenario, gene therapy will provide a powerful approach to treat and manage brain tumors. PMID:16457645

  12. Differential sensitivities of transcription factor target genes underlie cell type-specific gene expression profiles

    PubMed Central

    Johnson, Kirby D.; Kim, Shin-Il; Bresnick, Emery H.

    2006-01-01

    Changes in transcription factor levels and activities dictate developmental fate. Such a change might affect the full ensemble of target genes for a factor or only uniquely sensitive targets. We investigated the relationship among activity of the hematopoietic transcription factor GATA-1, chromatin occupancy, and target gene sensitivity. Graded activation of GATA-1 in GATA-1-null cells revealed high-, intermediate-, and low-sensitivity targets. GATA-1 activity requirements for occupancy and transcription often correlated. A GATA-1 amino-terminal deletion mutant severely deregulated the low-sensitivity gene Tac-2. Thus, cells expressing different levels of a cell type-specific activator can have qualitatively distinct target gene expression patterns, and factor mutations preferentially deregulate low-sensitivity genes. Unlike other target genes, GATA-1-mediated Tac-2 regulation was bimodal, with activation followed by repression, and the coregulator Friend of GATA-1 (FOG-1) selectively mediated repression. A GATA-1 mutant defective in FOG-1 binding occupied a Tac-2 regulatory region at levels higher than wild-type GATA-1, whereas FOG-1 facilitated chromatin occupancy at a distinct target site. These results indicate that FOG-1 is a determinant of GATA factor target gene sensitivity by either facilitating or opposing chromatin occupancy. PMID:17043224

  13. Ultrasound-Targeted Retroviral Gene Delivery

    NASA Astrophysics Data System (ADS)

    Taylor, Sarah L.; Rahim, Ahad A.; Bush, Nigel L.; Bamber, Jeffrey C.; Porter, Colin D.

    2007-05-01

    This study demonstrates the ability of focused ultrasound to target retroviral gene delivery. Key to our experiments was the use of non-infectious virus particles lacking the envelope protein required for receptor-mediated entry. The novelty of our approach is that spatial control at a distance is exerted upon viral delivery by subsequent exposure to ultrasound, leading to stable gene delivery. The technology is ideally suited to controlling gene delivery in vivo following systemic vector administration. Our data provide a solution to the critical issue of obtaining tissue specificity with retroviral vectors and impart stability of expression to ultrasound-mediated gene delivery.

  14. The Analysis of the Inflorescence miRNome of the Orchid Orchis italica Reveals a DEF-Like MADS-Box Gene as a New miRNA Target

    PubMed Central

    Aceto, Serena; Sica, Maria; De Paolo, Sofia; D'Argenio, Valeria; Cantiello, Piergiuseppe; Salvatore, Francesco; Gaudio, Luciano

    2014-01-01

    Plant microRNAs (miRNAs) are small, regulatory non-coding RNAs involved in a wide range of biological processes, from organ development to response to stimuli. In recent years, an increasing number of studies on model plant species have highlighted the evolutionary conservation of a high number of miRNA families and the existence of taxon-specific ones. However, few studies have examined miRNAs in non-model species such as orchids, which are characterized by highly diversified floral structures and pollination strategies. Therefore, we analysed a small RNA library of inflorescence tissue of the Mediterranean orchid Orchis italica to increase the knowledge on miRNAs in a non-model plant species. The high-throughput sequencing and analysis of a small RNA library of inflorescence of O. italica revealed 23 conserved and 161 putative novel miRNA families. Among the putative miRNA targets, experimental validation demonstrated that a DEF-like MADS-box transcript is cleaved by the homolog of miR5179 of O. italica. The presence of conserved miRNA families in the inflorescence of O. italica indicates that the basic developmental flower regulatory mechanisms mediated by miRNAs are maintained through evolution. Because, according to the “orchid code” theory, DEF-like genes exert a key function in the diversification of tepals and lip, the cleavage-mediated inhibitory activity of miR5179 on a OitaDEF-like transcript suggests that, in orchids, miRNAs play an important role in the diversification of the perianth organs. PMID:24832004

  15. Drug target prioritization by perturbed gene expression and network information

    PubMed Central

    Isik, Zerrin; Baldow, Christoph; Cannistraci, Carlo Vittorio; Schroeder, Michael

    2015-01-01

    Drugs bind to their target proteins, which interact with downstream effectors and ultimately perturb the transcriptome of a cancer cell. These perturbations reveal information about their source, i.e., drugs’ targets. Here, we investigate whether these perturbations and protein interaction networks can uncover drug targets and key pathways. We performed the first systematic analysis of over 500 drugs from the Connectivity Map. First, we show that the gene expression of drug targets is usually not significantly affected by the drug perturbation. Hence, expression changes after drug treatment on their own are not sufficient to identify drug targets. However, ranking of candidate drug targets by network topological measures prioritizes the targets. We introduce a novel measure, local radiality, which combines perturbed genes and functional interaction network information. The new measure outperforms other methods in target prioritization and proposes cancer-specific pathways from drugs to affected genes for the first time. Local radiality identifies more diverse targets with fewer neighbors and possibly less side effects. PMID:26615774

  16. Progress in gene targeting and gene therapy for retinitis pigmentosa

    SciTech Connect

    Farrar, G.J.; Humphries, M.M.; Erven, A.

    1994-09-01

    Previously, we localized disease genes involved in retinitis pigmentosa (RP), an inherited retinal degeneration, close to the rhodopsin and peripherin genes on 3q and 6p. Subsequently, we and others identified mutations in these genes in RP patients. Currently animal models for human retinopathies are being generated using gene targeting by homologous recombination in embryonic stem (ES) cells. Genomic clones for retinal genes including rhodopsin and peripherin have been obtained from a phage library carrying mouse DNA isogenic with the ES cell line (CC1.2). The peripherin clone has been sequenced to establish the genomic structure of the mouse gene. Targeting vectors for rhodopsin and peripherin including a neomycin cassette for positive selection and thymidine kinase genes enabling selection against random intergrants are under construction. Progress in vector construction will be presented. Simultaneously we are developing systems for delivery of gene therapies to retinal tissues utilizing replication-deficient adenovirus (Ad5). Efficacy of infection subsequent to various methods of intraocular injection and with varying viral titers is being assayed using an adenovirus construct containing a CMV promoter LacZ fusion as reporter and the range of tissues infected and the level of duration of LacZ expression monitored. Viral constructs with the LacZ reporter gene under the control of retinal specific promoters such as rhodopsin and IRBP cloned into pXCJL.1 are under construction. An update on developments in photoreceptor cell-directed expression of virally delivered genes will be presented.

  17. Improved Gene Targeting through Cell Cycle Synchronization

    PubMed Central

    Tsakraklides, Vasiliki; Brevnova, Elena; Stephanopoulos, Gregory; Shaw, A. Joe

    2015-01-01

    Gene targeting is a challenge in organisms where non-homologous end-joining is the predominant form of recombination. We show that cell division cycle synchronization can be applied to significantly increase the rate of homologous recombination during transformation. Using hydroxyurea-mediated cell cycle arrest, we obtained improved gene targeting rates in Yarrowia lipolytica, Arxula adeninivorans, Saccharomyces cerevisiae, Kluyveromyces lactis and Pichia pastoris demonstrating the broad applicability of the method. Hydroxyurea treatment enriches for S-phase cells that are active in homologous recombination and enables previously unattainable genomic modifications. PMID:26192309

  18. Gene targeting in primary human trophoblasts

    PubMed Central

    Rosario, Fredrick J; Sadovsky, Yoel; Jansson, Thomas

    2012-01-01

    Studies in primary human trophoblasts provide critical insights into placental function in normal and complicated pregnancies. Mechanistic studies in these cells require experimental tools to modulate gene expression. Lipid-based methods to transfect primary trophoblasts are fairly simple to use and allow for the efficient delivery of nucleic acids, but potential toxic effects limit these methods. Viral vectors are versatile transfection tools of native trophoblastic or foreign cDNAs, providing high transfection efficiency, low toxicity and stable DNA integration into the trophoblast genome. RNA interference (RNAi), using small interfering RNA (siRNA) or microRNA, constitutes a powerful approach to silence trophoblast genes. However, off-target effects, such as regulation of unintended complementary transcripts, inflammatory responses and saturation of the endogenous RNAi machinery, are significant concerns. Strategies to minimize off-target effects include using multiple individual siRNAs, elimination of pro-inflammatory sequences in the siRNA construct and chemical modification of a nucleotide in the guide strand or of the ribose moiety. Tools for efficient gene targeting in primary human trophoblasts are currently available, albeit not yet extensively validated. These methods are critical for exploring the function of human trophoblast genes and may provide a foundation for the future application of gene therapy that targets placental trophoblasts. PMID:22831880

  19. Targeted gene repair – in the arena

    PubMed Central

    Kmiec, Eric B.

    2003-01-01

    The development of targeted gene repair is under way and, despite some setbacks, shows promise as an alternative form of gene therapy. This approach uses synthetic DNA molecules to activate and direct the cell’s inherent DNA repair systems to correct inborn errors. The progress of this technique and its therapeutic potential are discussed in relation to the treatment of genetic diseases. PMID:12952907

  20. Targeting Herpetic Keratitis by Gene Therapy

    PubMed Central

    Elbadawy, Hossein Mostafa; Gailledrat, Marine; Desseaux, Carole; Ponzin, Diego; Ferrari, Stefano

    2012-01-01

    Ocular gene therapy is rapidly becoming a reality. By November 2012, approximately 28 clinical trials were approved to assess novel gene therapy agents. Viral infections such as herpetic keratitis caused by herpes simplex virus 1 (HSV-1) can cause serious complications that may lead to blindness. Recurrence of the disease is likely and cornea transplantation, therefore, might not be the ideal therapeutic solution. This paper will focus on the current situation of ocular gene therapy research against herpetic keratitis, including the use of viral and nonviral vectors, routes of delivery of therapeutic genes, new techniques, and key research strategies. Whereas the correction of inherited diseases was the initial goal of the field of gene therapy, here we discuss transgene expression, gene replacement, silencing, or clipping. Gene therapy of herpetic keratitis previously reported in the literature is screened emphasizing candidate gene therapy targets. Commonly adopted strategies are discussed to assess the relative advantages of the protective therapy using antiviral drugs and the common gene therapy against long-term HSV-1 ocular infections signs, inflammation and neovascularization. Successful gene therapy can provide innovative physiological and pharmaceutical solutions against herpetic keratitis. PMID:23326647

  1. The Differential Gene Expression Pattern of Mycobacterium tuberculosis in Response to Capreomycin and PA-824 versus First-Line TB Drugs Reveals Stress- and PE/PPE-Related Drug Targets

    PubMed Central

    Fu, Li M.; Tai, Shu C.

    2009-01-01

    Tuberculosis is a leading infectious disease causing millions of deaths each year. How to eradicate mycobacterial persistence has become a central research focus for developing next-generation TB drugs. Yet, the knowledge in this area is fundamentally limited and only a few drugs, notably capreomycin and PA-824, have been shown to be active against non-replicating persistent TB bacilli. In this study, we performed a new bioinformatics analysis on microarray-based gene expression data obtained from the public domain to explore genes that were differentially induced by drugs between the group of capreomycin and PA-824 and the group of mainly the first-line TB drugs. Our study has identified 42 genes specifically induced by capreomycin and PA-824. Many of these genes are related to stress responses. In terms of the distribution of identified genes in a specific category relative to the whole genome, only the categories of PE/PPE and conserved hypotheticals have statistical significance. Six among the 42 genes identified in this study are on the list of the top 100 persistence targets selected by the TB Structural Genomics Consortium. Further biological elucidation of their roles in mycobacterial persistence is warranted. PMID:20016672

  2. Molecular Analysis of Sarcoidosis Granulomas Reveals Antimicrobial Targets.

    PubMed

    Rotsinger, Joseph E; Celada, Lindsay J; Polosukhin, Vasiliy V; Atkinson, James B; Drake, Wonder P

    2016-07-01

    Sarcoidosis is a granulomatous disease of unknown cause. Prior molecular and immunologic studies have confirmed the presence of mycobacterial virulence factors, such as catalase peroxidase and superoxide dismutase A, within sarcoidosis granulomas. Molecular analysis of granulomas can identify targets of known antibiotics classes. Currently, major antibiotics are directed against DNA synthesis, protein synthesis, and cell wall formation. We conducted molecular analysis of 40 sarcoidosis diagnostic specimens and compared them with 33 disease control specimens for the presence of mycobacterial genes that encode antibiotic targets. We assessed for genes involved in DNA synthesis (DNA gyrase A [gyrA] and DNA gyrase B), protein synthesis (RNA polymerase subunit β), cell wall synthesis (embCAB operon and enoyl reductase), and catalase peroxidase. Immunohistochemical analysis was conducted to investigate the locale of mycobacterial genes such as gyrA within 12 sarcoidosis specimens and 12 disease controls. Mycobacterial DNA was detected in 33 of 39 sarcoidosis specimens by quantitative real-time polymerase chain reaction compared with 2 of 30 disease control specimens (P < 0.001, two-tailed Fisher's test). Twenty of 39 were positive for three or more mycobacterial genes, compared with 1 of 30 control specimens (P < 0.001, two-tailed Fisher's test). Immunohistochemistry analysis localized mycobacterial gyrA nucleic acids to sites of granuloma formation in 9 of 12 sarcoidosis specimens compared with 1 of 12 disease controls (P < 0.01). Microbial genes encoding enzymes that can be targeted by currently available antimycobacterial antibiotics are present in sarcoidosis specimens and localize to sites of granulomatous inflammation. Use of antimicrobials directed against target enzymes may be an innovative treatment alternative. PMID:26807608

  3. Efficient ectopic gene expression targeting chick mesoderm.

    PubMed

    Oberg, Kerby C; Pira, Charmaine U; Revelli, Jean-Pierre; Ratz, Beate; Aguilar-Cordova, Estuardo; Eichele, Gregor

    2002-07-01

    The chick model has been instrumental in illuminating genes that regulate early vertebrate development and pattern formation. Targeted ectopic gene expression is critical to dissect further the complicated gene interactions that are involved. In an effort to develop a consistent method to ectopically introduce and focally express genes in chick mesoderm, we evaluated and optimized several gene delivery methods, including implantation of 293 cells laden with viral vectors, direct adenoviral injection, and electroporation (EP). We targeted the mesoderm of chick wing buds between stages 19 and 21 (Hamburger and Hamilton stages) and used beta-galactosidase and green fluorescent protein (GFP) to document gene transfer. Expression constructs using the cytomegalovirus (CMV) promoter, the beta-actin promoter, and vectors with an internal ribosomal entry sequence linked to GFP (IRES-GFP) were also compared. After gene transfer, we monitored expression for up to 3 days. The functionality of ectopic expression was demonstrated with constructs containing the coding sequences for Shh, a secreted signaling protein, or Hoxb-8, a transcription factor, both of which can induce digit duplication when ectopically expressed in anterior limb mesoderm. We identified several factors that enhance mesodermal gene transfer. First, the use of a vector with the beta-actin promoter coupled to the 69% fragment of the bovine papilloma virus yielded superior mesodermal expression both by markers and functional results when compared with several CMV-driven vectors. Second, we found the use of mineral oil to be an important adjuvant for EP and direct viral injection to localize and contain vector within the mesoderm at the injection site. Lastly, although ectopic expression could be achieved with all three methods, we favored EP confined to the mesoderm with insulated microelectrodes (confined microelectroporation- CMEP), because vector construction is rapid, the method is efficient, and results

  4. Targeted gene silencing to induce permanent sterility.

    PubMed

    Dissen, G A; Lomniczi, A; Boudreau, R L; Chen, Y H; Davidson, B L; Ojeda, S R

    2012-08-01

    A non-surgical method to induce sterility would be a useful tool to control feral populations of animals. Our laboratories have experience with approaches aimed at targeting brain cells in vivo with vehicles that deliver a payload of either inhibitory RNAs or genes intended to correct cellular dysfunction. A combination/modification of these methods may provide a useful framework for the design of approaches that can be used to sterilize cats and dogs. For this approach to succeed, it has to meet several conditions: it needs to target a gene essential for fertility. It must involve a method that can selectively silence the gene of interest. It also needs to deliver the silencing agent via a minimally invasive method. Finally, the silencing effect needs to be sustained for many years, so that expansion of the targeted population can be effectively prevented. In this article, we discuss this subject and provide a succinct account of our previous experience with: (i) molecular reagents able to disrupt reproductive cyclicity when delivered to regions of the brain involved in the control of reproduction and (ii) molecular reagents able to ameliorate neuronal disease when delivered systemically using a novel approach of gene therapy. PMID:22827375

  5. Expression of Phosphocitrate-Targeted Genes in Osteoarthritis Menisci

    PubMed Central

    Sun, Yubo; Mauerhan, David R.; Steuerwald, Nury M.; Ingram, Jane; Kneisl, Jeffrey S.; Hanley, Edward N.

    2014-01-01

    Phosphocitrate (PC) inhibited calcium crystal-associated osteoarthritis (OA) in Hartley guinea pigs. However, the molecular mechanisms remain elusive. This study sought to determine PC targeted genes and the expression of select PC targeted genes in OA menisci to test hypothesis that PC exerts its disease modifying activity in part by reversing abnormal expressions of genes involved in OA. We found that PC downregulated the expression of numerous genes classified in immune response, inflammatory response, and angiogenesis, including chemokine (C-C motif) ligand 5, Fc fragment of IgG, low affinity IIIb receptor (FCGR3B), and leukocyte immunoglobulin-like receptor, subfamily B member 3 (LILRB3). In contrast, PC upregulated the expression of many genes classified in skeletal development, including collagen type II alpha1, fibroblast growth factor receptor 3 (FGFR3), and SRY- (sex determining region Y-) box 9 (SOX-9). Immunohistochemical examinations revealed higher levels of FCGR3B and LILRB3 and lower level of SOX-9 in OA menisci. These findings indicate that OA is a disease associated with immune system activation and decreased expression of SOX-9 gene in OA menisci. PC exerts its disease modifying activity on OA, at least in part, by targeting immune system activation and the production of extracellular matrix and selecting chondroprotective proteins. PMID:25525593

  6. Nucleotide substitutions revealing specific functions of Polycomb group genes.

    PubMed

    Bajusz, Izabella; Sipos, László; Pirity, Melinda K

    2015-04-01

    POLYCOMB group (PCG) proteins belong to the family of epigenetic regulators of genes playing important roles in differentiation and development. Mutants of PcG genes were isolated first in the fruit fly, Drosophila melanogaster, resulting in spectacular segmental transformations due to the ectopic expression of homeotic genes. Homologs of Drosophila PcG genes were also identified in plants and in vertebrates and subsequent experiments revealed the general role of PCG proteins in the maintenance of the repressed state of chromatin through cell divisions. The past decades of gene targeting experiments have allowed us to make significant strides towards understanding how the network of PCG proteins influences multiple aspects of cellular fate determination during development. Being involved in the transmission of specific expression profiles of different cell lineages, PCG proteins were found to control wide spectra of unrelated epigenetic processes in vertebrates, such as stem cell plasticity and renewal, genomic imprinting and inactivation of X-chromosome. PCG proteins also affect regulation of metabolic genes being important for switching programs between pluripotency and differentiation. Insight into the precise roles of PCG proteins in normal physiological processes has emerged from studies employing cell culture-based systems and genetically modified animals. Here we summarize the findings obtained from PcG mutant fruit flies and mice generated to date with a focus on PRC1 and PRC2 members altered by nucleotide substitutions resulting in specific alleles. We also include a compilation of lessons learned from these models about the in vivo functions of this complex protein family. With multiple knockout lines, sophisticated approaches to study the consequences of peculiar missense point mutations, and insights from complementary gain-of-function systems in hand, we are now in a unique position to significantly advance our understanding of the molecular basis of

  7. Gene Therapy Targeting Glaucoma: Where Are We?

    PubMed Central

    Liu, Xuyang; Rasmussen, Carol A.; Gabelt, B’Ann T.; Brandt, Curtis R.; Kaufman, Paul L.

    2010-01-01

    In a chronic disease such as glaucoma, a therapy that provides a long lasting local effect, with minimal systemic side effects, while circumventing the issue of patient compliance, is very attractive. The field of gene therapy is growing rapidly and ocular applications are expanding. Our understanding of the molecular pathogenesis of glaucoma is leading to greater specificity in ocular tissue targeting. Improvements in gene delivery techniques, refinement of vector construction methods, and development of better animal models combine to bring this potential therapy closer to reality. PMID:19539835

  8. A novel, dynamic pattern-based analysis of NF-κB binding during the priming phase of liver regeneration reveals switch-like functional regulation of target genes

    PubMed Central

    Cook, Daniel J.; Patra, Biswanath; Kuttippurathu, Lakshmi; Hoek, Jan B.; Vadigepalli, Rajanikanth

    2015-01-01

    Following partial hepatectomy, a coordinated series of molecular events occurs to regulate hepatocyte entry into the cell cycle to recover lost mass. In rats during the first 6 h following resection, hepatocytes are primed by a tightly controlled cytokine response to prepare hepatocytes to begin replication. Although it appears to be a critical element driving regeneration, the cytokine response to resection has not yet been fully characterized. Specifically, the role of one of the key response elements to cytokine signaling (NF-κB) remains incompletely characterized. In this study, we present a novel, genome-wide, pattern-based analysis characterizing NF-κB binding during the priming phase of liver regeneration. We interrogated the dynamic regulation of priming by NF-κB through categorizing NF-κB binding in different temporal profiles: immediate sustained response, early transient response, and delayed response to partial hepatectomy. We then identified functional regulation of NF-κB binding by relating the temporal response profile to differential gene expression. We found that NF-κB bound genes govern negative regulation of cell growth and inflammatory response immediately following hepatectomy. NF-κB also transiently regulates genes responsible for lipid biosynthesis and transport as well as induction of apoptosis following hepatectomy. By the end of the priming phase, NF-κB regulation of genes involved in inflammatory response, negative regulation of cell death, and extracellular structure organization became prominent. These results suggest that NF-κB regulates target genes through binding and unbinding in immediate, transient, and delayed patterns. Such dynamic switch-like patterns of NF-κB binding may govern different functional transitions that drive the onset of regeneration. PMID:26217230

  9. Targeted cancer exome sequencing reveals recurrent mutations in myeloproliferative neoplasms.

    PubMed

    Tenedini, E; Bernardis, I; Artusi, V; Artuso, L; Roncaglia, E; Guglielmelli, P; Pieri, L; Bogani, C; Biamonte, F; Rotunno, G; Mannarelli, C; Bianchi, E; Pancrazzi, A; Fanelli, T; Malagoli Tagliazucchi, G; Ferrari, S; Manfredini, R; Vannucchi, A M; Tagliafico, E

    2014-05-01

    With the intent of dissecting the molecular complexity of Philadelphia-negative myeloproliferative neoplasms (MPN), we designed a target enrichment panel to explore, using next-generation sequencing (NGS), the mutational status of an extensive list of 2000 cancer-associated genes and microRNAs. The genomic DNA of granulocytes and in vitro-expanded CD3+T-lymphocytes, as a germline control, was target-enriched and sequenced in a learning cohort of 20 MPN patients using Roche 454 technology. We identified 141 genuine somatic mutations, most of which were not previously described. To test the frequency of the identified variants, a larger validation cohort of 189 MPN patients was additionally screened for these mutations using Ion Torrent AmpliSeq NGS. Excluding the genes already described in MPN, for 8 genes (SCRIB, MIR662, BARD1, TCF12, FAT4, DAP3, POLG and NRAS), we demonstrated a mutation frequency between 3 and 8%. We also found that mutations at codon 12 of NRAS (NRASG12V and NRASG12D) were significantly associated, for primary myelofibrosis (PMF), with highest dynamic international prognostic scoring system (DIPSS)-plus score categories. This association was then confirmed in 66 additional PMF patients composing a final dataset of 168 PMF showing a NRAS mutation frequency of 4.7%, which was associated with a worse outcome, as defined by the DIPSS plus score. PMID:24150215

  10. Regulatory network of microRNAs, target genes, transcription factors and host genes in endometrial cancer.

    PubMed

    Xue, Lu-Chen; Xu, Zhi-Wen; Wang, Kun-Hao; Wang, Ning; Zhang, Xiao-Xu; Wang, Shang

    2015-01-01

    Genes and microRNAs (miRNAs) have important roles in human oncology. However, most of the biological factors are reported in disperse form which makes it hard to discover the pathology. In this study, genes and miRNAs involved in human endometrial cancer(EC) were collected and formed into regulatory networks following their interactive relations, including miRNAs targeting genes, transcription factors (TFs) regulating miRNAs and miRNAs included in their host genes. Networks are constructed hierarchically at three levels: differentially expressed, related and global. Among the three, the differentially expressed network is the most important and fundamental network that contains the key genes and miRNAs in EC. The target genes, TFs and miRNAs are differentially expressed in EC so that any mutation in them may impact on EC development. Some key pathways in networks were highlighted to analyze how they interactively influence other factors and carcinogenesis. Upstream and downstream pathways of the differentially expressed genes and miRNAs were compared and analyzed. The purpose of this study was to partially reveal the deep regulatory mechanisms in EC using a new method that combines comprehensive genes and miRNAs together with their relationships. It may contribute to cancer prevention and gene therapy of EC. PMID:25684474

  11. A Highly Efficient Gene-Targeting System for Aspergillus parasiticus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gene targeting via homologous recombination is often used to elucidate gene function. For filamentous fungi, the majority of transforming DNA integrates ectopically. Deletion of Aspergillus parasiticus ku70, a gene of the non-homologous end-joining pathway, drastically increased the gene targeting...

  12. Short DNA sequences inserted for gene targeting can accidentally interfere with off-target gene expression.

    PubMed

    Meier, Ingo D; Bernreuther, Christian; Tilling, Thomas; Neidhardt, John; Wong, Yong Wee; Schulze, Christian; Streichert, Thomas; Schachner, Melitta

    2010-06-01

    Targeting of genes in mice, a key approach to study development and disease, often leaves a neo cassette, loxP, or FRT sites inserted in the mouse genome. Insertion of neo can influence the expression of neighboring genes, but similar effects have not been reported for loxP sites. We therefore performed microarray analyses of mice in which the Ncam or the Tnr gene were targeted either by insertion of neo or loxP/FRT sites. In the case of Ncam, neo, but not loxP/FRT insertion, led to a 2-fold reduction in mRNA levels of 3 genes located at distances between 0.2 and 3.1 Mb from the target. In contrast, after introduction of loxP/FRT sites into introns of Tnr, we observed a 2.5- to 4-fold reduction in the transcript level of the Gas5 gene, 1.1 Mb away from Tnr, most probably due to disruption of a conserved regulatory element in Tnr. Insertion of short DNA sequences such as loxP/FRT can thus influence off-target mRNA levels if these sites are accidentally placed into regulatory elements. Our results imply that conditional knockout mice should be analyzed for genomic positional side effects that may influence the animals' phenotypes. PMID:20110269

  13. Phytochemicals targeting genes relevant for type 2 diabetes.

    PubMed

    Anuradha, Carani Venkatraman

    2013-06-01

    Nutrigenomic approaches based on ethnopharmacology and phytotherapy concepts have revealed that type 2 diabetes mellitus (T2DM) may be susceptible to dietary intervention. Interaction between bioactive food components and the genome may influence cell processes and modulate the onset and progression of the disease. T2DM, characterized by insulin resistance and beta cell dysfunction, is one of the leading causes of death and disability. Despite the great advances that have been made in the understanding and management of this complex, multifactorial disease, T2DM has become a worldwide epidemic in the 21st century. Population and family studies have revealed a strong genetic component of T2DM, and a number of candidate genes have been identified in humans. Variations in the gene sequences such as single nucleotide polymorphisms, explain the individual differences in traits like disease susceptibility and response to treatment. A clear understanding of how nutrients affect the expression of genes should facilitate the development of individualized intervention and, eventually, treatment strategies for T2DM. Review of the literature identified many phytochemicals/extracts from traditional medicinal plants that can target diabetogenic genes. This review focuses on the genetic aspects of T2DM, nutrient modification of genes relevant for diabetes, and future prospects of nutritional therapy of T2DM. PMID:23745945

  14. Rapid targeted gene disruption in Bacillus anthracis

    PubMed Central

    2013-01-01

    Background Anthrax is a zoonotic disease recognized to affect herbivores since Biblical times and has the widest range of susceptible host species of any known pathogen. The ease with which the bacterium can be weaponized and its recent deliberate use as an agent of terror, have highlighted the importance of gaining a deeper understanding and effective countermeasures for this important pathogen. High quality sequence data has opened the possibility of systematic dissection of how genes distributed on both the bacterial chromosome and associated plasmids have made it such a successful pathogen. However, low transformation efficiency and relatively few genetic tools for chromosomal manipulation have hampered full interrogation of its genome. Results Group II introns have been developed into an efficient tool for site-specific gene inactivation in several organisms. We have adapted group II intron targeting technology for application in Bacillus anthracis and generated vectors that permit gene inactivation through group II intron insertion. The vectors developed permit screening for the desired insertion through PCR or direct selection of intron insertions using a selection scheme that activates a kanamycin resistance marker upon successful intron insertion. Conclusions The design and vector construction described here provides a useful tool for high throughput experimental interrogation of the Bacillus anthracis genome and will benefit efforts to develop improved vaccines and therapeutics. PMID:24047152

  15. Transcriptome analysis reveals transmembrane targets on transplantable midbrain dopamine progenitors

    PubMed Central

    Jönsson, Marie E.; Björklund, Anders; Parish, Clare L.; Thompson, Lachlan H.

    2015-01-01

    An important challenge for the continued development of cell therapy for Parkinson’s disease (PD) is the establishment of procedures that better standardize cell preparations for use in transplantation. Although cell sorting has been an anticipated strategy, its application has been limited by lack of knowledge regarding transmembrane proteins that can be used to target and isolate progenitors for midbrain dopamine (mDA) neurons. We used a “FACS-array” approach to identify 18 genes for transmembrane proteins with high expression in mDA progenitors and describe the utility of four of these targets (Alcam, Chl1, Gfra1, and Igsf8) for isolating mDA progenitors from rat primary ventral mesencephalon through flow cytometry. Alcam and Chl1 facilitated a significant enrichment of mDA neurons following transplantation, while targeting of Gfra1 allowed for robust separation of dopamine and serotonin neurons. Importantly, we also show that mDA progenitors isolated on the basis of transmembrane proteins are capable of extensive, functional innervation of the host striatum and correction of motor impairment in a unilateral model of PD. These results are highly relevant for current efforts to establish safe and effective stem cell-based procedures for PD, where clinical translation will almost certainly require safety and standardization measures in order to deliver well-characterized cell preparations. PMID:25775569

  16. Identification of key target genes and pathways in laryngeal carcinoma

    PubMed Central

    Liu, Feng; Du, Jintao; Liu, Jun; Wen, Bei

    2016-01-01

    The purpose of the present study was to screen the key genes associated with laryngeal carcinoma and to investigate the molecular mechanism of laryngeal carcinoma progression. The gene expression profile of GSE10935 [Gene Expression Omnibus (GEO) accession number], including 12 specimens from laryngeal papillomas and 12 specimens from normal laryngeal epithelia controls, was downloaded from the GEO database. Differentially expressed genes (DEGs) were screened in laryngeal papillomas compared with normal controls using Limma package in R language, followed by Gene Ontology (GO) enrichment analysis and pathway enrichment analysis. Furthermore, the protein-protein interaction (PPI) network of DEGs was constructed using Cytoscape software and modules were analyzed using MCODE plugin from the PPI network. Furthermore, significant biological pathway regions (sub-pathway) were identified by using iSubpathwayMiner analysis. A total of 67 DEGs were identified, including 27 up-regulated genes and 40 down-regulated genes and they were involved in different GO terms and pathways. PPI network analysis revealed that Ras association (RalGDS/AF-6) domain family member 1 (RASSF1) was a hub protein. The sub-pathway analysis identified 9 significantly enriched sub-pathways, including glycolysis/gluconeogenesis and nitrogen metabolism. Genes such as phosphoglycerate kinase 1 (PGK1), carbonic anhydrase II (CA2), and carbonic anhydrase XII (CA12) whose node degrees were >10 were identified in the disease risk sub-pathway. Genes in the sub-pathway, such as RASSF1, PGK1, CA2 and CA12 were presumed to serve critical roles in laryngeal carcinoma. The present study identified DEGs and their sub-pathways in the disease, which may serve as potential targets for treatment of laryngeal carcinoma. PMID:27446427

  17. Spatiotemporal gene expression targeting with the TARGET and gene-switch systems in Drosophila.

    PubMed

    McGuire, Sean E; Mao, Zhengmei; Davis, Ronald L

    2004-02-17

    Targeted gene expression has become a standard technique for the study of biological questions in Drosophila. Until recently, transgene expression could be targeted in the dimension of either time or space, but not both. Several new systems have recently been developed to direct transgene expression simultaneously in both time and space. We describe here two such systems that we developed in our laboratory. The first system provides a general method for temporal and regional gene expression targeting (TARGET) with the conventional GAL4-upstream activator sequence (UAS) system and a temperature-sensitive GAL80 molecule, which represses GAL4 transcriptional activity at permissive temperatures. The second system, termed Gene-Switch, is based on a GAL4-progesterone receptor chimera that is hormone-inducible. We have used both systems for simultaneous spatial and temporal rescue of memory dysfunction in the rutabaga (rut) memory mutant of Drosophila. In this protocol, we provide guidelines for the use of these two novel systems, which should have general utility in studying Drosophila biology and in using the fly as a model for human disease. PMID:14970377

  18. A Genome-Wide Map of AAV-Mediated Human Gene Targeting

    PubMed Central

    Deyle, David R.; Hansen, R. Scott; Cornea, Anda M.; Li, Li B.; Burt, Amber A.; Alexander, Ian E.; Sandstrom, Richard S.; Stamatoyannopoulos, John A.; Wei, Chia-Lin; Russell, David W.

    2014-01-01

    To determine which genomic features promote homologous recombination, we created a genome-wide map of gene targeting sites. An adeno-associated virus vector was used to target identical loci introduced as transcriptionally active retroviral vector proviruses. A comparison of ~2,000 targeted and untargeted sites showed that targeting occurred throughout the human genome and was not influenced by the presence of nearby CpG islands, sequence repeats, or DNase I hypersensitive sites. Targeted sites were preferentially found within transcription units, especially when the target loci were transcribed in the opposite orientation to their surrounding chromosomal genes. The impact of DNA replication was determined by mapping replication forks, which revealed a preference for recombination at target loci transcribed towards an incoming fork. Our results constitute the first genome-wide screen of gene targeting in mammalian cells, and they demonstrate a strong recombinogenic effect of colliding polymerases. PMID:25282150

  19. Revealing potential molecular targets bridging colitis and colorectal cancer based on multidimensional integration strategy

    PubMed Central

    Hu, Yongfei; Li, Xiaobo; Wang, Xishan; Fan, Huihui; Wang, Guiyu; Wang, Dong

    2015-01-01

    Chronic inflammation may play a vital role in the pathogenesis of inflammation-associated tumors. However, the underlying mechanisms bridging ulcerative colitis (UC) and colorectal cancer (CRC) remain unclear. Here, we integrated multidimensional interaction resources, including gene expression profiling, protein-protein interactions (PPIs), transcriptional and post-transcriptional regulation data, and virus-host interactions, to tentatively explore potential molecular targets that functionally link UC and CRC at a systematic level. In this work, by deciphering the overlapping genes, crosstalking genes and pivotal regulators of both UC- and CRC-associated functional module pairs, we revealed a variety of genes (including FOS and DUSP1, etc.), transcription factors (including SMAD3 and ETS1, etc.) and miRNAs (including miR-155 and miR-196b, etc.) that may have the potential to complete the connections between UC and CRC. Interestingly, further analyses of the virus-host interaction network demonstrated that several virus proteins (including EBNA-LP of EBV and protein E7 of HPV) frequently inter-connected to UC- and CRC-associated module pairs with their validated targets significantly enriched in both modules of the host. Together, our results suggested that multidimensional integration strategy provides a novel approach to discover potential molecular targets that bridge the connections between UC and CRC, which could also be extensively applied to studies on other inflammation-related cancers. PMID:26461477

  20. Targeted gene knockout in chickens mediated by TALENs.

    PubMed

    Park, Tae Sub; Lee, Hong Jo; Kim, Ki Hyun; Kim, Jin-Soo; Han, Jae Yong

    2014-09-01

    Genetically modified animals are used for industrial applications as well as scientific research, and studies on these animals contribute to a better understanding of biological mechanisms. Gene targeting techniques have been developed to edit specific gene loci in the genome, but the conventional strategy of homologous recombination with a gene-targeted vector has low efficiency and many technical complications. Here, we generated specific gene knockout chickens through the use of transcription activator-like effector nuclease (TALEN)-mediated gene targeting. In this study, we accomplished targeted knockout of the ovalbumin (OV) gene in the chicken primordial germ cells, and OV gene mutant offspring were generated through test-cross analysis. TALENs successfully induced nucleotide deletion mutations of ORF shifts, resulting in loss of chicken OV gene function. Our results demonstrate that the TALEN technique used in the chicken primordial germ cell line is a powerful strategy to create specific genome-edited chickens safely for practical applications. PMID:25139993

  1. Targeted gene knockout in chickens mediated by TALENs

    PubMed Central

    Park, Tae Sub; Lee, Hong Jo; Kim, Ki Hyun; Kim, Jin-Soo; Han, Jae Yong

    2014-01-01

    Genetically modified animals are used for industrial applications as well as scientific research, and studies on these animals contribute to a better understanding of biological mechanisms. Gene targeting techniques have been developed to edit specific gene loci in the genome, but the conventional strategy of homologous recombination with a gene-targeted vector has low efficiency and many technical complications. Here, we generated specific gene knockout chickens through the use of transcription activator-like effector nuclease (TALEN)-mediated gene targeting. In this study, we accomplished targeted knockout of the ovalbumin (OV) gene in the chicken primordial germ cells, and OV gene mutant offspring were generated through test-cross analysis. TALENs successfully induced nucleotide deletion mutations of ORF shifts, resulting in loss of chicken OV gene function. Our results demonstrate that the TALEN technique used in the chicken primordial germ cell line is a powerful strategy to create specific genome-edited chickens safely for practical applications. PMID:25139993

  2. Reveal genes functionally associated with ACADS by a network study.

    PubMed

    Chen, Yulong; Su, Zhiguang

    2015-09-15

    Establishing a systematic network is aimed at finding essential human gene-gene/gene-disease pathway by means of network inter-connecting patterns and functional annotation analysis. In the present study, we have analyzed functional gene interactions of short-chain acyl-coenzyme A dehydrogenase gene (ACADS). ACADS plays a vital role in free fatty acid β-oxidation and regulates energy homeostasis. Modules of highly inter-connected genes in disease-specific ACADS network are derived by integrating gene function and protein interaction data. Among the 8 genes in ACADS web retrieved from both STRING and GeneMANIA, ACADS is effectively conjoined with 4 genes including HAHDA, HADHB, ECHS1 and ACAT1. The functional analysis is done via ontological briefing and candidate disease identification. We observed that the highly efficient-interlinked genes connected with ACADS are HAHDA, HADHB, ECHS1 and ACAT1. Interestingly, the ontological aspect of genes in the ACADS network reveals that ACADS, HAHDA and HADHB play equally vital roles in fatty acid metabolism. The gene ACAT1 together with ACADS indulges in ketone metabolism. Our computational gene web analysis also predicts potential candidate disease recognition, thus indicating the involvement of ACADS, HAHDA, HADHB, ECHS1 and ACAT1 not only with lipid metabolism but also with infant death syndrome, skeletal myopathy, acute hepatic encephalopathy, Reye-like syndrome, episodic ketosis, and metabolic acidosis. The current study presents a comprehensible layout of ACADS network, its functional strategies and candidate disease approach associated with ACADS network. PMID:26045367

  3. Comparative genomics of lactic acid bacteria reveals a niche-specific gene set

    PubMed Central

    2009-01-01

    Background The recently sequenced genome of Lactobacillus helveticus DPC4571 [1] revealed a dairy organism with significant homology (75% of genes are homologous) to a probiotic bacteria Lb. acidophilus NCFM [2]. This led us to hypothesise that a group of genes could be determined which could define an organism's niche. Results Taking 11 fully sequenced lactic acid bacteria (LAB) as our target, (3 dairy LAB, 5 gut LAB and 3 multi-niche LAB), we demonstrated that the presence or absence of certain genes involved in sugar metabolism, the proteolytic system, and restriction modification enzymes were pivotal in suggesting the niche of a strain. We identified 9 niche specific genes, of which 6 are dairy specific and 3 are gut specific. The dairy specific genes identified in Lactobacillus helveticus DPC4571 were lhv_1161 and lhv_1171, encoding components of the proteolytic system, lhv_1031 lhv_1152, lhv_1978 and lhv_0028 encoding restriction endonuclease genes, while bile salt hydrolase genes lba_0892 and lba_1078, and the sugar metabolism gene lba_1689 from Lb. acidophilus NCFM were identified as gut specific genes. Conclusion Comparative analysis revealed that if an organism had homologs to the dairy specific geneset, it probably came from a dairy environment, whilst if it had homologs to gut specific genes, it was highly likely to be of intestinal origin. We propose that this "barcode" of 9 genes will be a useful initial guide to researchers in the LAB field to indicate an organism's ability to occupy a specific niche. PMID:19265535

  4. Data mining of VDJ genes reveals interesting clues.

    PubMed

    Joshi, Rajani R; Gupta, Vinay K

    2006-01-01

    Hypervariability of the complementary determining regions in characteristic structure of Immunoglobulins and the distinct, cell-specific expressions of the genes coding for this important class of proteins pose intriguing problems in experimental and computational/informatics research requiring a special approach different from those for the other proteins. We present here an Average Linkage Hierarchical Clustering of the Homosapien VDJ genes and the Immunoglobulin polypeptides generated by them using special kind of data structures and correlation matrices in place of the microarray data. The results reveal interesting clues on the heterogeneity of exon - intron locations in these gene-families and its possible role in hypervariability of the Immunoglobulins. PMID:16842114

  5. Copy number and targeted mutational analysis reveals novel somatic events in metastatic prostate tumors.

    PubMed

    Robbins, Christiane M; Tembe, Waibov A; Baker, Angela; Sinari, Shripad; Moses, Tracy Y; Beckstrom-Sternberg, Stephen; Beckstrom-Sternberg, James; Barrett, Michael; Long, James; Chinnaiyan, Arul; Lowey, James; Suh, Edward; Pearson, John V; Craig, David W; Agus, David B; Pienta, Kenneth J; Carpten, John D

    2011-01-01

    Advanced prostate cancer can progress to systemic metastatic tumors, which are generally androgen insensitive and ultimately lethal. Here, we report a comprehensive genomic survey for somatic events in systemic metastatic prostate tumors using both high-resolution copy number analysis and targeted mutational survey of 3508 exons from 577 cancer-related genes using next generation sequencing. Focal homozygous deletions were detected at 8p22, 10q23.31, 13q13.1, 13q14.11, and 13q14.12. Key genes mapping within these deleted regions include PTEN, BRCA2, C13ORF15, and SIAH3. Focal high-level amplifications were detected at 5p13.2-p12, 14q21.1, 7q22.1, and Xq12. Key amplified genes mapping within these regions include SKP2, FOXA1, and AR. Furthermore, targeted mutational analysis of normal-tumor pairs has identified somatic mutations in genes known to be associated with prostate cancer including AR and TP53, but has also revealed novel somatic point mutations in genes including MTOR, BRCA2, ARHGEF12, and CHD5. Finally, in one patient where multiple independent metastatic tumors were available, we show common and divergent somatic alterations that occur at both the copy number and point mutation level, supporting a model for a common clonal progenitor with metastatic tumor-specific divergence. Our study represents a deep genomic analysis of advanced metastatic prostate tumors and has revealed candidate somatic alterations, possibly contributing to lethal prostate cancer. PMID:21147910

  6. Copy number and targeted mutational analysis reveals novel somatic events in metastatic prostate tumors

    PubMed Central

    Robbins, Christiane M.; Tembe, Waibov A.; Baker, Angela; Sinari, Shripad; Moses, Tracy Y.; Beckstrom-Sternberg, Stephen; Beckstrom-Sternberg, James; Barrett, Michael; Long, James; Chinnaiyan, Arul; Lowey, James; Suh, Edward; Pearson, John V.; Craig, David W.; Agus, David B.; Pienta, Kenneth J.; Carpten, John D.

    2011-01-01

    Advanced prostate cancer can progress to systemic metastatic tumors, which are generally androgen insensitive and ultimately lethal. Here, we report a comprehensive genomic survey for somatic events in systemic metastatic prostate tumors using both high-resolution copy number analysis and targeted mutational survey of 3508 exons from 577 cancer-related genes using next generation sequencing. Focal homozygous deletions were detected at 8p22, 10q23.31, 13q13.1, 13q14.11, and 13q14.12. Key genes mapping within these deleted regions include PTEN, BRCA2, C13ORF15, and SIAH3. Focal high-level amplifications were detected at 5p13.2-p12, 14q21.1, 7q22.1, and Xq12. Key amplified genes mapping within these regions include SKP2, FOXA1, and AR. Furthermore, targeted mutational analysis of normal-tumor pairs has identified somatic mutations in genes known to be associated with prostate cancer including AR and TP53, but has also revealed novel somatic point mutations in genes including MTOR, BRCA2, ARHGEF12, and CHD5. Finally, in one patient where multiple independent metastatic tumors were available, we show common and divergent somatic alterations that occur at both the copy number and point mutation level, supporting a model for a common clonal progenitor with metastatic tumor-specific divergence. Our study represents a deep genomic analysis of advanced metastatic prostate tumors and has revealed candidate somatic alterations, possibly contributing to lethal prostate cancer. PMID:21147910

  7. Enriching CRISPR-Cas9 targeted cells by co-targeting the HPRT gene.

    PubMed

    Liao, Shuren; Tammaro, Margaret; Yan, Hong

    2015-11-16

    The CRISPR-Cas9 system uses guide RNAs to direct the Cas9 endonuclease to cleave target sequences. It can, in theory, target essentially any sequence in a genome, but the efficiency of the predicted guide RNAs varies dramatically. If no targeted cells are obtained, it is also difficult to know why the experiment fails. We have developed a transient transfection based method to enrich successfully targeted cells by co-targeting the hypoxanthine phosphoribosyltransferase (HPRT) gene. Cells are transfected with two guide RNAs that target respectively HPRT and the gene of interest. HPRT targeted cells are selected by resistance to 6-thioguanine (6-TG) and then examined for potential alterations to the gene targeted by the co-transfected guide RNA. Alterations of many genes, such as AAVS1, Exo1 and Trex1, are highly enriched in the 6-TG resistant cells. This method works in both HCT116 cells and U2OS cells and can easily be scaled up to process multiple guide RNAs. When co-targeting fails, it is straightforward to determine whether the target gene is essential or the guide RNA is ineffective. HPRT co-targeting thus provides a simple, efficient and scalable way to enrich gene targeting events and to identify the cause of failure. PMID:26130722

  8. Novel prognostic genes of diffuse large B-cell lymphoma revealed by survival analysis of gene expression data

    PubMed Central

    Li, Chenglong; Zhu, Biao; Chen, Jiao; Huang, Xiaobing

    2015-01-01

    Objective This study aimed to identify prognostic genes for diffuse large B-cell lymphoma (DLBCL), using bioinformatic methods. Methods Five gene expression data sets were downloaded from the Gene Expression Omnibus database. Significance analysis of microarrays algorithm was used to identify differentially expressed genes (DEGs) from two data sets. Functional enrichment analysis was performed for the DEGs with the Database for Annotation, Visualization and Integration Discovery (DAVID). Survival analysis was performed with the Kaplan–Meier method using function survfit from package survival of R for the other three data sets. Cox univariate regression analysis was used to further screen out prognostic genes. Results Thirty-one common DEGs were identified in the two data sets, mainly enriched in the regulation of lymphocyte activation, immune response, and interleukin-mediated signaling pathway. Combined with 47 DLBCL-related genes acquired by literature retrieval, a total of 78 potential prognostic genes were obtained. Cases from the other three data sets were used in hierarchical clustering, and the 78 genes could cluster them into several subtypes with significant differences in survival curves. Cox univariate regression analysis revealed 45, 33, and eleven prognostic genes in the three data sets, respectively. Five common prognostic genes were revealed, including LCP2, TNFRSF9, FUT8, IRF4, and TLE1, among which LCP2, FUT8, and TLE1 were novel prognostic genes. Conclusion Five prognostic genes of DLBCL were identified in this study. They could not only be used for molecular subtyping of DLBCL but also be potential targets for treatment. PMID:26604798

  9. Molecular Expression Profile Reveals Potential Biomarkers and Therapeutic Targets in Canine Endometrial Lesions

    PubMed Central

    Voorwald, Fabiana Azevedo; Marchi, Fabio Albuquerque; Villacis, Rolando Andre Rios; Alves, Carlos Eduardo Fonseca; Toniollo, Gilson Hélio; Amorim, Renee Laufer

    2015-01-01

    Cystic endometrial hyperplasia (CEH), mucometra, and pyometra are common uterine diseases in intact dogs, with pyometra being a life threatening disease. This study aimed to determine the gene expression profile of these lesions and potential biomarkers for closed-cervix pyometra, the most severe condition. Total RNA was extracted from 69 fresh endometrium samples collected from 21 healthy female dogs during diestrus, 16 CEH, 15 mucometra and 17 pyometra (eight open and nine closed-cervixes). Global gene expression was detected using the Affymetrix Canine Gene 1.0 ST Array. Unsupervised analysis revealed two clusters, one mainly composed of diestrus and CEH samples and the other by 12/15 mucometra and all pyometra samples. When comparing pyometra with other groups, 189 differentially expressed genes were detected. SLPI, PTGS2/COX2, MMP1, S100A8, S100A9 and IL8 were among the top up-regulated genes detected in pyometra, further confirmed by external expression data. Notably, a particular molecular profile in pyometra from animals previously treated with exogenous progesterone compounds was observed in comparison with pyometra from untreated dogs as well as with other groups irrespective of exogenous hormone treatment status. In addition to S100A8 and S100A9 genes, overexpression of the inflammatory cytokines IL1B, TNF and IL6 as well as LTF were detected in the pyometra from treated animals. Interestingly, closed pyometra was more frequently detected in treated dogs (64% versus 33%), with IL1B, TNF, LBP and CXCL10 among the most relevant overexpressed genes. This molecular signature associated with potential biomarkers and therapeutic targets, such as CXCL10 and COX2, should guide future clinical studies. Based on the gene expression profile we suggested that pyometra from progesterone treated dogs is a distinct molecular entity. PMID:26222498

  10. Molecular Expression Profile Reveals Potential Biomarkers and Therapeutic Targets in Canine Endometrial Lesions.

    PubMed

    Voorwald, Fabiana Azevedo; Marchi, Fabio Albuquerque; Villacis, Rolando Andre Rios; Alves, Carlos Eduardo Fonseca; Toniollo, Gilson Hélio; Amorim, Renee Laufer; Drigo, Sandra Aparecida; Rogatto, Silvia Regina

    2015-01-01

    Cystic endometrial hyperplasia (CEH), mucometra, and pyometra are common uterine diseases in intact dogs, with pyometra being a life threatening disease. This study aimed to determine the gene expression profile of these lesions and potential biomarkers for closed-cervix pyometra, the most severe condition. Total RNA was extracted from 69 fresh endometrium samples collected from 21 healthy female dogs during diestrus, 16 CEH, 15 mucometra and 17 pyometra (eight open and nine closed-cervixes). Global gene expression was detected using the Affymetrix Canine Gene 1.0 ST Array. Unsupervised analysis revealed two clusters, one mainly composed of diestrus and CEH samples and the other by 12/15 mucometra and all pyometra samples. When comparing pyometra with other groups, 189 differentially expressed genes were detected. SLPI, PTGS2/COX2, MMP1, S100A8, S100A9 and IL8 were among the top up-regulated genes detected in pyometra, further confirmed by external expression data. Notably, a particular molecular profile in pyometra from animals previously treated with exogenous progesterone compounds was observed in comparison with pyometra from untreated dogs as well as with other groups irrespective of exogenous hormone treatment status. In addition to S100A8 and S100A9 genes, overexpression of the inflammatory cytokines IL1B, TNF and IL6 as well as LTF were detected in the pyometra from treated animals. Interestingly, closed pyometra was more frequently detected in treated dogs (64% versus 33%), with IL1B, TNF, LBP and CXCL10 among the most relevant overexpressed genes. This molecular signature associated with potential biomarkers and therapeutic targets, such as CXCL10 and COX2, should guide future clinical studies. Based on the gene expression profile we suggested that pyometra from progesterone treated dogs is a distinct molecular entity. PMID:26222498

  11. Reduction of Nfia gene expression and subsequent target genes by binge alcohol in the fetal brain.

    PubMed

    Mandal, Chanchal; Park, Ji Hyun; Lee, Hyung Tae; Seo, Hyemyung; Chung, Il Yup; Choi, Ihn Geun; Jung, Kyoung Hwa; Chai, Young Gyu

    2015-06-26

    The objective of the present study was to investigate the changes in gene expression in the fetal brain (forebrain and hippocampus) caused by maternal binge alcohol consumption. Pregnant C57BL/6J mice were treated intragastrically with distilled phosphate-buffered saline (PBS) or ethanol (2.9 g/kg) from embryonic day (ED) 8-12. Microarray analysis revealed that a significant number of genes were altered at ED 18 in the developing brain. Specifically, in hippocampus, nuclear factor one alpha (Nfia) and three N-methyl-D-aspartate (Nmda) receptors (Nmdar1, Nmdar2b, and Nmdar2d) were down-regulated. The transcription factor Nfia controls gliogenesis, cell proliferation and Nmda-induced neuronal survival by regulating the expression of target genes. Some of the Nfia-target gene (Aldh1a, Folh1, Gjb6, Fgf1, Neurod1, Sept4, and Ntsr2) expressions were also altered as expected. These results suggest that the altered expression of Nfia and Nmda receptors may be associated with the etiology of fetal alcohol syndrome (FAS). The data presented in this report will contribute to the understanding of the molecular mechanisms associated with the effects of alcohol in FASD individuals. PMID:25982323

  12. Transcriptional targeting of tumor endothelial cells for gene therapy

    PubMed Central

    Dong, Zhihong; Nör, Jacques E.

    2009-01-01

    It is well known that angiogenesis plays a critical role in the pathobiology of tumors. Recent clinical trials have shown that inhibition of angiogenesis can be an effective therapeutic strategy for patients with cancer. However, one of the outstanding issues in anti-angiogenic treatment for cancer is the development of toxicities related to off-target effects of drugs. Transcriptional targeting of tumor endothelial cells involves the use of specific promoters for selective expression of therapeutic genes in the endothelial cells lining the blood vessels of tumors. Recently, several genes that are expressed specifically in tumor-associated endothelial cells have been identified and characterized. These discoveries have enhanced the prospectus of transcriptionaly targeting tumor endothelial cells for cancer gene therapy. In this manuscript, we review the promoters, vectors, and therapeutic genes that have been used for transcriptional targeting of tumor endothelial cells, and discuss the prospects of such approaches for cancer gene therapy. PMID:19393703

  13. Homologous gene targeting in Caenorhabditis elegans by biolistic transformation

    PubMed Central

    Berezikov, Eugene; Bargmann, Cornelia I.; Plasterk, Ronald H. A.

    2004-01-01

    Targeted homologous recombination is a powerful approach for genome manipulation that is widely used for gene alteration and knockouts in mouse and yeast. In Caenorhabditis elegans, several methods of target-selected mutagenesis have been implemented but none of them provides the opportunity of introducing exact predefined changes into the genome. Although anecdotal cases of homologous gene targeting in C.elegans have been reported, no practical technique of gene targeting has been developed so far. In this work we demonstrate that transformation of C.elegans by microparticle bombardment (biolistic transformation) can result in homologous recombination between introduced DNA and the chromosomal locus. We describe a scaled up version of biolistic transformation that can be used as a method for homologous gene targeting in the worm. PMID:14982959

  14. siRNA Design Software for a Target Gene-Specific RNA Interference

    PubMed Central

    Naito, Yuki; Ui-Tei, Kumiko

    2012-01-01

    RNA interference (RNAi) is a mechanism through which small interfering RNA (siRNA) induces sequence-specific posttranscriptional gene silencing. RNAi is commonly recognized as a powerful tool not only for functional genomics but also for therapeutic applications. Twenty-one-nucleotide-long siRNA suppresses the expression of the intended gene whose transcript possesses perfect complementarity to the siRNA guide strand. Hence, its silencing effect has been assumed to be extremely specific. However, accumulated evidences revealed that siRNA could downregulate unintended genes with partial complementarities mainly to the seven-nucleotide seed region of siRNA. This phenomenon is referred to as off-target effect. We have revealed that the capability to induce off-target effect is strongly correlated to the thermodynamic stability in siRNA seed-target duplex. For understanding accurate target gene function and successful therapeutic application, it may be critical to select a target gene-specific siRNA with minimized off-target effect. Here we present our siRNA design software for a target-specific RNAi. In addition, we also introduce the software programs open to the public for designing functional siRNAs. PMID:22701467

  15. siRNA Design Software for a Target Gene-Specific RNA Interference.

    PubMed

    Naito, Yuki; Ui-Tei, Kumiko

    2012-01-01

    RNA interference (RNAi) is a mechanism through which small interfering RNA (siRNA) induces sequence-specific posttranscriptional gene silencing. RNAi is commonly recognized as a powerful tool not only for functional genomics but also for therapeutic applications. Twenty-one-nucleotide-long siRNA suppresses the expression of the intended gene whose transcript possesses perfect complementarity to the siRNA guide strand. Hence, its silencing effect has been assumed to be extremely specific. However, accumulated evidences revealed that siRNA could downregulate unintended genes with partial complementarities mainly to the seven-nucleotide seed region of siRNA. This phenomenon is referred to as off-target effect. We have revealed that the capability to induce off-target effect is strongly correlated to the thermodynamic stability in siRNA seed-target duplex. For understanding accurate target gene function and successful therapeutic application, it may be critical to select a target gene-specific siRNA with minimized off-target effect. Here we present our siRNA design software for a target-specific RNAi. In addition, we also introduce the software programs open to the public for designing functional siRNAs. PMID:22701467

  16. Gene-targeting pharmaceuticals for single-gene disorders.

    PubMed

    Beaudet, Arthur L; Meng, Linyan

    2016-04-15

    The concept of orphan drugs for treatment of orphan genetic diseases is perceived enthusiastically at present, and this is leading to research investment on the part of governments, disease-specific foundations and industry. This review attempts to survey the potential to use traditional pharmaceuticals as opposed to biopharmaceuticals to treat single-gene disorders. The available strategies include the use of antisense oligonucleotides (ASOs) to alter splicing or knock-down expression of a transcript, siRNAs to knock-down gene expression and drugs for nonsense mutation read-through. There is an approved drug for biallelic knock-down of the APOB gene as treatment for familial hypercholesterolemia. Both ASOs and siRNAs are being explored to knock-down the transthyretin gene to prevent the related form of amyloidosis. The use of ASOs to alter gene-splicing to treat spinal muscular atrophy is in phase 3 clinical trials. Work is progressing on the use of ASOs to activate the normally silent paternal copy of the imprinted UBE3A gene in neurons as a treatment for Angelman syndrome. A gene-activation or gene-specific ramp-up strategy would be generally helpful if such could be developed. There is exciting theoretical potential for converting biopharmaceutical strategies such gene correction and CRISPR-Cas9 editing to a synthetic pharmaceutical approach. PMID:26628634

  17. Transcriptionally targeted gene therapy to detect and treat cancer

    PubMed Central

    Wu, Lily; Johnson, Mai; Sato, Makoto

    2010-01-01

    The greatest challenge in cancer treatment is to achieve the highest levels of specificity and efficacy. Cancer gene therapy could be designed specifically to express therapeutic genes to induce cancer cell destruction. Cancer-specific promoters are useful tools to accomplish targeted expression; however, high levels of gene expression are needed to achieve therapeutic efficacy. Incorporating an imaging reporter gene in tandem with the therapeutic gene will allow tangible proof of principle that gene expression occurs at the correct location and at a sufficient level. Gene-based imaging can advance cancer detection and diagnosis. By combining the cancer-targeted imaging and therapeutic strategies, the exciting prospect of a ‘one-two punch’ to find hidden, disseminated cancer cells and destroy them simultaneously can potentially be realized. PMID:14557054

  18. Promoterless gene targeting without nucleases ameliorates haemophilia B in mice.

    PubMed

    Barzel, A; Paulk, N K; Shi, Y; Huang, Y; Chu, K; Zhang, F; Valdmanis, P N; Spector, L P; Porteus, M H; Gaensler, K M; Kay, M A

    2015-01-15

    Site-specific gene addition can allow stable transgene expression for gene therapy. When possible, this is preferred over the use of promiscuously integrating vectors, which are sometimes associated with clonal expansion and oncogenesis. Site-specific endonucleases that can induce high rates of targeted genome editing are finding increasing applications in biological discovery and gene therapy. However, two safety concerns persist: endonuclease-associated adverse effects, both on-target and off-target; and oncogene activation caused by promoter integration, even without nucleases. Here we perform recombinant adeno-associated virus (rAAV)-mediated promoterless gene targeting without nucleases and demonstrate amelioration of the bleeding diathesis in haemophilia B mice. In particular, we target a promoterless human coagulation factor IX (F9) gene to the liver-expressed mouse albumin (Alb) locus. F9 is targeted, along with a preceding 2A-peptide coding sequence, to be integrated just upstream to the Alb stop codon. While F9 is fused to Alb at the DNA and RNA levels, two separate proteins are synthesized by way of ribosomal skipping. Thus, F9 expression is linked to robust hepatic albumin expression without disrupting it. We injected an AAV8-F9 vector into neonatal and adult mice and achieved on-target integration into ∼0.5% of the albumin alleles in hepatocytes. We established that F9 was produced only from on-target integration, and ribosomal skipping was highly efficient. Stable F9 plasma levels at 7-20% of normal were obtained, and treated F9-deficient mice had normal coagulation times. In conclusion, transgene integration as a 2A-fusion to a highly expressed endogenous gene may obviate the requirement for nucleases and/or vector-borne promoters. This method may allow for safe and efficacious gene targeting in both infants and adults by greatly diminishing off-target effects while still providing therapeutic levels of expression from integration. PMID:25363772

  19. AAV-mediated gene targeting methods for human cells

    PubMed Central

    Khan, Iram F; Hirata, Roli K; Russell, David W

    2013-01-01

    Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types, with targeting frequencies ranging from 10−5 to 10−2 per infected cell. these targeting frequencies are 1–4 logs higher than those obtained by conventional transfection or electroporation approaches. a wide variety of different types of mutations can be introduced into chromosomal loci with high fidelity and without genotoxicity. Here we provide a detailed protocol for gene targeting in human cells with AAV vectors. We describe methods for vector design, stock preparation and titration. optimized transduction protocols are provided for human pluripotent stem cells, mesenchymal stem cells, fibroblasts and transformed cell lines, as well as a method for identifying targeted clones by southern blots. this protocol (from vector design through a single round of targeting and screening) can be completed in ~10 weeks; each subsequent round of targeting and screening should take an additional 7 weeks. PMID:21455185

  20. Deep sequencing reveals 50 novel genes for recessive cognitive disorders.

    PubMed

    Najmabadi, Hossein; Hu, Hao; Garshasbi, Masoud; Zemojtel, Tomasz; Abedini, Seyedeh Sedigheh; Chen, Wei; Hosseini, Masoumeh; Behjati, Farkhondeh; Haas, Stefan; Jamali, Payman; Zecha, Agnes; Mohseni, Marzieh; Püttmann, Lucia; Vahid, Leyla Nouri; Jensen, Corinna; Moheb, Lia Abbasi; Bienek, Melanie; Larti, Farzaneh; Mueller, Ines; Weissmann, Robert; Darvish, Hossein; Wrogemann, Klaus; Hadavi, Valeh; Lipkowitz, Bettina; Esmaeeli-Nieh, Sahar; Wieczorek, Dagmar; Kariminejad, Roxana; Firouzabadi, Saghar Ghasemi; Cohen, Monika; Fattahi, Zohreh; Rost, Imma; Mojahedi, Faezeh; Hertzberg, Christoph; Dehghan, Atefeh; Rajab, Anna; Banavandi, Mohammad Javad Soltani; Hoffer, Julia; Falah, Masoumeh; Musante, Luciana; Kalscheuer, Vera; Ullmann, Reinhard; Kuss, Andreas Walter; Tzschach, Andreas; Kahrizi, Kimia; Ropers, H Hilger

    2011-10-01

    Common diseases are often complex because they are genetically heterogeneous, with many different genetic defects giving rise to clinically indistinguishable phenotypes. This has been amply documented for early-onset cognitive impairment, or intellectual disability, one of the most complex disorders known and a very important health care problem worldwide. More than 90 different gene defects have been identified for X-chromosome-linked intellectual disability alone, but research into the more frequent autosomal forms of intellectual disability is still in its infancy. To expedite the molecular elucidation of autosomal-recessive intellectual disability, we have now performed homozygosity mapping, exon enrichment and next-generation sequencing in 136 consanguineous families with autosomal-recessive intellectual disability from Iran and elsewhere. This study, the largest published so far, has revealed additional mutations in 23 genes previously implicated in intellectual disability or related neurological disorders, as well as single, probably disease-causing variants in 50 novel candidate genes. Proteins encoded by several of these genes interact directly with products of known intellectual disability genes, and many are involved in fundamental cellular processes such as transcription and translation, cell-cycle control, energy metabolism and fatty-acid synthesis, which seem to be pivotal for normal brain development and function. PMID:21937992

  1. Genes but Not Genomes Reveal Bacterial Domestication of Lactococcus Lactis

    PubMed Central

    Passerini, Delphine; Beltramo, Charlotte; Coddeville, Michele; Quentin, Yves; Ritzenthaler, Paul

    2010-01-01

    Background The population structure and diversity of Lactococcus lactis subsp. lactis, a major industrial bacterium involved in milk fermentation, was determined at both gene and genome level. Seventy-six lactococcal isolates of various origins were studied by different genotyping methods and thirty-six strains displaying unique macrorestriction fingerprints were analyzed by a new multilocus sequence typing (MLST) scheme. This gene-based analysis was compared to genomic characteristics determined by pulsed-field gel electrophoresis (PFGE). Methodology/Principal Findings The MLST analysis revealed that L. lactis subsp. lactis is essentially clonal with infrequent intra- and intergenic recombination; also, despite its taxonomical classification as a subspecies, it displays a genetic diversity as substantial as that within several other bacterial species. Genome-based analysis revealed a genome size variability of 20%, a value typical of bacteria inhabiting different ecological niches, and that suggests a large pan-genome for this subspecies. However, the genomic characteristics (macrorestriction pattern, genome or chromosome size, plasmid content) did not correlate to the MLST-based phylogeny, with strains from the same sequence type (ST) differing by up to 230 kb in genome size. Conclusion/Significance The gene-based phylogeny was not fully consistent with the traditional classification into dairy and non-dairy strains but supported a new classification based on ecological separation between “environmental” strains, the main contributors to the genetic diversity within the subspecies, and “domesticated” strains, subject to recent genetic bottlenecks. Comparison between gene- and genome-based analyses revealed little relationship between core and dispensable genome phylogenies, indicating that clonal diversification and phenotypic variability of the “domesticated” strains essentially arose through substantial genomic flux within the dispensable genome

  2. Self-targeting by CRISPR: gene regulation or autoimmunity?

    PubMed Central

    Stern, Adi; Keren, Leeat; Wurtzel, Omri; Amitai, Gil; Sorek, Rotem

    2010-01-01

    CRISPR/Cas is a recently discovered prokaryotic immune system, which is based on small RNAs (“spacers”) that restrict phage and plasmid infection. It has been hypothesized that CRISPRs can also regulate self gene expression by utilizing spacers that target self genes. By analyzing CRISPRs from 330 organisms we found that one in every 250 spacers is self targeting, and that such self-targeting occurs in 18% of all CRISPR-bearing organisms. However, complete lack of conservation across species, combined with abundance of degraded repeats near self-targeting spacers, suggests that self-targeting is a consequence of autoimmunity rather than gene regulation. We propose that accidental incorporation of self nucleic-acids by CRISPR can incur an autoimmune fitness cost, which may explain the abundance of degraded CRISPR systems across prokaryotes. PMID:20598393

  3. Cell Targeting in Anti-Cancer Gene Therapy

    PubMed Central

    Lila, Mohd Azmi Mohd; Siew, John Shia Kwong; Zakaria, Hayati; Saad, Suria Mohd; Ni, Lim Shen; Abdullah, Jafri Malin

    2004-01-01

    Gene therapy is a promising approach towards cancer treatment. The main aim of the therapy is to destroy cancer cells, usually by apoptotic mechanisms, and preserving others. However, its application has been hindered by many factors including poor cellular uptake, non-specific cell targeting and undesirable interferences with other genes or gene products. A variety of strategies exist to improve cellular uptake efficiency of gene-based therapies. This paper highlights advancements in gene therapy research and its application in relation to anti-cancer treatment. PMID:22977356

  4. Chromatin looping as a target for altering erythroid gene expression.

    PubMed

    Krivega, Ivan; Dean, Ann

    2016-03-01

    The β-hemoglobinopathies are the most common monogenic disorders in humans, with symptoms arising after birth when the fetal γ-globin genes are silenced and the adult β-globin gene is activated. There is a growing appreciation that genome organization and the folding of chromosomes are key determinants of gene transcription. Underlying this function is the activity of transcriptional enhancers that increase the transcription of target genes over long linear distances. To accomplish this, enhancers engage in close physical contact with target promoters through chromosome folding or looping that is orchestrated by protein complexes that bind to both sites and stabilize their interaction. We find that enhancer activity can be redirected with concomitant changes in gene transcription. Both targeting the β-globin locus control region (LCR) to the γ-globin gene in adult erythroid cells by tethering and epigenetic unmasking of a silenced γ-globin gene lead to increased frequency of LCR/γ-globin contacts and reduced LCR/β-globin contacts. The outcome of these manipulations is robust, pancellular γ-globin transcription activation with a concomitant reduction in β-globin transcription. These examples show that chromosome looping may be considered a therapeutic target for gene activation in β-thalassemia and sickle cell disease. PMID:26918894

  5. A Flexible Approach for Highly Multiplexed Candidate Gene Targeted Resequencing

    PubMed Central

    Natsoulis, Georges; Bell, John M.; Xu, Hua; Buenrostro, Jason D.; Ordonez, Heather; Grimes, Susan; Newburger, Daniel; Jensen, Michael; Zahn, Jacob M.; Zhang, Nancy; Ji, Hanlee P.

    2011-01-01

    We have developed an integrated strategy for targeted resequencing and analysis of gene subsets from the human exome for variants. Our capture technology is geared towards resequencing gene subsets substantially larger than can be done efficiently with simplex or multiplex PCR but smaller in scale than exome sequencing. We describe all the steps from the initial capture assay to single nucleotide variant (SNV) discovery. The capture methodology uses in-solution 80-mer oligonucleotides. To provide optimal flexibility in choosing human gene targets, we designed an in silico set of oligonucleotides, the Human OligoExome, that covers the gene exons annotated by the Consensus Coding Sequencing Project (CCDS). This resource is openly available as an Internet accessible database where one can download capture oligonucleotides sequences for any CCDS gene and design custom capture assays. Using this resource, we demonstrated the flexibility of this assay by custom designing capture assays ranging from 10 to over 100 gene targets with total capture sizes from over 100 Kilobases to nearly one Megabase. We established a method to reduce capture variability and incorporated indexing schemes to increase sample throughput. Our approach has multiple applications that include but are not limited to population targeted resequencing studies of specific gene subsets, validation of variants discovered in whole genome sequencing surveys and possible diagnostic analysis of disease gene subsets. We also present a cost analysis demonstrating its cost-effectiveness for large population studies. PMID:21738606

  6. The genetic regulatory network centered on Pto-Wuschela and its targets involved in wood formation revealed by association studies

    PubMed Central

    Yang, Xiaohui; Wei, Zunzheng; Du, Qingzhang; Chen, Jinhui; Wang, Qingshi; Quan, Mingyang; Song, Yuepeng; Xie, Jianbo; Zhang, Deqiang

    2015-01-01

    Transcription factors (TFs) regulate gene expression and can strongly affect phenotypes. However, few studies have examined TF variants and TF interactions with their targets in plants. Here, we used genetic association in 435 unrelated individuals of Populus tomentosa to explore the variants in Pto-Wuschela and its targets to decipher the genetic regulatory network of Pto-Wuschela. Our bioinformatics and co-expression analysis identified 53 genes with the motif TCACGTGA as putative targets of Pto-Wuschela. Single-marker association analysis showed that Pto-Wuschela was associated with wood properties, which is in agreement with the observation that it has higher expression in stem vascular tissues in Populus. Also, SNPs in the 53 targets were associated with growth or wood properties under additive or dominance effects, suggesting these genes and Pto-Wuschela may act in the same genetic pathways that affect variation in these quantitative traits. Epistasis analysis indicated that 75.5% of these genes directly or indirectly interacted Pto-Wuschela, revealing the coordinated genetic regulatory network formed by Pto-Wuschela and its targets. Thus, our study provides an alternative method for dissection of the interactions between a TF and its targets, which will strength our understanding of the regulatory roles of TFs in complex traits in plants. PMID:26549216

  7. Integrative Molecular Profiling Reveals Asparagine Synthetase Is a Target in Castration-Resistant Prostate Cancer

    PubMed Central

    Sircar, Kanishka; Huang, Heng; Hu, Limei; Cogdell, David; Dhillon, Jasreman; Tzelepi, Vassiliki; Efstathiou, Eleni; Koumakpayi, Ismaël H.; Saad, Fred; Luo, Dijun; Bismar, Tarek A.; Aparicio, Ana; Troncoso, Patricia; Navone, Nora; Zhang, Wei

    2013-01-01

    The identification of new and effective therapeutic targets for the lethal, castration-resistant stage of prostate cancer (CRPC) has been challenging because of both the paucity of adequate frozen tissues and a lack of integrated molecular analysis. Therefore, in this study, we performed a genome-wide analysis of DNA copy number alterations from 34 unique surgical CRPC specimens and 5 xenografts, with matched transcriptomic profiling of 25 specimens. An integrated analysis of these data revealed that the asparagine synthetase (ASNS) gene showed a gain in copy number and was overexpressed at the transcript level. The overexpression of ASNS was validated by analyzing other public CRPC data sets. ASNS protein expression, as detected by reverse-phase protein lysate array, was tightly correlated with gene copy number. In addition, ASNS protein expression, as determined by IHC analysis, was associated with progression to a therapy-resistant disease state in TMAs that included 77 castration-resistant and 40 untreated prostate cancer patient samples. Knockdown of ASNS by small-interfering RNAs in asparagine-deprived media led to growth inhibition in both androgen-responsive (ie, LNCaP) and castration-resistant (ie, C4-2B) prostate cancer cell lines and in cells isolated from a CRPC xenograft (ie, MDA PCa 180-30). Together, our results suggest that ASNS is up-regulated in cases of CRPC and that depletion of asparagine using ASNS inhibitors will be a novel strategy for targeting CRPC cells. PMID:22245216

  8. Chimeric DNA methyltransferases target DNA methylation to specific DNA sequences and repress expression of target genes

    PubMed Central

    Li, Fuyang; Papworth, Monika; Minczuk, Michal; Rohde, Christian; Zhang, Yingying; Ragozin, Sergei; Jeltsch, Albert

    2007-01-01

    Gene silencing by targeted DNA methylation has potential applications in basic research and therapy. To establish targeted methylation in human cell lines, the catalytic domains (CDs) of mouse Dnmt3a and Dnmt3b DNA methyltransferases (MTases) were fused to different DNA binding domains (DBD) of GAL4 and an engineered Cys2His2 zinc finger domain. We demonstrated that (i) Dense DNA methylation can be targeted to specific regions in gene promoters using chimeric DNA MTases. (ii) Site-specific methylation leads to repression of genes controlled by various cellular or viral promoters. (iii) Mutations affecting any of the DBD, MTase or target DNA sequences reduce targeted methylation and gene silencing. (iv) Targeted DNA methylation is effective in repressing Herpes Simplex Virus type 1 (HSV-1) infection in cell culture with the viral titer reduced by at least 18-fold in the presence of an MTase fused to an engineered zinc finger DBD, which binds a single site in the promoter of HSV-1 gene IE175k. In short, we show here that it is possible to direct DNA MTase activity to predetermined sites in DNA, achieve targeted gene silencing in mammalian cell lines and interfere with HSV-1 propagation. PMID:17151075

  9. Genomic analysis of primordial dwarfism reveals novel disease genes.

    PubMed

    Shaheen, Ranad; Faqeih, Eissa; Ansari, Shinu; Abdel-Salam, Ghada; Al-Hassnan, Zuhair N; Al-Shidi, Tarfa; Alomar, Rana; Sogaty, Sameera; Alkuraya, Fowzan S

    2014-02-01

    Primordial dwarfism (PD) is a disease in which severely impaired fetal growth persists throughout postnatal development and results in stunted adult size. The condition is highly heterogeneous clinically, but the use of certain phenotypic aspects such as head circumference and facial appearance has proven helpful in defining clinical subgroups. In this study, we present the results of clinical and genomic characterization of 16 new patients in whom a broad definition of PD was used (e.g., 3M syndrome was included). We report a novel PD syndrome with distinct facies in two unrelated patients, each with a different homozygous truncating mutation in CRIPT. Our analysis also reveals, in addition to mutations in known PD disease genes, the first instance of biallelic truncating BRCA2 mutation causing PD with normal bone marrow analysis. In addition, we have identified a novel locus for Seckel syndrome based on a consanguineous multiplex family and identified a homozygous truncating mutation in DNA2 as the likely cause. An additional novel PD disease candidate gene XRCC4 was identified by autozygome/exome analysis, and the knockout mouse phenotype is highly compatible with PD. Thus, we add a number of novel genes to the growing list of PD-linked genes, including one which we show to be linked to a novel PD syndrome with a distinct facial appearance. PD is extremely heterogeneous genetically and clinically, and genomic tools are often required to reach a molecular diagnosis. PMID:24389050

  10. Molecular profiles of Quadriceps muscle in myostatin-null mice reveal PI3K and apoptotic pathways as myostatin targets

    PubMed Central

    Chelh, Ilham; Meunier, Bruno; Picard, Brigitte; Reecy, Mark James; Chevalier, Catherine; Hocquette, Jean-François; Cassar-Malek, Isabelle

    2009-01-01

    Background Myostatin (MSTN), a member of the TGF-β superfamily, has been identified as a negative regulator of skeletal muscle mass. Inactivating mutations in the MSTN gene are responsible for the development of a hypermuscular phenotype. In this study, we performed transcriptomic and proteomic analyses to detect altered expression/abundance of genes and proteins. These differentially expressed genes and proteins may represent new molecular targets of MSTN and could be involved in the regulation of skeletal muscle mass. Results Transcriptomic analysis of the Quadriceps muscles of 5-week-old MSTN-null mice (n = 4) and their controls (n = 4) was carried out using microarray (human and murine oligonucleotide sequences) of 6,473 genes expressed in muscle. Proteomic profiles were analysed using two-dimensional gel electrophoresis coupled with mass spectrometry. Comparison of the transcriptomic profiles revealed 192 up- and 245 down- regulated genes. Genes involved in the PI3K pathway, insulin/IGF pathway, carbohydrate metabolism and apoptosis regulation were up-regulated. Genes belonging to canonical Wnt, calcium signalling pathways and cytokine-receptor cytokine interaction were down-regulated. Comparison of the protein profiles revealed 20 up- and 18 down-regulated proteins spots. Knockout of the MSTN gene was associated with up-regulation of proteins involved in glycolytic shift of the muscles and down-regulation of proteins involved in oxidative energy metabolism. In addition, an increased abundance of survival/anti-apoptotic factors were observed. Conclusion All together, these results showed a differential expression of genes and proteins related to the muscle energy metabolism and cell survival/anti-apoptotic pathway (e.g. DJ-1, PINK1, 14-3-3ε protein, TCTP/GSK-3β). They revealed the PI3K and apoptotic pathways as MSTN targets and are in favour of a role of MSTN as a modulator of cell survival in vivo. PMID:19397818

  11. Identification of p53-target genes in Danio rerio

    PubMed Central

    Mandriani, Barbara; Castellana, Stefano; Rinaldi, Carmela; Manzoni, Marta; Venuto, Santina; Rodriguez-Aznar, Eva; Galceran, Juan; Nieto, M. Angela; Borsani, Giuseppe; Monti, Eugenio; Mazza, Tommaso; Merla, Giuseppe; Micale, Lucia

    2016-01-01

    To orchestrate the genomic response to cellular stress signals, p53 recognizes and binds to DNA containing specific and well-characterized p53-responsive elements (REs). Differences in RE sequences can strongly affect the p53 transactivation capacity and occur even between closely related species. Therefore, the identification and characterization of a species-specific p53 Binding sistes (BS) consensus sequence and of the associated target genes may help to provide new insights into the evolution of the p53 regulatory networks across different species. Although p53 functions were studied in a wide range of species, little is known about the p53-mediated transcriptional signature in Danio rerio. Here, we designed and biochemically validated a computational approach to identify novel p53 target genes in Danio rerio genome. Screening all the Danio rerio genome by pattern-matching-based analysis, we found p53 RE-like patterns proximal to 979 annotated Danio rerio genes. Prioritization analysis identified a subset of 134 candidate pattern-related genes, 31 of which have been investigated in further biochemical assays. Our study identified runx1, axin1, traf4a, hspa8, col4a5, necab2, and dnajc9 genes as novel direct p53 targets and 12 additional p53-controlled genes in Danio rerio genome. The proposed combinatorial approach resulted to be highly sensitive and robust for identifying new p53 target genes also in additional animal species. PMID:27581768

  12. Identification of p53-target genes in Danio rerio.

    PubMed

    Mandriani, Barbara; Castellana, Stefano; Rinaldi, Carmela; Manzoni, Marta; Venuto, Santina; Rodriguez-Aznar, Eva; Galceran, Juan; Nieto, M Angela; Borsani, Giuseppe; Monti, Eugenio; Mazza, Tommaso; Merla, Giuseppe; Micale, Lucia

    2016-01-01

    To orchestrate the genomic response to cellular stress signals, p53 recognizes and binds to DNA containing specific and well-characterized p53-responsive elements (REs). Differences in RE sequences can strongly affect the p53 transactivation capacity and occur even between closely related species. Therefore, the identification and characterization of a species-specific p53 Binding sistes (BS) consensus sequence and of the associated target genes may help to provide new insights into the evolution of the p53 regulatory networks across different species. Although p53 functions were studied in a wide range of species, little is known about the p53-mediated transcriptional signature in Danio rerio. Here, we designed and biochemically validated a computational approach to identify novel p53 target genes in Danio rerio genome. Screening all the Danio rerio genome by pattern-matching-based analysis, we found p53 RE-like patterns proximal to 979 annotated Danio rerio genes. Prioritization analysis identified a subset of 134 candidate pattern-related genes, 31 of which have been investigated in further biochemical assays. Our study identified runx1, axin1, traf4a, hspa8, col4a5, necab2, and dnajc9 genes as novel direct p53 targets and 12 additional p53-controlled genes in Danio rerio genome. The proposed combinatorial approach resulted to be highly sensitive and robust for identifying new p53 target genes also in additional animal species. PMID:27581768

  13. Genome-wide characterization of cis-acting DNA targets reveals the transcriptional regulatory framework of opaque2 in maize.

    PubMed

    Li, Chaobin; Qiao, Zhenyi; Qi, Weiwei; Wang, Qian; Yuan, Yue; Yang, Xi; Tang, Yuanping; Mei, Bing; Lv, Yuanda; Zhao, Han; Xiao, Han; Song, Rentao

    2015-03-01

    Opaque2 (O2) is a transcription factor that plays important roles during maize endosperm development. Mutation of the O2 gene improves the nutritional value of maize seeds but also confers pleiotropic effects that result in reduced agronomic quality. To reveal the transcriptional regulatory framework of O2, we studied the transcriptome of o2 mutants using RNA sequencing (RNA-Seq) and determined O2 DNA binding targets using chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-Seq). The RNA-Seq analysis revealed 1605 differentially expressed genes (DEGs) and 383 differentially expressed long, noncoding RNAs. The DEGs cover a wide range of functions related to nutrient reservoir activity, nitrogen metabolism, stress resistance, etc. ChIP-Seq analysis detected 1686 O2 DNA binding sites distributed over 1143 genes. Overlay of the RNA-Seq and ChIP-Seq results revealed 35 O2-modulated target genes. We identified four O2 binding motifs; among them, TGACGTGG appears to be the most conserved and strongest. We confirmed that, except for the 16- and 18-kD zeins, O2 directly regulates expression of all other zeins. O2 directly regulates two transcription factors, genes linked to carbon and amino acid metabolism and abiotic stress resistance. We built a hierarchical regulatory model for O2 that provides an understanding of its pleiotropic biological effects. PMID:25691733

  14. Nanoparticle-based targeted gene therapy for lung cancer

    PubMed Central

    Lee, Hung-Yen; Mohammed, Kamal A; Nasreen, Najmunnisa

    2016-01-01

    Despite striking insights on lung cancer progression, and cutting-edge therapeutic approaches the survival of patients with lung cancer, remains poor. In recent years, targeted gene therapy with nanoparticles is one of the most rapidly evolving and extensive areas of research for lung cancer. The major goal of targeted gene therapy is to bring forward a safe and efficient treatment to cancer patients via specifically targeting and deterring cancer cells in the body. To achieve high therapeutic efficacy of gene delivery, various carriers have been engineered and developed to provide protection to the genetic materials and efficient delivery to targeted cancer cells. Nanoparticles play an important role in the area of drug delivery and have been widely applied in cancer treatments for the purposes of controlled release and cancer cell targeting. Nanoparticles composed of artificial polymers, proteins, polysaccharides and lipids have been developed for the delivery of therapeutic deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences to target cancer. In addition, the effectiveness of cancer targeting has been enhanced by surface modification or conjugation with biomolecules on the surface of nanoparticles. In this review article we provide an overview on the latest developments in nanoparticle-based targeted gene therapy for lung cancers. Firstly, we outline the conventional therapies and discuss strategies for targeted gene therapy using nanoparticles. Secondly, we provide the most representative and recent researches in lung cancers including malignant pleural mesothelioma, mainly focusing on the application of Polymeric, Lipid-based, and Metal-based nanoparticles. Finally, we discuss current achievements and future challenges. PMID:27294004

  15. Nanoparticle-based targeted gene therapy for lung cancer.

    PubMed

    Lee, Hung-Yen; Mohammed, Kamal A; Nasreen, Najmunnisa

    2016-01-01

    Despite striking insights on lung cancer progression, and cutting-edge therapeutic approaches the survival of patients with lung cancer, remains poor. In recent years, targeted gene therapy with nanoparticles is one of the most rapidly evolving and extensive areas of research for lung cancer. The major goal of targeted gene therapy is to bring forward a safe and efficient treatment to cancer patients via specifically targeting and deterring cancer cells in the body. To achieve high therapeutic efficacy of gene delivery, various carriers have been engineered and developed to provide protection to the genetic materials and efficient delivery to targeted cancer cells. Nanoparticles play an important role in the area of drug delivery and have been widely applied in cancer treatments for the purposes of controlled release and cancer cell targeting. Nanoparticles composed of artificial polymers, proteins, polysaccharides and lipids have been developed for the delivery of therapeutic deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences to target cancer. In addition, the effectiveness of cancer targeting has been enhanced by surface modification or conjugation with biomolecules on the surface of nanoparticles. In this review article we provide an overview on the latest developments in nanoparticle-based targeted gene therapy for lung cancers. Firstly, we outline the conventional therapies and discuss strategies for targeted gene therapy using nanoparticles. Secondly, we provide the most representative and recent researches in lung cancers including malignant pleural mesothelioma, mainly focusing on the application of Polymeric, Lipid-based, and Metal-based nanoparticles. Finally, we discuss current achievements and future challenges. PMID:27294004

  16. Single molecule targeted sequencing for cancer gene mutation detection.

    PubMed

    Gao, Yan; Deng, Liwei; Yan, Qin; Gao, Yongqian; Wu, Zengding; Cai, Jinsen; Ji, Daorui; Li, Gailing; Wu, Ping; Jin, Huan; Zhao, Luyang; Liu, Song; Ge, Liangjin; Deem, Michael W; He, Jiankui

    2016-01-01

    With the rapid decline in cost of sequencing, it is now affordable to examine multiple genes in a single disease-targeted clinical test using next generation sequencing. Current targeted sequencing methods require a separate step of targeted capture enrichment during sample preparation before sequencing. Although there are fast sample preparation methods available in market, the library preparation process is still relatively complicated for physicians to use routinely. Here, we introduced an amplification-free Single Molecule Targeted Sequencing (SMTS) technology, which combined targeted capture and sequencing in one step. We demonstrated that this technology can detect low-frequency mutations using artificially synthesized DNA sample. SMTS has several potential advantages, including simple sample preparation thus no biases and errors are introduced by PCR reaction. SMTS has the potential to be an easy and quick sequencing technology for clinical diagnosis such as cancer gene mutation detection, infectious disease detection, inherited condition screening and noninvasive prenatal diagnosis. PMID:27193446

  17. Single molecule targeted sequencing for cancer gene mutation detection

    PubMed Central

    Gao, Yan; Deng, Liwei; Yan, Qin; Gao, Yongqian; Wu, Zengding; Cai, Jinsen; Ji, Daorui; Li, Gailing; Wu, Ping; Jin, Huan; Zhao, Luyang; Liu, Song; Ge, Liangjin; Deem, Michael W.; He, Jiankui

    2016-01-01

    With the rapid decline in cost of sequencing, it is now affordable to examine multiple genes in a single disease-targeted clinical test using next generation sequencing. Current targeted sequencing methods require a separate step of targeted capture enrichment during sample preparation before sequencing. Although there are fast sample preparation methods available in market, the library preparation process is still relatively complicated for physicians to use routinely. Here, we introduced an amplification-free Single Molecule Targeted Sequencing (SMTS) technology, which combined targeted capture and sequencing in one step. We demonstrated that this technology can detect low-frequency mutations using artificially synthesized DNA sample. SMTS has several potential advantages, including simple sample preparation thus no biases and errors are introduced by PCR reaction. SMTS has the potential to be an easy and quick sequencing technology for clinical diagnosis such as cancer gene mutation detection, infectious disease detection, inherited condition screening and noninvasive prenatal diagnosis. PMID:27193446

  18. Gene expression profiles of prostate cancer reveal involvement of multiple molecular pathways in the metastatic process

    PubMed Central

    Chandran, Uma R; Ma, Changqing; Dhir, Rajiv; Bisceglia, Michelle; Lyons-Weiler, Maureen; Liang, Wenjing; Michalopoulos, George; Becich, Michael; Monzon, Federico A

    2007-01-01

    Background Prostate cancer is characterized by heterogeneity in the clinical course that often does not correlate with morphologic features of the tumor. Metastasis reflects the most adverse outcome of prostate cancer, and to date there are no reliable morphologic features or serum biomarkers that can reliably predict which patients are at higher risk of developing metastatic disease. Understanding the differences in the biology of metastatic and organ confined primary tumors is essential for developing new prognostic markers and therapeutic targets. Methods Using Affymetrix oligonucleotide arrays, we analyzed gene expression profiles of 24 androgen-ablation resistant metastatic samples obtained from 4 patients and a previously published dataset of 64 primary prostate tumor samples. Differential gene expression was analyzed after removing potentially uninformative stromal genes, addressing the differences in cellular content between primary and metastatic tumors. Results The metastatic samples are highly heterogenous in expression; however, differential expression analysis shows that 415 genes are upregulated and 364 genes are downregulated at least 2 fold in every patient with metastasis. The expression profile of metastatic samples reveals changes in expression of a unique set of genes representing both the androgen ablation related pathways and other metastasis related gene networks such as cell adhesion, bone remodelling and cell cycle. The differentially expressed genes include metabolic enzymes, transcription factors such as Forkhead Box M1 (FoxM1) and cell adhesion molecules such as Osteopontin (SPP1). Conclusion We hypothesize that these genes have a role in the biology of metastatic disease and that they represent potential therapeutic targets for prostate cancer. PMID:17430594

  19. Targeted Deep Sequencing Reveals No Definitive Evidence for Somatic Mosaicism in Atrial Fibrillation

    PubMed Central

    Roberts, Jason D.; Longoria, James; Poon, Annie; Gollob, Michael H.; Dewland, Thomas A.; Kwok, Pui-Yan; Olgin, Jeffrey E.; Deo, Rahul C.; Marcus, Gregory M.

    2015-01-01

    Background Studies of ≤15 atrial fibrillation (AF) patients have identified atrial-specific mutations within connexin genes, suggesting that somatic mutations may account for sporadic cases of the arrhythmia. We sought to identify atrial somatic mutations among patients with and without AF using targeted deep next-generation sequencing of 560 genes, including genetic culprits implicated in AF, the Mendelian cardiomyopathies and channelopathies, and all ion channels within the genome. Methods and Results Targeted gene capture and next generation sequencing were performed on DNA from lymphocytes and left atrial appendages of 34 patients (25 with AF). Twenty AF patients had undergone cardiac surgery exclusively for pulmonary vein isolation, and 17 had no structural heart disease. Sequence alignment and variant calling were performed for each atrial-lymphocyte pair using the Burrows-Wheeler Aligner, the Genome Analysis Toolkit, and MuTect packages. Next generation sequencing yielded a median 265-fold coverage depth (IQR 164–369). Comparison of the 3 million base pairs from each atrial-lymphocyte pair revealed a single potential somatic missense mutation in 3 AF patients and 2 in a single control (12 vs. 11%; p=1). All potential discordant variants had low allelic fractions (range: 2.3–7.3%) and none were detected with conventional sequencing. Conclusions Using high-depth next generation sequencing and state-of-the art somatic mutation calling approaches, no pathogenic atrial somatic mutations could be confirmed among 25 AF patients in a comprehensive cardiac arrhythmia genetic panel. These findings indicate that atrial specific mutations are rare and that somatic mosaicism is unlikely to exert a prominent role in AF pathogenesis. PMID:25406240

  20. Targeted gene therapy and cell reprogramming in Fanconi anemia

    PubMed Central

    Rio, Paula; Baños, Rocio; Lombardo, Angelo; Quintana-Bustamante, Oscar; Alvarez, Lara; Garate, Zita; Genovese, Pietro; Almarza, Elena; Valeri, Antonio; Díez, Begoña; Navarro, Susana; Torres, Yaima; Trujillo, Juan P; Murillas, Rodolfo; Segovia, Jose C; Samper, Enrique; Surralles, Jordi; Gregory, Philip D; Holmes, Michael C; Naldini, Luigi; Bueren, Juan A

    2014-01-01

    Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies. PMID:24859981

  1. Immunologic advances reveal new targets in psoriasis and psoriatic arthritis.

    PubMed

    Mortezavi, Mahta; Ritchlin, Christopher

    2015-10-01

    Psoriatic arthritis (PsA) is a chronic inflammatory joint disorder with heterogeneous clinical features that may include plaque psoriasis, joint inflammation, enthesitis, dactylitis, and abnormal bone turn over. This disease is common, affecting up to 0.5% of the population with equal male and female prevalence. Until recently, few treatment options were available for PsA and patients suffered immense physical and social burden. Traditional disease modifying agents show limited efficacy in the treatment of PsA. Anti-tumor necrosis factor (TNF) drugs are effective for all the manifestations, yet recent studies show that up to 50% of patients either do not tolerate these medications or do not maintain a clinical response. The evolution in the treatment of PsA emerged from improved understanding of the pathophysiology of the disease with Th1 and Th17 cells taking center stage. Targeting TNF along with cytokines in the IL-23/TH17 pathway (IL-23, IL-17, and IL-22) holds great promise for improved treatment outcomes in PsA. PMID:26562469

  2. Bioimage analysis of Shigella infection reveals targeting of colonic crypts

    PubMed Central

    Arena, Ellen T.; Campbell-Valois, Francois-Xavier; Tinevez, Jean-Yves; Nigro, Giulia; Sachse, Martin; Moya-Nilges, Maryse; Nothelfer, Katharina; Marteyn, Benoit; Shorte, Spencer L.; Sansonetti, Philippe J.

    2015-01-01

    Few studies within the pathogenic field have used advanced imaging and analytical tools to quantitatively measure pathogenicity in vivo. In this work, we present a novel approach for the investigation of host–pathogen processes based on medium-throughput 3D fluorescence imaging. The guinea pig model for Shigella flexneri invasion of the colonic mucosa was used to monitor the infectious process over time with GFP-expressing S. flexneri. A precise quantitative imaging protocol was devised to follow individual S. flexneri in a large tissue volume. An extensive dataset of confocal images was obtained and processed to extract specific quantitative information regarding the progression of S. flexneri infection in an unbiased and exhaustive manner. Specific parameters included the analysis of S. flexneri positions relative to the epithelial surface, S. flexneri density within the tissue, and volume of tissue destruction. In particular, at early time points, there was a clear association of S. flexneri with crypts, key morphological features of the colonic mucosa. Numerical simulations based on random bacterial entry confirmed the bias of experimentally measured S. flexneri for early crypt targeting. The application of a correlative light and electron microscopy technique adapted for thick tissue samples further confirmed the location of S. flexneri within colonocytes at the mouth of crypts. This quantitative imaging approach is a novel means to examine host–pathogen systems in a tailored and robust manner, inclusive of the infectious agent. PMID:26056271

  3. Bioimage analysis of Shigella infection reveals targeting of colonic crypts.

    PubMed

    Arena, Ellen T; Campbell-Valois, Francois-Xavier; Tinevez, Jean-Yves; Nigro, Giulia; Sachse, Martin; Moya-Nilges, Maryse; Nothelfer, Katharina; Marteyn, Benoit; Shorte, Spencer L; Sansonetti, Philippe J

    2015-06-23

    Few studies within the pathogenic field have used advanced imaging and analytical tools to quantitatively measure pathogenicity in vivo. In this work, we present a novel approach for the investigation of host-pathogen processes based on medium-throughput 3D fluorescence imaging. The guinea pig model for Shigella flexneri invasion of the colonic mucosa was used to monitor the infectious process over time with GFP-expressing S. flexneri. A precise quantitative imaging protocol was devised to follow individual S. flexneri in a large tissue volume. An extensive dataset of confocal images was obtained and processed to extract specific quantitative information regarding the progression of S. flexneri infection in an unbiased and exhaustive manner. Specific parameters included the analysis of S. flexneri positions relative to the epithelial surface, S. flexneri density within the tissue, and volume of tissue destruction. In particular, at early time points, there was a clear association of S. flexneri with crypts, key morphological features of the colonic mucosa. Numerical simulations based on random bacterial entry confirmed the bias of experimentally measured S. flexneri for early crypt targeting. The application of a correlative light and electron microscopy technique adapted for thick tissue samples further confirmed the location of S. flexneri within colonocytes at the mouth of crypts. This quantitative imaging approach is a novel means to examine host-pathogen systems in a tailored and robust manner, inclusive of the infectious agent. PMID:26056271

  4. Evidence for Tissue-Specific JAK/STAT Target Genes in Drosophila Optic Lobe Development

    PubMed Central

    Wang, Hongbin; Chen, Xi; He, Teng; Zhou, Yanna; Luo, Hong

    2013-01-01

    The evolutionarily conserved JAK/STAT pathway plays important roles in development and disease processes in humans. Although the signaling process has been well established, we know relatively little about what the relevant target genes are that mediate JAK/STAT activation during development. Here, we have used genome-wide microarrays to identify JAK/STAT targets in the optic lobes of the Drosophila brain and identified 47 genes that are positively regulated by JAK/STAT. About two-thirds of the genes encode proteins that have orthologs in humans. The STAT targets in the optic lobe appear to be different from the targets identified in other tissues, suggesting that JAK/STAT signaling may regulate different target genes in a tissue-specific manner. Functional analysis of Nop56, a cell-autonomous STAT target, revealed an essential role for this gene in the growth and proliferation of neuroepithelial stem cells in the optic lobe and an inhibitory role in lamina neurogenesis. PMID:24077308

  5. SLC7A5 act as a potential leukemic transformation target gene in myelodysplastic syndrome

    PubMed Central

    Ma, Yan; Song, Jing; Chen, Bobin; Xu, Xiaoping; Lin, Guowei

    2016-01-01

    Objective Myelodysplastic syndromes (MDS) are a heterogenous group of clonal hematopoietic stem cell disorders characterized by increased risk of leukemic transformation. This study identifies microRNAs(miRNA) and miRNA targets that might represent leukemic transformation markers for MDS. Methods Based on our previously established nested case-control study cohort of MDS patients, we chose paired patients to undergo Angilent 8 × 15K human miRNA microarrays. Target prediction analysis was administrated using targetscan 5.1 software. We further investigated the function of target gene in MDS cell line using siRNA method, including cell proliferation, cell apoptosis, cell cycle and electron microscope. Results Finally we screened a subset of 7 miRNAs to be significantly differentially expressed between the case (at the end of follow up with leukemic transformation) and control group (at the end of follow up without leukemic transformation). Target prediction analysis revealed SLC7A5 was the common target gene of these 7 miRNAs. Further study on the function of SLC7A5 gene in SKM-1 cell line showed that downregulation of SLC7A5 inhibited SKM-1 cells proliferation, increased apoptosis and caused cell cycle arrest in the G0/G1 stage. Conclusion Our data indicate that SLC7A5 gene may act as a potential leukemic transformation target gene in MDS. PMID:26657287

  6. Transcriptional targets of the schizophrenia risk gene MIR137

    PubMed Central

    Collins, A L; Kim, Y; Bloom, R J; Kelada, S N; Sethupathy, P; Sullivan, P F

    2014-01-01

    Genome-wide association studies (GWAS) have strongly implicated MIR137 (the gene encoding the microRNA miR-137) in schizophrenia. A parsimonious hypothesis is that a pathway regulated by miR-137 is important in the etiology of schizophrenia. Full evaluation of this hypothesis requires more definitive knowledge about biological targets of miR-137, which is currently lacking. Our goals were to expand knowledge of the biology of miR-137 by identifying its empirical targets, and to test whether the resulting lists of direct and indirect targets were enriched for genes and pathways involved in risk for schizophrenia. We overexpressed miR-137 in a human neural stem cell line and analyzed gene expression changes at 24 and 48 h using RNA sequencing. Following miR-137 overexpression, 202 and 428 genes were differentially expressed after 24 and 48 h. Genes differentially expressed at 24 h were enriched for transcription factors and cell cycle genes, and differential expression at 48 h affected a wider variety of pathways. Pathways implicated in schizophrenia were upregulated in the 48 h findings (major histocompatibility complex, synapses, FMRP interacting RNAs and calcium channels). Critically, differentially expressed genes at 48 h were enriched for smaller association P-values in the largest published schizophrenia GWAS. This work provides empirical support for a role of miR-137 in the etiology of schizophrenia. PMID:24984191

  7. Synthetic lethal screening reveals FGFR as one of the combinatorial targets to overcome resistance to Met-targeted therapy

    PubMed Central

    Kim, Bogyou; Wang, Shangzi; Lee, Ji Min; Jeong, Yunju; Ahn, TaeJin; Son, Dae-Soon; Park, Hye Won; Yoo, Hyeon-seok; Song, Yun-Jeong; Lee, Eunjin; Oh, Young Mi; Lee, Saet Byoul; Choi, Jaehyun; Murray, Joseph C; Zhou, Yan; Song, Paul H; Kim, Kyung-Ah; Weiner, Louis M

    2014-01-01

    Met is a receptor tyrosine kinase that promotes cancer progression. In addition, Met has been implicated in resistance of tumors to various targeted therapies such as EGFR inhibitors in lung cancers, and has been prioritized as a key molecular target for cancer therapy. However, the underlying mechanism of resistance to Met targeting drugs is poorly understood. Here, we describe screening of 1310 genes to search for key regulators related to drug resistance to an anti-Met therapeutic antibody (SAIT301) by employing a siRNA-based synthetic lethal screening method. We found that knockdown of 69 genes in Met-amplified MKN45 cells sensitized the anti-tumor activity of SAIT301. Pathway analysis of these 69 genes implicated FGFR as a key regulator for anti-proliferative effects of Met targeting drugs. Inhibition of FGFR3 increased target cell apoptosis through the suppression of Bcl-xL expression, followed by reduced cancer cell growth in the presence of Met targeting drugs. Treatment of cells with the FGFR inhibitors substantially restored the efficacy of SAIT301 in SAIT301-resistant cells and enhanced the efficacy in SAIT301-sensitive cells. In addition to FGFR3, integrin β3 is another potential target for combination treatment with SAIT301. Suppression of integrin β3 decreased AKT phosphorylation in SAIT301-resistant cells and restores SAIT301 responsiveness in HCC1954 cells, which are resistant to SAIT301. Gene expression analysis using CCLE database shows cancer cells with high levels of FGFR and integrin β3 are resistant to crizotinib treatment, suggesting FGFR and integrin β3 could be used as predictive markers for Met targeted therapy and provide a potential therapeutic option to overcome acquired and innate resistance for the Met targeting drugs. PMID:24662823

  8. High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities.

    PubMed

    Hart, Traver; Chandrashekhar, Megha; Aregger, Michael; Steinhart, Zachary; Brown, Kevin R; MacLeod, Graham; Mis, Monika; Zimmermann, Michal; Fradet-Turcotte, Amelie; Sun, Song; Mero, Patricia; Dirks, Peter; Sidhu, Sachdev; Roth, Frederick P; Rissland, Olivia S; Durocher, Daniel; Angers, Stephane; Moffat, Jason

    2015-12-01

    The ability to perturb genes in human cells is crucial for elucidating gene function and holds great potential for finding therapeutic targets for diseases such as cancer. To extend the catalog of human core and context-dependent fitness genes, we have developed a high-complexity second-generation genome-scale CRISPR-Cas9 gRNA library and applied it to fitness screens in five human cell lines. Using an improved Bayesian analytical approach, we consistently discover 5-fold more fitness genes than were previously observed. We present a list of 1,580 human core fitness genes and describe their general properties. Moreover, we demonstrate that context-dependent fitness genes accurately recapitulate pathway-specific genetic vulnerabilities induced by known oncogenes and reveal cell-type-specific dependencies for specific receptor tyrosine kinases, even in oncogenic KRAS backgrounds. Thus, rigorous identification of human cell line fitness genes using a high-complexity CRISPR-Cas9 library affords a high-resolution view of the genetic vulnerabilities of a cell. PMID:26627737

  9. Gene Body Methylation can alter Gene Expression and is a Therapeutic Target in Cancer

    PubMed Central

    Yang, Xiaojing; Han, Han; De Carvalho, Daniel D.; Lay, Fides D.; Jones, Peter A.; Liang, Gangning

    2014-01-01

    SUMMARY DNA methylation in promoters is well known to silence genes and is the presumed therapeutic target of methylation inhibitors. Gene body methylation is positively correlated with expression yet its function is unknown. We show that 5-aza-2'-deoxycytidine treatment not only reactivates genes but decreases the over-expression of genes, many of which are involved in metabolic processes regulated by c-MYC. Down-regulation is caused by DNA demethylation of the gene bodies and restoration of high levels of expression requires remethylation by DNMT3B. Gene body methylation may therefore be an unexpected therapeutic target for DNA methylation inhibitors, resulting in the normalization of gene over-expression induced during carcinogenesis. Our results provide direct evidence for a causal relationship between gene body methylation and transcription. PMID:25263941

  10. Targeting of AID-mediated sequence diversification to immunoglobulin genes.

    PubMed

    Kothapalli, Naga Rama; Fugmann, Sebastian D

    2011-04-01

    Activation-induced cytidine deaminase (AID) is a key enzyme for antibody-mediated immune responses. Antibodies are encoded by the immunoglobulin genes and AID acts as a transcription-dependent DNA mutator on these genes to improve antibody affinity and effector functions. An emerging theme in field is that many transcribed genes are potential targets of AID, presenting an obvious danger to genomic integrity. Thus there are mechanisms in place to ensure that mutagenic outcomes of AID activity are specifically restricted to the immunoglobulin loci. Cis-regulatory targeting elements mediate this effect and their mode of action is probably a combination of immunoglobulin gene specific activation of AID and a perversion of faithful DNA repair towards error-prone outcomes. PMID:21295456

  11. Targeting of AID-mediated sequence diversification to immunoglobulin genes

    PubMed Central

    Kothapalli, Naga Rama; Fugmann, Sebastian D.

    2011-01-01

    Activation-induced cytidine deaminase (AID) is a key enzyme for antibody-mediated immune responses. Antibodies are encoded by the immunoglobulin genes and AID acts as a transcription-dependent DNA mutator on these genes to improve antibody affinity and effector functions. An emerging theme in field is that many transcribed genes are potential targets of AID, presenting an obvious danger to genomic integrity. Thus there are mechanisms in place to ensure that mutagenic outcomes of AID activity are specifically restricted to the immunoglobulin loci. Cis-regulatory targeting elements mediate this effect and their mode of action is likely a combination of immunoglobulin gene specific activation of AID and a perversion of faithful DNA repair towards error-prone outcomes. PMID:21295456

  12. Core Promoter Functions in the Regulation of Gene Expression of Drosophila Dorsal Target Genes*

    PubMed Central

    Zehavi, Yonathan; Kuznetsov, Olga; Ovadia-Shochat, Avital; Juven-Gershon, Tamar

    2014-01-01

    Developmental processes are highly dependent on transcriptional regulation by RNA polymerase II. The RNA polymerase II core promoter is the ultimate target of a multitude of transcription factors that control transcription initiation. Core promoters consist of core promoter motifs, e.g. the initiator, TATA box, and the downstream core promoter element (DPE), which confer specific properties to the core promoter. Here, we explored the importance of core promoter functions in the dorsal-ventral developmental gene regulatory network. This network includes multiple genes that are activated by different nuclear concentrations of Dorsal, an NFκB homolog transcription factor, along the dorsal-ventral axis. We show that over two-thirds of Dorsal target genes contain DPE sequence motifs, which is significantly higher than the proportion of DPE-containing promoters in Drosophila genes. We demonstrate that multiple Dorsal target genes are evolutionarily conserved and functionally dependent on the DPE. Furthermore, we have analyzed the activation of key Dorsal target genes by Dorsal, as well as by another Rel family transcription factor, Relish, and the dependence of their activation on the DPE motif. Using hybrid enhancer-promoter constructs in Drosophila cells and embryo extracts, we have demonstrated that the core promoter composition is an important determinant of transcriptional activity of Dorsal target genes. Taken together, our results provide evidence for the importance of core promoter composition in the regulation of Dorsal target genes. PMID:24634215

  13. Expression of PAX8 Target Genes in Papillary Thyroid Carcinoma.

    PubMed

    Rosignolo, Francesca; Sponziello, Marialuisa; Durante, Cosimo; Puppin, Cinzia; Mio, Catia; Baldan, Federica; Di Loreto, Carla; Russo, Diego; Filetti, Sebastiano; Damante, Giuseppe

    2016-01-01

    PAX8 is a thyroid-specific transcription factor whose expression is dysregulated in thyroid cancer. A recent study using a conditional knock-out mouse model identified 58 putative PAX8 target genes. In the present study, we evaluated the expression of 11 of these genes in normal and tumoral thyroid tissues from patients with papillary thyroid cancer (PTC). ATP1B1, GPC3, KCNIP3, and PRLR transcript levels in tumor tissues were significantly lower in PTCs than in NT, whereas LCN2, LGALS1 and SCD1 expression was upregulated in PTC compared with NT. Principal component analysis of the expression of the most markedly dysregulated PAX8 target genes was able to discriminate between PTC and NT. Immunohistochemistry was used to assess levels of proteins encoded by the two most dyregulated PAX8 target genes, LCN2 and GPC3. Interestingly, GPC3 was detectable in all of the NT samples but none of the PTC samples. Collectively, these findings point to significant PTC-associated dysregulation of several PAX8 target genes, supporting the notion that PAX8-regulated molecular cascades play important roles during thyroid tumorigenesis. PMID:27249794

  14. Expression of PAX8 Target Genes in Papillary Thyroid Carcinoma

    PubMed Central

    Rosignolo, Francesca; Sponziello, Marialuisa; Durante, Cosimo; Puppin, Cinzia; Mio, Catia; Baldan, Federica; Di Loreto, Carla; Russo, Diego; Filetti, Sebastiano; Damante, Giuseppe

    2016-01-01

    PAX8 is a thyroid-specific transcription factor whose expression is dysregulated in thyroid cancer. A recent study using a conditional knock-out mouse model identified 58 putative PAX8 target genes. In the present study, we evaluated the expression of 11 of these genes in normal and tumoral thyroid tissues from patients with papillary thyroid cancer (PTC). ATP1B1, GPC3, KCNIP3, and PRLR transcript levels in tumor tissues were significantly lower in PTCs than in NT, whereas LCN2, LGALS1 and SCD1 expression was upregulated in PTC compared with NT. Principal component analysis of the expression of the most markedly dysregulated PAX8 target genes was able to discriminate between PTC and NT. Immunohistochemistry was used to assess levels of proteins encoded by the two most dyregulated PAX8 target genes, LCN2 and GPC3. Interestingly, GPC3 was detectable in all of the NT samples but none of the PTC samples. Collectively, these findings point to significant PTC-associated dysregulation of several PAX8 target genes, supporting the notion that PAX8-regulated molecular cascades play important roles during thyroid tumorigenesis. PMID:27249794

  15. Non-targeted effects of virus-induced gene silencing vectors on host endogenous gene expression.

    PubMed

    Oláh, Enikő; Pesti, Réka; Taller, Dénes; Havelda, Zoltán; Várallyay, Éva

    2016-09-01

    Virus-induced gene silencing (VIGS) uses recombinant viruses to study gene function; however, the effect of the virus vector itself on the gene expression of the host is not always considered. In our work, we investigated non-targeted gene expression changes of the host in order to see how often these changes appear. Effects of various VIGS vector infections were analysed by monitoring gene expression levels of housekeeping genes by Northern blot analysis in four different hosts. We found that non-targeted changes happens very often. More importantly, these non-targeted effects can cause drastic changes in the gene-expression pattern of host genes that are usually used as references in these studies. We have also found that in a tobacco rattle virus (TRV)-based VIGS, the presence of foreign sequences in the cloning site of the vector can also have a non-targeted effect, and even the use of an internal control can lead to unpredicted changes. Our results show that although VIGS is a very powerful technique, the VIGS vector, as a pathogen of the host, can cause unwanted changes in its gene-expression pattern, highlighting the importance of careful selection of both the genes to be tested and those to be used as references in the planned experiments. PMID:27283101

  16. Cloning, characterization and targeting of the mouse HEXA gene

    SciTech Connect

    Wakamatsu, N.; Trasler, J.M.; Gravel, R.A.

    1994-09-01

    The HEXA gene, encoding the {alpha} subunit of {beta}-hexosaminidase A, is essential for the metabolism of ganglioside G{sub M2}, and defects in this gene cause Tay-Sachs disease in humans. To elucidate the role of the gene in the nervous system of the mouse and to establish a mouse model of Tay-Sachs disease, we have cloned and characterized the HEXA gene and targeted a disruption of the gene in mouse ES cells. The mouse HEXA gene spans {approximately}26 kb and consists of 14 exons, similar to the human gene. A heterogeneous transcription initiation site was identified 21-42 bp 5{prime} of the initiator ATG, with two of the sites fitting the consensus CTCA (A = start) as seen for some weak initiator systems. Promoter analysis showed that the first 150 bp 5{prime} of the ATG contained 85% of promoter activity observed in constructs containing up to 1050 bp of 5{prime} sequence. The active region contained a sequence matching that of the adenovirus major late promoter upstream element factor. A survey of mouse tissues showed that the highest mRNA levels were in (max to min): testis (5.5 x brain cortex), adrenal, epididymis, heart, brain, lung, kidney, and liver (0.3 x brain cortex). A 12 kb BstI/SalI fragment containing nine exons was disrupted with the insertion of the bacterial neo{sup r} gene in exon 11 and was targeted into 129/Sv ES cells by homologous recombination. Nine of 153 G418 resistant clones were correctly targeted as confirmed by Southern blotting. The heterozygous ES cells were microinjected into mouse blastocysts and implanted into pseudo-pregnant mice. Nine male chimeric mice, showing that 40-95% chimerism for the 129/Sv agouti coat color marker, are being bred in an effort to generate germline transmission of the disrupted HEXA gene.

  17. Fungal virulence genes as targets for antifungal chemotherapy.

    PubMed Central

    Perfect, J R

    1996-01-01

    Fungal virulence genes have now met the age of molecular pathogenesis. The definition of virulence genes needs to be broad so that it encompasses the focus on molecular antifungal targets and vaccine epitopes. However, in the broad but simple definition of a virulence gene, there will be many complex genetic and host interactions which investigators will need to carefully define. Nevertheless, with the increasing numbers of serious fungal infections produced by old and newly reported organisms, the paucity of present antifungal drugs, and the likelihood of increasing drug resistance, the need for investigations into understanding fungal virulence at the molecular level has never been more important. PMID:8807043

  18. Gene mutations and molecularly targeted therapies in acute myeloid leukemia

    PubMed Central

    Hatzimichael, Eleftheria; Georgiou, Georgios; Benetatos, Leonidas; Briasoulis, Evangelos

    2013-01-01

    Acute myelogenous leukemia (AML) can progress quickly and without treatment can become fatal in a short period of time. However, over the last 30 years fine-tuning of therapeutics have increased the rates of remission and cure. Cytogenetics and mutational gene profiling, combined with the option of allogeneic hematopoietic stem cell transplantation offered in selected patients have further optimized AML treatment on a risk stratification basis in younger adults. However there is still an unmet medical need for effective therapies in AML since disease relapses in almost half of adult patients becoming refractory to salvage therapy. Improvements in the understanding of molecular biology of cancer and identification of recurrent mutations in AML provide opportunities to develop targeted therapies and improve the clinical outcome. In the spectrum of identified gene mutations, primarily targetable lesions are gain of function mutations of tyrosine kinases FLT3, JAK2 and cKIT for which specific, dual and multi-targeted small molecule inhibitors have been developed. A number of targeted compounds such as sorafenib, quizartinib, lestaurtinib, midostaurin, pacritinib, PLX3397 and CCT137690 are in clinical development. For loss-of-function gene mutations, which are mostly biomarkers of favorable prognosis, combined therapeutic approaches can maximize the therapeutic efficacy of conventional therapy. Apart from mutated gene products, proteins aberrantly overexpressed in AML appear to be clinically significant therapeutic targets. Such a molecule for which targeted inhibitors are currently in clinical development is PLK1. We review characteristic gene mutations, discuss their biological functions and clinical significance and present small molecule compounds in clinical development, which are expected to have a role in treating AML subtypes with characteristic molecular alterations. PMID:23358589

  19. Homologous recombination is required for AAV-mediated gene targeting

    PubMed Central

    Vasileva, Ana; Linden, R. Michael; Jessberger, Rolf

    2006-01-01

    High frequencies of gene targeting can be achieved by infection of mammalian cells with recombinant adeno-associated virus (rAAV) vectors [D. W. Russell and R. K. Hirata (1998) Nature Genet., 18, 325–330; D. W. Russell and R. K. Hirata (2000) J. Virol., 74, 4612–4620; R. Hirata et al. (2002) Nat. Biotechnol., 20, 735–738], but the mechanism of targeting is unclear and random integration often occurs in parallel. We assessed the role of specific DNA repair and recombination pathways in rAAV gene targeting by measuring correction of a mutated enhanced green fluorescent protein (EGFP) gene in cells where homologous recombination (HR) or non-homologous end-joining (NHEJ) had been suppressed by RNAi. EGFP-negative cells were transduced with rAAV vectors carrying a different inactivating deletion in the EGFP, and in parallel with rAAV vectors carrying red fluorescent protein (RFP). Expression of RFP accounted for viral transduction efficiency and long-term random integration. Approximately 0.02% of the infected GFP-negative cells were stably converted to GFP positive cells. Silencing of the essential NHEJ component DNA-PK had no significant effect on the frequency of targeting at any time point examined. Silencing of the SNF2/SWI2 family members RAD54L or RAD54B, which are important for HR, reduced the rate of stable rAAV gene targeting ∼5-fold. Further, partial silencing of the Rad51 paralogue XRCC3 completely abolished stable long-term EGFP expression. These results show that rAAV gene targeting requires the Rad51/Rad54 pathway of HR. PMID:16822856

  20. Correction of human. beta. sup S -globin gene by gene targeting

    SciTech Connect

    Shesely, E.G.; Hyungsuk Kim; Shehee, W.R.; Smithies, O. ); Papayannopoulou, T. ); Popovich, B.W. )

    1991-05-15

    As a step toward using gene targeting for gene therapy, the authors have corrected a human {beta}{sup S}-globin gene to the normal {beta}{sup A} allele by homologous recombination in the mouse-human hybrid cell line BSM. BSM is derived from a mouse erythroleukemia cell line and carries a single human chromosome 11 with the {beta}{sup S}-globin allele. A {beta}{sup A}-globin targeting construct containing a unique oligomer and a neomycin-resistance gene was electroporated into the BSM cells, which were then placed under G418 selection. Then 126 resulting pools containing a total {approx}29,000 G418-resistant clones were screened by PCR for the presence of a targeted recombinant: 3 positive pools were identified. A targeted clone was isolated by replating one of the positive pools into smaller pools and rescreening by PCR, followed by dilution cloning. Southern blot analysis demonstrated that the isolated clone had been targeted as planned. The correction of the {beta}{sup S} allele to {beta}{sup A} was confirmed both by allele-specific PCR and by allele-specific antibodies. Expression studies comparing the uninduced and induced RNA levels in unmodified BSM cells and in the targeted clone showed no significant alteration in the ability of the targeted clone to undergo induction, despite the potentially disrupting presence of a transcriptionally active neomycin gene 5{prime} to the human {beta}{sup A}-globin gene. Thus gene targeting can correct a {beta}{sup S} allele to {beta}{sup A}, and the use of a selectable helper gene need not significantly interfere with the induction of the corrected gene.

  1. Chlorotoxin labeled magnetic nanovectors for targeted gene delivery to glioma.

    PubMed

    Kievit, Forrest M; Veiseh, Omid; Fang, Chen; Bhattarai, Narayan; Lee, Donghoon; Ellenbogen, Richard G; Zhang, Miqin

    2010-08-24

    Glioma accounts for 80% of brain tumors and currently remains one of the most lethal forms of cancers. Gene therapy could potentially improve the dismal prognosis of patients with glioma, but this treatment modality has not yet reached the bedside from the laboratory due to the lack of safe and effective gene delivery vehicles. In this study we investigate targeted gene delivery to C6 glioma cells in a xenograft mouse model using chlorotoxin (CTX) labeled nanoparticles. The developed nanovector consists of an iron oxide nanoparticle core, coated with a copolymer of chitosan, polyethylene glycol (PEG), and polyethylenimine (PEI). Green fluorescent protein (GFP) encoding DNA was bound to these nanoparticles, and CTX was then attached using a short PEG linker. Nanoparticles without CTX were also prepared as a control. Mice bearing C6 xenograft tumors were injected intravenously with the DNA-bound nanoparticles. Nanoparticle accumulation in the tumor site was monitored using magnetic resonance imaging and analyzed by histology, and GFP gene expression was monitored through Xenogen IVIS fluorescence imaging and confocal fluorescence microscopy. Interestingly, the CTX did not affect the accumulation of nanoparticles at the tumor site but specifically enhanced their uptake into cancer cells as evidenced by higher gene expression. These results indicate that this targeted gene delivery system may potentially improve treatment outcome of gene therapy for glioma and other deadly cancers. PMID:20731441

  2. Chlorotoxin Labeled Magnetic Nanovectors for Targeted Gene Delivery to Glioma

    PubMed Central

    Kievit, Forrest M.; Veiseh, Omid; Fang, Chen; Bhattarai, Narayan; Lee, Donghoon; Ellenbogen, Richard G.; Zhang, Miqin

    2010-01-01

    Glioma accounts for 80% of brain tumors, and currently remains one of the most lethal forms of cancers. Gene therapy could potentially improve the dismal prognosis of patients with glioma, but this treatment modality has not yet reached the bedside from the laboratory due to the lack of safe and effective gene delivery vehicles. In this study we investigate targeted gene delivery to C6 glioma cells in a xenograft mouse model using chlorotoxin (CTX) labeled nanoparticles. The developed nanovector consists of an iron oxide nanoparticle core, coated with a copolymer of chitosan, polyethylene glycol (PEG) and polyethylenimine (PEI). Green fluorescent protein (GFP) encoding DNA was bound to these nanoparticles, and CTX was then attached using a short PEG linker. Nanoparticles without CTX were also prepared as a control. Mice bearing C6 xenograft tumors were injected intravenously with the DNA bound nanoparticles. Nanoparticle accumulation in the tumor site was monitored using magnetic resonance imaging and analyzed by histology, and GFP gene expression was monitored through Xenogen IVIS fluorescence imaging and confocal fluorescence microscopy. Interestingly, the CTX did not affect the accumulation of nanoparticles at the tumor site, but specifically enhanced their uptake into cancer cells as evidenced by higher gene expression. These results indicate that this targeted gene delivery system may potentially improve treatment outcome of gene therapy for glioma and other deadly cancers. PMID:20731441

  3. Rescuing the Failing Heart by Targeted Gene Transfer

    PubMed Central

    Kawase, Yoshiaki; Ladage, Dennis; Hajjar, Roger J.

    2011-01-01

    Congestive heart failure is a major cause of morbidity and mortality in the US. While progress in conventional treatments is making steady and incremental gains to reduce heart failure mortality, there is a critical need to explore new therapeutic approaches. Gene therapy was initially applied in the clinical setting for inherited monogenic disorders. It is now apparent that gene therapy has broader potential that also includes acquired polygenic diseases, such as congestive heart failure. Recent advances in understanding of the molecular basis of myocardial dysfunction, together with the evolution of increasingly efficient gene transfer technology, has placed heart failure within reach of gene-based therapy. Furthermore, the recent successful and safe completion of a phase 2 trial targeting the sarcoplasmic reticulum calcium ATPase pump (SERCA2a) along with the start of more recent phase 1 trials usher a new era for gene therapy for the treatment of heart failure. PMID:21371634

  4. Essential genes as antimicrobial targets and cornerstones of synthetic biology.

    PubMed

    Juhas, Mario; Eberl, Leo; Church, George M

    2012-11-01

    Essential genes are absolutely required for the survival of any living entity. Investigation of essential genes is therefore expected to advance tremendously our understanding of the universal principles of life. Determination of a minimal set of essential genes needed to sustain life also plays an important role in the emerging field of synthetic biology, whose goals include creation of a stringently controlled minimal cell with predesigned phenotypic traits. In addition, due to their indispensability for survival of bacteria, genes encoding essential cellular functions have great potential in medicine as promising targets for the development of novel antimicrobials. Here, we review recent advances in the investigation of essential genes, with emphasis on the practical applications in medicine and synthetic biology. PMID:22951051

  5. Integrative Genomics Reveals Novel Molecular Pathways and Gene Networks for Coronary Artery Disease

    PubMed Central

    Mäkinen, Ville-Petteri; Civelek, Mete; Meng, Qingying; Zhang, Bin; Zhu, Jun; Levian, Candace; Huan, Tianxiao; Segrè, Ayellet V.; Ghosh, Sujoy; Vivar, Juan; Nikpay, Majid; Stewart, Alexandre F. R.; Nelson, Christopher P.; Willenborg, Christina; Erdmann, Jeanette; Blakenberg, Stefan; O'Donnell, Christopher J.; März, Winfried; Laaksonen, Reijo; Epstein, Stephen E.; Kathiresan, Sekar; Shah, Svati H.; Hazen, Stanley L.; Reilly, Muredach P.; Lusis, Aldons J.; Samani, Nilesh J.; Schunkert, Heribert; Quertermous, Thomas; McPherson, Ruth; Yang, Xia; Assimes, Themistocles L.

    2014-01-01

    The majority of the heritability of coronary artery disease (CAD) remains unexplained, despite recent successes of genome-wide association studies (GWAS) in identifying novel susceptibility loci. Integrating functional genomic data from a variety of sources with a large-scale meta-analysis of CAD GWAS may facilitate the identification of novel biological processes and genes involved in CAD, as well as clarify the causal relationships of established processes. Towards this end, we integrated 14 GWAS from the CARDIoGRAM Consortium and two additional GWAS from the Ottawa Heart Institute (25,491 cases and 66,819 controls) with 1) genetics of gene expression studies of CAD-relevant tissues in humans, 2) metabolic and signaling pathways from public databases, and 3) data-driven, tissue-specific gene networks from a multitude of human and mouse experiments. We not only detected CAD-associated gene networks of lipid metabolism, coagulation, immunity, and additional networks with no clear functional annotation, but also revealed key driver genes for each CAD network based on the topology of the gene regulatory networks. In particular, we found a gene network involved in antigen processing to be strongly associated with CAD. The key driver genes of this network included glyoxalase I (GLO1) and peptidylprolyl isomerase I (PPIL1), which we verified as regulatory by siRNA experiments in human aortic endothelial cells. Our results suggest genetic influences on a diverse set of both known and novel biological processes that contribute to CAD risk. The key driver genes for these networks highlight potential novel targets for further mechanistic studies and therapeutic interventions. PMID:25033284

  6. Bacteriophages and medical oncology: targeted gene therapy of cancer.

    PubMed

    Bakhshinejad, Babak; Karimi, Marzieh; Sadeghizadeh, Majid

    2014-08-01

    Targeted gene therapy of cancer is of paramount importance in medical oncology. Bacteriophages, viruses that specifically infect bacterial cells, offer a variety of potential applications in biomedicine. Their genetic flexibility to go under a variety of surface modifications serves as a basis for phage display methodology. These surface manipulations allow bacteriophages to be exploited for targeted delivery of therapeutic genes. Moreover, the excellent safety profile of these viruses paves the way for their potential use as cancer gene therapy platforms. The merge of phage display and combinatorial technology has led to the emergence of phage libraries turning phage display into a high throughput technology. Random peptide libraries, as one of the most frequently used phage libraries, provide a rich source of clinically useful peptide ligands. Peptides are known as a promising category of pharmaceutical agents in medical oncology that present advantages such as inexpensive synthesis, efficient tissue penetration and the lack of immunogenicity. Phage peptide libraries can be screened, through biopanning, against various targets including cancer cells and tissues that results in obtaining cancer-homing ligands. Cancer-specific peptides isolated from phage libraries show huge promise to be utilized for targeting of various gene therapy vectors towards malignant cells. Beyond doubt, bacteriophages will play a more impressive role in the future of medical oncology. PMID:25012686

  7. The Fusarium graminearum Genome Reveals More Secondary Metabolite Gene Clusters and Hints of Horizontal Gene Transfer

    PubMed Central

    Wong, Philip; Münsterkötter, Martin; Mewes, Hans-Werner; Schmeitzl, Clemens; Varga, Elisabeth; Berthiller, Franz; Adam, Gerhard; Güldener, Ulrich

    2014-01-01

    Fungal secondary metabolite biosynthesis genes are of major interest due to the pharmacological properties of their products (like mycotoxins and antibiotics). The genome of the plant pathogenic fungus Fusarium graminearum codes for a large number of candidate enzymes involved in secondary metabolite biosynthesis. However, the chemical nature of most enzymatic products of proteins encoded by putative secondary metabolism biosynthetic genes is largely unknown. Based on our analysis we present 67 gene clusters with significant enrichment of predicted secondary metabolism related enzymatic functions. 20 gene clusters with unknown metabolites exhibit strong gene expression correlation in planta and presumably play a role in virulence. Furthermore, the identification of conserved and over-represented putative transcription factor binding sites serves as additional evidence for cluster co-regulation. Orthologous cluster search provided insight into the evolution of secondary metabolism clusters. Some clusters are characteristic for the Fusarium phylum while others show evidence of horizontal gene transfer as orthologs can be found in representatives of the Botrytis or Cochliobolus lineage. The presented candidate clusters provide valuable targets for experimental examination. PMID:25333987

  8. Massive parallel IGHV gene sequencing reveals a germinal center pathway in origins of human multiple myeloma

    PubMed Central

    Bryant, Dean; Seckinger, Anja; Hose, Dirk; Zojer, Niklas; Sahota, Surinder S.

    2015-01-01

    Human multiple myeloma (MM) is characterized by accumulation of malignant terminally differentiated plasma cells (PCs) in the bone marrow (BM), raising the question when during maturation neoplastic transformation begins. Immunoglobulin IGHV genes carry imprints of clonal tumor history, delineating somatic hypermutation (SHM) events that generally occur in the germinal center (GC). Here, we examine MM-derived IGHV genes using massive parallel deep sequencing, comparing them with profiles in normal BM PCs. In 4/4 presentation IgG MM, monoclonal tumor-derived IGHV sequences revealed significant evidence for intraclonal variation (ICV) in mutation patterns. IGHV sequences of 2/2 normal PC IgG populations revealed dominant oligoclonal expansions, each expansion also displaying mutational ICV. Clonal expansions in MM and in normal BM PCs reveal common IGHV features. In such MM, the data fit a model of tumor origins in which neoplastic transformation is initiated in a GC B-cell committed to terminal differentiation but still targeted by on-going SHM. Strikingly, the data parallel IGHV clonal sequences in some monoclonal gammopathy of undetermined significance (MGUS) known to display on-going SHM imprints. Since MGUS generally precedes MM, these data suggest origins of MGUS and MM with IGHV gene mutational ICV from the same GC B-cell, arising via a distinctive pathway. PMID:25929340

  9. Comparative genomic analysis reveals a distant liver enhancer upstream of the COUP-TFII gene

    SciTech Connect

    Baroukh, Nadine; Ahituv, Nadav; Chang, Jessie; Shoukry, Malak; Afzal, Veena; Rubin, Edward M.; Pennacchio, Len A.

    2004-08-20

    COUP-TFII is a central nuclear hormone receptor that tightly regulates the expression of numerous target lipid metabolism genes in vertebrates. However, it remains unclear how COUP-TFII itself is transcriptionally controlled since studies with its promoter and upstream region fail to recapitulate the genes liver expression. In an attempt to identify liver enhancers in the vicinity of COUP-TFII, we employed a comparative genomic approach. Initial comparisons between humans and mice of the 3,470kb gene poor region surrounding COUP-TFII revealed 2,023 conserved non-coding elements. To prioritize a subset of these elements for functional studies, we performed further genomic comparisons with the orthologous pufferfish (Fugu rubripes) locus and uncovered two anciently conserved non-coding sequences (CNS) upstream of COUP-TFII (CNS-62kb and CNS-66kb). Testing these two elements using reporter constructs in liver (HepG2) cells revealed that CNS-66kb, but not CNS-62kb, yielded robust in vitro enhancer activity. In addition, an in vivo reporter assay using naked DNA transfer with CNS-66kb linked to luciferase displayed strong reproducible liver expression in adult mice, further supporting its role as a liver enhancer. Together, these studies further support the utility of comparative genomics to uncover gene regulatory sequences based on evolutionary conservation and provide the substrates to better understand the regulation and expression of COUP-TFII.

  10. Microbial population index and community structure in saline-alkaline soil using gene targeted metagenomics.

    PubMed

    Keshri, Jitendra; Mishra, Avinash; Jha, Bhavanath

    2013-03-30

    Population indices of bacteria and archaea were investigated from saline-alkaline soil and a possible microbe-environment pattern was established using gene targeted metagenomics. Clone libraries were constructed using 16S rRNA and functional gene(s) involved in carbon fixation (cbbL), nitrogen fixation (nifH), ammonia oxidation (amoA) and sulfur metabolism (apsA). Molecular phylogeny revealed the dominance of Actinobacteria, Firmicutes and Proteobacteria along with archaeal members of Halobacteraceae. The library consisted of novel bacterial (20%) and archaeal (38%) genera showing ≤95% similarity to previously retrieved sequences. Phylogenetic analysis indicated ability of inhabitant to survive in stress condition. The 16S rRNA gene libraries contained novel gene sequences and were distantly homologous with cultured bacteria. Functional gene libraries were found unique and most of the clones were distantly related to Proteobacteria, while clones of nifH gene library also showed homology with Cyanobacteria and Firmicutes. Quantitative real-time PCR exhibited that bacterial abundance was two orders of magnitude higher than archaeal. The gene(s) quantification indicated the size of the functional guilds harboring relevant key genes. The study provides insights on microbial ecology and different metabolic interactions occurring in saline-alkaline soil, possessing phylogenetically diverse groups of bacteria and archaea, which may be explored further for gene cataloging and metabolic profiling. PMID:23083746

  11. Target gene delivery from targeting ligand conjugated chitosan-PEI copolymer for cancer therapy.

    PubMed

    Nam, Joung-Pyo; Nah, Jae-Woon

    2016-01-01

    In this study, we designed a novel carrier which was having low cytotoxicity, site-specific target function, and high transfection efficiency using low molecular weight water soluble O-carboxymethyl chitosan (OCMCh), branched low molecular weight poly(ethyleneimine) (bPEI), and targeting ligand (epitope type, HER-2/neu). OCMCh/bPEI/targeting ligand, HPOCP copolymer, and targeting ligand-modified polyamphoteric polymer, and were prepared by chemical reaction and characterized by (1)H NMR and FT-IR. The binding affinity, protecting efficiency, and releasing ability of gene/HPOCP polyplex were confirmed by gel retardation assay. The pDNA(pEGFP)/HPOCP polyplexes showed high gene transfection efficiency in HCT 119 cell. In addition, siRNA/HPOCP polyplexes formed spherical shape and have particle sizes from 100 to 300nm. The siRNA/HPOCP polyplexes have lower cytotoxicity than PEI in the all of siRNA concentrations ranging from 0 to 2μg/μL in HEK 293 cells. The cell viability of siRNA/HPOCP polyplexes was performed in SK-Br3 cells with VEGF siRNA or BCL2 siRNA. In addition, confocal laser-scanning microscopy and flow cytometry assay were performed for cellular localization and cellular uptake efficiency of siRNA/HPOCP polyplexes. The results of the present study demonstrate that HPOCP copolymer is a good candidate as gene delivery carriers for gene delivery system or gene therapy. PMID:26453863

  12. Epigenetic Editing: targeted rewriting of epigenetic marks to modulate expression of selected target genes

    PubMed Central

    de Groote, Marloes L.; Verschure, Pernette J.; Rots, Marianne G.

    2012-01-01

    Despite significant advances made in epigenetic research in recent decades, many questions remain unresolved, especially concerning cause and consequence of epigenetic marks with respect to gene expression modulation (GEM). Technologies allowing the targeting of epigenetic enzymes to predetermined DNA sequences are uniquely suited to answer such questions and could provide potent (bio)medical tools. Toward the goal of gene-specific GEM by overwriting epigenetic marks (Epigenetic Editing, EGE), instructive epigenetic marks need to be identified and their writers/erasers should then be fused to gene-specific DNA binding domains. The appropriate epigenetic mark(s) to change in order to efficiently modulate gene expression might have to be validated for any given chromatin context and should be (mitotically) stable. Various insights in such issues have been obtained by sequence-specific targeting of epigenetic enzymes, as is presented in this review. Features of such studies provide critical aspects for further improving EGE. An example of this is the direct effect of the edited mark versus the indirect effect of recruited secondary proteins by targeting epigenetic enzymes (or their domains). Proof-of-concept of expression modulation of an endogenous target gene is emerging from the few EGE studies reported. Apart from its promise in correcting disease-associated epi-mutations, EGE represents a powerful tool to address fundamental epigenetic questions. PMID:23002135

  13. Dissecting Human Gene Functions Regulating Islet Development With Targeted Gene Transduction

    PubMed Central

    Pauerstein, Philip T.; Sugiyama, Takuya; Stanley, Susan E.; McLean, Graeme W.; Wang, Jing; Martín, Martín G.

    2015-01-01

    During pancreas development, endocrine precursors and their progeny differentiate, migrate, and cluster to form nascent islets. The transcription factor Neurogenin 3 (Neurog3) is required for islet development in mice, but its role in these dynamic morphogenetic steps has been inferred from fixed tissues. Moreover, little is known about the molecular genetic functions of NEUROG3 in human islet development. We developed methods for gene transduction by viral microinjection in the epithelium of cultured Neurog3-null mutant fetal pancreas, permitting genetic complementation in a developmentally relevant context. In addition, we developed methods for quantitative assessment of live-cell phenotypes in single developing islet cells. Delivery of wild-type NEUROG3 rescued islet differentiation, morphogenesis, and live cell deformation, whereas the patient-derived NEUROG3R107S allele partially restored indicators of islet development. NEUROG3P39X, a previously unreported patient allele, failed to restore islet differentiation or morphogenesis and was indistinguishable from negative controls, suggesting that it is a null mutation. Our systems also permitted genetic suppression analysis and revealed that targets of NEUROG3, including NEUROD1 and RFX6, can partially restore islet development in Neurog3-null mutant mouse pancreata. Thus, advances described here permitted unprecedented assessment of gene functions in regulating crucial dynamic aspects of islet development in the fetal pancreas. PMID:25901096

  14. Identification of novel regulatory factor X (RFX) target genes by comparative genomics in Drosophila species

    PubMed Central

    Laurençon, Anne; Dubruille, Raphaëlle; Efimenko, Evgeni; Grenier, Guillaume; Bissett, Ryan; Cortier, Elisabeth; Rolland, Vivien; Swoboda, Peter; Durand, Bénédicte

    2007-01-01

    Background Regulatory factor X (RFX) transcription factors play a key role in ciliary assembly in nematode, Drosophila and mouse. Using the tremendous advantages of comparative genomics in closely related species, we identified novel genes regulated by dRFX in Drosophila. Results We first demonstrate that a subset of known ciliary genes in Caenorhabditis elegans and Drosophila are regulated by dRFX and have a conserved RFX binding site (X-box) in their promoters in two highly divergent Drosophila species. We then designed an X-box consensus sequence and carried out a genome wide computer screen to identify novel genes under RFX control. We found 412 genes that share a conserved X-box upstream of the ATG in both species, with 83 genes presenting a more restricted consensus. We analyzed 25 of these 83 genes, 16 of which are indeed RFX target genes. Two of them have never been described as involved in ciliogenesis. In addition, reporter construct expression analysis revealed that three of the identified genes encode proteins specifically localized in ciliated endings of Drosophila sensory neurons. Conclusion Our X-box search strategy led to the identification of novel RFX target genes in Drosophila that are involved in sensory ciliogenesis. We also established a highly valuable Drosophila cilia and basal body dataset. These results demonstrate the accuracy of the X-box screen and will be useful for the identification of candidate genes for human ciliopathies, as several human homologs of RFX target genes are known to be involved in diseases, such as Bardet-Biedl syndrome. PMID:17875208

  15. Targeted gene delivery via N-acetylglucosamine receptor mediated endocytosis.

    PubMed

    Singh, Bijay; Maharjan, Sushila; Kim, You-Kyoung; Jiang, Tai; Islam, Mohammad Ariful; Kang, Sang-Kee; Cho, Myung-Haing; Choi, Yun-Jaie; Cho, Chong-Su

    2014-11-01

    Receptor-mediated endocytosis is a promising approach of gene delivery into the target cells via receptor-ligand interaction. Vimentins at the cell surface are recently known to bind N-acetylglucosamine (GlcNAc) residue, therefore, the cell surfaces of vimentin-expressing cells could be targeted by using the GlcNAc residue as a specific ligand for receptor-mediated gene delivery. Here, we have developed polymeric gene delivery vectors, based on poly(ethylene oxide)(PEO) and poly(aspartamide), namely poly[(aspartamide)(diethylenetriamine)]-b-[PEO-(GlcNAc)] (PADPG) and poly[(aspartamide)(diethylenetriamine)]-b-[PEO] (PADP) to elucidate the efficiency of GlcNAc ligand for gene delivery through receptor mediated endocytosis. To determine the efficiency of these polymeric vectors for specific gene delivery, the DNA condensation ability of PADPG and PADP and the subsequent formation of polymeric nanoparticles were confirmed by gel retardation assay and transmission electron microscopy respectively. Both PADPG and PADP had lower cytotoxicity than polyethylenimine 25 K (PEI 25 K). However, their transfection efficiency was comparatively lower than PEI 25 K due to hydrophilic property of PEO in the vectors. To observe the stability of polymeric nanoparticles, the transfection of PADPG and PADP was carried out in the presence of serum. Favorably, the interfering effect of serum on the transfection efficiency of PADPG and PADP was also very low. Finally, when the cell specificity of these polymeric vectors was investigated, PADPG had high gene transfection in vimentin-expressing cells than vimentin-deficiency cells. The high transfection efficiency of PADPG was attributed to the GlcNAc in the polymeric vector which interact specifically with vimentin in the cells for the receptor-mediated endocytosis. The competitive inhibition assay further proved the receptor-mediated endocytosis of PADPG. Thus, this study demonstrates that conjugation of GlcNAc is an effective and rational

  16. Genes Frequently Coexpressed with Hoxc8 Provide Insight into the Discovery of Target Genes.

    PubMed

    Kalyani, Ruthala; Lee, Ji-Yeon; Min, Hyehyun; Yoon, Heejei; Kim, Myoung Hee

    2016-05-31

    Identifying Hoxc8 target genes is at the crux of understanding the Hoxc8-mediated regulatory networks underlying its roles during development. However, identification of these genes remains difficult due to intrinsic factors of Hoxc8, such as low DNA binding specificity, context-dependent regulation, and unknown cofactors. Therefore, as an alternative, the present study attempted to test whether the roles of Hoxc8 could be inferred by simply analyzing genes frequently coexpressed with Hoxc8, and whether these genes include putative target genes. Using archived gene expression datasets in which Hoxc8 was differentially expressed, we identified a total of 567 genes that were positively coexpressed with Hoxc8 in at least four out of eight datasets. Among these, 23 genes were coexpressed in six datasets. Gene sets associated with extracellular matrix and cell adhesion were most significantly enriched, followed by gene sets for skeletal system development, morphogenesis, cell motility, and transcriptional regulation. In particular, transcriptional regulators, including paralogs of Hoxc8, known Hox co-factors, and transcriptional remodeling factors were enriched. We randomly selected Adam19, Ptpn13, Prkd1, Tgfbi, and Aldh1a3, and validated their coexpression in mouse embryonic tissues and cell lines following TGF-β2 treatment or ectopic Hoxc8 expression. Except for Aldh1a3, all genes showed concordant expression with that of Hoxc8, suggesting that the coexpressed genes might include direct or indirect target genes. Collectively, we suggest that the coexpressed genes provide a resource for constructing Hoxc8-mediated regulatory networks. PMID:27025388

  17. Genes Frequently Coexpressed with Hoxc8 Provide Insight into the Discovery of Target Genes

    PubMed Central

    Kalyani, Ruthala; Lee, Ji-Yeon; Min, Hyehyun; Yoon, Heejei; Kim, Myoung Hee

    2016-01-01

    Identifying Hoxc8 target genes is at the crux of understanding the Hoxc8-mediated regulatory networks underlying its roles during development. However, identification of these genes remains difficult due to intrinsic factors of Hoxc8, such as low DNA binding specificity, context-dependent regulation, and unknown cofactors. Therefore, as an alternative, the present study attempted to test whether the roles of Hoxc8 could be inferred by simply analyzing genes frequently coexpressed with Hoxc8, and whether these genes include putative target genes. Using archived gene expression datasets in which Hoxc8 was differentially expressed, we identified a total of 567 genes that were positively coexpressed with Hoxc8 in at least four out of eight datasets. Among these, 23 genes were coexpressed in six datasets. Gene sets associated with extracellular matrix and cell adhesion were most significantly enriched, followed by gene sets for skeletal system development, morphogenesis, cell motility, and transcriptional regulation. In particular, transcriptional regulators, including paralogs of Hoxc8, known Hox co-factors, and transcriptional remodeling factors were enriched. We randomly selected Adam19, Ptpn13, Prkd1, Tgfbi, and Aldh1a3, and validated their coexpression in mouse embryonic tissues and cell lines following TGF-β2 treatment or ectopic Hoxc8 expression. Except for Aldh1a3, all genes showed concordant expression with that of Hoxc8, suggesting that the coexpressed genes might include direct or indirect target genes. Collectively, we suggest that the coexpressed genes provide a resource for constructing Hoxc8-mediated regulatory networks. PMID:27025388

  18. Genome-wide gene expression profiling reveals unsuspected molecular alterations in pemphigus foliaceus

    PubMed Central

    Malheiros, Danielle; Panepucci, Rodrigo A; Roselino, Ana M; Araújo, Amélia G; Zago, Marco A; Petzl-Erler, Maria Luiza

    2014-01-01

    Pemphigus foliaceus (PF) is a complex autoimmune disease characterized by bullous skin lesions and the presence of antibodies against desmoglein 1. In this study we sought to contribute to a better understanding of the molecular processes in endemic PF, as the identification of factors that participate in the pathogenesis is a prerequisite for understanding its biological basis and may lead to novel therapeutic interventions. CD4+ T lymphocytes are central to the development of the disease. Therefore, we compared genome-wide gene expression profiles of peripheral CD4+ T cells of various PF patient subgroups with each other and with that of healthy individuals. The patient sample was subdivided into three groups: untreated patients with the generalized form of the disease, patients submitted to immunosuppressive treatment, and patients with the localized form of the disease. Comparisons between different subgroups resulted in 135, 54 and 64 genes differentially expressed. These genes are mainly related to lymphocyte adhesion and migration, apoptosis, cellular proliferation, cytotoxicity and antigen presentation. Several of these genes were differentially expressed when comparing lesional and uninvolved skin from the same patient. The chromosomal regions 19q13 and 12p13 concentrate differentially expressed genes and are candidate regions for PF susceptibility genes and disease markers. Our results reveal genes involved in disease severity, potential therapeutic targets and previously unsuspected processes involved in the pathogenesis. Besides, this study adds original information that will contribute to the understanding of PF's pathogenesis and of the still poorly defined in vivo functions of most of these genes. PMID:24813052

  19. Alteration of Caenorhabditis elegans gene expression by targeted transformation.

    PubMed Central

    Broverman, S; MacMorris, M; Blumenthal, T

    1993-01-01

    We have produced strains carrying a synthetic fusion of parts of two vitellogenin genes, vit-2 and vit-6, integrated into the Caenorhabditis elegans genome. In most of the 63 transformant strains, the plasmid sequences are integrated at random locations in the genome. However, in two strains the transgene integrated by homologous recombination into the endogenous vit-2 gene. In both cases the reciprocal exchange between the chromosome and the injected circular plasmid containing a promoter deletion led to switching of the plasmid-borne promoter and the endogenous promoter, with a reduction in vit-2 expression. Thus in nematodes, transforming DNA can integrate by homologous recombination to result in partial inactivation of the chromosomal locus. The simplicity of the event and its reasonably high frequency suggest that gene targeting by homologous recombination should be considered as a method for directed inactivation of C. elegans genes. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8506273

  20. Alteration of Caenorhabditis elegans gene expression by targeted transformation.

    PubMed

    Broverman, S; MacMorris, M; Blumenthal, T

    1993-05-15

    We have produced strains carrying a synthetic fusion of parts of two vitellogenin genes, vit-2 and vit-6, integrated into the Caenorhabditis elegans genome. In most of the 63 transformant strains, the plasmid sequences are integrated at random locations in the genome. However, in two strains the transgene integrated by homologous recombination into the endogenous vit-2 gene. In both cases the reciprocal exchange between the chromosome and the injected circular plasmid containing a promoter deletion led to switching of the plasmid-borne promoter and the endogenous promoter, with a reduction in vit-2 expression. Thus in nematodes, transforming DNA can integrate by homologous recombination to result in partial inactivation of the chromosomal locus. The simplicity of the event and its reasonably high frequency suggest that gene targeting by homologous recombination should be considered as a method for directed inactivation of C. elegans genes. PMID:8506273

  1. Imprinted genes as potential genetic and epigenetic toxicologic targets.

    PubMed Central

    Murphy, S K; Jirtle, R L

    2000-01-01

    Genomic imprinting is an epigenetic phenomenon in eutherian mammals that results in the differential expression of the paternally and maternally inherited alleles of a gene. Imprinted genes are necessary for normal mammalian development. This requirement has been proposed to have evolved because of an interparental genetic battle for the utilization of maternal resources during gestation and postnatally. The nonrandom requisite for monoallelic expression of a subset of genes has also resulted in the formation of susceptibility loci for neurobehavioral disorders, developmental disorders, and cancer. Since imprinting involves both cytosine methylation within CpG islands and changes in chromatin structure, imprinted genes are potential targets for dysregulation by epigenetic toxicants that modify DNA methylation and histone acetylation. PMID:10698719

  2. Mammalian Axoneme Central Pair Complex Proteins: Broader Roles Revealed by Gene Knockout Phenotypes

    PubMed Central

    Teves, Maria E.; Nagarkatti-Gude, David R.; Zhang, Zhibing; Strauss, Jerome F.

    2016-01-01

    The axoneme genes, their encoded proteins, their functions and the structures they form are largely conserved across species. Much of our knowledge of the function and structure of axoneme proteins in cilia and flagella is derived from studies on model organisms like the green algae, Chlamydomonas reinhardtii. The core structure of cilia and flagella is the axoneme, which in most motile cilia and flagella contains a 9 + 2 configuration of microtubules. The two central microtubules are the scaffold of the central pair complex (CPC). Mutations that disrupt CPC genes in Chlamydomonas and other model organisms result in defects in assembly, stability and function of the axoneme, leading to flagellar motility defects. However, targeted mutations generated in mice in the orthologous CPC genes have revealed significant differences in phenotypes of mutants compared to Chlamydomonas. Here we review observations that support the concept of cell-type specific roles for the CPC genes in mice, and an expanded repertoire of functions for the products of these genes in cilia, including non-motile cilia, and other microtubule-associated cellular functions. PMID:26785425

  3. Meta-analysis of primary target genes of peroxisome proliferator-activated receptors

    PubMed Central

    Heinäniemi, Merja; Uski, J Oskari; Degenhardt, Tatjana; Carlberg, Carsten

    2007-01-01

    Background Peroxisome proliferator-activated receptors (PPARs) are known for their critical role in the development of diseases, such as obesity, cardiovascular disease, type 2 diabetes and cancer. Here, an in silico screening method is presented, which incorporates experiment- and informatics-derived evidence, such as DNA-binding data of PPAR subtypes to a panel of PPAR response elements (PPREs), PPRE location relative to the transcription start site (TSS) and PPRE conservation across multiple species, for more reliable prediction of PPREs. Results In vitro binding and in vivo functionality evidence agrees with in silico predictions, validating the approach. The experimental analysis of 30 putative PPREs in eight validated PPAR target genes indicates that each gene contains at least one functional, strong PPRE that occurs without positional bias relative to the TSS. An extended analysis of the cross-species conservation of PPREs reveals limited conservation of PPRE patterns, although PPAR target genes typically contain strong or multiple medium strength PPREs. Human chromosome 19 was screened using this method, with validation of six novel PPAR target genes. Conclusion An in silico screening approach is presented, which allows increased sensitivity of PPAR binding site and target gene detection. PMID:17650321

  4. Next generation sequencing in synovial sarcoma reveals novel gene mutations.

    PubMed

    Vlenterie, Myrella; Hillebrandt-Roeffen, Melissa H S; Flucke, Uta E; Groenen, Patricia J T A; Tops, Bastiaan B J; Kamping, Eveline J; Pfundt, Rolph; de Bruijn, Diederik R H; Geurts van Kessel, Ad H M; van Krieken, Han J H J M; van der Graaf, Winette T A; Versleijen-Jonkers, Yvonne M H

    2015-10-27

    Over 95% of all synovial sarcomas (SS) share a unique translocation, t(X;18), however, they show heterogeneous clinical behavior. We analyzed multiple SS to reveal additional genetic alterations besides the translocation. Twenty-six SS from 22 patients were sequenced for 409 cancer-related genes using the Comprehensive Cancer Panel (Life Technologies, USA) on an Ion Torrent platform. The detected variants were verified by Sanger sequencing and compared to matched normal DNAs. Copy number variation was assessed in six tumors using the Oncoscan array (Affymetrix, USA). In total, eight somatic mutations were detected in eight samples. These mutations have not been reported previously in SS. Two of these, in KRAS and CCND1, represent known oncogenic mutations in other malignancies. Additional mutations were detected in RNF213, SEPT9, KDR, CSMD3, MLH1 and ERBB4. DNA alterations occurred more often in adult tumors. A distinctive loss of 6q was found in a metastatic lesion progressing under pazopanib, but not in the responding lesion. Our results emphasize t(X;18) as a single initiating event in SS and as the main oncogenic driver. Our results also show the occurrence of additional genetic events, mutations or chromosomal aberrations, occurring more frequently in SS with an onset in adults. PMID:26415226

  5. Next generation sequencing in synovial sarcoma reveals novel gene mutations

    PubMed Central

    Vlenterie, Myrella; Hillebrandt-Roeffen, Melissa H.S.; Flucke, Uta E.; Groenen, Patricia J.T.A.; Tops, Bastiaan B.J.; Kamping, Eveline J.; Pfundt, Rolph; de Bruijn, Diederik R.H.; van Kessel, Ad H.M. Geurts; van Krieken, Han J.H.J.M.; van der Graaf, Winette T.A.; Versleijen-Jonkers, Yvonne M.H.

    2015-01-01

    Over 95% of all synovial sarcomas (SS) share a unique translocation, t(X;18), however, they show heterogeneous clinical behavior. We analyzed multiple SS to reveal additional genetic alterations besides the translocation. Twenty-six SS from 22 patients were sequenced for 409 cancer-related genes using the Comprehensive Cancer Panel (Life Technologies, USA) on an Ion Torrent platform. The detected variants were verified by Sanger sequencing and compared to matched normal DNAs. Copy number variation was assessed in six tumors using the Oncoscan array (Affymetrix, USA). In total, eight somatic mutations were detected in eight samples. These mutations have not been reported previously in SS. Two of these, in KRAS and CCND1, represent known oncogenic mutations in other malignancies. Additional mutations were detected in RNF213, SEPT9, KDR, CSMD3, MLH1 and ERBB4. DNA alterations occurred more often in adult tumors. A distinctive loss of 6q was found in a metastatic lesion progressing under pazopanib, but not in the responding lesion. Our results emphasize t(X;18) as a single initiating event in SS and as the main oncogenic driver. Our results also show the occurrence of additional genetic events, mutations or chromosomal aberrations, occurring more frequently in SS with an onset in adults. PMID:26415226

  6. Gene Targeting of Mouse Tardbp Negatively Affects Masp2 Expression

    PubMed Central

    Dib, Samar; Xiao, Shangxi; Miletic, Denise; Robertson, Janice

    2014-01-01

    Amyotrophic Lateral Sclerosis (ALS) is a devastating adult onset neurodegenerative disease affecting both upper and lower motor neurons. TDP-43, encoded by the TARDBP gene, was identified as a component of motor neuron cytoplasmic inclusions in both familial and sporadic ALS and has become a pathological signature of the disease. TDP-43 is a nuclear protein involved in RNA metabolism, however in ALS, TDP-43 is mislocalized to the cytoplasm of affected motor neurons, suggesting that disease might be caused by TDP-43 loss of function. To investigate this hypothesis, we attempted to generate a mouse conditional knockout of the Tardbp gene using the classical Cre-loxP technology. Even though heterozygote mice for the targeted allele were successfully generated, we were unable to obtain homozygotes. Here we show that although the targeting vector was specifically designed to not overlap with Tardbp adjacent genes, the homologous recombination event affected the expression of a downstream gene, Masp2. This may explain the inability to obtain homozygote mice with targeted Tardbp. PMID:24740308

  7. Differential Gene Expression Reveals Candidate Genes for Drought Stress Response in Abies alba (Pinaceae)

    PubMed Central

    Ziegenhagen, Birgit; Liepelt, Sascha

    2015-01-01

    Increasing drought periods as a result of global climate change pose a threat to many tree species by possibly outpacing their adaptive capabilities. Revealing the genetic basis of drought stress response is therefore implemental for future conservation strategies and risk assessment. Access to informative genomic regions is however challenging, especially for conifers, partially due to their large genomes, which puts constraints on the feasibility of whole genome scans. Candidate genes offer a valuable tool to reduce the complexity of the analysis and the amount of sequencing work and costs. For this study we combined an improved drought stress phenotyping of needles via a novel terahertz water monitoring technique with Massive Analysis of cDNA Ends to identify candidate genes for drought stress response in European silver fir (Abies alba Mill.). A pooled cDNA library was constructed from the cotyledons of six drought stressed and six well-watered silver fir seedlings, respectively. Differential expression analyses of these libraries revealed 296 candidate genes for drought stress response in silver fir (247 up- and 49 down-regulated) of which a subset was validated by RT-qPCR of the twelve individual cotyledons. A majority of these genes code for currently uncharacterized proteins and hint on new genomic resources to be explored in conifers. Furthermore, we could show that some traditional reference genes from model plant species (GAPDH and eIF4A2) are not suitable for differential analysis and we propose a new reference gene, TPC1, for drought stress expression profiling in needles of conifer seedlings. PMID:25924061

  8. All-optical regulation of gene expression in targeted cells

    NASA Astrophysics Data System (ADS)

    Wang, Yisen; He, Hao; Li, Shiyang; Liu, Dayong; Lan, Bei; Hu, Minglie; Cao, Youjia; Wang, Chingyue

    2014-06-01

    Controllable gene expression is always a challenge and of great significance to biomedical research and clinical applications. Recently, various approaches based on extra-engineered light-sensitive proteins have been developed to provide optogenetic actuators for gene expression. Complicated biomedical techniques including exogenous genes engineering, transfection, and material delivery are needed. Here we present an all-optical method to regulate gene expression in targeted cells. Intrinsic or exogenous genes can be activated by a Ca2+-sensitive transcription factor nuclear factor of activated T cells (NFAT) driven by a short flash of femtosecond-laser irradiation. When applied to mesenchymal stem cells, expression of a differentiation regulator Osterix can be activated by this method to potentially induce differentiation of them. A laser-induced ``Ca2+-comb'' (LiCCo) by multi-time laser exposure is further developed to enhance gene expression efficiency. This noninvasive method hence provides an encouraging advance of gene expression regulation, with promising potential of applying in cell biology and stem-cell science.

  9. Evaluation of drug-targetable genes by defining modes of abnormality in gene expression.

    PubMed

    Park, Junseong; Lee, Jungsul; Choi, Chulhee

    2015-01-01

    In the post-genomic era, many researchers have taken a systematic approach to identifying abnormal genes associated with various diseases. However, the gold standard has not been established, and most of these abnormalities are difficult to be rehabilitated in real clinical settings. In addition to identifying abnormal genes, for a practical purpose, it is necessary to investigate abnormality diversity. In this context, this study is aimed to demonstrate simply restorable genes as useful drug targets. We devised the concept of "drug targetability" to evaluate several different modes of abnormal genes by predicting events after drug treatment. As a representative example, we applied our method to breast cancer. Computationally, PTPRF, PRKAR2B, MAP4K3, and RICTOR were calculated as highly drug-targetable genes for breast cancer. After knockdown of these top-ranked genes (i.e., high drug targetability) using siRNA, our predictions were validated by cell death and migration assays. Moreover, inhibition of RICTOR or PTPRF was expected to prolong lifespan of breast cancer patients according to patient information annotated in microarray data. We anticipate that our method can be widely applied to elaborate selection of novel drug targets, and, ultimately, to improve the efficacy of disease treatment. PMID:26336805

  10. Essential gene identification and drug target prioritization in Aspergillus fumigatus.

    PubMed

    Hu, Wenqi; Sillaots, Susan; Lemieux, Sebastien; Davison, John; Kauffman, Sarah; Breton, Anouk; Linteau, Annie; Xin, Chunlin; Bowman, Joel; Becker, Jeff; Jiang, Bo; Roemer, Terry

    2007-03-01

    Aspergillus fumigatus is the most prevalent airborne filamentous fungal pathogen in humans, causing severe and often fatal invasive infections in immunocompromised patients. Currently available antifungal drugs to treat invasive aspergillosis have limited modes of action, and few are safe and effective. To identify and prioritize antifungal drug targets, we have developed a conditional promoter replacement (CPR) strategy using the nitrogen-regulated A. fumigatus NiiA promoter (pNiiA). The gene essentiality for 35 A. fumigatus genes was directly demonstrated by this pNiiA-CPR strategy from a set of 54 genes representing broad biological functions whose orthologs are confirmed to be essential for growth in Candida albicans and Saccharomyces cerevisiae. Extending this approach, we show that the ERG11 gene family (ERG11A and ERG11B) is essential in A. fumigatus despite neither member being essential individually. In addition, we demonstrate the pNiiA-CPR strategy is suitable for in vivo phenotypic analyses, as a number of conditional mutants, including an ERG11 double mutant (erg11BDelta, pNiiA-ERG11A), failed to establish a terminal infection in an immunocompromised mouse model of systemic aspergillosis. Collectively, the pNiiA-CPR strategy enables a rapid and reliable means to directly identify, phenotypically characterize, and facilitate target-based whole cell assays to screen A. fumigatus essential genes for cognate antifungal inhibitors. PMID:17352532

  11. RFMirTarget: Predicting Human MicroRNA Target Genes with a Random Forest Classifier

    PubMed Central

    Mendoza, Mariana R.; da Fonseca, Guilherme C.; Loss-Morais, Guilherme; Alves, Ronnie; Margis, Rogerio; Bazzan, Ana L. C.

    2013-01-01

    MicroRNAs are key regulators of eukaryotic gene expression whose fundamental role has already been identified in many cell pathways. The correct identification of miRNAs targets is still a major challenge in bioinformatics and has motivated the development of several computational methods to overcome inherent limitations of experimental analysis. Indeed, the best results reported so far in terms of specificity and sensitivity are associated to machine learning-based methods for microRNA-target prediction. Following this trend, in the current paper we discuss and explore a microRNA-target prediction method based on a random forest classifier, namely RFMirTarget. Despite its well-known robustness regarding general classifying tasks, to the best of our knowledge, random forest have not been deeply explored for the specific context of predicting microRNAs targets. Our framework first analyzes alignments between candidate microRNA-target pairs and extracts a set of structural, thermodynamics, alignment, seed and position-based features, upon which classification is performed. Experiments have shown that RFMirTarget outperforms several well-known classifiers with statistical significance, and that its performance is not impaired by the class imbalance problem or features correlation. Moreover, comparing it against other algorithms for microRNA target prediction using independent test data sets from TarBase and starBase, we observe a very promising performance, with higher sensitivity in relation to other methods. Finally, tests performed with RFMirTarget show the benefits of feature selection even for a classifier with embedded feature importance analysis, and the consistency between relevant features identified and important biological properties for effective microRNA-target gene alignment. PMID:23922946

  12. Identification of novel Notch target genes in T cell leukaemia

    PubMed Central

    Chadwick, Nicholas; Zeef, Leo; Portillo, Virginia; Fennessy, Carl; Warrander, Fiona; Hoyle, Sarah; Buckle, Anne-Marie

    2009-01-01

    Background Dysregulated Notch signalling is believed to play an important role in the development and maintenance of T cell leukaemia. At a cellular level, Notch signalling promotes proliferation and inhibits apoptosis of T cell acute lymphoblastic leukaemia (T-ALL) cells. In this study we aimed to identify novel transcriptional targets of Notch signalling in the T-ALL cell line, Jurkat. Results RNA was prepared from Jurkat cells retrovirally transduced with an empty vector (GFP-alone) or vectors containing constitutively active forms of Notch (N1ΔE or N3ΔE), and used for Affymetrix microarray analysis. A subset of genes found to be regulated by Notch was chosen for real-time PCR validation and in some cases, validation at the protein level, using several Notch-transduced T-ALL and non-T-ALL leukaemic cell lines. As expected, several known transcriptional target of Notch, such as HES1 and Deltex, were found to be overexpressed in Notch-transduced cells, however, many novel transcriptional targets of Notch signalling were identified using this approach. These included the T cell costimulatory molecule CD28, the anti-apoptotic protein GIMAP5, and inhibitor of DNA binding 1 (1D1). Conclusion The identification of such downstream Notch target genes provides insights into the mechanisms of Notch function in T cell leukaemia, and may help identify novel therapeutic targets in this disease. PMID:19508709

  13. Structure-Based Analysis Reveals Cancer Missense Mutations Target Protein Interaction Interfaces

    PubMed Central

    Engin, H. Billur; Kreisberg, Jason F.; Carter, Hannah

    2016-01-01

    Recently it has been shown that cancer mutations selectively target protein-protein interactions. We hypothesized that mutations affecting distinct protein interactions involving established cancer genes could contribute to tumor heterogeneity, and that novel mechanistic insights might be gained into tumorigenesis by investigating protein interactions under positive selection in cancer. To identify protein interactions under positive selection in cancer, we mapped over 1.2 million nonsynonymous somatic cancer mutations onto 4,896 experimentally determined protein structures and analyzed their spatial distribution. In total, 20% of mutations on the surface of known cancer genes perturbed protein-protein interactions (PPIs), and this enrichment for PPI interfaces was observed for both tumor suppressors (Odds Ratio 1.28, P-value < 10−4) and oncogenes (Odds Ratio 1.17, P-value < 10−3). To study this further, we constructed a bipartite network representing structurally resolved PPIs from all available human complexes in the Protein Data Bank (2,864 proteins, 3,072 PPIs). Analysis of frequently mutated cancer genes within this network revealed that tumor-suppressors, but not oncogenes, are significantly enriched with functional mutations in homo-oligomerization regions (Odds Ratio 3.68, P-Value < 10−8). We present two important examples, TP53 and beta-2-microglobulin, for which the patterns of somatic mutations at interfaces provide insights into specifically perturbed biological circuits. In patients with TP53 mutations, patient survival correlated with the specific interactions that were perturbed. Moreover, we investigated mutations at the interface of protein-nucleotide interactions and observed an unexpected number of missense mutations but not silent mutations occurring within DNA and RNA binding sites. Finally, we provide a resource of 3,072 PPI interfaces ranked according to their mutation rates. Analysis of this list highlights 282 novel candidate cancer

  14. Structure-Based Analysis Reveals Cancer Missense Mutations Target Protein Interaction Interfaces.

    PubMed

    Engin, H Billur; Kreisberg, Jason F; Carter, Hannah

    2016-01-01

    Recently it has been shown that cancer mutations selectively target protein-protein interactions. We hypothesized that mutations affecting distinct protein interactions involving established cancer genes could contribute to tumor heterogeneity, and that novel mechanistic insights might be gained into tumorigenesis by investigating protein interactions under positive selection in cancer. To identify protein interactions under positive selection in cancer, we mapped over 1.2 million nonsynonymous somatic cancer mutations onto 4,896 experimentally determined protein structures and analyzed their spatial distribution. In total, 20% of mutations on the surface of known cancer genes perturbed protein-protein interactions (PPIs), and this enrichment for PPI interfaces was observed for both tumor suppressors (Odds Ratio 1.28, P-value < 10-4) and oncogenes (Odds Ratio 1.17, P-value < 10-3). To study this further, we constructed a bipartite network representing structurally resolved PPIs from all available human complexes in the Protein Data Bank (2,864 proteins, 3,072 PPIs). Analysis of frequently mutated cancer genes within this network revealed that tumor-suppressors, but not oncogenes, are significantly enriched with functional mutations in homo-oligomerization regions (Odds Ratio 3.68, P-Value < 10-8). We present two important examples, TP53 and beta-2-microglobulin, for which the patterns of somatic mutations at interfaces provide insights into specifically perturbed biological circuits. In patients with TP53 mutations, patient survival correlated with the specific interactions that were perturbed. Moreover, we investigated mutations at the interface of protein-nucleotide interactions and observed an unexpected number of missense mutations but not silent mutations occurring within DNA and RNA binding sites. Finally, we provide a resource of 3,072 PPI interfaces ranked according to their mutation rates. Analysis of this list highlights 282 novel candidate cancer genes

  15. Off-Target Effects of Psychoactive Drugs Revealed by Genome-Wide Assays in Yeast

    PubMed Central

    Ericson, Elke; Gebbia, Marinella; Heisler, Lawrence E.; Wildenhain, Jan; Tyers, Mike; Giaever, Guri; Nislow, Corey

    2008-01-01

    To better understand off-target effects of widely prescribed psychoactive drugs, we performed a comprehensive series of chemogenomic screens using the budding yeast Saccharomyces cerevisiae as a model system. Because the known human targets of these drugs do not exist in yeast, we could employ the yeast gene deletion collections and parallel fitness profiling to explore potential off-target effects in a genome-wide manner. Among 214 tested, documented psychoactive drugs, we identified 81 compounds that inhibited wild-type yeast growth and were thus selected for genome-wide fitness profiling. Many of these drugs had a propensity to affect multiple cellular functions. The sensitivity profiles of half of the analyzed drugs were enriched for core cellular processes such as secretion, protein folding, RNA processing, and chromatin structure. Interestingly, fluoxetine (Prozac) interfered with establishment of cell polarity, cyproheptadine (Periactin) targeted essential genes with chromatin-remodeling roles, while paroxetine (Paxil) interfered with essential RNA metabolism genes, suggesting potential secondary drug targets. We also found that the more recently developed atypical antipsychotic clozapine (Clozaril) had no fewer off-target effects in yeast than the typical antipsychotics haloperidol (Haldol) and pimozide (Orap). Our results suggest that model organism pharmacogenetic studies provide a rational foundation for understanding the off-target effects of clinically important psychoactive agents and suggest a rational means both for devising compound derivatives with fewer side effects and for tailoring drug treatment to individual patient genotypes. PMID:18688276

  16. Inferring gene targets of drugs and chemical compounds from gene expression profiles

    PubMed Central

    Noh, Heeju; Gunawan, Rudiyanto

    2016-01-01

    Motivation: Finding genes which are directly perturbed or targeted by drugs is of great interest and importance in drug discovery. Several network filtering methods have been created to predict the gene targets of drugs from gene expression data based on an ordinary differential equation model of the gene regulatory network (GRN). A critical step in these methods involves inferring the GRN from the expression data, which is a very challenging problem on its own. In addition, existing network filtering methods require computationally intensive parameter tuning or expression data from experiments with known genetic perturbations or both. Results: We developed a method called DeltaNet for the identification of drug targets from gene expression data. Here, the gene target predictions were directly inferred from the data without a separate step of GRN inference. DeltaNet formulation led to solving an underdetermined linear regression problem, for which we employed least angle regression (DeltaNet-LAR) or LASSO regularization (DeltaNet-LASSO). The predictions using DeltaNet for expression data of Escherichia coli, yeast, fruit fly and human were significantly more accurate than those using network filtering methods, namely mode of action by network identification (MNI) and sparse simultaneous equation model (SSEM). Furthermore, DeltaNet using LAR did not require any parameter tuning and could provide computational speed-up over existing methods. Conclusion: DeltaNet is a robust and numerically efficient tool for identifying gene perturbations from gene expression data. Importantly, the method requires little to no expert supervision, while providing accurate gene target predictions. Availability and implementation: DeltaNet is available on http://www.cabsel.ethz.ch/tools/DeltaNet. Contact: rudi.gunawan@chem.ethz.ch Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27153589

  17. Conserved miR164-targeted NAC genes negatively regulate drought resistance in rice

    PubMed Central

    Xie, Kabin; Xiong, Lizhong

    2014-01-01

    MicroRNAs constitute a large group of endogenous small RNAs of ~22 nt that emerge as vital regulators, mainly by targeting mRNAs for post-transcriptional repression. Previous studies have revealed that the miR164 family in Arabidopsis is comprised of three members which guide the cleavage of the mRNAs of five NAC genes to modulate developmental processes. However, the functions of the miR164-targeted NAC genes in crops are poorly deciphered. In this study, the conserved features of six miR164-targeted NAC genes (OMTN1–OMTN6) in rice are described, and evidence is provided that four of them confer a negative regulatory role in drought resistance. OMTN proteins have the characteristics of typical NAC transcriptional factors. The miR164 recognition sites of the OMTN genes are highly conserved in rice germplasms. Deletion of the recognition sites impaired the transactivation activity, indicating that the conserved recognition sites play a crucial role in maintaining the function of the OMTN proteins. The OMTN genes were responsive to abiotic stresses, and showed diverse spatio-temporal expression patterns in rice. Overexpression of OMTN2, OMTN3, OMTN4, and OMTN6 in rice led to negative effects on drought resistance at the reproductive stage. The expression of numerous genes related to stress response, development, and metabolism was altered in OMTN2-, OMTN3-, OMTN4-, and OMTN6-overexpressing plants. Most of the up-regulated genes in the OMTN-overexpressing plants were down-regulated by drought stress. The results suggest that the conserved miR164-targeted NAC genes may be negative regulators of drought tolerance in rice, in addition to their reported roles in development. PMID:24604734

  18. An Encapsulation of Gene Signatures for Hepatocellular Carcinoma, MicroRNA-132 Predicted Target Genes and the Corresponding Overlaps

    PubMed Central

    Chen, Gang; Ren, Fanghui; Liang, Haiwei; Dang, Yiwu; Rong, Minhua

    2016-01-01

    Objectives Previous studies have demonstrated that microRNA-132 plays a vital part in and is actively associated with several cancers, with its tumor-suppressive role in hepatocellular carcinoma confirmed. The current study employed multiple bioinformatics techniques to establish gene signatures for hepatocellular carcinoma, microRNA-132 predicted target genes and the corresponding overlaps. Methods Various assays were performed to explore the role and cellular functions of miR-132 in HCC and a successive panel of tasks was completed, including NLP analysis, miR-132 target genes prediction, comprehensive analyses (gene ontology analysis, pathway analysis, network analysis and connectivity analysis), and analytical integration. Later, HCC-related and miR-132-related potential targets, pathways, networks and highlighted hub genes were revealed as well as those of the overlapped section. Results MiR-132 was effective in both impeding cell growth and boosting apoptosis in HCC cell lines. A total of fifty-nine genes were obtained from the analytical integration, which were considered to be both HCC- and miR-132-related. Moreover, four specific pathways were unveiled in the network analysis of the overlaps, i.e. adherens junction, VEGF signaling pathway, neurotrophin signaling pathway, and MAPK signaling pathway. Conclusions The tumor-suppressive role of miR-132 in HCC has been further confirmed by in vitro experiments. Gene signatures in the study identified the potential molecular mechanisms of HCC, miR-132 and their established associations, which might be effective for diagnosis, individualized treatments and prognosis of HCC patients. However, combined detections of miR-132 with other bio-indicators in clinical practice and further in vitro experiments are needed. PMID:27467251

  19. Structural Code for DNA Recognition Revealed in Crystal Structures of Papillomavirus E2-DNA Targets

    NASA Astrophysics Data System (ADS)

    Rozenberg, Haim; Rabinovich, Dov; Frolow, Felix; Hegde, Rashmi S.; Shakked, Zippora

    1998-12-01

    Transcriptional regulation in papillomaviruses depends on sequence-specific binding of the regulatory protein E2 to several sites in the viral genome. Crystal structures of bovine papillomavirus E2 DNA targets reveal a conformational variant of B-DNA characterized by a roll-induced writhe and helical repeat of 10.5 bp per turn. A comparison between the free and the protein-bound DNA demonstrates that the intrinsic structure of the DNA regions contacted directly by the protein and the deformability of the DNA region that is not contacted by the protein are critical for sequence-specific protein/DNA recognition and hence for gene-regulatory signals in the viral system. We show that the selection of dinucleotide or longer segments with appropriate conformational characteristics, when positioned at correct intervals along the DNA helix, can constitute a structural code for DNA recognition by regulatory proteins. This structural code facilitates the formation of a complementary protein-DNA interface that can be further specified by hydrogen bonds and nonpolar interactions between the protein amino acids and the DNA bases.

  20. Signature gene expression reveals novel clues to the molecular mechanisms of dimorphic transition in Penicillium marneffei.

    PubMed

    Yang, Ence; Chow, Wang-Ngai; Wang, Gang; Woo, Patrick C Y; Lau, Susanna K P; Yuen, Kwok-Yung; Lin, Xiaorong; Cai, James J

    2014-10-01

    Systemic dimorphic fungi cause more than one million new infections each year, ranking them among the significant public health challenges currently encountered. Penicillium marneffei is a systemic dimorphic fungus endemic to Southeast Asia. The temperature-dependent dimorphic phase transition between mycelium and yeast is considered crucial for the pathogenicity and transmission of P. marneffei, but the underlying mechanisms are still poorly understood. Here, we re-sequenced P. marneffei strain PM1 using multiple sequencing platforms and assembled the genome using hybrid genome assembly. We determined gene expression levels using RNA sequencing at the mycelial and yeast phases of P. marneffei, as well as during phase transition. We classified 2,718 genes with variable expression across conditions into 14 distinct groups, each marked by a signature expression pattern implicated at a certain stage in the dimorphic life cycle. Genes with the same expression patterns tend to be clustered together on the genome, suggesting orchestrated regulations of the transcriptional activities of neighboring genes. Using qRT-PCR, we validated expression levels of all genes in one of clusters highly expressed during the yeast-to-mycelium transition. These included madsA, a gene encoding MADS-box transcription factor whose gene family is exclusively expanded in P. marneffei. Over-expression of madsA drove P. marneffei to undergo mycelial growth at 37°C, a condition that restricts the wild-type in the yeast phase. Furthermore, analyses of signature expression patterns suggested diverse roles of secreted proteins at different developmental stages and the potential importance of non-coding RNAs in mycelium-to-yeast transition. We also showed that RNA structural transition in response to temperature changes may be related to the control of thermal dimorphism. Together, our findings have revealed multiple molecular mechanisms that may underlie the dimorphic transition in P. marneffei

  1. Integrative analysis reveals disease-associated genes and biomarkers for prostate cancer progression

    PubMed Central

    2014-01-01

    Background Prostate cancer is one of the most common complex diseases with high leading cause of death in men. Identifications of prostate cancer associated genes and biomarkers are thus essential as they can gain insights into the mechanisms underlying disease progression and advancing for early diagnosis and developing effective therapies. Methods In this study, we presented an integrative analysis of gene expression profiling and protein interaction network at a systematic level to reveal candidate disease-associated genes and biomarkers for prostate cancer progression. At first, we reconstructed the human prostate cancer protein-protein interaction network (HPC-PPIN) and the network was then integrated with the prostate cancer gene expression data to identify modules related to different phases in prostate cancer. At last, the candidate module biomarkers were validated by its predictive ability of prostate cancer progression. Results Different phases-specific modules were identified for prostate cancer. Among these modules, transcription Androgen Receptor (AR) nuclear signaling and Epidermal Growth Factor Receptor (EGFR) signalling pathway were shown to be the pathway targets for prostate cancer progression. The identified candidate disease-associated genes showed better predictive ability of prostate cancer progression than those of published biomarkers. In context of functional enrichment analysis, interestingly candidate disease-associated genes were enriched in the nucleus and different functions were encoded for potential transcription factors, for examples key players as AR, Myc, ESR1 and hidden player as Sp1 which was considered as a potential novel biomarker for prostate cancer. Conclusions The successful results on prostate cancer samples demonstrated that the integrative analysis is powerful and useful approach to detect candidate disease-associate genes and modules which can be used as the potential biomarkers for prostate cancer progression. The

  2. Targeted massively parallel sequencing of angiosarcomas reveals frequent activation of the mitogen activated protein kinase pathway

    PubMed Central

    Murali, Rajmohan; Chandramohan, Raghu; Möller, Inga; Scholz, Simone L.; Berger, Michael; Huberman, Kety; Viale, Agnes; Pirun, Mono; Socci, Nicholas D.; Bouvier, Nancy; Bauer, Sebastian; Artl, Monika; Schilling, Bastian; Schimming, Tobias; Sucker, Antje; Schwindenhammer, Benjamin; Grabellus, Florian; Speicher, Michael R.; Schaller, Jörg; Hillen, Uwe; Schadendorf, Dirk; Mentzel, Thomas; Cheng, Donavan T.; Wiesner, Thomas; Griewank, Klaus G.

    2015-01-01

    Angiosarcomas are rare malignant mesenchymal tumors of endothelial differentiation. The clinical behavior is usually aggressive and the prognosis for patients with advanced disease is poor with no effective therapies. The genetic bases of these tumors have been partially revealed in recent studies reporting genetic alterations such as amplifications of MYC (primarily in radiation-associated angiosarcomas), inactivating mutations in PTPRB and R707Q hotspot mutations of PLCG1. Here, we performed a comprehensive genomic analysis of 34 angiosarcomas using a clinically-approved, hybridization-based targeted next-generation sequencing assay for 341 well-established oncogenes and tumor suppressor genes. Over half of the angiosarcomas (n = 18, 53%) harbored genetic alterations affecting the MAPK pathway, involving mutations in KRAS, HRAS, NRAS, BRAF, MAPK1 and NF1, or amplifications in MAPK1/CRKL, CRAF or BRAF. The most frequently detected genetic aberrations were mutations in TP53 in 12 tumors (35%) and losses of CDKN2A in 9 tumors (26%). MYC amplifications were generally mutually exclusive of TP53 alterations and CDKN2A loss and were identified in 8 tumors (24%), most of which (n = 7, 88%) arose post-irradiation. Previously reported mutations in PTPRB (n = 10, 29%) and one (3%) PLCG1 R707Q mutation were also identified. Our results demonstrate that angiosarcomas are a genetically heterogeneous group of tumors, harboring a wide range of genetic alterations. The high frequency of genetic events affecting the MAPK pathway suggests that targeted therapies inhibiting MAPK signaling may be promising therapeutic avenues in patients with advanced angiosarcomas. PMID:26440310

  3. Targeted gene knockout by direct delivery of ZFN proteins

    PubMed Central

    Gaj, Thomas; Guo, Jing; Kato, Yoshio; Sirk, Shannon J.; Barbas, Carlos F.

    2012-01-01

    Zinc-finger nucleases (ZFNs) are versatile reagents that have redefined genome engineering. Realizing the full potential of this technology requires the development of safe and effective methods for delivering ZFNs into cells. We demonstrate the intrinsic cell-penetrating capabilities of the standard ZFN architecture and show that direct delivery of ZFNs as proteins leads to efficient endogenous gene disruption in a variety of mammalian cell types with minimal off-target effects. PMID:22751204

  4. Analysis of the siRNA-Mediated Gene Silencing Process Targeting Three Homologous Genes Controlling Soybean Seed Oil Quality

    PubMed Central

    Lu, Sha; Yin, Xiaoyan; Spollen, William; Zhang, Ning; Xu, Dong; Schoelz, James; Bilyeu, Kristin; Zhang, Zhanyuan J.

    2015-01-01

    In the past decade, RNA silencing has gained significant attention because of its success in genomic scale research and also in the genetic improvement of crop plants. However, little is known about the molecular basis of siRNA processing in association with its target transcript. To reveal this process for improving hpRNA-mediated gene silencing in crop plants, the soybean GmFAD3 gene family was chosen as a test model. We analyzed RNAi mutant soybean lines in which three members of the GmFAD3 gene family were silenced. The silencing levels of FAD3A, FAD3B and FAD3C were correlated with the degrees of sequence homology between the inverted repeat of hpRNA and the GmFAD3 transcripts in the RNAi lines. Strikingly, transgenes in two of the three RNAi lines were heavily methylated, leading to a dramatic reduction of hpRNA-derived siRNAs. Small RNAs corresponding to the loop portion of the hairpin transcript were detected while much lower levels of siRNAs were found outside of the target region. siRNAs generated from the 318-bp inverted repeat were found to be diced much more frequently at stem sequences close to the loop and associated with the inferred cleavage sites on the target transcripts, manifesting “hot spots”. The top candidate hpRNA-derived siRNA share certain sequence features with mature miRNA. This is the first comprehensive and detailed study revealing the siRNA-mediated gene silencing mechanism in crop plants using gene family GmFAD3 as a test model. PMID:26061033

  5. Integration of TP53, DREAM, MMB-FOXM1 and RB-E2F target gene analyses identifies cell cycle gene regulatory networks

    PubMed Central

    Fischer, Martin; Grossmann, Patrick; Padi, Megha; DeCaprio, James A.

    2016-01-01

    Cell cycle (CC) and TP53 regulatory networks are frequently deregulated in cancer. While numerous genome-wide studies of TP53 and CC-regulated genes have been performed, significant variation between studies has made it difficult to assess regulation of any given gene of interest. To overcome the limitation of individual studies, we developed a meta-analysis approach to identify high confidence target genes that reflect their frequency of identification in independent datasets. Gene regulatory networks were generated by comparing differential expression of TP53 and CC-regulated genes with chromatin immunoprecipitation studies for TP53, RB1, E2F, DREAM, B-MYB, FOXM1 and MuvB. RNA-seq data from p21-null cells revealed that gene downregulation by TP53 generally requires p21 (CDKN1A). Genes downregulated by TP53 were also identified as CC genes bound by the DREAM complex. The transcription factors RB, E2F1 and E2F7 bind to a subset of DREAM target genes that function in G1/S of the CC while B-MYB, FOXM1 and MuvB control G2/M gene expression. Our approach yields high confidence ranked target gene maps for TP53, DREAM, MMB-FOXM1 and RB-E2F and enables prediction and distinction of CC regulation. A web-based atlas at www.targetgenereg.org enables assessing the regulation of any human gene of interest. PMID:27280975

  6. Integration of TP53, DREAM, MMB-FOXM1 and RB-E2F target gene analyses identifies cell cycle gene regulatory networks.

    PubMed

    Fischer, Martin; Grossmann, Patrick; Padi, Megha; DeCaprio, James A

    2016-07-27

    Cell cycle (CC) and TP53 regulatory networks are frequently deregulated in cancer. While numerous genome-wide studies of TP53 and CC-regulated genes have been performed, significant variation between studies has made it difficult to assess regulation of any given gene of interest. To overcome the limitation of individual studies, we developed a meta-analysis approach to identify high confidence target genes that reflect their frequency of identification in independent datasets. Gene regulatory networks were generated by comparing differential expression of TP53 and CC-regulated genes with chromatin immunoprecipitation studies for TP53, RB1, E2F, DREAM, B-MYB, FOXM1 and MuvB. RNA-seq data from p21-null cells revealed that gene downregulation by TP53 generally requires p21 (CDKN1A). Genes downregulated by TP53 were also identified as CC genes bound by the DREAM complex. The transcription factors RB, E2F1 and E2F7 bind to a subset of DREAM target genes that function in G1/S of the CC while B-MYB, FOXM1 and MuvB control G2/M gene expression. Our approach yields high confidence ranked target gene maps for TP53, DREAM, MMB-FOXM1 and RB-E2F and enables prediction and distinction of CC regulation. A web-based atlas at www.targetgenereg.org enables assessing the regulation of any human gene of interest. PMID:27280975

  7. Targeted disruption of the Lowe syndrome gene (OCRL-1)

    SciTech Connect

    Jaenne, P.A.; Olivos, I.; Grinberg, A.

    1994-09-01

    The oculocerebrorenal syndrome of Lowe (OCRL) is a rare X-linked disease characterized by congenital cataract formation, mental retardation and renal tubular dysfunction (Fanconi syndrome). The gene for OCRL (OCRL-1) has recently been identified through positional cloning techniques and is highly homologous to a previously reported gene encoding a 75 kDa inositol polyphosphate-5-phosphatase. Thus OCRL might be caused by an alteration in inositol metabolism. In order to further investigate the role of OCRL-1 in Lowe`s syndrome, we decided to use targeted disruption to create mice lacking a functional OCRL-1 protein. The murine homologue of OCRL-1 (Ocrl-1) was cloned from a 129Sv genomic library. Two targeting vectors were created from the 3{prime}-end of the gene by fusing a neomycin resistance gene (PGK-Neo) into two exons. The first vector employed a classic positive negative selection scheme whereas the second vector included a polyadenylation trap. The vectors were electroporated into CCE or J1 ES cells and recombinants were screened by Southern blotting. Targeted cells were obtained at a frequency of 1/50 (for CCE) and 1/16 (for J1 using the polyadenylation trap). Using antibodies made to an OCRL-1 fusion protein, we could demonstrate a lack of Ocrl-1 protein product in the targeted ES cell lines. Therefore, we had created a null allele at the Ocrl-1 locus. The targeted ES clones were injected into 3.5 dpc C57B1/6 blastocysts and chimeric mice were obtained. Male chimeras have been made from five targeted cell lines. The males were mated with C57B1/6 females and germline transmission has been obtained from males derived from two of the five cell lines (one from CCE and one from J1 targeted ES cells). Preliminary analysis of male Ocrl-1{sup {minus}} mice suggests the presence of a proximal renal tubular dysfunction but the absence of detectable cataracts. We are presently continuing our phenotypic analyses.

  8. Small interfering RNAs targeting the rabies virus nucleoprotein gene.

    PubMed

    Yang, Yu-Jiao; Zhao, Ping-Sen; Zhang, Tao; Wang, Hua-Lei; Liang, Hong-Ru; Zhao, Li-Li; Wu, Hong-Xia; Wang, Tie-Cheng; Yang, Song-Tao; Xia, Xian-Zhu

    2012-10-01

    Rabies virus (RABV) infection continues to be a global threat to human and animal health, yet no curative therapy has been developed. RNA interference (RNAi) therapy, which silences expression of specific target genes, represents a promising approach for treating viral infections in mammalian hosts. We designed six small interfering (si)RNAs (N473, N580, N783, N796, N799 and N1227) that target the conserved region of the RABV challenge virus standard (CVS)-11 strain nucleoprotein (N) gene. Using a plasmid-based transient expression model, we demonstrated that N796, N580 and N799 were capable of significantly inhibiting viral replication in vitro and in vivo. These three siRNAs effectively suppressed RABV expression in infected baby hamster kidney-21 (BHK-21) cells, as evidenced by direct immunofluorescence assay, viral titer measurements, real-time PCR, and Western blotting. In addition, liposome-mediated siRNA expression plasmid delivery to RABV-infected mice significantly increased survival, compared to a non-liposome-mediated delivery method. Collectively, our results showed that the three siRNAs, N796, N580 and N799, targeting the N gene could potently inhibit RABV CVS-11 reproduction. These siRNAs have the potential to be developed into new and effective prophylactic anti-RABV drugs. PMID:22884777

  9. Treating psoriasis by targeting its susceptibility gene Rel.

    PubMed

    Fan, Tingting; Wang, Shaowen; Yu, Linjiang; Yi, Huqiang; Liu, Ruiling; Geng, Wenwen; Wan, Xiaochun; Ma, Yifan; Cai, Lintao; Chen, Youhai H; Ruan, Qingguo

    2016-04-01

    Psoriasis is a chronic inflammatory disorder of the skin. Accumulating evidence indicates that the Rel gene, a member of the NF-κB family, is a risk factor for the disease. We sought to investigate whether psoriasis can be prevented by directly targeting the Rel gene transcript, i.e., the c-Rel mRNA. Using chemically-modified c-Rel specific siRNA (siRel) and poly(ethylene glycol)-b-poly(l-lysine)-b-poly(l-leucine) (PEG-PLL-PLLeu) micelles, we successfully knocked down the expression of c-Rel, and showed that the expression of cytokine IL-23, a direct target of c-Rel that can drive the development of IL-17-producing T cells, was markedly inhibited. More importantly, treating mice with siRel not only prevented but also ameliorated imiquimod (IMQ)-induced psoriasis. Mechanistic studies showed that siRel treatment down-regulated the expression of multiple inflammatory cytokines. Taken together, these results indicate that the susceptibility gene Rel can be targeted to treat and prevent psoriasis. PMID:26993753

  10. Target Gene Abundance Contributes to the Efficiency of siRNA-Mediated Gene Silencing

    PubMed Central

    Hong, Sun Woo; Jiang, Yuanyuan; Kim, Soyoun; Li, Chiang J.

    2014-01-01

    The gene-silencing activity of a small interfering RNA (siRNA) is determined by various factors. Considering that RNA interference (RNAi) is an unparalleled technology in both basic research and therapeutic applications, thorough understanding of the factors determining RNAi activity is critical. This report presents observations that siRNAs targeting KRT7 show cell-line-dependent activity, which correlates with the expression level of KRT7 mRNA. By modulating the target mRNA level, it was confirmed that highly expressed genes are more susceptible to siRNA-mediated gene silencing. Finally, several genes that show different expression levels in a cell-line dependent manner were tested, which verified the expression-level-dependent siRNA activities. These results strongly suggest that the abundance of target mRNA is a critical factor that determines the efficiency of the siRNA-mediated gene silencing in a given cellular context. This report should provide practical guidelines for designing RNAi experiments and for selecting targetable genes in RNAi therapeutics studies. PMID:24527979

  11. Salivary epithelial cells: an unassuming target site for gene therapeutics

    PubMed Central

    Perez, Paola; Rowzee, Anne M.; Zheng, Changyu; Adriaansen, Janik; Baum, Bruce J.

    2010-01-01

    Salivary glands are classical exocrine glands whose external secretions result in the production of saliva. However, in addition to the secretion of exocrine proteins, salivary epithelial cells are also capable of secreting proteins internally, into the bloodstream. This brief review examines the potential for using salivary epithelial cells as a target site for in situ gene transfer, with an ultimate goal of producing therapeutic proteins for treating both systemic and upper gastrointestinal tract disorders. The review discusses the protein secretory pathways reported to be present in salivary epithelial cells, the viral gene transfer vectors shown useful for transducing these cells, model transgenic secretory proteins examined, and some clinical conditions that might benefit from such salivary gland gene transfer. PMID:20219693

  12. The down regulation of target genes by photo activated DNA nanoscissors.

    PubMed

    Tsai, Tsung-Lin; Shieh, Dar-Bin; Yeh, Chen-Sheng; Tzeng, Yonhua; Htet, Khant; Chuang, Kao-Shu; Hwu, Jih Ru; Su, Wu-Chou

    2010-09-01

    An artificial, targeted, light-activated nanoscissor (ATLANS) was developed for precision photonic cleavage of DNA at selectable target sequences. The ATLANS is comprised of nanoparticle core and a monolayer of hydrazone-modified triplex-forming oligonucleotides (TFOs), which recognize and capture the targeted DNA duplex. Upon photo-illumination (lambda = 460 nm), the attached hydrazone scissor specifically cleaves the targeted DNA at a pre-designed nucleotide pair. Electrophoretic mobility shift and co-precipitation assays revealed sequence-specific binding with the short-fragment and long-form plasmid DNA of both TFO and TFO-nanoparticle probes. Upon photo-illumination, ATLANS introduced a precise double-stranded break 12bp downstream the TFO binding sequence and down-regulated the target gene in HeLa cell system. Gold nanoparticles multiplexed the cutting efficiency and potential for simultaneous manipulation of multiple targets, as well as protected DNA from non-specific photo-damage. This photon-mediated DNA manipulation technology will facilitate high spatial and temporal precision in simultaneous silencing at the genome level, and advanced simultaneous manipulation of multiple targeted genes. PMID:20605206

  13. Tumor targeting and microenvironment-responsive nanoparticles for gene delivery.

    PubMed

    Huang, Shixian; Shao, Kun; Kuang, Yuyang; Liu, Yang; Li, Jianfeng; An, Sai; Guo, Yubo; Ma, Haojun; He, Xi; Jiang, Chen

    2013-07-01

    A tumor targeting nanoparticle system has been successfully developed to response to the lowered tumor extracellular pH (pHe) and upregulated matrix metalloproteinase 2 (MMP2) in the tumor microenvironment. The nanoparticles are modified with activatable cell-penetrating peptide (designated as dtACPP) that's dual-triggered by the lowered pHe and MMP2. In dtACPP, the internalization function of cell-penetrating peptide (CPP) is quenched by a pH-sensitive masking peptide, linking by a MMP2 substrate. The masking peptide is negatively charged to quench the cationic CPP well after systemic administration. Hence, dtACPP-modified nanoparticles possesses passive tumor targetability via the enhanced permeability and retention (EPR) effect. Once reaching the tumor microenvironment, the pre-existing attraction would be eliminated due to the lowered pHe, accompanying the linker cleaved by MMP2, dtACPP would be activated to expose CPP to drive the nanoparticles' internalization into the intratumoral cells. The studies of plasmid DNA loading, toxicity assessment, cellular uptake, tumor targeting delivery, and gene transfection demonstrate that dtACPP-modified nanoparticle system is a potential candidate for tumor targeting gene delivery. PMID:23562171

  14. Methods of Combinatorial Optimization to Reveal Factors Affecting Gene Length

    PubMed Central

    Bolshoy, Alexander; Tatarinova, Tatiana

    2012-01-01

    In this paper we present a novel method for genome ranking according to gene lengths. The main outcomes described in this paper are the following: the formulation of the genome ranking problem, presentation of relevant approaches to solve it, and the demonstration of preliminary results from prokaryotic genomes ordering. Using a subset of prokaryotic genomes, we attempted to uncover factors affecting gene length. We have demonstrated that hyperthermophilic species have shorter genes as compared with mesophilic organisms, which probably means that environmental factors affect gene length. Moreover, these preliminary results show that environmental factors group together in ranking evolutionary distant species. PMID:23300345

  15. Analysis of Deregulated microRNAs and Their Target Genes in Gastric Cancer

    PubMed Central

    Kupcinskas, Juozas; Link, Alexander; Kiudelis, Gediminas; Jonaitis, Laimas; Jarmalaite, Sonata; Kupcinskas, Limas; Malfertheiner, Peter; Skieceviciene, Jurgita

    2015-01-01

    Background MicroRNAs (miRNAs) are widely studied non-coding RNAs that modulate gene expression. MiRNAs are deregulated in different tumors including gastric cancer (GC) and have potential diagnostic and prognostic implications. The aim of our study was to determine miRNA profile in GC tissues, followed by evaluation of deregulated miRNAs in plasma of GC patients. Using available databases and bioinformatics methods we also aimed to evaluate potential target genes of confirmed differentially expressed miRNA and validate these findings in GC tissues. Methods The study included 51 GC patients and 51 controls. Initially, we screened miRNA expression profile in 13 tissue samples of GC and 12 normal gastric tissues with TaqMan low density array (TLDA). In the second stage, differentially expressed miRNAs were validated in a replication cohort using qRT-PCR in tissue and plasma samples. Subsequently, we analyzed potential target genes of deregulated miRNAs using bioinformatics approach, determined their expression in GC tissues and performed correlation analysis with targeting miRNAs. Results Profiling with TLDA revealed 15 deregulated miRNAs in GC tissues compared to normal gastric mucosa. Replication analysis confirmed that miR-148a-3p, miR-204-5p, miR-223-3p and miR-375 were consistently deregulated in GC tissues. Analysis of GC patients’ plasma samples showed significant down-regulation of miR-148a-3p, miR-375 and up-regulation of miR-223-3p compared to healthy subjects. Further, using bioinformatic tools we identified targets of replicated miRNAs and performed disease-associated gene enrichment analysis. Ultimately, we evaluated potential target gene BCL2 and DNMT3B expression by qRT-PCR in GC tissue, which correlated with targeting miRNA expression. Conclusions Our study revealed miRNA profile in GC tissues and showed that miR-148a-3p, miR-223-3p and miR-375 are deregulated in GC plasma samples, but these circulating miRNAs showed relatively weak diagnostic

  16. Identification of gene targets against dormant phase Mycobacterium tuberculosis infections

    PubMed Central

    Murphy, Dennis J; Brown, James R

    2007-01-01

    Background Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), infects approximately 2 billion people worldwide and is the leading cause of mortality due to infectious disease. Current TB therapy involves a regimen of four antibiotics taken over a six month period. Patient compliance, cost of drugs and increasing incidence of drug resistant M. tuberculosis strains have added urgency to the development of novel TB therapies. Eradication of TB is affected by the ability of the bacterium to survive up to decades in a dormant state primarily in hypoxic granulomas in the lung and to cause recurrent infections. Methods The availability of M. tuberculosis genome-wide DNA microarrays has lead to the publication of several gene expression studies under simulated dormancy conditions. However, no single model best replicates the conditions of human pathogenicity. In order to identify novel TB drug targets, we performed a meta-analysis of multiple published datasets from gene expression DNA microarray experiments that modeled infection leading to and including the dormant state, along with data from genome-wide insertional mutagenesis that examined gene essentiality. Results Based on the analysis of these data sets following normalization, several genome wide trends were identified and used to guide the selection of targets for therapeutic development. The trends included the significant up-regulation of genes controlled by devR, down-regulation of protein and ATP synthesis, and the adaptation of two-carbon metabolism to the hypoxic and nutrient limited environment of the granuloma. Promising targets for drug discovery were several regulatory elements (devR/devS, relA, mprAB), enzymes involved in redox balance and respiration, sulfur transport and fixation, pantothenate, isoprene, and NAD biosynthesis. The advantages and liabilities of each target are discussed in the context of enzymology, bacterial pathways, target tractability, and drug development

  17. Eye can read your mind: decoding gaze fixations to reveal categorical search targets.

    PubMed

    Zelinsky, Gregory J; Peng, Yifan; Samaras, Dimitris

    2013-01-01

    Is it possible to infer a person's goal by decoding their fixations on objects? Two groups of participants categorically searched for either a teddy bear or butterfly among random category distractors, each rated as high, medium, or low in similarity to the target classes. Target-similar objects were preferentially fixated in both search tasks, demonstrating information about target category in looking behavior. Different participants then viewed the searchers' scanpaths, superimposed over the target-absent displays, and attempted to decode the target category (bear/butterfly). Bear searchers were classified perfectly; butterfly searchers were classified at 77%. Bear and butterfly Support Vector Machine (SVM) classifiers were also used to decode the same preferentially fixated objects and found to yield highly comparable classification rates. We conclude that information about a person's search goal exists in fixation behavior, and that this information can be behaviorally decoded to reveal a search target-essentially reading a person's mind by analyzing their fixations. PMID:24338446

  18. Genome-wide identification of miR-200 targets reveals a regulatory network controlling cell invasion.

    PubMed

    Bracken, Cameron P; Li, Xiaochun; Wright, Josephine A; Lawrence, David M; Pillman, Katherine A; Salmanidis, Marika; Anderson, Matthew A; Dredge, B Kate; Gregory, Philip A; Tsykin, Anna; Neilsen, Corine; Thomson, Daniel W; Bert, Andrew G; Leerberg, Joanne M; Yap, Alpha S; Jensen, Kirk B; Khew-Goodall, Yeesim; Goodall, Gregory J

    2014-09-17

    The microRNAs of the miR-200 family maintain the central characteristics of epithelia and inhibit tumor cell motility and invasiveness. Using the Ago-HITS-CLIP technology for transcriptome-wide identification of direct microRNA targets in living cells, along with extensive validation to verify the reliability of the approach, we have identified hundreds of miR-200a and miR-200b targets, providing insights into general features of miRNA target site selection. Gene ontology analysis revealed a predominant effect of miR-200 targets in widespread coordinate control of actin cytoskeleton dynamics. Functional characterization of the miR-200 targets indicates that they constitute subnetworks that underlie the ability of cancer cells to migrate and invade, including coordinate effects on Rho-ROCK signaling, invadopodia formation, MMP activity, and focal adhesions. Thus, the miR-200 family maintains the central characteristics of the epithelial phenotype by acting on numerous targets at multiple levels, encompassing both cytoskeletal effectors that control actin filament organization and dynamics, and upstream signals that locally regulate the cytoskeleton to maintain cell morphology and prevent cell migration. PMID:25069772

  19. Genome-wide identification of miR-200 targets reveals a regulatory network controlling cell invasion

    PubMed Central

    Bracken, Cameron P; Li, Xiaochun; Wright, Josephine A; Lawrence, David M; Pillman, Katherine A; Salmanidis, Marika; Anderson, Matthew A; Dredge, B Kate; Gregory, Philip A; Tsykin, Anna; Neilsen, Corine; Thomson, Daniel W; Bert, Andrew G; Leerberg, Joanne M; Yap, Alpha S; Jensen, Kirk B; Khew-Goodall, Yeesim; Goodall, Gregory J

    2014-01-01

    The microRNAs of the miR-200 family maintain the central characteristics of epithelia and inhibit tumor cell motility and invasiveness. Using the Ago-HITS-CLIP technology for transcriptome-wide identification of direct microRNA targets in living cells, along with extensive validation to verify the reliability of the approach, we have identified hundreds of miR-200a and miR-200b targets, providing insights into general features of miRNA target site selection. Gene ontology analysis revealed a predominant effect of miR-200 targets in widespread coordinate control of actin cytoskeleton dynamics. Functional characterization of the miR-200 targets indicates that they constitute subnetworks that underlie the ability of cancer cells to migrate and invade, including coordinate effects on Rho-ROCK signaling, invadopodia formation, MMP activity, and focal adhesions. Thus, the miR-200 family maintains the central characteristics of the epithelial phenotype by acting on numerous targets at multiple levels, encompassing both cytoskeletal effectors that control actin filament organization and dynamics, and upstream signals that locally regulate the cytoskeleton to maintain cell morphology and prevent cell migration. PMID:25069772

  20. Regulation, overexpression, and target gene identification of Potato Homeobox 15 (POTH15) - a class-I KNOX gene in potato.

    PubMed

    Mahajan, Ameya S; Kondhare, Kirtikumar R; Rajabhoj, Mohit P; Kumar, Amit; Ghate, Tejashree; Ravindran, Nevedha; Habib, Farhat; Siddappa, Sundaresha; Banerjee, Anjan K

    2016-07-01

    Potato Homeobox 15 (POTH15) is a KNOX-I (Knotted1-like homeobox) family gene in potato that is orthologous to Shoot Meristemless (STM) in Arabidopsis. Despite numerous reports on KNOX genes from different species, studies in potato are limited. Here, we describe photoperiodic regulation of POTH15, its overexpression phenotype, and identification of its potential targets in potato (Solanum tuberosum ssp. andigena). qRT-PCR analysis showed a higher abundance of POTH15 mRNA in shoot tips and stolons under tuber-inducing short-day conditions. POTH15 promoter activity was detected in apical and axillary meristems, stolon tips, tuber eyes, and meristems of tuber sprouts, indicating its role in meristem maintenance and leaf development. POTH15 overexpression altered multiple morphological traits including leaf and stem development, leaflet number, and number of nodes and branches. In particular, the rachis of the leaf was completely reduced and leaves appeared as a bouquet of leaflets. Comparative transcriptomic analysis of 35S::GUS and two POTH15 overexpression lines identified more than 6000 differentially expressed genes, including 2014 common genes between the two overexpression lines. Functional analysis of these genes revealed their involvement in responses to hormones, biotic/abiotic stresses, transcription regulation, and signal transduction. qRT-PCR of selected candidate target genes validated their differential expression in both overexpression lines. Out of 200 randomly chosen POTH15 targets, 173 were found to have at least one tandem TGAC core motif, characteristic of KNOX interaction, within 3.0kb in the upstream sequence of the transcription start site. Overall, this study provides insights to the role of POTH15 in controlling diverse developmental processes in potato. PMID:27217546

  1. Exome sequencing reveals novel mutation targets in diffuse large B-cell lymphomas derived from Chinese patients

    PubMed Central

    de Miranda, Noel F. C. C.; Georgiou, Konstantinos; Chen, Longyun; Wu, Chenglin; Gao, Zhibo; Zaravinos, Apostolos; Lisboa, Susana; Enblad, Gunilla; Teixeira, Manuel R.; Zeng, Yixin

    2014-01-01

    Next-generation sequencing studies on diffuse large B-cell lymphomas (DLBCLs) have revealed novel targets of genetic aberrations but also high intercohort heterogeneity. Previous studies have suggested that the prevalence of disease subgroups and cytogenetic profiles differ between Western and Asian patients. To characterize the coding genome of Chinese DLBCL, we performed whole-exome sequencing of DNA derived from 31 tumors and respective peripheral blood samples. The mutation prevalence of B2M, CD70, DTX1, LYN, TMSB4X, and UBE2A was investigated in an additional 105 tumor samples. We discovered 11 novel targets of recurrent mutations in DLBCL that included functionally relevant genes such as LYN and TMSB4X. Additional genes were found mutated at high frequency (≥10%) in the Chinese cohort including DTX1, which was the most prevalent mutation target in the Notch pathway. We furthermore demonstrated that mutations in DTX1 impair its function as a negative regulator of Notch. Novel and previous unappreciated targets of somatic mutations in DLBCL identified in this study support the existence of additional/alternative tumorigenic pathways in these tumors. The observed differences with previous reports might be explained by the genetic heterogeneity of DLBCL, the germline genetic makeup of Chinese individuals, and/or exposure to distinct etiological agents. PMID:25171927

  2. MicroRNAs and Their Target Genes in Gingival Tissues

    PubMed Central

    Stoecklin-Wasmer, C.; Guarnieri, P.; Celenti, R.; Demmer, R.T.; Kebschull, M.; Papapanou, P.N.

    2012-01-01

    To gain insights into the in vivo function of miRNAs in the context of periodontitis, we examined the occurrence of miRNAs in healthy and diseased gingival tissues and validated their in silico-predicted targets through mRNA profiling using whole-genome microarrays in the same specimens. Eighty-six individuals with periodontitis contributed 198 gingival papillae: 158 ‘diseased’ (bleeding-on-probing, PD > 4 mm, and AL ≥ 3 mm) and 40 ‘healthy’ (no bleeding, PD ≤ 4 mm, and AL ≤ 2 mm). Expression of 1,205 miRNAs was assessed by microarrays, followed by selected confirmation by quantitative RT-PCR. Predicted miRNA targets were identified and tested for enrichment by Gene Set Enrichment Analysis (GSEA). Enriched gene sets were grouped in functional categories by DAVID and Ingenuity Pathway Analysis. One hundred fifty-nine miRNAs were significantly differentially expressed between healthy and diseased gingiva. Four miRNAs (hsa-miR-451, hsa-miR-223, hsa-miR-486-5p, hsa-miR-3917) were significantly overexpressed, and 7 (hsa-miR-1246, hsa-miR-1260, hsa-miR-141, hsa-miR-1260b, hsa-miR-203, hsa-miR-210, hsa-miR-205*) were underexpressed by > 2-fold in diseased vs. healthy gingiva. GSEA and additional filtering identified 60 enriched miRNA gene sets with target genes involved in immune/inflammatory responses and tissue homeostasis. This is the first study that concurrently examined miRNA and mRNA expression in gingival tissues and will inform mechanistic experimentation to dissect the role of miRNAs in periodontal tissue homeostasis and pathology. PMID:22879578

  3. Hierarchical Clustering of Breast Cancer Methylomes Revealed Differentially Methylated and Expressed Breast Cancer Genes

    PubMed Central

    Lin, I-Hsuan; Chen, Dow-Tien; Chang, Yi-Feng; Lee, Yu-Ling; Su, Chia-Hsin; Cheng, Ching; Tsai, Yi-Chien; Ng, Swee-Chuan; Chen, Hsiao-Tan; Lee, Mei-Chen; Chen, Hong-Wei; Suen, Shih-Hui; Chen, Yu-Cheng; Liu, Tze-Tze; Chang, Chuan-Hsiung; Hsu, Ming-Ta

    2015-01-01

    Oncogenic transformation of normal cells often involves epigenetic alterations, including histone modification and DNA methylation. We conducted whole-genome bisulfite sequencing to determine the DNA methylomes of normal breast, fibroadenoma, invasive ductal carcinomas and MCF7. The emergence, disappearance, expansion and contraction of kilobase-sized hypomethylated regions (HMRs) and the hypomethylation of the megabase-sized partially methylated domains (PMDs) are the major forms of methylation changes observed in breast tumor samples. Hierarchical clustering of HMR revealed tumor-specific hypermethylated clusters and differential methylated enhancers specific to normal or breast cancer cell lines. Joint analysis of gene expression and DNA methylation data of normal breast and breast cancer cells identified differentially methylated and expressed genes associated with breast and/or ovarian cancers in cancer-specific HMR clusters. Furthermore, aberrant patterns of X-chromosome inactivation (XCI) was found in breast cancer cell lines as well as breast tumor samples in the TCGA BRCA (breast invasive carcinoma) dataset. They were characterized with differentially hypermethylated XIST promoter, reduced expression of XIST, and over-expression of hypomethylated X-linked genes. High expressions of these genes were significantly associated with lower survival rates in breast cancer patients. Comprehensive analysis of the normal and breast tumor methylomes suggests selective targeting of DNA methylation changes during breast cancer progression. The weak causal relationship between DNA methylation and gene expression observed in this study is evident of more complex role of DNA methylation in the regulation of gene expression in human epigenetics that deserves further investigation. PMID:25706888

  4. RNA-Seq analysis reveals a six-gene SoxR regulon in Streptomyces coelicolor.

    PubMed

    Naseer, Nawar; Shapiro, Joshua A; Chander, Monica

    2014-01-01

    The redox-regulated transcription factor SoxR is conserved in diverse bacteria, but emerging studies suggest that this protein plays distinct physiological roles in different bacteria. SoxR regulates a global oxidative stress response (involving > 100 genes) against exogenous redox-cycling drugs in Escherichia coli and related enterics. In the antibiotic producers Streptomyces coelicolor and Pseudomonas aeruginosa, however, SoxR regulates a smaller number of genes that encode membrane transporters and proteins with homology to antibiotic-tailoring enzymes. In both S. coelicolor and P. aeruginosa, SoxR-regulated genes are expressed in stationary phase during the production of endogenously-produced redox-active antibiotics. These observations suggest that SoxR evolved to sense endogenous secondary metabolites and activate machinery to process and transport them in antibiotic-producing bacteria. Previous bioinformatics analysis that searched the genome for SoxR-binding sites in putative promoters defined a five-gene SoxR regulon in S. coelicolor including an ABC transporter, two oxidoreductases, a monooxygenase and an epimerase/dehydratase. Since this in silico screen may have missed potential SoxR-targets, we conducted a whole genome transcriptome comparison of wild type S. coelicolor and a soxR-deficient mutant in stationary phase using RNA-Seq. Our analysis revealed a sixth SoxR-regulated gene in S. coelicolor that encodes a putative quinone oxidoreductase. Knowledge of the full complement of genes regulated by SoxR will facilitate studies to elucidate the function of this regulatory molecule in antibiotic producers. PMID:25162599

  5. Comprehensive analysis of imprinted genes in maize reveals allelic variation for imprinting and limited conservation with other species.

    PubMed

    Waters, Amanda J; Bilinski, Paul; Eichten, Steven R; Vaughn, Matthew W; Ross-Ibarra, Jeffrey; Gehring, Mary; Springer, Nathan M

    2013-11-26

    In plants, a subset of genes exhibit imprinting in endosperm tissue such that expression is primarily from the maternal or paternal allele. Imprinting may arise as a consequence of mechanisms for silencing of transposons during reproduction, and in some cases imprinted expression of particular genes may provide a selective advantage such that it is conserved across species. Separate mechanisms for the origin of imprinted expression patterns and maintenance of these patterns may result in substantial variation in the targets of imprinting in different species. Here we present deep sequencing of RNAs isolated from reciprocal crosses of four diverse maize genotypes, providing a comprehensive analysis that allows evaluation of imprinting at more than 95% of endosperm-expressed genes. We find that over 500 genes exhibit statistically significant parent-of-origin effects in maize endosperm tissue, but focused our analyses on a subset of these genes that had >90% expression from the maternal allele (69 genes) or from the paternal allele (108 genes) in at least one reciprocal cross. Over 10% of imprinted genes show evidence of allelic variation for imprinting. A comparison of imprinting in maize and rice reveals that 13% of genes with syntenic orthologs in both species exhibit conserved imprinting. Genes that exhibit conserved imprinting between maize and rice have elevated nonsynonymous to synonymous substitution ratios compared with other imprinted genes, suggesting a history of more rapid evolution. Together, these data suggest that imprinting only has functional relevance at a subset of loci that currently exhibit imprinting in maize. PMID:24218619

  6. Targeted disruption of the NIT8 gene in Chlamydomonas reinhardtii.

    PubMed Central

    Nelson, J A; Lefebvre, P A

    1995-01-01

    We have used homologous recombination to disrupt the nuclear gene NIT8 in Chlamydomonas reinhardtii. This is the first report of targeted gene disruption of an endogenous locus in C. reinhardtii and only the second for a photosynthetic eukaryote. NIT8 encodes a protein necessary for nitrate and nitrite assimilation by C. reinhardtii. A disruption vector was constructed by placing the CRY1-1 selectable marker gene, which confers emetine resistance, within the NIT8 coding region. nit8 mutants are unable to grow on nitrate as their sole nitrogen source (Nit-) and are resistant to killing by chlorate. One of 2,000 transformants obtained after selection on emetine-chlorate medium contained a homologous insertion of five copies of the disruption plasmid into the NIT8 gene, producing an emetine-resistant, chlorate-resistant Nit- phenotype. The mutant phenotype was rescued by the wild-type NIT8 gene upon transformation. Seven other mutations at the nit8 locus, presumably resulting from homologous recombination with the disruption plasmid, were identified but were shown to be accompanied by deletions of the surrounding genomic region. PMID:7565729

  7. [Driver gene mutation and targeted therapy of lung cancer].

    PubMed

    Mitsudomi, Tetsuya

    2013-03-01

    Although cancers may have many genetic alterations, there are only a few mutations actually associated with essential traits of cancer cells such as cell proliferation or evasion from apoptosis. Because cancer cells are "addicted" to these "drive genes" , pharmacologic inhibition of these gene function is highly effective. Epidermal growth factor receptor(EGFR)-tyrosine kinase inhibitor(TKI)(such as gefitinib or erlotinib)treatment of lung cancer harboring EGFR gene mutation is one of the prototypes of such therapies. Several clinical trials clearly demonstrated that progression-free survival of patients treated with EGFR-TKI is significantly longer than that of those treated by conventional platinum doublet chemotherapy. EGFR-TKI therapy dramatically changed the paradigm of lung cancer treatment. Furthermore, in 2012, crizotinib was approved for lung cancer treatment with anaplastic lymphoma kinase(ALK)gene translocation. Targeted therapies for lung cancers "addicted" to other driver gene mutations including ROS1, RET or HER2 are also under development. Through these personalized approaches, lung cancer is changing from an acute fatal disease to a more chronic disease, and eventually we might be able to cure it. PMID:23507588

  8. IRF1 marks activated genes in SLE and can induce target gene expression

    PubMed Central

    Zhang, Zhe; Shi, Lihua; Song, Li; Ephrem, Elshaddai; Petri, Michelle; Sullivan, Kathleen E.

    2014-01-01

    Objective IRF1 both mediates responses to type I interferons and the induction of interferons. It has been implicated in murine lupus models as a critical mediator of inflammation. A previous study of chromatin modifications in SLE patient monocytes implicated IRF1 as associated with increased histone acetylation in SLE patients. This study directly investigated IRF1 binding sites on chromatin using ChIP-seq. Methods Nine female SLE patients and seven female controls were examined. Monocytes were purified from peripheral blood and subjected to library preparation using a validated antibody to IRF1. The effect of IRF1 on target gene expression was confirmed using an overexpression system in cell lines and co-immunoprecipitation was used to define protein interactions. Results IRF1 binding around transcribed regions was increased in SLE patient monocytes but histone modifications at potential IRF1 binding sites without detectable IRF1 binding were also increased. IRF1 overexpression was sufficient to drive transcription of target genes. IRF1 overexpression was also able to alter histone modifications at a focus set of target genes and the use of an IRF1 inhibitor decreased both expression and histone modifications at target genes. IRF1 was found to interact with a select set of histone modifying enzymes and other transcription factors. Conclusions IRF1 is an important signaling protein in the interferon pathway. IRF1 not only activates gene expression as a transcription factor but may perpetuate disease by leading to a dysregulated epigenome. PMID:25418955

  9. Smooth Muscle Cell Genome Browser: Enabling the Identification of Novel Serum Response Factor Target Genes

    PubMed Central

    Lee, Moon Young; Park, Chanjae; Berent, Robyn M.; Park, Paul J.; Fuchs, Robert; Syn, Hannah; Chin, Albert; Townsend, Jared; Benson, Craig C.; Redelman, Doug; Shen, Tsai-wei; Park, Jong Kun; Miano, Joseph M.; Sanders, Kenton M.; Ro, Seungil

    2015-01-01

    Genome-scale expression data on the absolute numbers of gene isoforms offers essential clues in cellular functions and biological processes. Smooth muscle cells (SMCs) perform a unique contractile function through expression of specific genes controlled by serum response factor (SRF), a transcription factor that binds to DNA sites known as the CArG boxes. To identify SRF-regulated genes specifically expressed in SMCs, we isolated SMC populations from mouse small intestine and colon, obtained their transcriptomes, and constructed an interactive SMC genome and CArGome browser. To our knowledge, this is the first online resource that provides a comprehensive library of all genetic transcripts expressed in primary SMCs. The browser also serves as the first genome-wide map of SRF binding sites. The browser analysis revealed novel SMC-specific transcriptional variants and SRF target genes, which provided new and unique insights into the cellular and biological functions of the cells in gastrointestinal (GI) physiology. The SRF target genes in SMCs, which were discovered in silico, were confirmed by proteomic analysis of SMC-specific Srf knockout mice. Our genome browser offers a new perspective into the alternative expression of genes in the context of SRF binding sites in SMCs and provides a valuable reference for future functional studies. PMID:26241044

  10. Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes

    PubMed Central

    Biankin, Andrew V.; Waddell, Nicola; Kassahn, Karin S.; Gingras, Marie-Claude; Muthuswamy, Lakshmi B.; Johns, Amber L.; Miller, David K.; Wilson, Peter J.; Patch, Ann-Marie; Wu, Jianmin; Chang, David K.; Cowley, Mark J.; Gardiner, Brooke B.; Song, Sarah; Harliwong, Ivon; Idrisoglu, Senel; Nourse, Craig; Nourbakhsh, Ehsan; Manning, Suzanne; Wani, Shivangi; Gongora, Milena; Pajic, Marina; Scarlett, Christopher J.; Gill, Anthony J.; Pinho, Andreia V.; Rooman, Ilse; Anderson, Matthew; Holmes, Oliver; Leonard, Conrad; Taylor, Darrin; Wood, Scott; Xu, Qinying; Nones, Katia; Fink, J. Lynn; Christ, Angelika; Bruxner, Tim; Cloonan, Nicole; Kolle, Gabriel; Newell, Felicity; Pinese, Mark; Mead, R. Scott; Humphris, Jeremy L.; Kaplan, Warren; Jones, Marc D.; Colvin, Emily K.; Nagrial, Adnan M.; Humphrey, Emily S.; Chou, Angela; Chin, Venessa T.; Chantrill, Lorraine A.; Mawson, Amanda; Samra, Jaswinder S.; Kench, James G.; Lovell, Jessica A.; Daly, Roger J.; Merrett, Neil D.; Toon, Christopher; Epari, Krishna; Nguyen, Nam Q.; Barbour, Andrew; Zeps, Nikolajs; Kakkar, Nipun; Zhao, Fengmei; Wu, Yuan Qing; Wang, Min; Muzny, Donna M.; Fisher, William E.; Brunicardi, F. Charles; Hodges, Sally E.; Reid, Jeffrey G.; Drummond, Jennifer; Chang, Kyle; Han, Yi; Lewis, Lora R.; Dinh, Huyen; Buhay, Christian J.; Beck, Timothy; Timms, Lee; Sam, Michelle; Begley, Kimberly; Brown, Andrew; Pai, Deepa; Panchal, Ami; Buchner, Nicholas; De Borja, Richard; Denroche, Robert E.; Yung, Christina K.; Serra, Stefano; Onetto, Nicole; Mukhopadhyay, Debabrata; Tsao, Ming-Sound; Shaw, Patricia A.; Petersen, Gloria M.; Gallinger, Steven; Hruban, Ralph H.; Maitra, Anirban; Iacobuzio-Donahue, Christine A.; Schulick, Richard D.; Wolfgang, Christopher L.; Morgan, Richard A.; Lawlor, Rita T.; Capelli, Paola; Corbo, Vincenzo; Scardoni, Maria; Tortora, Giampaolo; Tempero, Margaret A.; Mann, Karen M.; Jenkins, Nancy A.; Perez-Mancera, Pedro A.; Adams, David J.; Largaespada, David A.; Wessels, Lodewyk F. A.; Rust, Alistair G.; Stein, Lincoln D.; Tuveson, David A.; Copeland, Neal G.; Musgrove, Elizabeth A.; Scarpa, Aldo; Eshleman, James R.; Hudson, Thomas J.; Sutherland, Robert L.; Wheeler, David A.; Pearson, John V.; McPherson, John D.; Gibbs, Richard A.; Grimmond, Sean M.

    2012-01-01

    Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis. PMID:23103869

  11. Identification of Genetic Causes of Inherited Peripheral Neuropathies by Targeted Gene Panel Sequencing.

    PubMed

    Nam, Soo Hyun; Hong, Young Bin; Hyun, Young Se; Nam, Da Eun; Kwak, Geon; Hwang, Sun Hee; Choi, Byung-Ok; Chung, Ki Wha

    2016-05-31

    Inherited peripheral neuropathies (IPN), which are a group of clinically and genetically heterogeneous peripheral nerve disorders including Charcot-Marie-Tooth disease (CMT), exhibit progressive degeneration of muscles in the extremities and loss of sensory function. Over 70 genes have been reported as genetic causatives and the number is still growing. We prepared a targeted gene panel for IPN diagnosis based on next generation sequencing (NGS). The gene panel was designed to detect mutations in 73 genes reported to be genetic causes of IPN or related peripheral neuropathies, and to detect duplication of the chromosome 17p12 region, the major genetic cause of CMT1A. We applied the gene panel to 115 samples from 63 non-CMT1A families, and isolated 15 pathogenic or likely-pathogenic mutations in eight genes from 25 patients (17 families). Of them, eight mutations were unreported variants. Of particular interest, this study revealed several very rare mutations in the SPTLC2, DCTN1, and MARS genes. In addition, the effectiveness of the detection of CMT1A was confirmed by comparing five 17p12-nonduplicated controls and 15 CMT1A cases. In conclusion, we developed a gene panel for one step genetic diagnosis of IPN. It seems that its time- and cost-effectiveness are superior to previous tiered-genetic diagnosis algorithms, and it could be applied as a genetic diagnostic system for inherited peripheral neuropathies. PMID:27025386

  12. Identification of Genetic Causes of Inherited Peripheral Neuropathies by Targeted Gene Panel Sequencing

    PubMed Central

    Nam, Soo Hyun; Hong, Young Bin; Hyun, Young Se; Nam, Da Eun; Kwak, Geon; Hwang, Sun Hee; Choi, Byung-Ok; Chung, Ki Wha

    2016-01-01

    Inherited peripheral neuropathies (IPN), which are a group of clinically and genetically heterogeneous peripheral nerve disorders including Charcot-Marie-Tooth disease (CMT), exhibit progressive degeneration of muscles in the extremities and loss of sensory function. Over 70 genes have been reported as genetic causatives and the number is still growing. We prepared a targeted gene panel for IPN diagnosis based on next generation sequencing (NGS). The gene panel was designed to detect mutations in 73 genes reported to be genetic causes of IPN or related peripheral neuropathies, and to detect duplication of the chromosome 17p12 region, the major genetic cause of CMT1A. We applied the gene panel to 115 samples from 63 non-CMT1A families, and isolated 15 pathogenic or likely-pathogenic mutations in eight genes from 25 patients (17 families). Of them, eight mutations were unreported variants. Of particular interest, this study revealed several very rare mutations in the SPTLC2, DCTN1, and MARS genes. In addition, the effectiveness of the detection of CMT1A was confirmed by comparing five 17p12-nonduplicated controls and 15 CMT1A cases. In conclusion, we developed a gene panel for one step genetic diagnosis of IPN. It seems that its time- and cost-effectiveness are superior to previous tiered-genetic diagnosis algorithms, and it could be applied as a genetic diagnostic system for inherited peripheral neuropathies. PMID:27025386

  13. Quantitative determination of target gene with electrical sensor

    NASA Astrophysics Data System (ADS)

    Zhang, Xuzhi; Li, Qiufen; Jin, Xianshi; Jiang, Cheng; Lu, Yong; Tavallaie, Roya; Gooding, J. Justin

    2015-07-01

    Integrating loop-mediated isothermal amplification (LAMP) with capacitively coupled contactless conductivity detection (C4D), we have developed an electrical sensor for the simultaneous amplification and detection of specific sequence DNA. Using the O26-wzy gene as a model, the amount of initial target gene could be determined via the threshold time obtained by monitoring the progression of the LAMP reaction in real time. Using the optimal conditions, a detection limit of 12.5 copy/μL can be obtained within 30 min. Monitoring the LAMP reaction by C4D has not only all the advantages that existing electrochemical methods have, but also additional attractive features including being completely free of carryover contamination risk, high simplicity and extremely low cost. These benefits all arise from the fact that the electrodes are separated from the reaction solution, that is C4D is a contactless method. Hence in proof of principle, the new strategy promises a robust, simple, cost-effective and sensitive method for quantitative determination of a target gene, that is applicable either to specialized labs or at point-of-care.

  14. Induction of hepatocellular carcinoma by in vivo gene targeting

    PubMed Central

    Wang, Pei-Rong; Xu, Mei; Toffanin, Sara; Li, Yi; Llovet, Josep M.; Russell, David W.

    2012-01-01

    The distinct phenotypic and prognostic subclasses of human hepatocellular carcinoma (HCC) are difficult to reproduce in animal experiments. Here we have used in vivo gene targeting to insert an enhancer-promoter element at an imprinted chromosome 12 locus in mice, thereby converting ∼1 in 20,000 normal hepatocytes into a focus of HCC with a single genetic modification. A 300-kb chromosomal domain containing multiple mRNAs, snoRNAs, and microRNAs was activated surrounding the integration site. An identical domain was activated at the syntenic locus in a specific molecular subclass of spontaneous human HCCs with a similar histological phenotype, which was associated with partial loss of DNA methylation. These findings demonstrate the accuracy of in vivo gene targeting in modeling human cancer and suggest future applications in studying various tumors in diverse animal species. In addition, similar insertion events produced by randomly integrating vectors could be a concern for liver-directed human gene therapy. PMID:22733778

  15. Sgs1 and Exo1 suppress targeted chromosome duplication during ends-in and ends-out gene targeting.

    PubMed

    Štafa, Anamarija; Miklenić, Marina; Zunar, Bojan; Lisnić, Berislav; Symington, Lorraine S; Svetec, Ivan-Krešimir

    2014-10-01

    Gene targeting is extremely efficient in the yeast Saccharomyces cerevisiae. It is performed by transformation with a linear, non-replicative DNA fragment carrying a selectable marker and containing ends homologous to the particular locus in a genome. However, even in S. cerevisiae, transformation can result in unwanted (aberrant) integration events, the frequency and spectra of which are quite different for ends-out and ends-in transformation assays. It has been observed that gene replacement (ends-out gene targeting) can result in illegitimate integration, integration of the transforming DNA fragment next to the target sequence and duplication of a targeted chromosome. By contrast, plasmid integration (ends-in gene targeting) is often associated with multiple targeted integration events but illegitimate integration is extremely rare and a targeted chromosome duplication has not been reported. Here we systematically investigated the influence of design of the ends-out assay on the success of targeted genetic modification. We have determined transformation efficiency, fidelity of gene targeting and spectra of all aberrant events in several ends-out gene targeting assays designed to insert, delete or replace a particular sequence in the targeted region of the yeast genome. Furthermore, we have demonstrated for the first time that targeted chromosome duplications occur even during ends-in gene targeting. Most importantly, the whole chromosome duplication is POL32 dependent pointing to break-induced replication (BIR) as the underlying mechanism. Moreover, the occurrence of duplication of the targeted chromosome was strikingly increased in the exo1Δ sgs1Δ double mutant but not in the respective single mutants demonstrating that the Exo1 and Sgs1 proteins independently suppress whole chromosome duplication during gene targeting. PMID:25089886

  16. Bioactivity-guided genome mining reveals the lomaiviticin biosynthetic gene cluster in Salinispora tropica

    PubMed Central

    Kersten, Roland D.; Lane, Amy L.; Nett, Markus; Richter, Taylor K. S.; Duggan, Brendan M.; Dorrestein, Pieter C.

    2013-01-01

    The use of genome sequences has become routine in guiding the discovery and identification of microbial natural products and their biosynthetic pathways. In silico prediction of molecular features, such as metabolic building blocks, physico-chemical properties or biological functions, from orphan gene clusters has opened up the characterization of many new chemo- and genotypes in genome mining approaches. Here, we guided our genome mining of two predicted enediyne pathways in Salinispora tropica CNB-440 by a DNA interference bioassay to isolate DNA-targeting enediyne polyketides. An organic extract of S. tropica showed DNA-interference activity that surprisingly was not abolished in genetic mutants of the targeted enediyne pathways, ST_pks1 and spo. Instead we showed that the product of the orphan type II polyketide synthase pathway, ST_pks2, is solely responsible for the DNA-interfering activity of the parent strain. Subsequent comparative metabolic profiling revealed the lomaiviticins, glycosylated diazofluorene polyketides, as the ST_pks2 products. This study marks the first report of the 59 open reading frame lomaiviticin gene cluster (lom) and supports the biochemical logic of their dimeric construction via a pathway related to the kinamycin monomer. PMID:23649992

  17. Development of a successive targeting liposome with multi-ligand for efficient targeting gene delivery

    PubMed Central

    Ma, Kun; Shen, Haijun; Shen, Song; Xie, Men; Mao, Chuanbin; Qiu, Liyan; Jin, Yi

    2012-01-01

    Background A successful gene delivery system needs to breakthrough several barriers to allow efficient transgenic expression. In the present study, successive targeting liposomes (STL) were constructed by integrating various targeting groups into a nanoparticle to address this issue. Methods Polyethylenimine (PEI) 1800-triamcinolone acetonide (TA) with nuclear targeting capability was synthesized by a two-step reaction. Lactobionic acid was connected with cholesterol to obtain a compound of [(2-lactoylamido) ethylamino]formic acid cholesterol ester (CHEDLA) with hepatocyte-targeting capability. The liposome was modified with PEI 1800-TA and CHEDLA to prepare successive targeting liposome (STL). Its physicochemical properties and transfection efficiency were investigated both in vitro and in vivo. Results The diameter of STL was approximately 100 nm with 20 mV of potential. The confocal microscopy observation and potential assay verified that lipid bilayer of STL was decorated with PEI 1800-TA. Cytotoxicity of STL was significantly lower than that of PEI 1800-TA and PEI 25K. The transfection efficiency of 10% CHEDLA STL in HepG2 cells was the higher than of the latter two with serum. Its transfection efficiency was greatly reduced with excessive free galactose, indicating that STL was absorbed via galactose receptor-mediated endocytosis. The in vivo study in mice showed that 10% CHEDLA STL had better transgenic expression in liver than the other carriers. Conclusions STL with multi-ligand was able to overcome the various barriers to target nucleus and special cells and present distinctive transgenic expression. Therefore, it has a great potential for gene therapy as a nonviral carrier. PMID:21574214

  18. Real-time imaging of Huntingtin aggregates diverting target search and gene transcription.

    PubMed

    Li, Li; Liu, Hui; Dong, Peng; Li, Dong; Legant, Wesley R; Grimm, Jonathan B; Lavis, Luke D; Betzig, Eric; Tjian, Robert; Liu, Zhe

    2016-01-01

    The presumptive altered dynamics of transient molecular interactions in vivo contributing to neurodegenerative diseases have remained elusive. Here, using single-molecule localization microscopy, we show that disease-inducing Huntingtin (mHtt) protein fragments display three distinct dynamic states in living cells - 1) fast diffusion, 2) dynamic clustering and 3) stable aggregation. Large, stable aggregates of mHtt exclude chromatin and form 'sticky' decoy traps that impede target search processes of key regulators involved in neurological disorders. Functional domain mapping based on super-resolution imaging reveals an unexpected role of aromatic amino acids in promoting protein-mHtt aggregate interactions. Genome-wide expression analysis and numerical simulation experiments suggest mHtt aggregates reduce transcription factor target site sampling frequency and impair critical gene expression programs in striatal neurons. Together, our results provide insights into how mHtt dynamically forms aggregates and disrupts the finely-balanced gene control mechanisms in neuronal cells. PMID:27484239

  19. Targeted genes and interacting proteins of hypoxia inducible factor-1

    PubMed Central

    Liu, Wei; Shen, Shao-Ming; Zhao, Xu-Yun; Chen, Guo-Qiang

    2012-01-01

    Heterodimeric transcription factor hypoxia inducible factor-1 (HIF-1) functions as a master regulator of oxygen homeostasis in almost all nucleated mammalian cells. The fundamental process adapted to cellular oxygen alteration largely depends on the refined regulation on its alpha subunit, HIF-1α. Recent studies have unraveled expanding and critical roles of HIF-1α, involving in a multitude of developmental, physiological, and pathophysiological processes. This review will focus on the current knowledge of HIF-1α-targeting genes and its interacting proteins, as well as the concomitant functional relationships between them. PMID:22773957

  20. Gene Profiling of Mta1 Identifies Novel Gene Targets and Functions

    PubMed Central

    Eswaran, Jeyanthy; Kumar, Rakesh

    2011-01-01

    Background Metastasis-associated protein 1 (MTA1), a master dual co-regulatory protein is found to be an integral part of NuRD (Nucleosome Remodeling and Histone Deacetylation) complex, which has indispensable transcriptional regulatory functions via histone deacetylation and chromatin remodeling. Emerging literature establishes MTA1 to be a valid DNA-damage responsive protein with a significant role in maintaining the optimum DNA-repair activity in mammalian cells exposed to genotoxic stress. This DNA-damage responsive function of MTA1 was reported to be a P53-dependent and independent function. Here, we investigate the influence of P53 on gene regulation function of Mta1 to identify novel gene targets and functions of Mta1. Methods Gene expression analysis was performed on five different mouse embryonic fibroblasts (MEFs) samples (i) the Mta1 wild type, (ii) Mta1 knock out (iii) Mta1 knock out in which Mta1 was reintroduced (iv) P53 knock out (v) P53 knock out in which Mta1 was over expressed using Affymetrix Mouse Exon 1.0 ST arrays. Further Hierarchical Clustering, Gene Ontology analysis with GO terms satisfying corrected p-value<0.1, and the Ingenuity Pathway Analysis were performed. Finally, RT-qPCR was carried out on selective candidate genes. Significance/Conclusion This study represents a complete genome wide screen for possible target genes of a coregulator, Mta1. The comparative gene profiling of Mta1 wild type, Mta1 knockout and Mta1 re-expression in the Mta1 knockout conditions define “bona fide” Mta1 target genes. Further extensive analyses of the data highlights the influence of P53 on Mta1 gene regulation. In the presence of P53 majority of the genes regulated by Mta1 are related to inflammatory and anti-microbial responses whereas in the absence of P53 the predominant target genes are involved in cancer signaling. Thus, the presented data emphasizes the known functions of Mta1 and serves as a rich resource which could help us identify novel Mta

  1. Target genes discovery through copy number alteration analysis in human hepatocellular carcinoma.

    PubMed

    Gu, De-Leung; Chen, Yen-Hsieh; Shih, Jou-Ho; Lin, Chi-Hung; Jou, Yuh-Shan; Chen, Chian-Feng

    2013-12-21

    High-throughput short-read sequencing of exomes and whole cancer genomes in multiple human hepatocellular carcinoma (HCC) cohorts confirmed previously identified frequently mutated somatic genes, such as TP53, CTNNB1 and AXIN1, and identified several novel genes with moderate mutation frequencies, including ARID1A, ARID2, MLL, MLL2, MLL3, MLL4, IRF2, ATM, CDKN2A, FGF19, PIK3CA, RPS6KA3, JAK1, KEAP1, NFE2L2, C16orf62, LEPR, RAC2, and IL6ST. Functional classification of these mutated genes suggested that alterations in pathways participating in chromatin remodeling, Wnt/β-catenin signaling, JAK/STAT signaling, and oxidative stress play critical roles in HCC tumorigenesis. Nevertheless, because there are few druggable genes used in HCC therapy, the identification of new therapeutic targets through integrated genomic approaches remains an important task. Because a large amount of HCC genomic data genotyped by high density single nucleotide polymorphism arrays is deposited in the public domain, copy number alteration (CNA) analyses of these arrays is a cost-effective way to reveal target genes through profiling of recurrent and overlapping amplicons, homozygous deletions and potentially unbalanced chromosomal translocations accumulated during HCC progression. Moreover, integration of CNAs with other high-throughput genomic data, such as aberrantly coding transcriptomes and non-coding gene expression in human HCC tissues and rodent HCC models, provides lines of evidence that can be used to facilitate the identification of novel HCC target genes with the potential of improving the survival of HCC patients. PMID:24379610

  2. CRISPR/Cas9-mediated targeted gene mutagenesis in Spodoptera litura.

    PubMed

    Bi, Hong-Lun; Xu, Jun; Tan, An-Jiang; Huang, Yong-Ping

    2016-06-01

    Custom-designed nuclease technologies such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) system provide attractive genome editing tools for insect functional genetics. The targeted gene mutagenesis mediated by the CRISPR/Cas9 system has been achieved in several insect orders including Diptera, Lepidoptera and Coleoptera. However, little success has been reported in agricultural pests due to the lack of genomic information and embryonic microinjection techniques in these insect species. Here we report that the CRISPR/Cas9 system induced efficient gene mutagenesis in an important Lepidopteran pest Spodoptera litura. We targeted the S. litura Abdominal-A (Slabd-A) gene which is an important embryonic development gene and plays a significant role in determining the identities of the abdominal segments of insects. Direct injection of Cas9 messenger RNA and Slabd-A-specific single guide RNA (sgRNA) into S. litura embryos successfully induced the typical abd-A deficient phenotype, which shows anomalous segmentation and ectopic pigmentation during the larval stage. A polymerase chain reaction-based analysis revealed that the Cas9/sgRNA complex effectively induced a targeted mutagenesis in S. litura. These results demonstrate that the CRISPR/Cas9 system is a powerful tool for genome manipulation in Lepidopteran pests such as S. litura. PMID:27061764

  3. Targeting human melanoma neoantigens by T cell receptor gene therapy.

    PubMed

    Leisegang, Matthias; Kammertoens, Thomas; Uckert, Wolfgang; Blankenstein, Thomas

    2016-03-01

    In successful cancer immunotherapy, T cell responses appear to be directed toward neoantigens created by somatic mutations; however, direct evidence that neoantigen-specific T cells cause regression of established cancer is lacking. Here, we generated T cells expressing a mutation-specific transgenic T cell receptor (TCR) to target different immunogenic mutations in cyclin-dependent kinase 4 (CDK4) that naturally occur in human melanoma. Two mutant CDK4 isoforms (R24C, R24L) similarly stimulated T cell responses in vitro and were analyzed as therapeutic targets for TCR gene therapy. In a syngeneic HLA-A2-transgenic mouse model of large established tumors, we found that both mutations differed dramatically as targets for TCR-modified T cells in vivo. While T cells expanded efficiently and produced IFN-γ in response to R24L, R24C failed to induce an effective antitumor response. Such differences in neoantigen quality might explain why cancer immunotherapy induces tumor regression in some individuals, while others do not respond, despite similar mutational load. We confirmed the validity of the in vivo model by showing that the melan-A-specific (MART-1-specific) TCR DMF5 induces rejection of tumors expressing analog, but not native, MART-1 epitopes. The described model allows identification of those neoantigens in human cancer that serve as suitable T cell targets and may help to predict clinical efficacy. PMID:26808500

  4. Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes

    SciTech Connect

    Li, T; Huang, S; Zhao, XF; Wright, DA; Carpenter, S; Spalding, MH; Weeks, DP; Yang, B

    2011-08-08

    Recent studies indicate that the DNA recognition domain of transcription activator-like (TAL) effectors can be combined with the nuclease domain of FokI restriction enzyme to produce TAL effector nucleases (TALENs) that, in pairs, bind adjacent DNA target sites and produce double-strand breaks between the target sequences, stimulating non-homologous end-joining and homologous recombination. Here, we exploit the four prevalent TAL repeats and their DNA recognition cipher to develop a 'modular assembly' method for rapid production of designer TALENs (dTALENs) that recognize unique DNA sequence up to 23 bases in any gene. We have used this approach to engineer 10 dTALENs to target specific loci in native yeast chromosomal genes. All dTALENs produced high rates of site-specific gene disruptions and created strains with expected mutant phenotypes. Moreover, dTALENs stimulated high rates (up to 34%) of gene replacement by homologous recombination. Finally, dTALENs caused no detectable cytotoxicity and minimal levels of undesired genetic mutations in the treated yeast strains. These studies expand the realm of verified TALEN activity from cultured human cells to an intact eukaryotic organism and suggest that low-cost, highly dependable dTALENs can assume a significant role for gene modifications of value in human and animal health, agriculture and industry.

  5. The cavefish genome reveals candidate genes for eye loss

    PubMed Central

    McGaugh, Suzanne E.; Gross, Joshua B.; Aken, Bronwen; Blin, Maryline; Borowsky, Richard; Chalopin, Domitille; Hinaux, Hélène; Jeffery, William R.; Keene, Alex; Ma, Li; Minx, Patrick; Murphy, Daniel; O’Quin, Kelly E.; Rétaux, Sylvie; Rohner, Nicolas; Searle, Steve M. J.; Stahl, Bethany A.; Tabin, Cliff; Volff, Jean-Nicolas; Yoshizawa, Masato; Warren, Wesley C.

    2014-01-01

    Natural populations subjected to strong environmental selection pressures offer a window into the genetic underpinnings of evolutionary change. Cavefish populations, Astyanax mexicanus (Teleostei: Characiphysi), exhibit repeated, independent evolution for a variety of traits including eye degeneration, pigment loss, increased size and number of taste buds and mechanosensory organs, and shifts in many behavioural traits. Surface and cave forms are interfertile making this system amenable to genetic interrogation; however, lack of a reference genome has hampered efforts to identify genes responsible for changes in cave forms of A. mexicanus. Here we present the first de novo genome assembly for Astyanax mexicanus cavefish, contrast repeat elements to other teleost genomes, identify candidate genes underlying quantitative trait loci (QTL), and assay these candidate genes for potential functional and expression differences. We expect the cavefish genome to advance understanding of the evolutionary process, as well as, analogous human disease including retinal dysfunction. PMID:25329095

  6. The cavefish genome reveals candidate genes for eye loss.

    PubMed

    McGaugh, Suzanne E; Gross, Joshua B; Aken, Bronwen; Blin, Maryline; Borowsky, Richard; Chalopin, Domitille; Hinaux, Hélène; Jeffery, William R; Keene, Alex; Ma, Li; Minx, Patrick; Murphy, Daniel; O'Quin, Kelly E; Rétaux, Sylvie; Rohner, Nicolas; Searle, Steve M J; Stahl, Bethany A; Tabin, Cliff; Volff, Jean-Nicolas; Yoshizawa, Masato; Warren, Wesley C

    2014-01-01

    Natural populations subjected to strong environmental selection pressures offer a window into the genetic underpinnings of evolutionary change. Cavefish populations, Astyanax mexicanus (Teleostei: Characiphysi), exhibit repeated, independent evolution for a variety of traits including eye degeneration, pigment loss, increased size and number of taste buds and mechanosensory organs, and shifts in many behavioural traits. Surface and cave forms are interfertile making this system amenable to genetic interrogation; however, lack of a reference genome has hampered efforts to identify genes responsible for changes in cave forms of A. mexicanus. Here we present the first de novo genome assembly for Astyanax mexicanus cavefish, contrast repeat elements to other teleost genomes, identify candidate genes underlying quantitative trait loci (QTL), and assay these candidate genes for potential functional and expression differences. We expect the cavefish genome to advance understanding of the evolutionary process, as well as, analogous human disease including retinal dysfunction. PMID:25329095

  7. A novel molecular mechanism involved in multiple myeloma development revealed by targeting MafB to haematopoietic progenitors

    PubMed Central

    Vicente-Dueñas, Carolina; Romero-Camarero, Isabel; González-Herrero, Inés; Alonso-Escudero, Esther; Abollo-Jiménez, Fernando; Jiang, Xiaoyu; Gutierrez, Norma C; Orfao, Alberto; Marín, Nieves; Villar, Luisa María; Criado, Ma Carmen Fernández; Pintado, Belén; Flores, Teresa; Alonso-López, Diego; De Las Rivas, Javier; Jiménez, Rafael; Criado, Francisco Javier García; Cenador, María Begoña García; Lossos, Izidore S; Cobaleda, César; Sánchez-García, Isidro

    2012-01-01

    Understanding the cellular origin of cancer can help to improve disease prevention and therapeutics. Human plasma cell neoplasias are thought to develop from either differentiated B cells or plasma cells. However, when the expression of Maf oncogenes (associated to human plasma cell neoplasias) is targeted to mouse B cells, the resulting animals fail to reproduce the human disease. Here, to explore early cellular changes that might take place in the development of plasma cell neoplasias, we engineered transgenic mice to express MafB in haematopoietic stem/progenitor cells (HS/PCs). Unexpectedly, we show that plasma cell neoplasias arise in the MafB-transgenic mice. Beyond their clinical resemblance to human disease, these neoplasias highly express genes that are known to be upregulated in human multiple myeloma. Moreover, gene expression profiling revealed that MafB-expressing HS/PCs were more similar to B cells and tumour plasma cells than to any other subset, including wild-type HS/PCs. Consistent with this, genome-scale DNA methylation profiling revealed that MafB imposes an epigenetic program in HS/PCs, and that this program is preserved in mature B cells of MafB-transgenic mice, demonstrating a novel molecular mechanism involved in tumour initiation. Our findings suggest that, mechanistically, the haematopoietic progenitor population can be the target for transformation in MafB-associated plasma cell neoplasias. PMID:22903061

  8. Suicide Gene-Engineered Stromal Cells Reveal a Dynamic Regulation of Cancer Metastasis

    PubMed Central

    Shen, Keyue; Luk, Samantha; Elman, Jessica; Murray, Ryan; Mukundan, Shilpaa; Parekkadan, Biju

    2016-01-01

    Cancer-associated fibroblasts (CAFs) are a major cancer-promoting component in the tumor microenvironment (TME). The dynamic role of human CAFs in cancer progression has been ill-defined because human CAFs lack a unique marker needed for a cell-specific, promoter-driven knockout model. Here, we developed an engineered human CAF cell line with an inducible suicide gene to enable selective in vivo elimination of human CAFs at different stages of xenograft tumor development, effectively circumventing the challenge of targeting a cell-specific marker. Suicide-engineered CAFs were highly sensitive to apoptosis induction in vitro and in vivo by the addition of a simple small molecule inducer. Selection of timepoints for targeted CAF apoptosis in vivo during the progression of a human breast cancer xenograft model was guided by a bi-phasic host cytokine response that peaked at early timepoints after tumor implantation. Remarkably, we observed that the selective apoptosis of CAFs at these early timepoints did not affect primary tumor growth, but instead increased the presence of tumor-associated macrophages and the metastatic spread of breast cancer cells to the lung and bone. The study revealed a dynamic relationship between CAFs and cancer metastasis that has counter-intuitive ramifications for CAF-targeted therapy. PMID:26893143

  9. Suicide Gene-Engineered Stromal Cells Reveal a Dynamic Regulation of Cancer Metastasis

    NASA Astrophysics Data System (ADS)

    Shen, Keyue; Luk, Samantha; Elman, Jessica; Murray, Ryan; Mukundan, Shilpaa; Parekkadan, Biju

    2016-02-01

    Cancer-associated fibroblasts (CAFs) are a major cancer-promoting component in the tumor microenvironment (TME). The dynamic role of human CAFs in cancer progression has been ill-defined because human CAFs lack a unique marker needed for a cell-specific, promoter-driven knockout model. Here, we developed an engineered human CAF cell line with an inducible suicide gene to enable selective in vivo elimination of human CAFs at different stages of xenograft tumor development, effectively circumventing the challenge of targeting a cell-specific marker. Suicide-engineered CAFs were highly sensitive to apoptosis induction in vitro and in vivo by the addition of a simple small molecule inducer. Selection of timepoints for targeted CAF apoptosis in vivo during the progression of a human breast cancer xenograft model was guided by a bi-phasic host cytokine response that peaked at early timepoints after tumor implantation. Remarkably, we observed that the selective apoptosis of CAFs at these early timepoints did not affect primary tumor growth, but instead increased the presence of tumor-associated macrophages and the metastatic spread of breast cancer cells to the lung and bone. The study revealed a dynamic relationship between CAFs and cancer metastasis that has counter-intuitive ramifications for CAF-targeted therapy.

  10. Candidate EDA targets revealed by expression profiling of primary keratinocytes from Tabby mutant mice

    PubMed Central

    Esibizione, Diana; Cui, Chang-Yi; Schlessinger, David

    2009-01-01

    EDA, the gene mutated in anhidrotic ectodermal dysplasia, encodes ectodysplasin, a TNF superfamily member that activates NF-kB mediated transcription. To identify EDA target genes, we have earlier used expression profiling to infer genes differentially expressed at various developmental time points in Tabby (Eda-deficient) compared to wild-type mouse skin. To increase the resolution to find genes whose expression may be restricted to epidermal cells, we have now extended studies to primary keratinocyte cultures established from E19 wild-type and Tabby skin. Using microarrays bearing 44,000 gene probes, we found 385 preliminary candidate genes whose expression was significantly affected by Eda loss. By comparing expression profiles to those from Eda-A1 transgenic skin, we restricted the list to 38 “candidate EDA targets”, 14 of which were already known to be expressed in hair follicles or epidermis. We confirmed expression changes for 3 selected genes, Tbx1, Bmp7, and Jag1, both in keratinocytes and in whole skin, by Q-PCR and Western blotting analyses. Thus, by the analysis of keratinocytes, novel candidate pathways downstream of EDA were detected. PMID:18848976

  11. Pathway analysis of senescence-associated miRNA targets reveals common processes to different senescence induction mechanisms.

    PubMed

    Lafferty-Whyte, Kyle; Cairney, Claire J; Jamieson, Nigel B; Oien, Karin A; Keith, W Nicol

    2009-04-01

    Multiple mechanisms of senescence induction exist including telomere attrition, oxidative stress, oncogene expression and DNA damage signalling. The regulation of the cellular changes required to respond to these stimuli and create the complex senescent cell phenotype has many different mechanisms. MiRNAs present one mechanism by which genes with diverse functions on multiple pathways can be simultaneously regulated. In this study we investigated 12 miRNAs previously identified as senescence regulators. Using pathway analysis of their target genes we tested the relevance of miRNA regulation in the induction of senescence. Our analysis highlighted the potential of these senescence-associated miRNAs (SA-miRNAs) to regulate the cell cycle, cytoskeletal remodelling and proliferation signalling logically required to create a senescent cell. The reanalysis of publicly available gene expression data from studies exploring different senescence stimuli also revealed their potential to regulate core senescence processes, regardless of stimuli. We also identified stimulus specific apoptosis survival pathways theoretically regulated by the SA-miRNAs. Furthermore the observation that miR-499 and miR-34c had the potential to regulate all 4 of the senescence induction types we studied highlights their future potential as novel drug targets for senescence induction. PMID:19419692

  12. Screening targeted testis‑specific genes for molecular assessment of aberrant sperm quality.

    PubMed

    Liu, Xue Xia; Shen, Xiao Fang; Liu, Fu-Jun

    2016-08-01

    Teratospermia is a heterogeneous and complex disorder, which is closely associated with male fertility. Genes and gene products associated with teratospermia may serve as targeted biomarkers that help understand the underlying mechanisms of male infertility; however, systematic information on the subject remains to be elucidated. The present study performed a comparative bioinformatics analysis to identify biomarkers associated with sperm quality, particular focusing on testis‑specific biomarkers. A stepwise screening approach identified 1,085 testis/epididymis‑specific genes and 3,406 teratospermia‑associated genes, resulting in 348 testis‑specific genes associated with aberrant sperm quality. These genes were functionally associated with the reproduction process. Gene products corresponding to heat shock protein family A (Hsp70) member 4 like (HSPA4L) and phosphoglycerate kinase 2 were characterized at the cellular level in human testes and ejaculated spermatozoa. HSPA4L expression in sperm was revealed to be associated with sperm quality. The present study provided a novel insight into the understanding of sperm quality, and a potential method for the diagnosis and assessment of sperm quality in the event of male infertility. PMID:27356588

  13. Screening targeted testis-specific genes for molecular assessment of aberrant sperm quality

    PubMed Central

    Liu, Xue Xia; Shen, Xiao Fang; Liu, Fu-Jun

    2016-01-01

    Teratospermia is a heterogeneous and complex disorder, which is closely associated with male fertility. Genes and gene products associated with teratospermia may serve as targeted biomarkers that help understand the underlying mechanisms of male infertility; however, systematic information on the subject remains to be elucidated. The present study performed a comparative bioinformatics analysis to identify biomarkers associated with sperm quality, particular focusing on testis-specific biomarkers. A stepwise screening approach identified 1,085 testis/epididymis-specific genes and 3,406 teratospermia-associated genes, resulting in 348 testis-specific genes associated with aberrant sperm quality. These genes were functionally associated with the reproduction process. Gene products corresponding to heat shock protein family A (Hsp70) member 4 like (HSPA4L) and phosphoglycerate kinase 2 were characterized at the cellular level in human testes and ejaculated spermatozoa. HSPA4L expression in sperm was revealed to be associated with sperm quality. The present study provided a novel insight into the understanding of sperm quality, and a potential method for the diagnosis and assessment of sperm quality in the event of male infertility. PMID:27356588

  14. Transcriptional regulation of gene expression during osmotic stress responses by the mammalian target of rapamycin.

    PubMed

    Ortells, M Carmen; Morancho, Beatriz; Drews-Elger, Katherine; Viollet, Benoit; Laderoute, Keith R; López-Rodríguez, Cristina; Aramburu, Jose

    2012-05-01

    Although stress can suppress growth and proliferation, cells can induce adaptive responses that allow them to maintain these functions under stress. While numerous studies have focused on the inhibitory effects of stress on cell growth, less is known on how growth-promoting pathways influence stress responses. We have approached this question by analyzing the effect of mammalian target of rapamycin (mTOR), a central growth controller, on the osmotic stress response. Our results showed that mammalian cells exposed to moderate hypertonicity maintained active mTOR, which was required to sustain their cell size and proliferative capacity. Moreover, mTOR regulated the induction of diverse osmostress response genes, including targets of the tonicity-responsive transcription factor NFAT5 as well as NFAT5-independent genes. Genes sensitive to mTOR-included regulators of stress responses, growth and proliferation. Among them, we identified REDD1 and REDD2, which had been previously characterized as mTOR inhibitors in other stress contexts. We observed that mTOR facilitated transcription-permissive conditions for several osmoresponsive genes by enhancing histone H4 acetylation and the recruitment of RNA polymerase II. Altogether, these results reveal a previously unappreciated role of mTOR in regulating transcriptional mechanisms that control gene expression during cellular stress responses. PMID:22287635

  15. A comparative cDNA microarray analysis reveals a spectrum of genes regulated by Pax6 in mouse lens

    PubMed Central

    Chauhan, Bharesh K.; Reed, Nathan A.; Yang, Ying; Čermák, Lukáš; Reneker, Lixing; Duncan, Melinda K.; Cvekl, Aleš

    2007-01-01

    Background Pax6 is a transcription factor that is required for induction, growth, and maintenance of the lens; however, few direct target genes of Pax6 are known. Results In this report, we describe the results of a cDNA microarray analysis of lens transcripts from transgenic mice over-expressing Pax6 in lens fibre cells in order to narrow the field of potential direct Pax6 target genes. This study revealed that the transcript levels were significantly altered for 508 of the 9700 genes analysed, including five genes encoding the cell adhesion molecules β1-integrin, JAM1, L1 CAM, NCAM-140 and neogenin. Notably, comparisons between the genes differentially expressed in Pax6 heterozygous and Pax6 over-expressing lenses identified 13 common genes, including paralemmin, GDIβ, ATF1, Hrp12 and Brg1. Immunohistochemistry and Western blotting demonstrated that Brg1 is expressed in the embryonic and neonatal (2-week-old) but not in 14-week adult lenses, and confirmed altered expression in transgenic lenses over-expressing Pax6. Furthermore, EMSA demonstrated that the BRG1 promoter contains Pax6 binding sites, further supporting the proposition that it is directly regulated by Pax6. Conclusions These results provide a list of genes with possible roles in lens biology and cataracts that are directly or indirectly regulated by Pax6. PMID:12485166

  16. AAV9-mediated central nervous system–targeted gene delivery via cisterna magna route in mice

    PubMed Central

    Lukashchuk, Vera; Lewis, Katherine E; Coldicott, Ian; Grierson, Andrew J; Azzouz, Mimoun

    2016-01-01

    Current barriers to the use of adeno-associated virus serotype 9 (AAV9) in clinical trials for treating neurological disorders are its high expression in many off-target tissues such as liver and heart, and lack of cell specificity within the central nervous system (CNS) when using ubiquitous promoters such as human cytomegalovirus (CMV) or chicken-β-actin hybrid (CAG). To enhance targeting the transgene expression in CNS cells, self-complementary (sc) AAV9 vectors, scAAV9-GFP vectors carrying neuronal Hb9 and synapsin 1, and nonspecific CMV and CAG promoters were constructed. We demonstrate that synapsin 1 and Hb9 promoters exclusively targeted neurons in vitro, although their strengths were up to 10-fold lower than that of CMV. In vivo analyses of mouse tissue after scAAV9-GFP vector delivery via the cisterna magna revealed a significant advantage of synapsin 1 promoter over both Hb9 variants in targeting neurons throughout the brain, since Hb9 promoters were driving gene expression mainly within the motor-related areas of the brain stem. In summary, this study demonstrates that cisterna magna administration is a safe alternative to intracranial or intracerebroventricular vector delivery route using scAAV9, and introduces a novel utility of the Hb9 promoter for the targeted gene expression for both in vivo and in vitro applications. PMID:26942208

  17. [Targeted modification of CCR5 gene in rabbits by TALEN].

    PubMed

    Tang, Chengcheng; Zhang, Quanjun; Li, Xiaoping; Fan, Nana; Yang, Yi; Quan, Longquan; Lai, Liangxue

    2014-04-01

    The lack of suitable animal model for HIV-1 infection has become a bottleneck for the development of AIDS vaccines and drugs. Wild-type rabbits can be infected by HIV-1 persistently and HIV-1 can be efficiently replicated resulting in syncytia in rabbit cell line co-expressing human CD4 and CCR5.Therefore, a rabbit highly expressing human CD4 and CCR5 may be an ideal animal model for AIDS disease study. In the present report, by using the efficient gene targeting technology, transcription activator-like effector nuclease (TALEN), we explored the feasibility of generating a HIV-1 model by knocking in human CD4 and CCR5 into rabbit genome. First we constructed two TALEN vectors targeting rabbit CCR5 gene and a vector with homologous arms. TALEN mRNAs and donor DNA were then co-injected into fertilized oocytes. After 3?5 days, 24 embryos were collected and used to conduct mutation analysis with PCR and sequencing. All the 24 embryos were detected with CCR5 knockouts and 5 were human CD4 and CCR5 knockins. Our results laid a foundation for establishing a new animal model for the study of AIDS. PMID:24846981

  18. Single-cell analysis reveals gene-expression heterogeneity in syntrophic dual-culture of Desulfovibrio vulgaris with Methanosarcina barkeri

    NASA Astrophysics Data System (ADS)

    Qi, Zhenhua; Pei, Guangsheng; Chen, Lei; Zhang, Weiwen

    2014-12-01

    Microbial syntrophic metabolism has been well accepted as the heart of how methanogenic and other anaerobic microbial communities function. In this work, we applied a single-cell RT-qPCR approach to reveal gene-expression heterogeneity in a model syntrophic system of Desulfovibrio vulgaris and Methanosarcina barkeri, as compared with the D. vulgaris monoculture. Using the optimized primers and single-cell analytical protocol, we quantitatively determine gene-expression levels of 6 selected target genes in each of the 120 single cells of D. vulgaris isolated from its monoculture and dual-culture with M. barkeri. The results demonstrated very significant cell-to-cell gene-expression heterogeneity for the selected D. vulgaris genes in both the monoculture and the syntrophic dual-culture. Interestingly, no obvious increase in gene-expression heterogeneity for the selected genes was observed for the syntrophic dual-culture when compared with its monoculture, although the community structure and cell-cell interactions have become more complicated in the syntrophic dual-culture. In addition, the single-cell RT-qPCR analysis also provided further evidence that the gene cluster (DVU0148-DVU0150) may be involved syntrophic metabolism between D. vulgaris and M. barkeri. Finally, the study validated that single-cell RT-qPCR analysis could be a valuable tool in deciphering gene functions and metabolism in mixed-cultured microbial communities.

  19. Global Transcriptome Analysis Reveals That Poly(ADP-Ribose) Polymerase 1 Regulates Gene Expression through EZH2.

    PubMed

    Martin, Kayla A; Cesaroni, Matteo; Denny, Michael F; Lupey, Lena N; Tempera, Italo

    2015-12-01

    Posttranslational modifications, such as poly(ADP-ribosyl)ation (PARylation), regulate chromatin-modifying enzymes, ultimately affecting gene expression. This study explores the role of poly(ADP-ribose) polymerase (PARP) on global gene expression in a lymphoblastoid B cell line. We found that inhibition of PARP catalytic activity with olaparib resulted in global gene deregulation, affecting approximately 11% of the genes expressed. Gene ontology analysis revealed that PARP could exert these effects through transcription factors and chromatin-remodeling enzymes, including the polycomb repressive complex 2 (PRC2) member EZH2. EZH2 mediates the trimethylation of histone H3 at lysine 27 (H3K27me3), a modification associated with chromatin compaction and gene silencing. Both pharmacological inhibition of PARP and knockdown of PARP1 induced the expression of EZH2, which resulted in increased global H3K27me3. Chromatin immunoprecipitation confirmed that PARP1 inhibition led to H3K27me3 deposition at EZH2 target genes, which resulted in gene silencing. Moreover, increased EZH2 expression is attributed to the loss of the occupancy of the transcription repressor E2F4 at the EZH2 promoter following PARP inhibition. Together, these data show that PARP plays an important role in global gene regulation and identifies for the first time a direct role of PARP1 in regulating the expression and function of EZH2. PMID:26370511

  20. Global Transcriptome Analysis Reveals That Poly(ADP-Ribose) Polymerase 1 Regulates Gene Expression through EZH2

    PubMed Central

    Martin, Kayla A.; Cesaroni, Matteo; Denny, Michael F.; Lupey, Lena N.

    2015-01-01

    Posttranslational modifications, such as poly(ADP-ribosyl)ation (PARylation), regulate chromatin-modifying enzymes, ultimately affecting gene expression. This study explores the role of poly(ADP-ribose) polymerase (PARP) on global gene expression in a lymphoblastoid B cell line. We found that inhibition of PARP catalytic activity with olaparib resulted in global gene deregulation, affecting approximately 11% of the genes expressed. Gene ontology analysis revealed that PARP could exert these effects through transcription factors and chromatin-remodeling enzymes, including the polycomb repressive complex 2 (PRC2) member EZH2. EZH2 mediates the trimethylation of histone H3 at lysine 27 (H3K27me3), a modification associated with chromatin compaction and gene silencing. Both pharmacological inhibition of PARP and knockdown of PARP1 induced the expression of EZH2, which resulted in increased global H3K27me3. Chromatin immunoprecipitation confirmed that PARP1 inhibition led to H3K27me3 deposition at EZH2 target genes, which resulted in gene silencing. Moreover, increased EZH2 expression is attributed to the loss of the occupancy of the transcription repressor E2F4 at the EZH2 promoter following PARP inhibition. Together, these data show that PARP plays an important role in global gene regulation and identifies for the first time a direct role of PARP1 in regulating the expression and function of EZH2. PMID:26370511

  1. Aberrant gene expression in mucosa adjacent to tumor reveals a molecular crosstalk in colon cancer

    PubMed Central

    2014-01-01

    Background A colorectal tumor is not an isolated entity growing in a restricted location of the body. The patient’s gut environment constitutes the framework where the tumor evolves and this relationship promotes and includes a complex and tight correlation of the tumor with inflammation, blood vessels formation, nutrition, and gut microbiome composition. The tumor influence in the environment could both promote an anti-tumor or a pro-tumor response. Methods A set of 98 paired adjacent mucosa and tumor tissues from colorectal cancer (CRC) patients and 50 colon mucosa from healthy donors (246 samples in total) were included in this work. RNA extracted from each sample was hybridized in Affymetrix chips Human Genome U219. Functional relationships between genes were inferred by means of systems biology using both transcriptional regulation networks (ARACNe algorithm) and protein-protein interaction networks (BIANA software). Results Here we report a transcriptomic analysis revealing a number of genes activated in adjacent mucosa from CRC patients, not activated in mucosa from healthy donors. A functional analysis of these genes suggested that this active reaction of the adjacent mucosa was related to the presence of the tumor. Transcriptional and protein-interaction networks were used to further elucidate this response of normal gut in front of the tumor, revealing a crosstalk between proteins secreted by the tumor and receptors activated in the adjacent colon tissue; and vice versa. Remarkably, Slit family of proteins activated ROBO receptors in tumor whereas tumor-secreted proteins transduced a cellular signal finally activating AP-1 in adjacent tissue. Conclusions The systems-level approach provides new insights into the micro-ecology of colorectal tumorogenesis. Disrupting this intricate molecular network of cell-cell communication and pro-inflammatory microenvironment could be a therapeutic target in CRC patients. PMID:24597571

  2. Network Analysis Reveals Sex- and Antibiotic Resistance-Associated Antivirulence Targets in Clinical Uropathogens

    PubMed Central

    2015-01-01

    Increasing antibiotic resistance among uropathogenic Escherichia coli (UPEC) is driving interest in therapeutic targeting of nonconserved virulence factor (VF) genes. The ability to formulate efficacious combinations of antivirulence agents requires an improved understanding of how UPEC deploy these genes. To identify clinically relevant VF combinations, we applied contemporary network analysis and biclustering algorithms to VF profiles from a large, previously characterized inpatient clinical cohort. These mathematical approaches identified four stereotypical VF combinations with distinctive relationships to antibiotic resistance and patient sex that are independent of traditional phylogenetic grouping. Targeting resistance- or sex-associated VFs based upon these contemporary mathematical approaches may facilitate individualized anti-infective therapies and identify synergistic VF combinations in bacterial pathogens. PMID:26985454

  3. Inference of Target Gene Regulation via miRNAs during Cell Senescence by Using the MiRaGE Server.

    PubMed

    Taguchi, Y-H

    2012-08-01

    miRNAs have recently been shown to play a key role in cell senescence, by downregulating target genes. Thus, inference of those miRNAs that critically downregulate target genes is important. However, inference of target gene regulation by miRNAs is difficult and is often achieved simply by investigating significant upregulation during cell senescence. Here, we inferred the regulation of target genes by miRNAs, using the recently developed MiRaGE server, together with the change in miRNA expression during fibroblast IMR90 cell senescence. We revealed that the simultaneous consideration of 2 criteria, the up(down)regulation and the down(up) regulatiion of target genes, yields more feasible miRNA, i.e., those that are most frequently reported to be down/upregulated and/or to possess biological backgrounds that induce cell senescence. Thus, when analyzing miRNAs that critically contribute to cell senescence, it is important to consider the level of target gene regulation, simultaneously with the change in miRNA expression. PMID:23185711

  4. Homozygosity Mapping and Targeted Sanger Sequencing Reveal Genetic Defects Underlying Inherited Retinal Disease in Families from Pakistan

    PubMed Central

    Waheed, Nadia Khalida; Siddiqui, Sorath Noorani; Mustafa, Bilal; Ayub, Humaira; Ali, Liaqat; Ahmad, Shakeel; Micheal, Shazia; Hussain, Alamdar; Shah, Syed Tahir Abbas; Ali, Syeda Hafiza Benish; Ahmed, Waqas; Khan, Yar Muhammad; den Hollander, Anneke I.; Haer-Wigman, Lonneke; Collin, Rob W. J.; Khan, Muhammad Imran; Qamar, Raheel; Cremers, Frans P. M.

    2015-01-01

    Background Homozygosity mapping has facilitated the identification of the genetic causes underlying inherited diseases, particularly in consanguineous families with multiple affected individuals. This knowledge has also resulted in a mutation dataset that can be used in a cost and time effective manner to screen frequent population-specific genetic variations associated with diseases such as inherited retinal disease (IRD). Methods We genetically screened 13 families from a cohort of 81 Pakistani IRD families diagnosed with Leber congenital amaurosis (LCA), retinitis pigmentosa (RP), congenital stationary night blindness (CSNB), or cone dystrophy (CD). We employed genome-wide single nucleotide polymorphism (SNP) array analysis to identify homozygous regions shared by affected individuals and performed Sanger sequencing of IRD-associated genes located in the sizeable homozygous regions. In addition, based on population specific mutation data we performed targeted Sanger sequencing (TSS) of frequent variants in AIPL1, CEP290, CRB1, GUCY2D, LCA5, RPGRIP1 and TULP1, in probands from 28 LCA families. Results Homozygosity mapping and Sanger sequencing of IRD-associated genes revealed the underlying mutations in 10 families. TSS revealed causative variants in three families. In these 13 families four novel mutations were identified in CNGA1, CNGB1, GUCY2D, and RPGRIP1. Conclusions Homozygosity mapping and TSS revealed the underlying genetic cause in 13 IRD families, which is useful for genetic counseling as well as therapeutic interventions that are likely to become available in the near future. PMID:25775262

  5. In situ Expression of Functional Genes Reveals Nitrogen Cycling at High Temperatures in Terrestrial Hydrothermal Systems

    NASA Astrophysics Data System (ADS)

    Loiacono, S. T.; Meyer-Dombard, D. R.

    2011-12-01

    An essential element for life, nitrogen occurs in all living organisms and is critical for the synthesis of amino acids, proteins, nucleic acids, and other forms of biomass. Thus, nitrogen cycling likely plays a vital role in microbial metabolic processes as well as nutrient availability. For microorganisms in "extreme" environments, this means developing adaptations that allow them to survive in harsh conditions and still perform the metabolisms essential to sustain life. Recent studies have screened biofilms and thermal sediments of Yellowstone National Park (YNP) thermal features for the presence of nifH genes, which code for a key enzyme in the nitrogen fixation process [1-4]. Furthermore, analysis of nitrogen isotopes in biofilms across a temperature and chemical gradient revealed that nitrogen fixation likely varies across the chemosynthetic/photosynthetic ecotone [5]. Although research has evaluated and confirmed the presence of nifH genes in various thermophilic microbial communities, the existence of a gene in the DNA of an organism does not verify its use. Instead, other methods, such as culturing, isotope tracer assays, and gene expression studies are required to provide direct evidence of biological nitrogen fixation. Culturing and isotope tracer approaches have successfully revealed high-temperature biological nitrogen fixation in both marine hydrothermal vent microbial communities [6] and in acidic, terrestrial hydrothermal sediment [3]. Transcriptomics-based techniques (using mRNA extracted from samples to confirm in situ expression of targeted genes) have been much more limited in number, and only a few studies have, to date, investigated in situ expression of the nifH gene in thermophilic microbial communities [2, 7]. This study explores the presence and expression of nifH genes in several features of the Lower Geyser Basin (LGB) of YNP. Nucleic acids from chemosynthetic and photosynthetic microbial communities were extracted and then amplified

  6. Gene Targeting to the Uteroplacental Circulation of Pregnant Guinea Pigs.

    PubMed

    Mehta, Vedanta; Ofir, Keren; Swanson, Anna; Kloczko, Ewa; Boyd, Michael; Barker, Hannah; Avdic-Belltheus, Adnan; Martin, John; Zachary, Ian; Peebles, Donald; David, Anna L

    2016-08-01

    Our study aimed to target adenoviral gene therapy to the uteroplacental circulation of pregnant guinea pigs in order to develop a novel therapy for fetal growth restriction. Four methods of delivery of an adenovirus encoding β-galactosidase (Ad.LacZ) were evaluated: intravascular injection using phosphate-buffered saline (PBS) into (1) uterine artery (UtA) or (2) internal iliac artery or external administration in (3) PBS or (4) pluronic F-127 gel (Sigma Aldrich). Postmortem examination was performed 4 to 7 days after gene transfer. Tissue transduction was assessed by X-gal histochemistry and enzyme-linked immunosorbent assay. External vascular application of the adenovirus vector in combination with pluronic gel had 91.7% success rate in terms of administration (85% maternal survival) and gave the best results for maternal/fetal survival and local transduction efficiency without any spread to maternal or fetal tissues. This study suggests an optimal method of gene delivery to the UtAs of a small rodent for preclinical studies. PMID:26865541

  7. Tackling centrosome biology through gene targeting in chicken B cells.

    PubMed

    Chavali, Pavithra L; Gergely, Fanni

    2015-01-01

    The centrosome proteome comprises hundreds of proteins whose function at the organelle and in the cellular context is unknown. Loss-of-function studies present a powerful tool to probe the roles of these individual constituents and hence improve our insight into key questions of centrosome biology such as how centrosomes are built, how they duplicate, and which cellular processes they partake in. In cultured cells ribonucleic acid (RNA) interference remains the most widely used method to achieve protein depletion, but due to the remarkable stability of many centrosome components depletion is often incomplete. In such instances genome editing provides a viable alternative. The exceptionally high homologous recombination rate of chicken DT40 cells makes this lymphocytic cell line ideal for genetic manipulation. Here we describe methods for the design and generation of knockouts and in situ tagging of genes in these cells. Furthermore, we report an optimized technique that allows isolation of centrosomes from DT40 cells for use in in vitro functional assays and proteomic analysis. Gene editing by CRISPR-Cas9 technology is fast replacing RNA interference as a method of choice for loss-of-function studies, but the combination of the fast cell cycle, the robustness in culture and ease of gene targeting, will continue to make DT40 cells a useful model system for studies of vertebrate protein function. PMID:26175435

  8. Targeted Gene Therapy of Cancer: Second Amendment toward Holistic Therapy.

    PubMed

    Barar, Jaleh; Omidi, Yadollah

    2013-01-01

    It seems solid tumors are developing smart organs with specialized cells creating specified bio-territory, the so called "tumor microenvironment (TME)", in which there is reciprocal crosstalk among cancer cells, immune system cells and stromal cells. TME as an intricate milieu also consists of cancer stem cells (CSCs) that can resist against chemotherapies. In solid tumors, metabolism and vascularization appears to be aberrant and tumor interstitial fluid (TIF) functions as physiologic barrier. Thus, chemotherapy, immunotherapy and gene therapy often fail to provide cogent clinical outcomes. It looms that it is the time to accept the fact that initiation of cancer could be generation of another form of life that involves a cluster of thousands of genes, while we have failed to observe all aspects of it. Hence, the current treatment modalities need to be re-visited to cover all key aspects of disease using combination therapy based on the condition of patients. Perhaps personalized cluster of genes need to be simultaneously targeted. PMID:23878787

  9. Listening to the noise: random fluctuations reveal gene network parameters

    SciTech Connect

    Munsky, Brian; Khammash, Mustafa

    2009-01-01

    The cellular environment is abuzz with noise. The origin of this noise is attributed to the inherent random motion of reacting molecules that take part in gene expression and post expression interactions. In this noisy environment, clonal populations of cells exhibit cell-to-cell variability that frequently manifests as significant phenotypic differences within the cellular population. The stochastic fluctuations in cellular constituents induced by noise can be measured and their statistics quantified. We show that these random fluctuations carry within them valuable information about the underlying genetic network. Far from being a nuisance, the ever-present cellular noise acts as a rich source of excitation that, when processed through a gene network, carries its distinctive fingerprint that encodes a wealth of information about that network. We demonstrate that in some cases the analysis of these random fluctuations enables the full identification of network parameters, including those that may otherwise be difficult to measure. This establishes a potentially powerful approach for the identification of gene networks and offers a new window into the workings of these networks.

  10. p53-directed translational control can shape and expand the universe of p53 target genes

    PubMed Central

    Zaccara, S; Tebaldi, T; Pederiva, C; Ciribilli, Y; Bisio, A; Inga, A

    2014-01-01

    The increasing number of genome-wide transcriptome analyses focusing on p53-induced cellular responses in many cellular contexts keeps adding to the already numerous p53-regulated transcriptional networks. To investigate post-transcriptional controls as an additional dimension of p53-directed gene expression responses, we performed a translatome analysis through polysomal profiling on MCF7 cells upon 16 hours of doxorubicin or nutlin-3a treatment. The comparison between the transcriptome and the translatome revealed a considerable level of uncoupling, characterized by genes whose transcription variations did not correlate with translation variations. Interestingly, uncoupled genes were associated with apoptosis, DNA and RNA metabolism and cell cycle functions, suggesting that post-transcriptional control can modulate classical p53-regulated responses. Furthermore, even for well-established p53 targets that were differentially expressed both at the transcriptional and translational levels, quantitative differences between the transcriptome, subpolysomal and polysomal RNAs were evident. As we searched mechanisms underlying gene expression uncoupling, we identified the p53-dependent modulation of six RNA-binding proteins, where hnRNPD (AUF1) and CPEB4 are direct p53 transcriptional targets, whereas SRSF1, DDX17, YBX1 and TARDBP are indirect targets (genes modulated preferentially in the subpolysomal or polysomal mRNA level) modulated at the translational level in a p53-dependent manner. In particular, YBX1 translation appeared to be reduced by p53 via two different mechanisms, one related to mTOR inhibition and the other to miR-34a expression. Overall, we established p53 as a master regulator of translational control and identified new p53-regulated genes affecting translation that can contribute to p53-dependent cellular responses. PMID:24926617

  11. p53-directed translational control can shape and expand the universe of p53 target genes.

    PubMed

    Zaccara, S; Tebaldi, T; Pederiva, C; Ciribilli, Y; Bisio, A; Inga, A

    2014-10-01

    The increasing number of genome-wide transcriptome analyses focusing on p53-induced cellular responses in many cellular contexts keeps adding to the already numerous p53-regulated transcriptional networks. To investigate post-transcriptional controls as an additional dimension of p53-directed gene expression responses, we performed a translatome analysis through polysomal profiling on MCF7 cells upon 16 hours of doxorubicin or nutlin-3a treatment. The comparison between the transcriptome and the translatome revealed a considerable level of uncoupling, characterized by genes whose transcription variations did not correlate with translation variations. Interestingly, uncoupled genes were associated with apoptosis, DNA and RNA metabolism and cell cycle functions, suggesting that post-transcriptional control can modulate classical p53-regulated responses. Furthermore, even for well-established p53 targets that were differentially expressed both at the transcriptional and translational levels, quantitative differences between the transcriptome, subpolysomal and polysomal RNAs were evident. As we searched mechanisms underlying gene expression uncoupling, we identified the p53-dependent modulation of six RNA-binding proteins, where hnRNPD (AUF1) and CPEB4 are direct p53 transcriptional targets, whereas SRSF1, DDX17, YBX1 and TARDBP are indirect targets (genes modulated preferentially in the subpolysomal or polysomal mRNA level) modulated at the translational level in a p53-dependent manner. In particular, YBX1 translation appeared to be reduced by p53 via two different mechanisms, one related to mTOR inhibition and the other to miR-34a expression. Overall, we established p53 as a master regulator of translational control and identified new p53-regulated genes affecting translation that can contribute to p53-dependent cellular responses. PMID:24926617

  12. Genomic Analysis Reveals Disruption of Striatal Neuronal Development and Therapeutic Targets in Human Huntington’s Disease Neural Stem Cells

    PubMed Central

    Ring, Karen L.; An, Mahru C.; Zhang, Ningzhe; O’Brien, Robert N.; Ramos, Eliana Marisa; Gao, Fuying; Atwood, Robert; Bailus, Barbara J.; Melov, Simon; Mooney, Sean D.; Coppola, Giovanni; Ellerby, Lisa M.

    2015-01-01

    Summary We utilized induced pluripotent stem cells (iPSCs) derived from Huntington’s disease (HD) patients as a human model of HD and determined that the disease phenotypes only manifest in the differentiated neural stem cell (NSC) stage, not in iPSCs. To understand the molecular basis for the CAG repeat expansion-dependent disease phenotypes in NSCs, we performed transcriptomic analysis of HD iPSCs and HD NSCs compared to isogenic controls. Differential gene expression and pathway analysis pointed to transforming growth factor β (TGF-β) and netrin-1 as the top dysregulated pathways. Using data-driven gene coexpression network analysis, we identified seven distinct coexpression modules and focused on two that were correlated with changes in gene expression due to the CAG expansion. Our HD NSC model revealed the dysregulation of genes involved in neuronal development and the formation of the dorsal striatum. The striatal and neuronal networks disrupted could be modulated to correct HD phenotypes and provide therapeutic targets. PMID:26651603

  13. Integrative genomic analysis in K562 chronic myelogenous leukemia cells reveals that proximal NCOR1 binding positively regulates genes that govern erythroid differentiation and Imatinib sensitivity

    PubMed Central

    Long, Mark D.; van den Berg, Patrick R.; Russell, James L.; Singh, Prashant K.; Battaglia, Sebastiano; Campbell, Moray J.

    2015-01-01

    To define the functions of NCOR1 we developed an integrative analysis that combined ENCODE and NCI-60 data, followed by in vitro validation. NCOR1 and H3K9me3 ChIP-Seq, FAIRE-seq and DNA CpG methylation interactions were related to gene expression using bootstrapping approaches. Most NCOR1 combinations (24/44) were associated with significantly elevated level expression of protein coding genes and only very few combinations related to gene repression. DAVID's biological process annotation revealed that elevated gene expression was uniquely associated with acetylation and ETS binding. A matrix of gene and drug interactions built on NCI-60 data identified that Imatinib significantly targeted the NCOR1 governed transcriptome. Stable knockdown of NCOR1 in K562 cells slowed growth and significantly repressed genes associated with NCOR1 cistrome, again, with the GO terms acetylation and ETS binding, and significantly dampened sensitivity to Imatinib-induced erythroid differentiation. Mining public microarray data revealed that NCOR1-targeted genes were significantly enriched in Imatinib response gene signatures in cell lines and chronic myelogenous leukemia (CML) patients. These approaches integrated cistrome, transcriptome and drug sensitivity relationships to reveal that NCOR1 function is surprisingly most associated with elevated gene expression, and that these targets, both in CML cell lines and patients, associate with sensitivity to Imatinib. PMID:26117541

  14. Integrated multi-omics analyses reveal the pleiotropic nature of the control of gene expression by Puf3p

    PubMed Central

    Kershaw, Christopher J.; Costello, Joseph L.; Talavera, David; Rowe, William; Castelli, Lydia M.; Sims, Paul F. G.; Grant, Christopher M.; Ashe, Mark P.; Hubbard, Simon J.; Pavitt, Graham D.

    2015-01-01

    The PUF family of RNA-binding proteins regulate gene expression post-transcriptionally. Saccharomyces cerevisiae Puf3p is characterised as binding nuclear-encoded mRNAs specifying mitochondrial proteins. Extensive studies of its regulation of COX17 demonstrate its role in mRNA decay. Using integrated genome-wide approaches we define an expanded set of Puf3p target mRNAs and quantitatively assessed the global impact of loss of PUF3 on gene expression using mRNA and polysome profiling and quantitative proteomics. In agreement with prior studies, our sequencing of affinity-purified Puf3-TAP associated mRNAs (RIP-seq) identified mRNAs encoding mitochondrially-targeted proteins. Additionally, we also found 720  new mRNA targets that predominantly encode proteins that enter the nucleus. Comparing transcript levels in wild-type and puf3∆ cells revealed that only a small fraction of mRNA levels alter, suggesting Puf3p determines mRNA stability for only a limited subset of its target mRNAs. Finally, proteomic and translatomic studies suggest that loss of Puf3p has widespread, but modest, impact on mRNA translation. Taken together our integrated multi-omics data point to multiple classes of Puf3p targets, which display coherent post-transcriptional regulatory properties and suggest Puf3p plays a broad, but nuanced, role in the fine-tuning of gene expression. PMID:26493364

  15. Small RNA and Degradome Sequencing Reveal Complex Roles of miRNAs and Their Targets in Developing Wheat Grains

    PubMed Central

    Geng, Yuke; Hao, Chenyang; Chen, Xinhong; Zhang, Xueyong

    2015-01-01

    Plant microRNAs (miRNAs) have been shown to play critical roles in plant development. In this study, we employed small RNA combined with degradome sequencing to survey development-related miRNAs and their validated targets during wheat grain development. A total of 186 known miRNAs and 37 novel miRNAs were identified in four small RNA libraries. Moreover, a miRNA-like long hairpin locus was first identified to produce 21~22-nt phased siRNAs that act in trans to cleave target mRNAs. A comparison of the miRNAomes revealed that 55 miRNA families were differentially expressed during the grain development. Predicted and validated targets of these development-related miRNAs are involved in different cellular responses and metabolic processes including cell proliferation, auxin signaling, nutrient metabolism and gene expression. This study provides insight into the complex roles of miRNAs and their targets in regulating wheat grain development. PMID:26426440

  16. Ontogeny of Hepatic Energy Metabolism Genes in Mice as Revealed by RNA-Sequencing

    PubMed Central

    Renaud, Helen J.; Cui, Yue Julia; Lu, Hong; Zhong, Xiao-bo; Klaassen, Curtis D.

    2014-01-01

    The liver plays a central role in metabolic homeostasis by coordinating synthesis, storage, breakdown, and redistribution of nutrients. Hepatic energy metabolism is dynamically regulated throughout different life stages due to different demands for energy during growth and development. However, changes in gene expression patterns throughout ontogeny for factors important in hepatic energy metabolism are not well understood. We performed detailed transcript analysis of energy metabolism genes during various stages of liver development in mice. Livers from male C57BL/6J mice were collected at twelve ages, including perinatal and postnatal time points (n = 3/age). The mRNA was quantified by RNA-Sequencing, with transcript abundance estimated by Cufflinks. One thousand sixty energy metabolism genes were examined; 794 were above detection, of which 627 were significantly changed during at least one developmental age compared to adult liver. Two-way hierarchical clustering revealed three major clusters dependent on age: GD17.5–Day 5 (perinatal-enriched), Day 10–Day 20 (pre-weaning-enriched), and Day 25–Day 60 (adolescence/adulthood-enriched). Clustering analysis of cumulative mRNA expression values for individual pathways of energy metabolism revealed three patterns of enrichment: glycolysis, ketogenesis, and glycogenesis were all perinatally-enriched; glycogenolysis was the only pathway enriched during pre-weaning ages; whereas lipid droplet metabolism, cholesterol and bile acid metabolism, gluconeogenesis, and lipid metabolism were all enriched in adolescence/adulthood. This study reveals novel findings such as the divergent expression of the fatty acid β-oxidation enzymes Acyl-CoA oxidase 1 and Carnitine palmitoyltransferase 1a, indicating a switch from mitochondrial to peroxisomal β-oxidation after weaning; as well as the dynamic ontogeny of genes implicated in obesity such as Stearoyl-CoA desaturase 1 and Elongation of very long chain fatty acids-like 3

  17. In silico pathway analysis in cervical carcinoma reveals potential new targets for treatment

    PubMed Central

    van Dam, Peter A.; van Dam, Pieter-Jan H. H.; Rolfo, Christian; Giallombardo, Marco; van Berckelaer, Christophe; Trinh, Xuan Bich; Altintas, Sevilay; Huizing, Manon; Papadimitriou, Kostas; Tjalma, Wiebren A. A.; van Laere, Steven

    2016-01-01

    An in silico pathway analysis was performed in order to improve current knowledge on the molecular drivers of cervical cancer and detect potential targets for treatment. Three publicly available Affymetrix gene expression data-sets (GSE5787, GSE7803, GSE9750) were retrieved, vouching for a total of 9 cervical cancer cell lines (CCCLs), 39 normal cervical samples, 7 CIN3 samples and 111 cervical cancer samples (CCSs). Predication analysis of microarrays was performed in the Affymetrix sets to identify cervical cancer biomarkers. To select cancer cell-specific genes the CCSs were compared to the CCCLs. Validated genes were submitted to a gene set enrichment analysis (GSEA) and Expression2Kinases (E2K). In the CCSs a total of 1,547 probe sets were identified that were overexpressed (FDR < 0.1). Comparing to CCCLs 560 probe sets (481 unique genes) had a cancer cell-specific expression profile, and 315 of these genes (65%) were validated. GSEA identified 5 cancer hallmarks enriched in CCSs (P < 0.01 and FDR < 0.25) showing that deregulation of the cell cycle is a major component of cervical cancer biology. E2K identified a protein-protein interaction (PPI) network of 162 nodes (including 20 drugable kinases) and 1626 edges. This PPI-network consists of 5 signaling modules associated with MYC signaling (Module 1), cell cycle deregulation (Module 2), TGFβ-signaling (Module 3), MAPK signaling (Module 4) and chromatin modeling (Module 5). Potential targets for treatment which could be identified were CDK1, CDK2, ABL1, ATM, AKT1, MAPK1, MAPK3 among others. The present study identified important driver pathways in cervical carcinogenesis which should be assessed for their potential therapeutic drugability. PMID:26701206

  18. In silico pathway analysis in cervical carcinoma reveals potential new targets for treatment.

    PubMed

    van Dam, Peter A; van Dam, Pieter-Jan H H; Rolfo, Christian; Giallombardo, Marco; van Berckelaer, Christophe; Trinh, Xuan Bich; Altintas, Sevilay; Huizing, Manon; Papadimitriou, Kostas; Tjalma, Wiebren A A; van Laere, Steven

    2016-01-19

    An in silico pathway analysis was performed in order to improve current knowledge on the molecular drivers of cervical cancer and detect potential targets for treatment. Three publicly available Affymetrix gene expression data-sets (GSE5787, GSE7803, GSE9750) were retrieved, vouching for a total of 9 cervical cancer cell lines (CCCLs), 39 normal cervical samples, 7 CIN3 samples and 111 cervical cancer samples (CCSs). Predication analysis of microarrays was performed in the Affymetrix sets to identify cervical cancer biomarkers. To select cancer cell-specific genes the CCSs were compared to the CCCLs. Validated genes were submitted to a gene set enrichment analysis (GSEA) and Expression2Kinases (E2K). In the CCSs a total of 1,547 probe sets were identified that were overexpressed (FDR < 0.1). Comparing to CCCLs 560 probe sets (481 unique genes) had a cancer cell-specific expression profile, and 315 of these genes (65%) were validated. GSEA identified 5 cancer hallmarks enriched in CCSs (P < 0.01 and FDR < 0.25) showing that deregulation of the cell cycle is a major component of cervical cancer biology. E2K identified a protein-protein interaction (PPI) network of 162 nodes (including 20 drugable kinases) and 1626 edges. This PPI-network consists of 5 signaling modules associated with MYC signaling (Module 1), cell cycle deregulation (Module 2), TGFβ-signaling (Module 3), MAPK signaling (Module 4) and chromatin modeling (Module 5). Potential targets for treatment which could be identified were CDK1, CDK2, ABL1, ATM, AKT1, MAPK1, MAPK3 among others. The present study identified important driver pathways in cervical carcinogenesis which should be assessed for their potential therapeutic drugability. PMID:26701206

  19. Functional gene pyrosequencing reveals core proteobacterial denitrifiers in boreal lakes

    PubMed Central

    Saarenheimo, Jatta; Tiirola, Marja Annika; Rissanen, Antti J.

    2015-01-01

    Denitrification is an important microbial process in aquatic ecosystems that can reduce the effects of eutrophication. Here, quantification and pyrosequencing of nirS, nirK, and nosZ genes encoding for nitrite and nitrous oxide reductases was performed in sediment samples from four boreal lakes to determine the structure and seasonal stability of denitrifying microbial populations. Sediment quality and nitrate concentrations were linked to the quantity and diversity of denitrification genes, the abundance of denitrifying populations (nirS and nosZ genes) correlated with coupled nitrification-denitrification (Dn), and the denitrification of the overlying water NO3- (Dw) correlated with the nirS/nirK ratio. The number of core nirS, nirK, and nosZ operational taxonomical units (OTUs) was low (6, 7, and 3, respectively), and most of these core OTUs were shared among the lakes. Dominant nirK sequences matched best with those of the order Rhizobiales, which was one of the main bacterial orders present in the sediment microbiomes, whereas the dominant nirS sequences were affiliated with the order Burkholderiales. Over half of the nosZ sequences belonged to a single OTU of the order Burkholderiales, but coupled nitrification–denitrification rate correlated with another dominant nosZ OTU assigned to the order Rhodospirillales. The study indicates that a few core proteobacterial clusters may drive denitrification in boreal lake sediments, as the same Alpha- and Betaproteobacteria denitrifier clusters were present in different lakes and seasons. PMID:26191058

  20. Identification of the Drosophila Mes4 gene as a novel target of the transcription factor DREF

    SciTech Connect

    Suyari, Osamu; Ida, Hiroyuki; Yoshioka, Yasuhide; Kato, Yasuko; Hashimoto, Reina; Yamaguchi, Masamitsu

    2009-05-01

    The Mes4 gene has been identified as one of the maternal Dorsal target genes in Drosophila. In the present study, we found a DNA replication-related element (DRE, 5'-TATCGATA) in the Mes4 promoter recognized by the DRE-binding factor (DREF). Luciferase transient expression assays in S2 cells using Mes4 promoter-luciferase fusion plasmids revealed that the DRE sequence is essential for Mes4 promoter activity. Requirement of DRE for Mes4 promoter activity was further confirmed by anti-{beta}-galactosidase antibody-staining of various tissues from transgenic flies carrying Mes4 promoter-lacZ fusion genes. Furthermore, wild type Mes4 promoter activity was decreased by 40% in DREF-depleted S2 cells. These results indicate that DREF positively regulates Mes4 gene expression. Band mobility shift analyses using Kc cell nuclear extracts further indicated that the DRE sequence in the Mes4 promoter is especially important for binding to DREF. Moreover, specific binding of DREF to the involved genomic region could be demonstrated by chromatin immunoprecipitation assays using anti-DREF antibodies. These results, taken together, indicate that the DRE/DREF system activates transcription of the Mes4 gene. In addition, knockdown of the Mes4 gene in wing imaginal discs using the GAL4-UAS system caused an atrophied wing phenotype, suggesting that Mes4 is required for wing morphogenesis.

  1. The Integrator complex controls the termination of transcription at diverse classes of gene targets

    PubMed Central

    Skaar, Jeffrey R; Ferris, Andrea L; Wu, Xiaolin; Saraf, Anita; Khanna, Kum Kum; Florens, Laurence; Washburn, Michael P; Hughes, Stephen H; Pagano, Michele

    2015-01-01

    Complexes containing INTS3 and either NABP1 or NABP2 were initially characterized in DNA damage responses, but their biochemical function remained unknown. Using affinity purifications and HIV Integration targeting-sequencing (HIT-Seq), we find that these complexes are part of the Integrator complex, which binds RNA Polymerase II and regulates specific target genes. Integrator cleaves snRNAs as part of their processing to their mature form in a mechanism that is intimately coupled with transcription termination. However, HIT-Seq reveals that Integrator also binds to the 3′ end of replication-dependent histones and promoter proximal regions of genes with polyadenylated transcripts. Depletion of Integrator subunits results in transcription termination failure, disruption of histone mRNA processing, and polyadenylation of snRNAs and histone mRNAs. Furthermore, promoter proximal binding of Integrator negatively regulates expression of genes whose transcripts are normally polyadenylated. Integrator recruitment to all three gene classes is DSIF-dependent, suggesting that Integrator functions as a termination complex at DSIF-dependent RNA Polymerase II pause sites. PMID:25675981

  2. DICER Inactivation Identifies Pancreatic β-Cell “Disallowed” Genes Targeted by MicroRNAs

    PubMed Central

    Martinez-Sanchez, Aida; Nguyen-Tu, Marie-Sophie

    2015-01-01

    Pancreatic β-cells are the body's sole source of circulating insulin and essential for the maintenance of blood glucose homeostasis. Levels of up to 66 “disallowed” genes, which are strongly expressed and play housekeeping roles in most other mammalian tissues, are unusually low in β-cells. The molecular mechanisms involved in repressing these genes are largely unknown. Here, we explore the role in gene disallowance of microRNAs (miRNAs), a type of small noncoding RNAs that silence gene expression at the posttranscriptional level and are essential for β-cell development and function. To selectively deplete miRNAs from adult β-cells, the miRNA-processing enzyme DICER was inactivated by deletion of the RNase III domain with a tamoxifen-inducible Pdx1CreER transgene. In this model, β-cell dysfunction was apparent 2 weeks after recombination and preceded a decrease in insulin content and loss of β-cell mass. Of the 14 disallowed genes studied, quantitative RT-quantitative real-time PCR revealed that 6 genes (Fcgrt, Igfbp4, Maf, Oat, Pdgfra, and Slc16a1) were up-regulated (1.4- to 2.1-fold, P < .05) at this early stage. Expression of luciferase constructs bearing the 3′-untranslated regions of the corresponding mRNAs in wild-type or DICER-null β-cells demonstrated that Fcgrt, Oat, and Pdgfra are miRNA direct targets. We thus reveal a role for miRNAs in the regulation of disallowed genes in β-cells and provide evidence for a novel means through which noncoding RNAs control the functional identity of these cells independently of actions on β-cell mass. PMID:26038943

  3. Connectivity Map-based discovery of parbendazole reveals targetable human osteogenic pathway

    PubMed Central

    Brum, Andrea M.; van de Peppel, Jeroen; van der Leije, Cindy S.; Schreuders-Koedam, Marijke; Eijken, Marco; van der Eerden, Bram C. J.; van Leeuwen, Johannes P. T. M.

    2015-01-01

    Osteoporosis is a common skeletal disorder characterized by low bone mass leading to increased bone fragility and fracture susceptibility. In this study, we have identified pathways that stimulate differentiation of bone forming osteoblasts from human mesenchymal stromal cells (hMSCs). Gene expression profiling was performed in hMSCs differentiated toward osteoblasts (at 6 h). Significantly regulated genes were analyzed in silico, and the Connectivity Map (CMap) was used to identify candidate bone stimulatory compounds. The signature of parbendazole matches the expression changes observed for osteogenic hMSCs. Parbendazole stimulates osteoblast differentiation as indicated by increased alkaline phosphatase activity, mineralization, and up-regulation of bone marker genes (alkaline phosphatase/ALPL, osteopontin/SPP1, and bone sialoprotein II/IBSP) in a subset of the hMSC population resistant to the apoptotic effects of parbendazole. These osteogenic effects are independent of glucocorticoids because parbendazole does not up-regulate glucocorticoid receptor (GR) target genes and is not inhibited by the GR antagonist mifepristone. Parbendazole causes profound cytoskeletal changes including degradation of microtubules and increased focal adhesions. Stabilization of microtubules by pretreatment with Taxol inhibits osteoblast differentiation. Parbendazole up-regulates bone morphogenetic protein 2 (BMP-2) gene expression and activity. Cotreatment with the BMP-2 antagonist DMH1 limits, but does not block, parbendazole-induced mineralization. Using the CMap we have identified a previously unidentified lineage-specific, bone anabolic compound, parbendazole, which induces osteogenic differentiation through a combination of cytoskeletal changes and increased BMP-2 activity. PMID:26420877

  4. Connectivity Map-based discovery of parbendazole reveals targetable human osteogenic pathway.

    PubMed

    Brum, Andrea M; van de Peppel, Jeroen; van der Leije, Cindy S; Schreuders-Koedam, Marijke; Eijken, Marco; van der Eerden, Bram C J; van Leeuwen, Johannes P T M

    2015-10-13

    Osteoporosis is a common skeletal disorder characterized by low bone mass leading to increased bone fragility and fracture susceptibility. In this study, we have identified pathways that stimulate differentiation of bone forming osteoblasts from human mesenchymal stromal cells (hMSCs). Gene expression profiling was performed in hMSCs differentiated toward osteoblasts (at 6 h). Significantly regulated genes were analyzed in silico, and the Connectivity Map (CMap) was used to identify candidate bone stimulatory compounds. The signature of parbendazole matches the expression changes observed for osteogenic hMSCs. Parbendazole stimulates osteoblast differentiation as indicated by increased alkaline phosphatase activity, mineralization, and up-regulation of bone marker genes (alkaline phosphatase/ALPL, osteopontin/SPP1, and bone sialoprotein II/IBSP) in a subset of the hMSC population resistant to the apoptotic effects of parbendazole. These osteogenic effects are independent of glucocorticoids because parbendazole does not up-regulate glucocorticoid receptor (GR) target genes and is not inhibited by the GR antagonist mifepristone. Parbendazole causes profound cytoskeletal changes including degradation of microtubules and increased focal adhesions. Stabilization of microtubules by pretreatment with Taxol inhibits osteoblast differentiation. Parbendazole up-regulates bone morphogenetic protein 2 (BMP-2) gene expression and activity. Cotreatment with the BMP-2 antagonist DMH1 limits, but does not block, parbendazole-induced mineralization. Using the CMap we have identified a previously unidentified lineage-specific, bone anabolic compound, parbendazole, which induces osteogenic differentiation through a combination of cytoskeletal changes and increased BMP-2 activity. PMID:26420877

  5. Strategies on the nuclear-targeted delivery of genes

    PubMed Central

    Yao, Jing; Fan, Ying; Li, Yuanke; Huang, Leaf

    2016-01-01

    To improve the nuclear-targeted delivery of non-viral vectors, extensive effort has been carried out on the development of smart vectors which could overcome multiple barriers. The nuclear envelope presents a major barrier to transgene delivery. Viruses are capable of crossing the nuclear envelope to efficiently deliver their genome into the nucleus through the specialized protein components. However, non-viral vectors are preferred over viral ones because of the safety concerns associated with the latter. Non-viral delivery systems have been designed to include various types of components to enable nuclear translocation at the periphery of the nucleus. This review summarizes the progress of research regarding nuclear transport mechanisms. “Smart” non-viral vectors that have been modified by peptides and other small molecules are able to facilitate the nuclear translocation and enhance the efficacy of gene expression. The resulting technology may also enhance delivery of other macromolecules to the nucleus. PMID:23964565

  6. Targeted disruption of the mouse Lipoma Preferred Partner gene

    SciTech Connect

    Vervenne, Hilke B.V.K.; Crombez, Koen R.M.O.; Delvaux, Els L.; Janssens, Veerle; Ven, Wim J.M. van de Petit, Marleen M.R.

    2009-02-06

    LPP (Lipoma Preferred Partner) is a zyxin-related cell adhesion protein that is involved in the regulation of cell migration. We generated mice with a targeted disruption of the Lpp gene and analysed the importance of Lpp for embryonic development and adult functions. Aberrant Mendelian inheritance in heterozygous crosses suggested partial embryonic lethality of Lpp{sup -/-} females. Fertility of Lpp{sup -/-} males was proven to be normal, however, females from Lpp{sup -/-} x Lpp{sup -/-} crosses produced a strongly reduced number of offspring, probably due to a combination of female embryonic lethality and aberrant pregnancies. Apart from these developmental and reproductive abnormalities, Lpp{sup -/-} mice that were born reached adulthood without displaying any additional macroscopic defects. On the other hand, Lpp{sup -/-} mouse embryonic fibroblasts exhibited reduced migration capacity, reduced viability, and reduced expression of some Lpp interaction partners. Finally, we discovered a short nuclear form of Lpp, expressed mainly in testis via an alternative promoter.

  7. In Vivo RNAi Screen Reveals Neddylation Genes as Novel Regulators of Hedgehog Signaling

    PubMed Central

    Su, Ying; Liu, Min; Ospina, Jason K.; Yang, Shengyuan; Zhu, Alan Jian

    2011-01-01

    Hedgehog (Hh) signaling is highly conserved in all metazoan animals and plays critical roles in many developmental processes. Dysregulation of the Hh signaling cascade has been implicated in many diseases, including cancer. Although key components of the Hh pathway have been identified, significant gaps remain in our understanding of the regulation of individual Hh signaling molecules. Here, we report the identification of novel regulators of the Hh pathway, obtained from an in vivo RNA interference (RNAi) screen in Drosophila. By selectively targeting critical genes functioning in post-translational modification systems utilizing ubiquitin (Ub) and Ub-like proteins, we identify two novel genes (dUba3 and dUbc12) that negatively regulate Hh signaling activity. We provide in vivo and in vitro evidence illustrating that dUba3 and dUbc12 are essential components of the neddylation pathway; they function in an enzyme cascade to conjugate the ubiquitin-like NEDD8 modifier to Cullin proteins. Neddylation activates the Cullin-containing ubiquitin ligase complex, which in turn promotes the degradation of Cubitus interruptus (Ci), the downstream transcription factor of the Hh pathway. Our study reveals a conserved molecular mechanism of the neddylation pathway in Drosophila and sheds light on the complex post-translational regulations in Hh signaling. PMID:21931660

  8. Comparative gene expression analysis of Dtg, a novel target gene of Dpp signaling pathway in the early Drosophila melanogaster embryo.

    PubMed

    Hodar, Christian; Zuñiga, Alejandro; Pulgar, Rodrigo; Travisany, Dante; Chacon, Carlos; Pino, Michael; Maass, Alejandro; Cambiazo, Verónica

    2014-02-10

    In the early Drosophila melanogaster embryo, Dpp, a secreted molecule that belongs to the TGF-β superfamily of growth factors, activates a set of downstream genes to subdivide the dorsal region into amnioserosa and dorsal epidermis. Here, we examined the expression pattern and transcriptional regulation of Dtg, a new target gene of Dpp signaling pathway that is required for proper amnioserosa differentiation. We showed that the expression of Dtg was controlled by Dpp and characterized a 524-bp enhancer that mediated expression in the dorsal midline, as well as, in the differentiated amnioserosa in transgenic reporter embryos. This enhancer contained a highly conserved region of 48-bp in which bioinformatic predictions and in vitro assays identified three Mad binding motifs. Mutational analysis revealed that these three motifs were necessary for proper expression of a reporter gene in transgenic embryos, suggesting that short and highly conserved genomic sequences may be indicative of functional regulatory regions in D. melanogaster genes. Dtg orthologs were not detected in basal lineages of Dipterans, which unlike D. melanogaster develop two extra-embryonic membranes, amnion and serosa, nevertheless Dtg orthologs were identified in the transcriptome of Musca domestica, in which dorsal ectoderm patterning leads to the formation of a single extra-embryonic membrane. These results suggest that Dtg was recruited as a new component of the network that controls dorsal ectoderm patterning in the lineage leading to higher Cyclorrhaphan flies, such as D. melanogaster and M. domestica. PMID:24321690

  9. Mechanisms of double-strand-break repair during gene targeting in mammalian cells.

    PubMed Central

    Ng, P; Baker, M D

    1999-01-01

    In the present study, the mechanism of double-strand-break (DSB) repair during gene targeting at the chromosomal immunoglobulin mu-locus in a murine hybridoma was examined. The gene-targeting assay utilized specially designed insertion vectors genetically marked in the region of homology to the chromosomal mu-locus by six diagnostic restriction enzyme site markers. The restriction enzyme markers permitted the contribution of vector-borne and chromosomal mu-sequences in the recombinant product to be determined. The use of the insertion vectors in conjunction with a plating procedure in which individual integrative homologous recombination events were retained for analysis revealed several important features about the mammalian DSB repair process:The presence of the markers within the region of shared homology did not affect the efficiency of gene targeting.In the majority of recombinants, the vector-borne marker proximal to the DSB was absent, being replaced with the corresponding chromosomal restriction enzyme site. This result is consistent with either formation and repair of a vector-borne gap or an "end" bias in mismatch repair of heteroduplex DNA (hDNA) that favored the chromosomal sequence. Formation of hDNA was frequently associated with gene targeting and, in most cases, began approximately 645 bp from the DSB and could encompass a distance of at least 1469 bp.The hDNA was efficiently repaired prior to DNA replication.The repair of adjacent mismatches in hDNA occurred predominantly on the same strand, suggesting the involvement of a long-patch repair mechanism. PMID:10049929

  10. A dual-targeting drug co-delivery system for tumor chemo- and gene combined therapy.

    PubMed

    Zhang, Fangrong; Li, Min; Su, Yujie; Zhou, Jianping; Wang, Wei

    2016-07-01

    Regulation of gene expression using p53 is a promising strategy for treatment of numerous cancers, and chemotherapeutic drug dichloroacetate (DCA) induces apoptosis and growth inhibition in tumor, without apparent toxicity in normal tissues. Combining DCA and p53 gene could be an effective way to treat tumors. The progress towards broad applications of DCA/p53 combination requires the development of safe and efficient vectors that target to specific cells. In this study, we developed a DSPE-PEG-AA (1,2-distearoryl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol-2000)] ammonium salt-anisamide) modified reconstituted high-density lipoprotein-based DCA/p53-loaded nanoparticles (DSPE-PEG-AA/rHDL/DCA-PEI/p53 complexes), which was fabricated as a drug/gene dual-targeting co-delivery system for potential cancer therapy. Here, DCA-PEI was utilized to effectively condense the p53 plasmid, to incorporate the plasmid into rHDL and to act as an antitumor drug to inhibit tumor cell growth. The DSPE-PEG-AA/rHDL/DCA-PEI/p53 complexes exhibited desirable and homogenous particle size, neutral surface charge and low cytotoxicity for normal cells in vitro. The results of confocal laser scanning microscopy (CLSM) and flow cytometry confirmed that the scavenger receptor class B type I (SR-BI) and sigma receptor mediated dual-targeting function of the complexes inducing efficient cytoplasmic drug delivery and gene transfection in human lung adenocarcinoma cell line A549. And in vivo investigation on nude mice bearing A549 tumor xenografts revealed that DSPE-PEG-AA/rHDL/DCA-PEI/p53 complexes possessed specific tumor targeting and strong antitumor activity. The work described here demonstrated that the DSPE-PEG-AA/rHDL/DCA-PEI/p53 complexes might offer a promising tool for effective cancer therapy. PMID:27127046

  11. Preparation and characterization of magnetic gene vectors for targeting gene delivery

    NASA Astrophysics Data System (ADS)

    Zheng, S. W.; Liu, G.; Hong, R. Y.; Li, H. Z.; Li, Y. G.; Wei, D. G.

    2012-10-01

    The PEI-CMD-MNPs were successfully prepared by the surface modification of magnetic Fe3O4 nanoparticles with carboxymethyl dextran (CMD) and polyethyleneimine (PEI). The PEI-CMD-MNPs polyplexes exhibited a typical superparamagnetic behavior and were well stable over the entire range of pH and NaCl concentration. These PEI-CMD-MNPs were used as magnetic gene vectors for targeting gene delivery. The prepared MNPs at different surface modification stages were characterized using Fourier transform infrared (FT-IR), thermogravimetric analysis (TGA), field emissions canning electron microscopy (FE-SEM), powder X-ray diffraction (XRD) and dynamic laser light scattering (DLS) analysis. The magnetic properties were studied by vibrating sample magnetometer (VSM). To evaluate the performance of the magnetic nanoparticles as gene transfer vector, the PEI-CMD-MNPs were used to delivery green fluorescent protein (GFP) gene into BHK21 cells. The expression of GFP gene was detected by fluorescence microscope. DNA-PEI-CMD-MNPs polyplexes absorbed by the cells were also monitored by Magnetic resonance imaging (MRI). The transfection efficiency and gene expression efficiency of that transfected with a magnet were much higher than that of standard transfection.

  12. Inherited cobalamin malabsorption. Mutations in three genes reveal functional and ethnic patterns

    PubMed Central

    2012-01-01

    Background Inherited malabsorption of cobalamin (Cbl) causes hematological and neurological abnormalities that can be fatal. Three genes have been implicated in Cbl malabsorption; yet, only about 10% of ~400-500 reported cases have been molecularly studied to date. Recessive mutations in CUBN or AMN cause Imerslund-Gräsbeck Syndrome (IGS), while recessive mutations in GIF cause Intrinsic Factor Deficiency (IFD). IGS and IFD differ in that IGS usually presents with proteinuria, which is not observed in IFD. The genetic heterogeneity and numerous differential diagnoses make clinical assessment difficult. Methods We present a large genetic screening study of 154 families or patients with suspected hereditary Cbl malabsorption. Patients and their families have been accrued over a period spanning >12 years. Systematic genetic testing of the three genes CUBN, AMN, and GIF was accomplished using a combination of single strand conformation polymorphism and DNA and RNA sequencing. In addition, six genes that were contenders for a role in inherited Cbl malabsorption were studied in a subset of these patients. Results Our results revealed population-specific mutations, mutational hotspots, and functionally distinct regions in the three causal genes. We identified mutations in 126/154 unrelated cases (82%). Fifty-three of 126 cases (42%) were mutated in CUBN, 45/126 (36%) were mutated in AMN, and 28/126 (22%) had mutations in GIF. We found 26 undescribed mutations in CUBN, 19 in AMN, and 7 in GIF for a total of 52 novel defects described herein. We excluded six other candidate genes as culprits and concluded that additional genes might be involved. Conclusions Cbl malabsorption is found worldwide and genetically complex. However, our results indicate that population-specific founder mutations are quite common. Consequently, targeted genetic testing has become feasible if ethnic ancestry is considered. These results will facilitate clinical and molecular genetic testing of

  13. Polyploid genome of Camelina sativa revealed by isolation of fatty acid synthesis genes

    PubMed Central

    2010-01-01

    Background Camelina sativa, an oilseed crop in the Brassicaceae family, has inspired renewed interest due to its potential for biofuels applications. Little is understood of the nature of the C. sativa genome, however. A study was undertaken to characterize two genes in the fatty acid biosynthesis pathway, fatty acid desaturase (FAD) 2 and fatty acid elongase (FAE) 1, which revealed unexpected complexity in the C. sativa genome. Results In C. sativa, Southern analysis indicates the presence of three copies of both FAD2 and FAE1 as well as LFY, a known single copy gene in other species. All three copies of both CsFAD2 and CsFAE1 are expressed in developing seeds, and sequence alignments show that previously described conserved sites are present, suggesting that all three copies of both genes could be functional. The regions downstream of CsFAD2 and upstream of CsFAE1 demonstrate co-linearity with the Arabidopsis genome. In addition, three expressed haplotypes were observed for six predicted single-copy genes in 454 sequencing analysis and results from flow cytometry indicate that the DNA content of C. sativa is approximately three-fold that of diploid Camelina relatives. Phylogenetic analyses further support a history of duplication and indicate that C. sativa and C. microcarpa might share a parental genome. Conclusions There is compelling evidence for triplication of the C. sativa genome, including a larger chromosome number and three-fold larger measured genome size than other Camelina relatives, three isolated copies of FAD2, FAE1, and the KCS17-FAE1 intergenic region, and three expressed haplotypes observed for six predicted single-copy genes. Based on these results, we propose that C. sativa be considered an allohexaploid. The characterization of fatty acid synthesis pathway genes will allow for the future manipulation of oil composition of this emerging biofuel crop; however, targeted manipulations of oil composition and general development of C. sativa should

  14. Deletion of the DNA Ligase IV Gene in Candida glabrata Significantly Increases Gene-Targeting Efficiency

    PubMed Central

    Cen, Yuke; Fiori, Alessandro

    2015-01-01

    Candida glabrata is reported as the second most prevalent human opportunistic fungal pathogen in the United States. Over the last decades, its incidence increased, whereas that of Candida albicans decreased slightly. One of the main reasons for this shift is attributed to the inherent tolerance of C. glabrata toward the commonly used azole antifungal drugs. Despite a close phylogenetic distance to Saccharomyces cerevisiae, homologous recombination works with poor efficiency in C. glabrata compared to baker's yeast, in fact limiting targeted genetic alterations of the pathogen's genome. It has been shown that nonhomologous DNA end joining is dominant over specific gene targeting in C. glabrata. To improve the homologous recombination efficiency, we have generated a strain in which the LIG4 gene has been deleted, which resulted in a significant increase in correct gene targeting. The very specific function of Lig4 in mediating nonhomologous end joining is the reason for the absence of clear side effects, some of which affect the ku80 mutant, another mutant with reduced nonhomologous end joining. We also generated a LIG4 reintegration cassette. Our results show that the lig4 mutant strain may be a valuable tool for the C. glabrata research community. PMID:26048009

  15. Dissection of the Process of Brain Metastasis Reveals Targets and Mechanisms for Molecular-based Intervention.

    PubMed

    Weidle, Ulrich H; Birzele, Fabian; Kollmorgen, Gwendlyn; Rüger, Rüdiger

    2016-01-01

    Brain metastases outnumber the incidence of brain tumors by a factor of ten. Patients with brain metastases have a dismal prognosis and current treatment modalities achieve only a modest clinical benefit. We discuss the process of brain metastasis with respect to mechanisms and involved targets to outline options for therapeutic intervention and focus on breast and lung cancer, as well as melanoma. We describe the process of penetration of the blood-brain-barrier (BBB) by disseminated tumor cells, establishment of a metastatic niche, colonization and outgrowth in the brain parenchyma. Furthermore, the role of angiogenesis in colonization of the brain parenchyma, interactions of extravasated tumor cells with microglia and astrocytes, as well as their propensity for neuromimicry, is discussed. We outline targets suitable for prevention of metastasis and summarize targets suitable for treatment of established brain metastases. Finally, we highlight the implications of findings revealing druggable mutations in brain metastases that cannot be identified in matching primary tumors. PMID:27365375

  16. Targeted Next-generation Sequencing Reveals Novel EYS Mutations in Chinese Families with Autosomal Recessive Retinitis Pigmentosa

    PubMed Central

    Chen, Xue; Liu, Xiaoxing; Sheng, Xunlun; Gao, Xiang; Zhang, Xiumei; Li, Zili; Li, Huiping; Liu, Yani; Rong, Weining; Zhao, Kanxing; Zhao, Chen

    2015-01-01

    EYS mutations demonstrate great genotypic and phenotypic varieties, and are one of the major causes for patients with autosomal recessive retinitis pigmentosa (ARRP). Here, we aim to determine the genetic lesions with phenotypic correlations in two Chinese families with ARRP. Medical histories and ophthalmic documentations were obtained from all participants from the two pedigrees. Targeted next-generation sequencing (NGS) on 189 genes was performed to screen for RP causative mutations in the two families. Two biallelic mutations in EYS, p.[R164*];[C2139Y] and p.[W2640*];[F2954S], were identified in the two families, respectively. EYS p.R164* and p.F2954S are novel alleles associated with RP, while p.C2139Y and p.W2640* are known mutations. Crystal structure modeling on the protein eyes shut homolog encoded by the EYS gene revealed abnormal hydrogen bonds generated by p.C2139Y and p.F2954S, which would likely affect the solubility and cause significant structural changes of the two mutated proteins. In conclusion, our study expands the genotypic spectrums for EYS mutations, and may provide novel insights into the relevant pathogenesis for RP. We also demonstrate targeted NGS approach as a valuable tool for genetic diagnosis. PMID:25753737

  17. Efficient Gene Transfer and Targeted Mutagenesis in Fusobacterium nucleatum

    PubMed Central

    Haake, Susan Kinder; Yoder, Sean; Gerardo, Sharon Hunt

    2006-01-01

    Fusobacterium nucleatum is a Gram-negative anaerobe important in dental biofilm ecology and infectious diseases with significant societal impact. The lack of efficient genetic systems has hampered molecular analyses in this microorganism. We previously reported construction of a shuttle plasmid, pHS17, using the native fusobacterial plasmid pFN1 and an erythromycin resistance cassette. However, the host range of pHS17 was restricted to F. nucleatum, ATCC 10953 and the transformation efficiency was limited. This study was undertaken to improve genetic systems for molecular analysis in F. nucleatum. We identified a second F. nucleatum strain, ATCC 23726, which is transformed with improved efficiency compared to ATCC 10953. Two novel second generation pFN1-based shuttle plasmids, pHS23 and pHS30, were developed and enable transformation of ATCC 23726 at 6.2 x 104 and 1.5 x 106 transformants/microgram of plasmid DNA, respectively. The transformation efficiency of pHS30, which harbors a catP gene conferring resistance to chloramphenicol, was more than 1,000-fold greater than that of pHS17. The improved transformation efficiency facilitated disruption of the chromosomal rnr gene using a suicide plasmid pHS19, the first demonstration of targeted mutagenesis in F. nucleatum. These results provide significant advances in the development of systems for molecular analysis in F. nucleatum. PMID:16115683

  18. Survey of activated kinase proteins reveals potential targets for cholangiocarcinoma treatment.

    PubMed

    Dokduang, Hasaya; Juntana, Sirinun; Techasen, Anchalee; Namwat, Nisana; Yongvanit, Puangrat; Khuntikeo, Narong; Riggins, Gregory J; Loilome, Watcharin

    2013-12-01

    Improving therapy for patients with cholangiocarcinoma (CCA) presents a significant challenge. This is made more difficult by a lack of a clear understanding of potential molecular targets, such as deregulated kinases. In this work, we profiled the activated kinases in CCA in order to apply them as the targets for CCA therapy. Human phospho-receptor tyrosine kinases (RTKs) and phospho-kinase array analyses revealed that multiple kinases are activated in both CCA cell lines and human CCA tissues that included cell growth, apoptosis, cell to cell interaction, movement, and angiogenesis RTKs. Predominately, the kinases activated downstream were those in the PI3K/Akt, Ras/MAPK, JAK/STAT, and Wnt/β-catenin signaling pathways. Western blot analysis confirms that Erk1/2 and Akt activation were increased in CCA tissues when compared with their normal adjacent tissue. The inhibition of kinase activation using multi-targeted kinase inhibitors, sorafenib and sunitinib led to significant cell growth inhibition and apoptosis induction via suppression of Erk1/2 and Akt activation, whereas drugs with specificity to a single kinase showed less potency. In conclusion, our study reveals the involvement of multiple kinase proteins in CCA growth that might serve as therapeutic targets for combined kinase inhibition. PMID:23812726

  19. A modular gene targeting system for sequential transgene stacking in plants.

    PubMed

    Kumar, Sandeep; AlAbed, Diaa; Worden, Andrew; Novak, Stephen; Wu, Huixia; Ausmus, Carla; Beck, Margaret; Robinson, Heather; Minnicks, Tatyana; Hemingway, Daren; Lee, Ryan; Skaggs, Nicole; Wang, Lizhen; Marri, Pradeep; Gupta, Manju

    2015-08-10

    A modular, selection-based method was developed for site-specific integration of transgenes into a genomic locus to create multigene stacks. High-frequency gene targeting was obtained using zinc finger nuclease (ZFN)-mediated double-strand break (DSB) formation at a pre-defined target genomic location using a unique intron directly downstream of a promoter driving a selectable marker gene to facilitate homology between target and donor sequences. In this system, only insertion into the target locus leads to a functional selectable marker, and regeneration from random insertions of the promoterless donor construct are reduced on selection media. A new stack of transgenes can potentially be loaded with each successive cycle of gene targeting by exchanging the selectable marker gene using the intron homology. This system was tested in maize using the pat selectable marker gene, whereby up to 30% of the plants regenerated on Bialaphos-containing medium were observed to have the donor construct integrated into the target locus. Unlike previous gene targeting methods that utilize defective or partial genes for selecting targeted events, the present method exchanges fully functional genes with every cycle of targeting, thereby allowing the recycling of selectable marker genes, hypothetically for multiple generations of gene targeting. PMID:25913173

  20. Targeted gene delivery to human airway epithelial cells with synthetic vectors incorporating novel targeting peptides selected by phage display.

    PubMed

    Writer, Michele J; Marshall, Barry; Pilkington-Miksa, Michael A; Barker, Susie E; Jacobsen, Marianne; Kritz, Angelika; Bell, Paul C; Lester, Douglas H; Tabor, Alethea B; Hailes, Helen C; Klein, Nigel; Hart, Stephen L

    2004-05-01

    Human airway epithelial cell targeting peptides were identified by biopanning on 1HAEo-cells, a well characterised epithelial cell line. Bound phage were recovered after three rounds of binding, high stringency washing and elution, leading to the production of an enriched phage peptide population. DNA sequencing of 56 clones revealed 14 unique sequences. Subsequent binding analysis revealed that 13 of these peptides bound 1HAEo-cells with high affinity. Three peptides, SERSMNF, YGLPHKF and PSGAARA were represented at high frequency. Three clearly defined families of peptide were identified on the basis of sequence motifs including (R/K)SM, L(P/Q)HK and PSG(A/T)ARA. Two peptides, LPHKSMP and LQHKSMP contained two motifs. Further detailed sequence analysis by comparison of peptide sequences with the SWISSPROT protein database revealed that some of the peptides closely resembled the cell binding proteins of viral and bacterial pathogens including Herpes Simplex Virus, rotavirus, Mycoplasma pneumoniae and rhinovirus, the latter two being respiratory pathogens, as well as peptide YGLPHKF having similarity to a protein of unknown function from the respiratory pathogen Legionella pneumophila. Peptides were incorporated into gene delivery formulations with the cationic lipid Lipofectin and plasmid DNA and shown to confer a high degree of transfection efficiency and specificity in 1HAEo-cells. Improved transfection efficiency and specificity was also observed in human endothelial cells, fibroblasts and keratinocytes. Therefore, on the basis of clone frequency after biopanning, cell binding affinity, peptide sequence conservation and pathogenic similarity, we have identified 3 novel peptide families and 5 specific peptides that have the potential for gene transfer to respiratory epithelium in vivo as well as providing useful in vitro transfection reagents for primary human cell types of scientific and commercial interest. PMID:15506167

  1. Identification of Novel Cellular Targets in Biliary Tract Cancers Using Global Gene Expression Technology

    PubMed Central

    Hansel, Donna E.; Rahman, Ayman; Hidalgo, Manuel; Thuluvath, Paul J.; Lillemoe, Keith D.; Shulick, Richard; Ku, Ja-Lok; Park, Jae-Gahb; Miyazaki, Kohje; Ashfaq, Raheela; Wistuba, Ignacio I.; Varma, Ram; Hawthorne, Lesleyann; Geradts, Joseph; Argani, Pedram; Maitra, Anirban

    2003-01-01

    Biliary tract carcinoma carries a poor prognosis, and difficulties with clinical management in patients with advanced disease are often due to frequent late-stage diagnosis, lack of serum markers, and limited information regarding biliary tumor pathogenesis. RNA-based global analyses of gene expression have led to the identification of a large number of up-regulated genes in several cancer types. We have used the recently developed Affymetrix U133A gene expression microarrays containing nearly 22,000 unique transcripts to obtain global gene expression profiles from normal biliary epithelial scrapings (n = 5), surgically resected biliary carcinomas (n = 11), and biliary cancer cell lines (n = 9). Microarray hybridization data were normalized using dCHIP (http://www.dCHIP.org) to identify differentially up-regulated genes in primary biliary cancers and biliary cancer cell lines and their expression profiles was compared to that of normal epithelial scrapings using the dCHIP software as well as Significance Analysis of Microarrays or SAM (http://www-stat.stanford.edu/∼tibs/SAM/). Comparison of the dCHIP and SAM datasets revealed an overlapping list of 282 genes expressed at greater than threefold levels in the cancers compared to normal epithelium (t-test P <0.1 in dCHIP, and median false discovery rate <10 in SAM). Several pathways integral to tumorigenesis were up-regulated in the biliary cancers, including proliferation and cell cycle antigens (eg, cyclins D2 and E2, cdc2/p34, and geminin), transcription factors (eg, homeobox B7 and islet-1), growth factors and growth factor receptors (eg, hepatocyte growth factor, amphiregulin, and insulin-like growth factor 1 receptor), and enzymes modulating sensitivity to chemotherapeutic agents (eg, cystathionine β synthase, dCMP deaminase, and CTP synthase). In addition, we identified several “pathway” genes that are rapidly emerging as novel therapeutic targets in cancer (eg, cytosolic phospholipase A2, an upstream

  2. Identification of microRNA-regulated gene networks by expression analysis of target genes.

    PubMed

    Gennarino, Vincenzo Alessandro; D'Angelo, Giovanni; Dharmalingam, Gopuraja; Fernandez, Serena; Russolillo, Giorgio; Sanges, Remo; Mutarelli, Margherita; Belcastro, Vincenzo; Ballabio, Andrea; Verde, Pasquale; Sardiello, Marco; Banfi, Sandro

    2012-06-01

    MicroRNAs (miRNAs) and transcription factors control eukaryotic cell proliferation, differentiation, and metabolism through their specific gene regulatory networks. However, differently from transcription factors, our understanding of the processes regulated by miRNAs is currently limited. Here, we introduce gene network analysis as a new means for gaining insight into miRNA biology. A systematic analysis of all human miRNAs based on Co-expression Meta-analysis of miRNA Targets (CoMeTa) assigns high-resolution biological functions to miRNAs and provides a comprehensive, genome-scale analysis of human miRNA regulatory networks. Moreover, gene cotargeting analyses show that miRNAs synergistically regulate cohorts of genes that participate in similar processes. We experimentally validate the CoMeTa procedure through focusing on three poorly characterized miRNAs, miR-519d/190/340, which CoMeTa predicts to be associated with the TGFβ pathway. Using lung adenocarcinoma A549 cells as a model system, we show that miR-519d and miR-190 inhibit, while miR-340 enhances TGFβ signaling and its effects on cell proliferation, morphology, and scattering. Based on these findings, we formalize and propose co-expression analysis as a general paradigm for second-generation procedures to recognize bona fide targets and infer biological roles and network communities of miRNAs. PMID:22345618

  3. Identification of microRNA-regulated gene networks by expression analysis of target genes

    PubMed Central

    Gennarino, Vincenzo Alessandro; D'Angelo, Giovanni; Dharmalingam, Gopuraja; Fernandez, Serena; Russolillo, Giorgio; Sanges, Remo; Mutarelli, Margherita; Belcastro, Vincenzo; Ballabio, Andrea; Verde, Pasquale; Sardiello, Marco; Banfi, Sandro

    2012-01-01

    MicroRNAs (miRNAs) and transcription factors control eukaryotic cell proliferation, differentiation, and metabolism through their specific gene regulatory networks. However, differently from transcription factors, our understanding of the processes regulated by miRNAs is currently limited. Here, we introduce gene network analysis as a new means for gaining insight into miRNA biology. A systematic analysis of all human miRNAs based on Co-expression Meta-analysis of miRNA Targets (CoMeTa) assigns high-resolution biological functions to miRNAs and provides a comprehensive, genome-scale analysis of human miRNA regulatory networks. Moreover, gene cotargeting analyses show that miRNAs synergistically regulate cohorts of genes that participate in similar processes. We experimentally validate the CoMeTa procedure through focusing on three poorly characterized miRNAs, miR-519d/190/340, which CoMeTa predicts to be associated with the TGFβ pathway. Using lung adenocarcinoma A549 cells as a model system, we show that miR-519d and miR-190 inhibit, while miR-340 enhances TGFβ signaling and its effects on cell proliferation, morphology, and scattering. Based on these findings, we formalize and propose co-expression analysis as a general paradigm for second-generation procedures to recognize bona fide targets and infer biological roles and network communities of miRNAs. PMID:22345618

  4. Comparative Plasmodium gene overexpression reveals distinct perturbation of sporozoite transmission by profilin.

    PubMed

    Sato, Yuko; Hliscs, Marion; Dunst, Josefine; Goosmann, Christian; Brinkmann, Volker; Montagna, Georgina N; Matuschewski, Kai

    2016-07-15

    Plasmodium relies on actin-based motility to migrate from the site of infection and invade target cells. Using a substrate-dependent gliding locomotion, sporozoites are able to move at fast speed (1-3 μm/s). This motility relies on a minimal set of actin regulatory proteins and occurs in the absence of detectable filamentous actin (F-actin). Here we report an overexpression strategy to investigate whether perturbations of F-actin steady-state levels affect gliding locomotion and host invasion. We selected two vital Plasmodium berghei G-actin-binding proteins, C-CAP and profilin, in combination with three stage-specific promoters and mapped the phenotypes afforded by overexpression in all three extracellular motile stages. We show that in merozoites and ookinetes, additional expression does not impair life cycle progression. In marked contrast, overexpression of C-CAP and profilin in sporozoites impairs circular gliding motility and salivary gland invasion. The propensity for productive motility correlates with actin accumulation at the parasite tip, as revealed by combinations of an actin-stabilizing drug and transgenic parasites. Strong expression of profilin, but not C-CAP, resulted in complete life cycle arrest. Comparative overexpression is an alternative experimental genetic strategy to study essential genes and reveals effects of regulatory imbalances that are not uncovered from deletion-mutant phenotyping. PMID:27226484

  5. Comprehensive reanalysis of transcription factor knockout expression data in Saccharomyces cerevisiae reveals many new targets.

    PubMed

    Reimand, Jüri; Vaquerizas, Juan M; Todd, Annabel E; Vilo, Jaak; Luscombe, Nicholas M

    2010-08-01

    Transcription factor (TF) perturbation experiments give valuable insights into gene regulation. Genome-scale evidence from microarray measurements may be used to identify regulatory interactions between TFs and targets. Recently, Hu and colleagues published a comprehensive study covering 269 TF knockout mutants for the yeast Saccharomyces cerevisiae. However, the information that can be extracted from this valuable dataset is limited by the method employed to process the microarray data. Here, we present a reanalysis of the original data using improved statistical techniques freely available from the BioConductor project. We identify over 100,000 differentially expressed genes-nine times the total reported by Hu et al. We validate the biological significance of these genes by assessing their functions, the occurrence of upstream TF-binding sites, and the prevalence of protein-protein interactions. The reanalysed dataset outperforms the original across all measures, indicating that we have uncovered a vastly expanded list of relevant targets. In summary, this work presents a high-quality reanalysis that maximizes the information contained in the Hu et al. compendium. The dataset is available from ArrayExpress (accession: E-MTAB-109) and it will be invaluable to any scientist interested in the yeast transcriptional regulatory system. PMID:20385592

  6. Id-1 gene and gene products as therapeutic targets for treatment of breast cancer and other types of carcinoma

    SciTech Connect

    Desprez, Pierre-Yves; Campisi, Judith

    2014-08-19

    A method for treatment of breast cancer and other types of cancer. The method comprises targeting and modulating Id-1 gene expression, if any, for the Id-1 gene, or gene products in breast or other epithelial cancers in a patient by delivering products that modulate Id-1 gene expression. When expressed, Id-1 gene is a prognostic indicator that cancer cells are invasive and metastatic.

  7. Gene invasion in distant eukaryotic lineages: discovery of mutually exclusive genetic elements reveals marine biodiversity.

    PubMed

    Monier, Adam; Sudek, Sebastian; Fast, Naomi M; Worden, Alexandra Z

    2013-09-01

    Inteins are rare, translated genetic parasites mainly found in bacteria and archaea, while spliceosomal introns are distinctly eukaryotic features abundant in most nuclear genomes. Using targeted metagenomics, we discovered an intein in an Atlantic population of the photosynthetic eukaryote, Bathycoccus, harbored by the essential spliceosomal protein PRP8 (processing factor 8 protein). Although previously thought exclusive to fungi, we also identified PRP8 inteins in parasitic (Capsaspora) and predatory (Salpingoeca) protists. Most new PRP8 inteins were at novel insertion sites that, surprisingly, were not in the most conserved regions of the gene. Evolutionarily, Dikarya fungal inteins at PRP8 insertion site a appeared more related to the Bathycoccus intein at a unique insertion site, than to other fungal and opisthokont inteins. Strikingly, independent analyses of Pacific and Atlantic samples revealed an intron at the same codon as the Bathycoccus PRP8 intein. The two elements are mutually exclusive and neither was found in cultured Bathycoccus or other picoprasinophyte genomes. Thus, wild Bathycoccus contain one of few non-fungal eukaryotic inteins known and a rare polymorphic intron. Our data indicate at least two Bathycoccus ecotypes exist, associated respectively with oceanic or mesotrophic environments. We hypothesize that intein propagation is facilitated by marine viruses; and, while intron gain is still poorly understood, presence of a spliceosomal intron where a locus lacks an intein raises the possibility of new, intein-primed mechanisms for intron gain. The discovery of nucleus-encoded inteins and associated sequence polymorphisms in uncultivated marine eukaryotes highlights their diversity and reveals potential sexual boundaries between populations indistinguishable by common marker genes. PMID:23635865

  8. Gene invasion in distant eukaryotic lineages: discovery of mutually exclusive genetic elements reveals marine biodiversity

    PubMed Central

    Monier, Adam; Sudek, Sebastian; Fast, Naomi M; Worden, Alexandra Z

    2013-01-01

    Inteins are rare, translated genetic parasites mainly found in bacteria and archaea, while spliceosomal introns are distinctly eukaryotic features abundant in most nuclear genomes. Using targeted metagenomics, we discovered an intein in an Atlantic population of the photosynthetic eukaryote, Bathycoccus, harbored by the essential spliceosomal protein PRP8 (processing factor 8 protein). Although previously thought exclusive to fungi, we also identified PRP8 inteins in parasitic (Capsaspora) and predatory (Salpingoeca) protists. Most new PRP8 inteins were at novel insertion sites that, surprisingly, were not in the most conserved regions of the gene. Evolutionarily, Dikarya fungal inteins at PRP8 insertion site a appeared more related to the Bathycoccus intein at a unique insertion site, than to other fungal and opisthokont inteins. Strikingly, independent analyses of Pacific and Atlantic samples revealed an intron at the same codon as the Bathycoccus PRP8 intein. The two elements are mutually exclusive and neither was found in cultured Bathycoccus or other picoprasinophyte genomes. Thus, wild Bathycoccus contain one of few non-fungal eukaryotic inteins known and a rare polymorphic intron. Our data indicate at least two Bathycoccus ecotypes exist, associated respectively with oceanic or mesotrophic environments. We hypothesize that intein propagation is facilitated by marine viruses; and, while intron gain is still poorly understood, presence of a spliceosomal intron where a locus lacks an intein raises the possibility of new, intein-primed mechanisms for intron gain. The discovery of nucleus-encoded inteins and associated sequence polymorphisms in uncultivated marine eukaryotes highlights their diversity and reveals potential sexual boundaries between populations indistinguishable by common marker genes. PMID:23635865

  9. Comparative genomics reveals tissue-specific regulation of prolactin receptor gene expression.

    PubMed

    Schennink, Anke; Trott, Josephine F; Manjarin, Rodrigo; Lemay, Danielle G; Freking, Bradley A; Hovey, Russell C

    2015-02-01

    Prolactin (PRL), acting via the PRL receptor (PRLR), controls hundreds of biological processes across a range of species. Endocrine PRL elicits well-documented effects on target tissues such as the mammary glands and reproductive organs in addition to coordinating whole-body homeostasis during states such as lactation or adaptive responses to the environment. While changes in PRLR expression likely facilitates these tissue-specific responses to circulating PRL, the mechanisms regulating this regulation in non-rodent species has received limited attention. We performed a wide-scale analysis of PRLR 5' transcriptional regulation in pig tissues. Apart from the abundantly expressed and widely conserved exon 1, we identified alternative splicing of transcripts from an additional nine first exons of the porcine PRLR (pPRLR) gene. Notably, exon 1.5 transcripts were expressed most abundantly in the heart, while expression of exon 1.3-containing transcripts was greatest in the kidneys and small intestine. Expression of exon 1.3 mRNAs within the kidneys was most abundant in the renal cortex, and increased during gestation. A comparative analysis revealed a human homologue to exon 1.3, hE1N2, which was also principally transcribed in the kidneys and small intestines, and an exon hE1N3 was only expressed in the kidneys of humans. Promoter alignment revealed conserved motifs within the proximal promoter upstream of exon 1.3, including putative binding sites for hepatocyte nuclear factor-1 and Sp1. Together, these results highlight the diverse, conserved and tissue-specific regulation of PRLR expression in the targets for PRL, which may function to coordinate complex physiological states such as lactation and osmoregulation. PMID:25358647

  10. DNA capture reveals transoceanic gene flow in endangered river sharks

    PubMed Central

    Li, Chenhong; Corrigan, Shannon; Yang, Lei; Straube, Nicolas; Harris, Mark; Hofreiter, Michael; White, William T.; Naylor, Gavin J. P.

    2015-01-01

    For over a hundred years, the “river sharks” of the genus Glyphis were only known from the type specimens of species that had been collected in the 19th century. They were widely considered extinct until populations of Glyphis-like sharks were rediscovered in remote regions of Borneo and Northern Australia at the end of the 20th century. However, the genetic affinities between the newly discovered Glyphis-like populations and the poorly preserved, original museum-type specimens have never been established. Here, we present the first (to our knowledge) fully resolved, complete phylogeny of Glyphis that includes both archival-type specimens and modern material. We used a sensitive DNA hybridization capture method to obtain complete mitochondrial genomes from all of our samples and show that three of the five described river shark species are probably conspecific and widely distributed in Southeast Asia. Furthermore we show that there has been recent gene flow between locations that are separated by large oceanic expanses. Our data strongly suggest marine dispersal in these species, overturning the widely held notion that river sharks are restricted to freshwater. It seems that species in the genus Glyphis are euryhaline with an ecology similar to the bull shark, in which adult individuals live in the ocean while the young grow up in river habitats with reduced predation pressure. Finally, we discovered a previously unidentified species within the genus Glyphis that is deeply divergent from all other lineages, underscoring the current lack of knowledge about the biodiversity and ecology of these mysterious sharks. PMID:26460025

  11. DNA capture reveals transoceanic gene flow in endangered river sharks.

    PubMed

    Li, Chenhong; Corrigan, Shannon; Yang, Lei; Straube, Nicolas; Harris, Mark; Hofreiter, Michael; White, William T; Naylor, Gavin J P

    2015-10-27

    For over a hundred years, the "river sharks" of the genus Glyphis were only known from the type specimens of species that had been collected in the 19th century. They were widely considered extinct until populations of Glyphis-like sharks were rediscovered in remote regions of Borneo and Northern Australia at the end of the 20th century. However, the genetic affinities between the newly discovered Glyphis-like populations and the poorly preserved, original museum-type specimens have never been established. Here, we present the first (to our knowledge) fully resolved, complete phylogeny of Glyphis that includes both archival-type specimens and modern material. We used a sensitive DNA hybridization capture method to obtain complete mitochondrial genomes from all of our samples and show that three of the five described river shark species are probably conspecific and widely distributed in Southeast Asia. Furthermore we show that there has been recent gene flow between locations that are separated by large oceanic expanses. Our data strongly suggest marine dispersal in these species, overturning the widely held notion that river sharks are restricted to freshwater. It seems that species in the genus Glyphis are euryhaline with an ecology similar to the bull shark, in which adult individuals live in the ocean while the young grow up in river habitats with reduced predation pressure. Finally, we discovered a previously unidentified species within the genus Glyphis that is deeply divergent from all other lineages, underscoring the current lack of knowledge about the biodiversity and ecology of these mysterious sharks. PMID:26460025

  12. PDX1 regulation of FABP1 and novel target genes in human intestinal epithelial Caco-2 cells

    PubMed Central

    Chen, Chin; Fang, Rixun; Chou, Lin-Chiang; Lowe, Anson W.; Sibley, Eric

    2012-01-01

    The transcription factor pancreatic and duodenal homeobox 1 (PDX1) plays an essential role in pancreatic development and in maintaining proper islet function via target gene regulation. Few intestinal PDX1 targets, however, have been described. We sought to define novel PDX1-regulated intestinal genes. Caco-2 human intestinal epithelial cells were engineered to overexpress PDX1 and gene expression profiles relative to control cells were assessed. Expression of 80 genes significantly increased while that of 49 genes significantly decreased more than 4-fold following PDX1 overexpression in differentiated Caco-2 cells. Analysis of the differentially regulated genes with known functional annotations revealed genes encoding transcription factors, growth factors, kinases, digestive glycosidases, nutrient transporters, nutrient binding proteins, and structural components. The gene for fatty acid binding protein 1, liver, FABP1, is repressed by PDX1 in Caco-2 cells. PDX1 overexpression in Caco-2 cells also results in repression of promoter activity driven by the 0.6 kb FABP1 promoter. PDX1 regulation of promoter activity is consistent with the decrease in FABP1 RNA abundance resulting from PDX1 overexpression and identifies FABP1 as a candidate PDX1 target. PDX1 repression of FABP1, LCT, and SI suggests a role for PDX1 in patterning anterior intestinal development. PMID:22640736

  13. Genome-wide analysis of Musashi-2 targets reveals novel functions in governing epithelial cell migration

    PubMed Central

    Bennett, Christopher G.; Riemondy, Kent; Chapnick, Douglas A.; Bunker, Eric; Liu, Xuedong; Kuersten, Scott; Yi, Rui

    2016-01-01

    The Musashi-2 (Msi2) RNA-binding protein maintains stem cell self-renewal and promotes oncogenesis by enhancing cell proliferation in hematopoietic and gastrointestinal tissues. However, it is unclear how Msi2 recognizes and regulates mRNA targets in vivo and whether Msi2 primarily controls cell growth in all cell types. Here we identified Msi2 targets with HITS-CLIP and revealed that Msi2 primarily recognizes mRNA 3′UTRs at sites enriched in multiple copies of UAG motifs in epithelial progenitor cells. RNA-seq and ribosome profiling demonstrated that Msi2 promotes targeted mRNA decay without affecting translation efficiency. Unexpectedly, the most prominent Msi2 targets identified are key regulators that govern cell motility with a high enrichment in focal adhesion and extracellular matrix-receptor interaction, in addition to regulators of cell growth and survival. Loss of Msi2 stimulates epithelial cell migration, increases the number of focal adhesions and also compromises cell growth. These findings provide new insights into the molecular mechanisms of Msi2's recognition and repression of targets and uncover a key function of Msi2 in restricting epithelial cell migration. PMID:27034466

  14. Transcriptome analysis reveals genes commonly induced by Botrytis cinerea infection, cold, drought and oxidative stresses in Arabidopsis.

    PubMed

    Sham, Arjun; Al-Azzawi, Ahmed; Al-Ameri, Salma; Al-Mahmoud, Bassam; Awwad, Falah; Al-Rawashdeh, Ahmed; Iratni, Rabah; AbuQamar, Synan

    2014-01-01

    Signaling pathways controlling biotic and abiotic stress responses may interact synergistically or antagonistically. To identify the similarities and differences among responses to diverse stresses, we analyzed previously published microarray data on the transcriptomic responses of Arabidopsis to infection with Botrytis cinerea (a biotic stress), and to cold, drought, and oxidative stresses (abiotic stresses). Our analyses showed that at early stages after B. cinerea inoculation, 1498 genes were up-regulated (B. cinerea up-regulated genes; BUGs) and 1138 genes were down-regulated (B. cinerea down-regulated genes; BDGs). We showed a unique program of gene expression was activated in response each biotic and abiotic stress, but that some genes were similarly induced or repressed by all of the tested stresses. Of the identified BUGs, 25%, 6% and 12% were also induced by cold, drought and oxidative stress, respectively; whereas 33%, 7% and 5.5% of the BDGs were also down-regulated by the same abiotic stresses. Coexpression and protein-protein interaction network analyses revealed a dynamic range in the expression levels of genes encoding regulatory proteins. Analysis of gene expression in response to electrophilic oxylipins suggested that these compounds are involved in mediating responses to B. cinerea infection and abiotic stress through TGA transcription factors. Our results suggest an overlap among genes involved in the responses to biotic and abiotic stresses in Arabidopsis. Changes in the transcript levels of genes encoding components of the cyclopentenone signaling pathway in response to biotic and abiotic stresses suggest that the oxylipin signal transduction pathway plays a role in plant defense. Identifying genes that are commonly expressed in response to environmental stresses, and further analyzing the functions of their encoded products, will increase our understanding of the plant stress response. This information could identify targets for genetic

  15. Transcriptome Analysis Reveals Genes Commonly Induced by Botrytis cinerea Infection, Cold, Drought and Oxidative Stresses in Arabidopsis

    PubMed Central

    Al-Ameri, Salma; Al-Mahmoud, Bassam; Awwad, Falah; Al-Rawashdeh, Ahmed; Iratni, Rabah; AbuQamar, Synan

    2014-01-01

    Signaling pathways controlling biotic and abiotic stress responses may interact synergistically or antagonistically. To identify the similarities and differences among responses to diverse stresses, we analyzed previously published microarray data on the transcriptomic responses of Arabidopsis to infection with Botrytis cinerea (a biotic stress), and to cold, drought, and oxidative stresses (abiotic stresses). Our analyses showed that at early stages after B. cinerea inoculation, 1498 genes were up-regulated (B. cinerea up-regulated genes; BUGs) and 1138 genes were down-regulated (B. cinerea down-regulated genes; BDGs). We showed a unique program of gene expression was activated in response each biotic and abiotic stress, but that some genes were similarly induced or repressed by all of the tested stresses. Of the identified BUGs, 25%, 6% and 12% were also induced by cold, drought and oxidative stress, respectively; whereas 33%, 7% and 5.5% of the BDGs were also down-regulated by the same abiotic stresses. Coexpression and protein-protein interaction network analyses revealed a dynamic range in the expression levels of genes encoding regulatory proteins. Analysis of gene expression in response to electrophilic oxylipins suggested that these compounds are involved in mediating responses to B. cinerea infection and abiotic stress through TGA transcription factors. Our results suggest an overlap among genes involved in the responses to biotic and abiotic stresses in Arabidopsis. Changes in the transcript levels of genes encoding components of the cyclopentenone signaling pathway in response to biotic and abiotic stresses suggest that the oxylipin signal transduction pathway plays a role in plant defense. Identifying genes that are commonly expressed in response to environmental stresses, and further analyzing the functions of their encoded products, will increase our understanding of the plant stress response. This information could identify targets for genetic

  16. Estrogen receptor α can selectively repress dioxin receptor-mediated gene expression by targeting DNA methylation.

    PubMed

    Marques, Maud; Laflamme, Liette; Gaudreau, Luc

    2013-09-01

    Selective inhibitory crosstalk has been known to occur within the signaling pathways of the dioxin (AhR) and estrogen (ERα) receptors. More specifically, ERα represses a cytochrome P450-encoding gene (CYP1A1) that converts cellular estradiol into a metabolite that inhibits the cell cycle, while it has no effect on a P450-encoding gene (CYP1B1) that converts estrodiol into a genotoxic product. Here we show that ERα represses CYP1A1 by targeting the Dnmt3B DNA methyltransferase and concomitant DNA methylation of the promoter. We also find that histone H2A.Z can positively contribute to CYP1A1 gene expression, and its presence at that gene is inversely correlated with DNA methylation. Taken together, our results provide a framework for how ERα can repress transcription, and how that impinges on the production of an enzyme that generates genotoxic estradiol metabolites, and potential breast cancer progression. Finally, our results reveal a new mechanism for how H2A.Z can positively influence gene expression, which is by potentially competing with DNA methylation events in breast cancer cells. PMID:23828038

  17. Estrogen receptor α can selectively repress dioxin receptor-mediated gene expression by targeting DNA methylation

    PubMed Central

    Marques, Maud; Laflamme, Liette; Gaudreau, Luc

    2013-01-01

    Selective inhibitory crosstalk has been known to occur within the signaling pathways of the dioxin (AhR) and estrogen (ERα) receptors. More specifically, ERα represses a cytochrome P450-encoding gene (CYP1A1) that converts cellular estradiol into a metabolite that inhibits the cell cycle, while it has no effect on a P450-encoding gene (CYP1B1) that converts estrodiol into a genotoxic product. Here we show that ERα represses CYP1A1 by targeting the Dnmt3B DNA methyltransferase and concomitant DNA methylation of the promoter. We also find that histone H2A.Z can positively contribute to CYP1A1 gene expression, and its presence at that gene is inversely correlated with DNA methylation. Taken together, our results provide a framework for how ERα can repress transcription, and how that impinges on the production of an enzyme that generates genotoxic estradiol metabolites, and potential breast cancer progression. Finally, our results reveal a new mechanism for how H2A.Z can positively influence gene expression, which is by potentially competing with DNA methylation events in breast cancer cells. PMID:23828038

  18. Comparison of gene activation by two TAL effectors from Xanthomonas axonopodis pv. manihotis reveals candidate host susceptibility genes in cassava.

    PubMed

    Cohn, Megan; Morbitzer, Robert; Lahaye, Thomas; Staskawicz, Brian J

    2016-08-01

    Xanthomonas axonopodis pv. manihotis (Xam) employs transcription activator-like (TAL) effectors to promote bacterial growth and symptom formation during infection of cassava. TAL effectors are secreted via the bacterial type III secretion system into plant cells, where they are directed to the nucleus, bind DNA in plant promoters and activate the expression of downstream genes. The DNA-binding activity of TAL effectors is carried out by a central domain which contains a series of repeat variable diresidues (RVDs) that dictate the sequence of bound nucleotides. TAL14Xam668 promotes virulence in Xam strain Xam668 and has been shown to activate multiple cassava genes. In this study, we used RNA sequencing to identify the full target repertoire of TAL14Xam668 in cassava, which includes over 50 genes. A subset of highly up-regulated genes was tested for activation by TAL14CIO151 from Xam strain CIO151. Although TAL14CIO151 and TAL14Xam668 differ by only a single RVD, they display differential activation of gene targets. TAL14CIO151 complements the TAL14Xam668 mutant defect, implying that shared target genes are important for TAL14Xam668 -mediated disease susceptibility. Complementation with closely related TAL effectors is a novel approach to the narrowing down of biologically relevant susceptibility genes of TAL effectors with multiple targets. This study provides an example of how TAL effector target activation by two strains within a single species of Xanthomonas can be dramatically affected by a small change in RVD-nucleotide affinity at a single site, and reflects the parameters of RVD-nucleotide interaction determined using designer TAL effectors in transient systems. PMID:26575863

  19. Global investigation of the co-evolution of MIRNA genes and microRNA targets during soybean domestication.

    PubMed

    Liu, Tengfei; Fang, Chao; Ma, Yanming; Shen, Yanting; Li, Congcong; Li, Qing; Wang, Min; Liu, Shulin; Zhang, Jixiang; Zhou, Zhengkui; Yang, Rui; Wang, Zheng; Tian, Zhixi

    2016-02-01

    Although the selection of coding genes during plant domestication has been well studied, the evolution of MIRNA genes (MIRs) and the interaction between microRNAs (miRNAs) and their targets in this process are poorly understood. Here, we present a genome-wide survey of the selection of MIRs and miRNA targets during soybean domestication and improvement. Our results suggest that, overall, MIRs have higher evolutionary rates than miRNA targets. Nonetheless, they do demonstrate certain similar evolutionary patterns during soybean domestication: MIRs and miRNA targets with high expression and duplication status, and with greater numbers of partners, exhibit lower nucleotide divergence than their counterparts without these characteristics, suggesting that expression level, duplication status, and miRNA-target interaction are essential for evolution of MIRs and miRNA targets. Further investigation revealed that miRNA-target pairs that are subjected to strong purifying selection have greater similarities than those that exhibited genetic diversity. Moreover, mediated by domestication and improvement, the similarities of a large number of miRNA-target pairs in cultivated soybean populations were increased compared to those in wild soybeans, whereas a small number of miRNA-target pairs exhibited decreased similarity, which may be associated with the adoption of particular domestication traits. Taken together, our results shed light on the co-evolution of MIRs and miRNA targets during soybean domestication. PMID:26714457

  20. Cytochrome P450 genes in coronary artery diseases: Codon usage analysis reveals genomic GC adaptation.

    PubMed

    Malakar, Arup Kumar; Halder, Binata; Paul, Prosenjit; Chakraborty, Supriyo

    2016-09-15

    Establishing codon usage biases are imperative for understanding the etiology of coronary artery diseases (CAD) as well as the genetic factors associated with these diseases. The aim of this study was to evaluate the contribution of 18 responsible cytochrome P450 (CYP) genes for the risk of CAD. Effective number of codon (Nc) showed a negative correlation with both GC3 and synonymous codon usage order (SCUO) suggesting an antagonistic relationship between codon usage and Nc of genes. The dinucleotide analysis revealed that CG and TA dinucleotides have the lowest odds ratio in these genes. Principal component analysis showed that GC composition has a profound effect in separating the genes along the first major axis. Our findings revealed that mutational pressure and natural selection could possibly be the major factors responsible for codon bias in these genes. The study not only offers an insight into the mechanisms of genomic GC adaptation, but also illustrates the complexity of CYP genes in CAD. PMID:27275533

  1. Chemogenetic Characterization of Inositol Phosphate Metabolic Pathway Reveals Druggable Enzymes for Targeting Kinetoplastid Parasites.

    PubMed

    Cestari, Igor; Haas, Paige; Moretti, Nilmar Silvio; Schenkman, Sergio; Stuart, Ken

    2016-05-19

    Kinetoplastids cause Chagas disease, human African trypanosomiasis, and leishmaniases. Current treatments for these diseases are toxic and inefficient, and our limited knowledge of drug targets and inhibitors has dramatically hindered the development of new drugs. Here we used a chemogenetic approach to identify new kinetoplastid drug targets and inhibitors. We conditionally knocked down Trypanosoma brucei inositol phosphate (IP) pathway genes and showed that almost every pathway step is essential for parasite growth and infection. Using a genetic and chemical screen, we identified inhibitors that target IP pathway enzymes and are selective against T. brucei. Two series of these inhibitors acted on T. brucei inositol polyphosphate multikinase (IPMK) preventing Ins(1,4,5)P3 and Ins(1,3,4,5)P4 phosphorylation. We show that IPMK is functionally conserved among kinetoplastids and that its inhibition is also lethal for Trypanosoma cruzi. Hence, IP enzymes are viable drug targets in kinetoplastids, and IPMK inhibitors may aid the development of new drugs. PMID:27133314

  2. Co-modulation analysis of gene regulation in breast cancer reveals complex interplay between ESR1 and ERBB2 genes

    PubMed Central

    2015-01-01

    Background Gene regulation is dynamic across cellular conditions and disease subtypes. From the aspect of regulation under modulation, regulation strength between a pair of genes can be modulated by (dependent on) expression abundance of another gene (modulator gene). Previous studies have demonstrated the involvement of genes modulated by single modulator genes in cancers, including breast cancer. However, analysis of multi-modulator co-modulation that can further delineate the landscape of complex gene regulation is, to our knowledge, unexplored previously. In the present study we aim to explore the joint effects of multiple modulator genes in modulating global gene regulation and dissect the biological functions in breast cancer. Results To carry out the analysis, we proposed the Covariability-based Multiple Regression (CoMRe) method. The method is mainly built on a multiple regression model that takes expression levels of multiple modulators as inputs and regulation strength between genes as output. Pairs of genes were divided into groups based on their co-modulation patterns. Analyzing gene expression profiles from 286 breast cancer patients, CoMRe investigated ten candidate modulator genes that interacted and jointly determined global gene regulation. Among the candidate modulators, ESR1, ERBB2, and ADAM12 were found modulating the most numbers of gene pairs. The largest group of gene pairs was composed of ones that were modulated by merely ESR1. Functional annotation revealed that the group was significantly related to tumorigenesis and estrogen signaling in breast cancer. ESR1−ERBB2 co-modulation was the largest group modulated by more than one modulators. Similarly, the group was functionally associated with hormone stimulus, suggesting that functions of the two modulators are performed, at least partially, through modulation. The findings were validated in majorities of patients (> 99%) of two independent breast cancer datasets. Conclusions We have

  3. ALK is a MYCN target gene and regulates cell migration and invasion in neuroblastoma.

    PubMed

    Hasan, Md Kamrul; Nafady, Asmaa; Takatori, Atsushi; Kishida, Satoshi; Ohira, Miki; Suenaga, Yusuke; Hossain, Shamim; Akter, Jesmin; Ogura, Atsushi; Nakamura, Yohko; Kadomatsu, Kenji; Nakagawara, Akira

    2013-01-01

    Human anaplastic lymphoma kinase (ALK) has been identified as an oncogene that is mutated or amplified in NBLs. To obtain a better understanding of the molecular events associated with ALK in the pathogenesis of NBL, it is necessary to clarify how ALK gene contributes to NBL progression. In the present study, we found that ALK expression was significantly high in NBL clinical samples with amplified MYCN (n = 126, P < 0.01) and in developing tumors of MYCN-transgenic mice. Indeed, promoter analysis revealed that ALK is a direct transcriptional target of MYCN. Overexpression and knockdown of ALK demonstrated its function in cell proliferation, migration and invasion. Moreover, treatment with an ALK inhibitor, TAE-684, efficiently suppressed such biological effects in MYCN amplified cells and tumor growth of the xenograft in mice. Our present findings explore the fundamental understanding of ALK in order to develop novel therapeutic tools by targeting ALK for aggressive NBL treatment. PMID:24356251

  4. AB46. Screening and identification for the target genes of androgen receptor in mouse Sertoli cells

    PubMed Central

    Gui, Yaoting; Mou, Lisha; Zhang, Qiaoxia; Yang, Lihua; Wang, Yadong; Cai, Zhiming

    2014-01-01

    ubiquitylation, while downregulation of UBE2B blocked the testosterone-induced H2A ubiquitylation. The ubiquitylation of H2A was markedly decreased in the testes of S-AR mice by immunohistochemistry. Digital gene expression analysis showed that 113 genes were significantly down-regulated and 71 were up-regulated by UBE2B in TM4 cells. These results suggest that Ube2b, as a direct AR transcriptional target in Sertoli cells, mediates the function of AR in spermatogenesis by promoting H2A ubiquitylation. Our previous digital gene expression analysis data also showed that heat shock transcription factors 1 (HSF1) was regulated by androgen in mouse Sertoli cells. We found that the expression of Hsf1 was increased in the testes of S-AR mice compared with wild-type mice by quantitative real-time PCR and the expression of HSF1 in the S-ARSertoli cells was significantly increased as examined by immunofluorescence analysis. Besides, in vitro study showed that testosterone repressed the expression of Hsf1 in TM4 cells. Moreover, luciferase assay, electrophoretic mobility shift assay and chromatin immunoprecipitation assay showed that testosterone repressed Hsf1 expression by facilitating the binding of androgen receptor to Hsf1 promoter. Our experiment also demonstrated that testosterone downregulated the expression of heat shock protein HSP105 and HSP60 by inhibiting the transcription of Hsf1. Taken together, these results reveal that HSF1 is a novel target of androgen receptor in mouse Sertoli Cells, and testosterone and its receptor regulate the process of spermatogenesis partially by inhibiting HSF1 expression.

  5. A model of gene-gene and gene-environment interactions and its implications for targeting environmental interventions by genotype

    PubMed Central

    Wallace, Helen M

    2006-01-01

    Background The potential public health benefits of targeting environmental interventions by genotype depend on the environmental and genetic contributions to the variance of common diseases, and the magnitude of any gene-environment interaction. In the absence of prior knowledge of all risk factors, twin, family and environmental data may help to define the potential limits of these benefits in a given population. However, a general methodology to analyze twin data is required because of the potential importance of gene-gene interactions (epistasis), gene-environment interactions, and conditions that break the 'equal environments' assumption for monozygotic and dizygotic twins. Method A new model for gene-gene and gene-environment interactions is developed that abandons the assumptions of the classical twin study, including Fisher's (1918) assumption that genes act as risk factors for common traits in a manner necessarily dominated by an additive polygenic term. Provided there are no confounders, the model can be used to implement a top-down approach to quantifying the potential utility of genetic prediction and prevention, using twin, family and environmental data. The results describe a solution space for each disease or trait, which may or may not include the classical twin study result. Each point in the solution space corresponds to a different model of genotypic risk and gene-environment interaction. Conclusion The results show that the potential for reducing the incidence of common diseases using environmental interventions targeted by genotype may be limited, except in special cases. The model also confirms that the importance of an individual's genotype in determining their risk of complex diseases tends to be exaggerated by the classical twin studies method, owing to the 'equal environments' assumption and the assumption of no gene-environment interaction. In addition, if phenotypes are genetically robust, because of epistasis, a largely environmental

  6. Advances in plant gene-targeted and functional markers: a review

    PubMed Central

    2013-01-01

    Public genomic databases have provided new directions for molecular marker development and initiated a shift in the types of PCR-based techniques commonly used in plant science. Alongside commonly used arbitrarily amplified DNA markers, other methods have been developed. Targeted fingerprinting marker techniques are based on the well-established practices of arbitrarily amplified DNA methods, but employ novel methodological innovations such as the incorporation of gene or promoter elements in the primers. These markers provide good reproducibility and increased resolution by the concurrent incidence of dominant and co-dominant bands. Despite their promising features, these semi-random markers suffer from possible problems of collision and non-homology analogous to those found with randomly generated fingerprints. Transposable elements, present in abundance in plant genomes, may also be used to generate fingerprints. These markers provide increased genomic coverage by utilizing specific targeted sites and produce bands that mostly seem to be homologous. The biggest drawback with most of these techniques is that prior genomic information about retrotransposons is needed for primer design, prohibiting universal applications. Another class of recently developed methods exploits length polymorphism present in arrays of multi-copy gene families such as cytochrome P450 and β-tubulin genes to provide cross-species amplification and transferability. A specific class of marker makes use of common features of plant resistance genes to generate bands linked to a given phenotype, or to reveal genetic diversity. Conserved DNA-based strategies have limited genome coverage and may fail to reveal genetic diversity, while resistance genes may be under specific evolutionary selection. Markers may also be generated from functional and/or transcribed regions of the genome using different gene-targeting approaches coupled with the use of RNA information. Such techniques have the

  7. Single chromosome transcriptional profiling reveals chromosome-level regulation of gene expression

    PubMed Central

    Levesque, Marshall J.; Raj, Arjun

    2013-01-01

    Here we report iceFISH, a multiplex imaging method for measuring gene expression and chromosome structure simultaneously on single chromosomes. We demonstrate that chromosomal translocations can alter transcription chromosome-wide, finding substantial differences in transcriptional frequency between genes located on a translocated chromosome in comparison to the normal chromosome in the same cell. Examination of correlations between genes on a single chromosome revealed a cis chromosome-level transcriptional interaction spanning 14.3 megabases. PMID:23416756

  8. An Integrative Transcriptomic Analysis for Identifying Novel Target Genes Corresponding to Severity Spectrum in Spinal Muscular Atrophy

    PubMed Central

    Yang, Chung-Wei; Chen, Chien-Lin; Chou, Wei-Chun; Lin, Ho-Chen; Jong, Yuh-Jyh; Tsai, Li-Kai; Chuang, Chun-Yu

    2016-01-01

    Spinal muscular atrophy (SMA) is an inherited neuromuscular disease resulting from a recessive mutation in the SMN1 gene. This disease affects multiple organ systems with varying degrees of severity. Exploration of the molecular pathological changes occurring in different cell types in SMA is crucial for developing new therapies. This study collected 39 human microarray datasets from ArrayExpress and GEO databases to build an integrative transcriptomic analysis for recognizing novel SMA targets. The transcriptomic analysis was conducted through combining weighted correlation network analysis (WGCNA) for gene module detection, gene set enrichment analysis (GSEA) for functional categorization and filtration, and Cytoscape (visual interaction gene network analysis) for target gene identification. Seven novel target genes (Bmp4, Serpine1, Gata6, Ptgs2, Bcl2, IL6 and Cntn1) of SMA were revealed, and are all known in the regulation of TNFα for controlling neural, cardiac and bone development. Sequentially, the differentially expressed patterns of these 7 target genes in mouse tissues (e.g., spinal cord, heart, muscles and bone) were validated in SMA mice of different severities (pre-symptomatic, mildly symptomatic, and severely symptomatic). In severely symptomatic SMA mice, TNFα was up-regulated with attenuation of Bmp4 and increase of Serpine1 and Gata6 (a pathway in neural and cardiac development), but not in pre-symptomatic and mildly symptomatic SMA mice. The severely symptomatic SMA mice also had the elevated levels of Ptgs2 and Bcl2 (a pathway in skeletal development) as well as IL6 and Cntn1 (a pathway in nervous system development). Thus, the 7 genes identified in this study might serve as potential target genes for future investigations of disease pathogenesis and SMA therapy. PMID:27331400

  9. An Integrative Transcriptomic Analysis for Identifying Novel Target Genes Corresponding to Severity Spectrum in Spinal Muscular Atrophy.

    PubMed

    Yang, Chung-Wei; Chen, Chien-Lin; Chou, Wei-Chun; Lin, Ho-Chen; Jong, Yuh-Jyh; Tsai, Li-Kai; Chuang, Chun-Yu

    2016-01-01

    Spinal muscular atrophy (SMA) is an inherited neuromuscular disease resulting from a recessive mutation in the SMN1 gene. This disease affects multiple organ systems with varying degrees of severity. Exploration of the molecular pathological changes occurring in different cell types in SMA is crucial for developing new therapies. This study collected 39 human microarray datasets from ArrayExpress and GEO databases to build an integrative transcriptomic analysis for recognizing novel SMA targets. The transcriptomic analysis was conducted through combining weighted correlation network analysis (WGCNA) for gene module detection, gene set enrichment analysis (GSEA) for functional categorization and filtration, and Cytoscape (visual interaction gene network analysis) for target gene identification. Seven novel target genes (Bmp4, Serpine1, Gata6, Ptgs2, Bcl2, IL6 and Cntn1) of SMA were revealed, and are all known in the regulation of TNFα for controlling neural, cardiac and bone development. Sequentially, the differentially expressed patterns of these 7 target genes in mouse tissues (e.g., spinal cord, heart, muscles and bone) were validated in SMA mice of different severities (pre-symptomatic, mildly symptomatic, and severely symptomatic). In severely symptomatic SMA mice, TNFα was up-regulated with attenuation of Bmp4 and increase of Serpine1 and Gata6 (a pathway in neural and cardiac development), but not in pre-symptomatic and mildly symptomatic SMA mice. The severely symptomatic SMA mice also had the elevated levels of Ptgs2 and Bcl2 (a pathway in skeletal development) as well as IL6 and Cntn1 (a pathway in nervous system development). Thus, the 7 genes identified in this study might serve as potential target genes for future investigations of disease pathogenesis and SMA therapy. PMID:27331400

  10. Pan-viral specificity of IFN-induced genes reveals new roles for cGAS in innate immunity

    NASA Astrophysics Data System (ADS)

    Schoggins, John W.; MacDuff, Donna A.; Imanaka, Naoko; Gainey, Maria D.; Shrestha, Bimmi; Eitson, Jennifer L.; Mar, Katrina B.; Richardson, R. Blake; Ratushny, Alexander V.; Litvak, Vladimir; Dabelic, Rea; Manicassamy, Balaji; Aitchison, John D.; Aderem, Alan; Elliott, Richard M.; García-Sastre, Adolfo; Racaniello, Vincent; Snijder, Eric J.; Yokoyama, Wayne M.; Diamond, Michael S.; Virgin, Herbert W.; Rice, Charles M.

    2014-01-01

    The type I interferon (IFN) response protects cells from viral infection by inducing hundreds of interferon-stimulated genes (ISGs), some of which encode direct antiviral effectors. Recent screening studies have begun to catalogue ISGs with antiviral activity against several RNA and DNA viruses. However, antiviral ISG specificity across multiple distinct classes of viruses remains largely unexplored. Here we used an ectopic expression assay to screen a library of more than 350 human ISGs for effects on 14 viruses representing 7 families and 11 genera. We show that 47 genes inhibit one or more viruses, and 25 genes enhance virus infectivity. Comparative analysis reveals that the screened ISGs target positive-sense single-stranded RNA viruses more effectively than negative-sense single-stranded RNA viruses. Gene clustering highlights the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS, also known as MB21D1) as a gene whose expression also broadly inhibits several RNA viruses. In vitro, lentiviral delivery of enzymatically active cGAS triggers a STING-dependent, IRF3-mediated antiviral program that functions independently of canonical IFN/STAT1 signalling. In vivo, genetic ablation of murine cGAS reveals its requirement in the antiviral response to two DNA viruses, and an unappreciated contribution to the innate control of an RNA virus. These studies uncover new paradigms for the preferential specificity of IFN-mediated antiviral pathways spanning several virus families.

  11. MicroRNA Expression Is Altered in an Ovalbumin-Induced Asthma Model and Targeting miR-155 with Antagomirs Reveals Cellular Specificity

    PubMed Central

    Plank, Maximilian W.; Maltby, Steven; Tay, Hock L.; Stewart, Jessica; Eyers, Fiona; Hansbro, Philip M.; Foster, Paul S.

    2015-01-01

    MicroRNAs are post-transcriptional regulators of gene expression that are differentially regulated during development and in inflammatory diseases. A role for miRNAs in allergic asthma is emerging and further investigation is required to determine whether they may serve as potential therapeutic targets. We profiled miRNA expression in murine lungs from an ovalbumin-induced allergic airways disease model, and compared expression to animals receiving dexamethasone treatment and non-allergic controls. Our analysis identified 29 miRNAs that were significantly altered during allergic inflammation. Target prediction analysis revealed novel genes with altered expression in allergic airways disease and suggests synergistic miRNA regulation of target mRNAs. To assess the impacts of one induced miRNA on pathology, we targeted miR-155-5p using a specific antagomir. Antagomir administration successfully reduced miR-155-5p expression with high specificity, but failed to alter the disease phenotype. Interestingly, further investigation revealed that antagomir delivery has variable efficacy across different immune cell types, effectively targeting myeloid cell populations, but exhibiting poor uptake in lymphocytes. Our findings demonstrate that antagomir-based targeting of miRNA function in the lung is highly specific, but highlights cell-specificity as a key limitation to be considered for antagomir-based strategies as therapeutics. PMID:26693910

  12. A Molecular-Level Landscape of Diet-Gut Microbiome Interactions: Toward Dietary Interventions Targeting Bacterial Genes

    PubMed Central

    Ni, Yueqiong; Li, Jun

    2015-01-01

    ABSTRACT As diet is considered the major regulator of the gut ecosystem, the overall objective of this work was to demonstrate that a detailed knowledge of the phytochemical composition of food could add to our understanding of observed changes in functionality and activity of the gut microbiota. We used metatranscriptomic data from a human dietary intervention study to develop a network that consists of >400 compounds present in the administered plant-based diet linked to 609 microbial targets in the gut. Approximately 20% of the targeted bacterial proteins showed significant changes in their gene expression levels, while functional and topology analyses revealed that proteins in metabolic networks with high centrality are the most “vulnerable” targets. This global view and the mechanistic understanding of the associations between microbial gene expression and dietary molecules could be regarded as a promising methodological approach for targeting specific bacterial proteins that impact human health. PMID:26507230

  13. Global Gene Expression Analysis of the Zoonotic Parasite Trichinella spiralis Revealed Novel Genes in Host Parasite Interaction

    PubMed Central

    Jiang, Ning; Wang, Jielin; Tang, Bin; Lu, Huijun; Peng, Shuai; Chang, Zhiguang; Tang, Yizhi; Yin, Jigang; Liu, Mingyuan; Tan, Yan; Chen, Qijun

    2012-01-01

    Background Trichinellosis is a typical food-borne zoonotic disease which is epidemic worldwide and the nematode Trichinella spiralis is the main pathogen. The life cycle of T. spiralis contains three developmental stages, i.e. adult worms, new borne larva (new borne L1 larva) and muscular larva (infective L1 larva). Stage-specific gene expression in the parasites has been investigated with various immunological and cDNA cloning approaches, whereas the genome-wide transcriptome and expression features of the parasite have been largely unknown. The availability of the genome sequence information of T. spiralis has made it possible to deeply dissect parasite biology in association with global gene expression and pathogenesis. Methodology and Principal Findings In this study, we analyzed the global gene expression patterns in the three developmental stages of T. spiralis using digital gene expression (DGE) analysis. Almost 15 million sequence tags were generated with the Illumina RNA-seq technology, producing expression data for more than 9,000 genes, covering 65% of the genome. The transcriptome analysis revealed thousands of differentially expressed genes within the genome, and importantly, a panel of genes encoding functional proteins associated with parasite invasion and immuno-modulation were identified. More than 45% of the genes were found to be transcribed from both strands, indicating the importance of RNA-mediated gene regulation in the development of the parasite. Further, based on gene ontological analysis, over 3000 genes were functionally categorized and biological pathways in the three life cycle stage were elucidated. Conclusions and Significance The global transcriptome of T. spiralis in three developmental stages has been profiled, and most gene activity in the genome was found to be developmentally regulated. Many metabolic and biological pathways have been revealed. The findings of the differential expression of several protein families facilitate

  14. Gene-sharing networks reveal organizing principles of transcriptomes in Arabidopsis and other multicellular organisms.

    PubMed

    Li, Song; Pandey, Sona; Gookin, Timothy E; Zhao, Zhixin; Wilson, Liza; Assmann, Sarah M

    2012-04-01

    Understanding tissue-related gene expression patterns can provide important insights into gene, tissue, and organ function. Transcriptome analyses often have focused on housekeeping or tissue-specific genes or on gene coexpression. However, by analyzing thousands of single-gene expression distributions in multiple tissues of Arabidopsis thaliana, rice (Oryza sativa), human (Homo sapiens), and mouse (Mus musculus), we found that these organisms primarily operate by gene sharing, a phenomenon where, in each organism, most genes exhibit a high expression level in a few key tissues. We designed an analytical pipeline to characterize this phenomenon and then derived Arabidopsis and human gene-sharing networks, in which tissues are connected solely based on the extent of shared preferentially expressed genes. The results show that tissues or cell types from the same organ system tend to group together to form network modules. Tissues that are in consecutive developmental stages or have common physiological functions are connected in these networks, revealing the importance of shared preferentially expressed genes in conferring specialized functions of each tissue type. The networks provide predictive power for each tissue type regarding gene functions of both known and heretofore unknown genes, as shown by the identification of four new genes with functions in guard cell and abscisic acid response. We provide a Web interface that enables, based on the extent of gene sharing, both prediction of tissue-related functions for any Arabidopsis gene of interest and predictions concerning the relatedness of tissues. Common gene-sharing patterns observed in the four model organisms suggest that gene sharing evolved as a fundamental organizing principle of gene expression in diverse multicellular eukaryotes. PMID:22517316

  15. Predicting miRNA Targets by Integrating Gene Regulatory Knowledge with Expression Profiles

    PubMed Central

    Zhang, Weijia; Le, Thuc Duy; Liu, Lin; Zhou, Zhi-Hua; Li, Jiuyong

    2016-01-01

    Motivation microRNAs (miRNAs) play crucial roles in post-transcriptional gene regulation of both plants and mammals, and dysfunctions of miRNAs are often associated with tumorigenesis and development through the effects on their target messenger RNAs (mRNAs). Identifying miRNA functions is critical for understanding cancer mechanisms and determining the efficacy of drugs. Computational methods analyzing high-throughput data offer great assistance in understanding the diverse and complex relationships between miRNAs and mRNAs. However, most of the existing methods do not fully utilise the available knowledge in biology to reduce the uncertainty in the modeling process. Therefore it is desirable to develop a method that can seamlessly integrate existing biological knowledge and high-throughput data into the process of discovering miRNA regulation mechanisms. Results In this article we present an integrative framework, CIDER (Causal miRNA target Discovery with Expression profile and Regulatory knowledge), to predict miRNA targets. CIDER is able to utilise a variety of gene regulation knowledge, including transcriptional and post-transcriptional knowledge, and to exploit gene expression data for the discovery of miRNA-mRNA regulatory relationships. The benefits of our framework is demonstrated by both simulation study and the analysis of the epithelial-to-mesenchymal transition (EMT) and the breast cancer (BRCA) datasets. Our results reveal that even a limited amount of either Transcription Factor (TF)-miRNA or miRNA-mRNA regulatory knowledge improves the performance of miRNA target prediction, and the combination of the two types of knowledge enhances the improvement further. Another useful property of the framework is that its performance increases monotonically with the increase of regulatory knowledge. PMID:27064982

  16. Potentiation of gene targeting in human cells by expression of Saccharomyces cerevisiae Rad52.

    PubMed

    Di Primio, Cristina; Galli, Alvaro; Cervelli, Tiziana; Zoppè, Monica; Rainaldi, Giuseppe

    2005-01-01

    When exogenous DNA is stably introduced in mammalian cells, it is typically integrated in random positions, and only a minor fraction enters a pathway of homologous recombination (HR). The complex Rad51/Rad52 is a major player in the management of exogenous DNA in eukaryotic organisms and plays a critical role in the choice of repair system. In Saccharomyces cerevisiae, the pathway of choice is HR, mediated by Rad52 (ScRad52), which differs slightly from its human homologue. Here, we present an approach that utilizes ScRad52 to enhance HR in human cells containing a specific substrate for recombination. Clones of HeLa cells were produced expressing functional ScRad52. These cells showed enhanced resistance to DNA damaging treatments and revealed a different distribution of Rad51 foci (a marker of recombination complex formation). More significantly, ScRad52 expression resulted in an up to 37-fold increase in gene targeting by HR. In the same cells, random integration of exogenous DNA was significantly reduced, consistent with the view that HR and non-homologous end joining are alternative competing pathways. Expression of ScRad52 could offer a major improvement for experiments requiring gene targeting by HR, both in basic research and in gene therapy studies. PMID:16106043

  17. Potentiation of gene targeting in human cells by expression of Saccharomyces cerevisiae Rad52

    PubMed Central

    Di Primio, Cristina; Galli, Alvaro; Cervelli, Tiziana; Zoppè, Monica; Rainaldi, Giuseppe

    2005-01-01

    When exogenous DNA is stably introduced in mammalian cells, it is typically integrated in random positions, and only a minor fraction enters a pathway of homologous recombination (HR). The complex Rad51/Rad52 is a major player in the management of exogenous DNA in eukaryotic organisms and plays a critical role in the choice of repair system. In Saccharomyces cerevisiae, the pathway of choice is HR, mediated by Rad52 (ScRad52), which differs slightly from its human homologue. Here, we present an approach that utilizes ScRad52 to enhance HR in human cells containing a specific substrate for recombination. Clones of HeLa cells were produced expressing functional ScRad52. These cells showed enhanced resistance to DNA damaging treatments and revealed a different distribution of Rad51 foci (a marker of recombination complex formation). More significantly, ScRad52 expression resulted in an up to 37-fold increase in gene targeting by HR. In the same cells, random integration of exogenous DNA was significantly reduced, consistent with the view that HR and non-homologous end joining are alternative competing pathways. Expression of ScRad52 could offer a major improvement for experiments requiring gene targeting by HR, both in basic research and in gene therapy studies. PMID:16106043

  18. Controllability analysis of the directed human protein interaction network identifies disease genes and drug targets.

    PubMed

    Vinayagam, Arunachalam; Gibson, Travis E; Lee, Ho-Joon; Yilmazel, Bahar; Roesel, Charles; Hu, Yanhui; Kwon, Young; Sharma, Amitabh; Liu, Yang-Yu; Perrimon, Norbert; Barabási, Albert-László

    2016-05-01

    The protein-protein interaction (PPI) network is crucial for cellular information processing and decision-making. With suitable inputs, PPI networks drive the cells to diverse functional outcomes such as cell proliferation or cell death. Here, we characterize the structural controllability of a large directed human PPI network comprising 6,339 proteins and 34,813 interactions. This network allows us to classify proteins as "indispensable," "neutral," or "dispensable," which correlates to increasing, no effect, or decreasing the number of driver nodes in the network upon removal of that protein. We find that 21% of the proteins in the PPI network are indispensable. Interestingly, these indispensable proteins are the primary targets of disease-causing mutations, human viruses, and drugs, suggesting that altering a network's control property is critical for the transition between healthy and disease states. Furthermore, analyzing copy number alterations data from 1,547 cancer patients reveals that 56 genes that are frequently amplified or deleted in nine different cancers are indispensable. Among the 56 genes, 46 of them have not been previously associated with cancer. This suggests that controllability analysis is very useful in identifying novel disease genes and potential drug targets. PMID:27091990

  19. De Novo Transcriptome and Small RNA Analysis of Two Chinese Willow Cultivars Reveals Stress Response Genes in Salix matsudana

    PubMed Central

    Zeng, Yanfei; He, Caiyun; Duan, Aiguo; Zhang, Jianguo

    2014-01-01

    Salix matsudana Koidz. is a deciduous, rapidly growing, and drought resistant tree and is one of the most widely distributed and commonly cultivated willow species in China. Currently little transcriptomic and small RNAomic data are available to reveal the genes involve in the stress resistant in S. matsudana. Here, we report the RNA-seq analysis results of both transcriptome and small RNAome data using Illumina deep sequencing of shoot tips from two willow variants(Salix. matsudana and Salix matsudana Koidz. cultivar ‘Tortuosa’). De novo gene assembly was used to generate the consensus transcriptome and small RNAome, which contained 106,403 unique transcripts with an average length of 944 bp and a total length of 100.45 MB, and 166 known miRNAs representing 35 miRNA families. Comparison of transcriptomes and small RNAomes combined with quantitative real-time PCR from the two Salix libraries revealed a total of 292 different expressed genes(DEGs) and 36 different expressed miRNAs (DEMs). Among the DEGs and DEMs, 196 genes and 24 miRNAs were up regulated, 96 genes and 12 miRNA were down regulated in S. matsudana. Functional analysis of DEGs and miRNA targets showed that many genes were involved in stress resistance in S. matsudana. Our global gene expression profiling presents a comprehensive view of the transcriptome and small RNAome which provide valuable information and sequence resources for uncovering the stress response genes in S. matsudana. Moreover the transcriptome and small RNAome data provide a basis for future study of genetic resistance in Salix. PMID:25275458

  20. Proliferating cell nuclear antigen (Pcna) as a direct downstream target gene of Hoxc8

    SciTech Connect

    Min, Hyehyun; Lee, Ji-Yeon; Bok, Jinwoong; Chung, Hyun Joo; Kim, Myoung Hee

    2010-02-19

    Hoxc8 is a member of Hox family transcription factors that play crucial roles in spatiotemporal body patterning during embryogenesis. Hox proteins contain a conserved 61 amino acid homeodomain, which is responsible for recognition and binding of the proteins onto Hox-specific DNA binding motifs and regulates expression of their target genes. Previously, using proteome analysis, we identified Proliferating cell nuclear antigen (Pcna) as one of the putative target genes of Hoxc8. Here, we asked whether Hoxc8 regulates Pcna expression by directly binding to the regulatory sequence of Pcna. In mouse embryos at embryonic day 11.5, the expression pattern of Pcna was similar to that of Hoxc8 along the anteroposterior body axis. Moreover, Pcna transcript levels as well as cell proliferation rate were increased by overexpression of Hoxc8 in C3H10T1/2 mouse embryonic fibroblast cells. Characterization of 2.3 kb genomic sequence upstream of Pcna coding region revealed that the upstream sequence contains several Hox core binding sequences and one Hox-Pbx binding sequence. Direct binding of Hoxc8 proteins to the Pcna regulatory sequence was verified by chromatin immunoprecipitation assay. Taken together, our data suggest that Pcna is a direct downstream target of Hoxc8.

  1. Simple F Test Reveals Gene-Gene Interactions in Case-Control Studies

    PubMed Central

    Chen, Guanjie; Yuan, Ao; Zhou, Jie; Bentley, Amy R.; Adeyemo, Adebowale; Rotimi, Charles N.

    2012-01-01

    Missing heritability is still a challenge for Genome Wide Association Studies (GWAS). Gene-gene interactions may partially explain this residual genetic influence and contribute broadly to complex disease. To analyze the gene-gene interactions in case-control studies of complex disease, we propose a simple, non-parametric method that utilizes the F-statistic. This approach consists of three steps. First, we examine the joint distribution of a pair of SNPs in cases and controls separately. Second, an F-test is used to evaluate the ratio of dependence in cases to that of controls. Finally, results are adjusted for multiple tests. This method was used to evaluate gene-gene interactions that are associated with risk of Type 2 Diabetes among African Americans in the Howard University Family Study. We identified 18 gene-gene interactions (P < 0.0001). Compared with the commonly-used logistical regression method, we demonstrate that the F-ratio test is an efficient approach to measuring gene-gene interactions, especially for studies with limited sample size. PMID:22837643

  2. Gene family analysis of the Arabidopsis pollen transcriptome reveals biological implications for cell growth, division control, and gene expression regulation.

    PubMed

    Pina, Cristina; Pinto, Francisco; Feijó, José A; Becker, Jörg D

    2005-06-01

    Upon germination, pollen forms a tube that elongates dramatically through female tissues to reach and fertilize ovules. While essential for the life cycle of higher plants, the genetic basis underlying most of the process is not well understood. We previously used a combination of flow cytometry sorting of viable hydrated pollen grains and GeneChip array analysis of one-third of the Arabidopsis (Arabidopsis thaliana) genome to define a first overview of the pollen transcriptome. We now extend that study to approximately 80% of the genome of Arabidopsis by using Affymetrix Arabidopsis ATH1 arrays and perform comparative analysis of gene family and gene ontology representation in the transcriptome of pollen and vegetative tissues. Pollen grains have a smaller and overall unique transcriptome (6,587 genes expressed) with greater proportions of selectively expressed (11%) and enriched (26%) genes than any vegetative tissue. Relative gene ontology category representations in pollen and vegetative tissues reveal a functional skew of the pollen transcriptome toward signaling, vesicle transport, and the cytoskeleton, suggestive of a commitment to germination and tube growth. Cell cycle analysis reveals an accumulation of G2/M-associated factors that may play a role in the first mitotic division of the zygote. Despite the relative underrepresentation of transcription-associated transcripts, nonclassical MADS box genes emerge as a class with putative unique roles in pollen. The singularity of gene expression control in mature pollen grains is further highlighted by the apparent absence of small RNA pathway components. PMID:15908605

  3. Topological robustness analysis of protein interaction networks reveals key targets for overcoming chemotherapy resistance in glioma

    NASA Astrophysics Data System (ADS)

    Azevedo, Hátylas; Moreira-Filho, Carlos Alberto

    2015-11-01

    Biological networks display high robustness against random failures but are vulnerable to targeted attacks on central nodes. Thus, network topology analysis represents a powerful tool for investigating network susceptibility against targeted node removal. Here, we built protein interaction networks associated with chemoresistance to temozolomide, an alkylating agent used in glioma therapy, and analyzed their modular structure and robustness against intentional attack. These networks showed functional modules related to DNA repair, immunity, apoptosis, cell stress, proliferation and migration. Subsequently, network vulnerability was assessed by means of centrality-based attacks based on the removal of node fractions in descending orders of degree, betweenness, or the product of degree and betweenness. This analysis revealed that removing nodes with high degree and high betweenness was more effective in altering networks’ robustness parameters, suggesting that their corresponding proteins may be particularly relevant to target temozolomide resistance. In silico data was used for validation and confirmed that central nodes are more relevant for altering proliferation rates in temozolomide-resistant glioma cell lines and for predicting survival in glioma patients. Altogether, these results demonstrate how the analysis of network vulnerability to topological attack facilitates target prioritization for overcoming cancer chemoresistance.

  4. Topological robustness analysis of protein interaction networks reveals key targets for overcoming chemotherapy resistance in glioma

    PubMed Central

    Azevedo, Hátylas; Moreira-Filho, Carlos Alberto

    2015-01-01

    Biological networks display high robustness against random failures but are vulnerable to targeted attacks on central nodes. Thus, network topology analysis represents a powerful tool for investigating network susceptibility against targeted node removal. Here, we built protein interaction networks associated with chemoresistance to temozolomide, an alkylating agent used in glioma therapy, and analyzed their modular structure and robustness against intentional attack. These networks showed functional modules related to DNA repair, immunity, apoptosis, cell stress, proliferation and migration. Subsequently, network vulnerability was assessed by means of centrality-based attacks based on the removal of node fractions in descending orders of degree, betweenness, or the product of degree and betweenness. This analysis revealed that removing nodes with high degree and high betweenness was more effective in altering networks’ robustness parameters, suggesting that their corresponding proteins may be particularly relevant to target temozolomide resistance. In silico data was used for validation and confirmed that central nodes are more relevant for altering proliferation rates in temozolomide-resistant glioma cell lines and for predicting survival in glioma patients. Altogether, these results demonstrate how the analysis of network vulnerability to topological attack facilitates target prioritization for overcoming cancer chemoresistance. PMID:26582089

  5. Multifunctional disulfide-based cationic dextran conjugates for intravenous gene delivery targeting ovarian cancer cells.

    PubMed

    Song, Yanyan; Lou, Bo; Zhao, Peng; Lin, Chao

    2014-07-01

    A folate-decorated, disulfide-based cationic dextran conjugate having dextran as the main chain and disulfide-linked 1,4-bis(3-aminopropyl)piperazine (BAP) residues as the grafts was designed and successfully prepared as a multifunctional gene delivery vector for targeted gene delivery to ovarian cancer SKOV-3 cells in vitro and in vivo. Initially, a new bioreducible cationic polyamide (denoted as pSSBAP) was prepared by polycondensation reaction of bis(p-nitrophenyl)-3,3'-dithiodipropanoate, a disulfide-containing monomer, and BAP. It was found that the pSSBAP was highly efficient for in vitro gene delivery against MCF-7 and SKOV-3 cell lines. Subsequently, two cationic dextran conjugates with different amounts of BAP residues (denoted as Dex-SSBAP6 and Dex-SSBAP30, respectively) were synthesized by coupling BAP to disulfide-linked carboxylated dextran or coupling pSSBAP-oligomer to p-nitrophenyl carbonated dextran. Both two conjugates were able to bind DNA to form nanosized polyplexes with an improved colloidal stability in physiological conditions. The polyplexes, however, were rapidly dissociated to liberate DNA in a reducing environment. In vitro transfection experiments revealed that the polyplexes of Dex-SSBAP30 efficiently transfected SKOV-3 cells, yielding transfection efficiency that is comparable to that of linear polyethylenimine or lipofectamine 2000. AlamarBlue assay showed that the conjugates had low cytotoxicity in vitro at a high concentration of 100 mg/L. Further, Dex-SSBAP30 has primary amine side groups and thus allows for folate (FA) conjugation, yielding FA-coupled Dex-SSBAP30 (Dex-SSBAP30-FA). It was found that Dex-SSBAP30-FA was efficient for targeted gene delivery to SKOV-3 tumor xenografted in a nude mouse model by intravenous injection, inducing a higher level of gene expression in the tumor as compared to Dex-SSBAP30 lacking FA and comparable gene expression to linear polyethylenimine as one of the most efficient polymeric vectors for

  6. Exploring What's Missing: What Do Target Absent Trials Reveal About Autism Search Superiority?

    PubMed

    Keehn, Brandon; Joseph, Robert M

    2016-05-01

    We used eye-tracking to investigate the roles of enhanced discrimination and peripheral selection in superior visual search in autism spectrum disorder (ASD). Children with ASD were faster at visual search than their typically developing peers. However, group differences in performance and eye-movements did not vary with the level of difficulty of discrimination or selection. Rather, consistent with prior ASD research, group differences were mainly the effect of faster performance on target-absent trials. Eye-tracking revealed a lack of left-visual-field search asymmetry in ASD, which may confer an additional advantage when the target is absent. Lastly, ASD symptomatology was positively associated with search superiority, the mechanisms of which may shed light on the atypical brain organization that underlies social-communicative impairment in ASD. PMID:26762114

  7. Late Multiple Organ Surge in Interferon-Regulated Target Genes Characterizes Staphylococcal Enterotoxin B Lethality

    PubMed Central

    Ferreyra, Gabriela A.; Elinoff, Jason M.; Demirkale, Cumhur Y.; Starost, Matthew F.; Buckley, Marilyn; Munson, Peter J.; Krakauer, Teresa; Danner, Robert L.

    2014-01-01

    Background Bacterial superantigens are virulence factors that cause toxic shock syndrome. Here, the genome-wide, temporal response of mice to lethal intranasal staphylococcal enterotoxin B (SEB) challenge was investigated in six tissues. Results The earliest responses and largest number of affected genes occurred in peripheral blood mononuclear cells (PBMC), spleen, and lung tissues with the highest content of both T-cells and monocyte/macrophages, the direct cellular targets of SEB. In contrast, the response of liver, kidney, and heart was delayed and involved fewer genes, but revealed a dominant genetic program that was seen in all 6 tissues. Many of the 85 uniquely annotated transcripts participating in this shared genomic response have not been previously linked to SEB. Nine of the 85 genes were subsequently confirmed by RT-PCR in every tissue/organ at 24 h. These 85 transcripts, up-regulated in all tissues, annotated to the interferon (IFN)/antiviral-response and included genes belonging to the DNA/RNA sensing system, DNA damage repair, the immunoproteasome, and the ER/metabolic stress-response and apoptosis pathways. Overall, this shared program was identified as a type I and II interferon (IFN)-response and the promoters of these genes were highly enriched for IFN regulatory matrices. Several genes whose secreted products induce the IFN pathway were up-regulated at early time points in PBMCs, spleen, and/or lung. Furthermore, IFN regulatory factors including Irf1, Irf7 and Irf8, and Zbp1, a DNA sensor/transcription factor that can directly elicit an IFN innate immune response, participated in this host-wide SEB signature. Conclusion Global gene-expression changes across multiple organs implicated a host-wide IFN-response in SEB-induced death. Therapies aimed at IFN-associated innate immunity may improve outcome in toxic shock syndromes. PMID:24551153

  8. Cdk12 Is A Gene-Selective RNA Polymerase II Kinase That Regulates a Subset of the Transcriptome, Including Nrf2 Target Genes

    PubMed Central

    Li, Xuan; Chatterjee, Nirmalya; Spirohn, Kerstin; Boutros, Michael; Bohmann, Dirk

    2016-01-01

    The Nrf2 transcription factor is well conserved throughout metazoan evolution and serves as a central regulator of adaptive cellular responses to oxidative stress. We carried out an RNAi screen in Drosophila S2 cells to better understand the regulatory mechanisms governing Nrf2 target gene expression. This paper describes the identification and characterization of the RNA polymerase II (Pol II) kinase Cdk12 as a factor that is required for Nrf2 target gene expression in cell culture and in vivo. Cdk12 is, however, not essential for bulk mRNA transcription and cells lacking CDK12 function are viable and able to proliferate. Consistent with previous findings on the DNA damage and heat shock responses, it emerges that Cdk12 may be specifically required for stress activated gene expression. Transcriptome analysis revealed that antioxidant gene expression is compromised in flies with reduced Cdk12 function, which makes them oxidative stress sensitive. In addition to supporting Reactive Oxygen Species (ROS) induced gene activation, Cdk12 suppresses genes that support metabolic functions in stressed conditions. We suggest that Cdk12 acts as a gene-selective Pol II kinase that engages a global shift in gene expression to switch cells from a metabolically active state to “stress-defence mode” when challenged by external stress. PMID:26911346

  9. Isolation of Hox cluster genes from insects reveals an accelerated sequence evolution rate.

    PubMed

    Hadrys, Heike; Simon, Sabrina; Kaune, Barbara; Schmitt, Oliver; Schöner, Anja; Jakob, Wolfgang; Schierwater, Bernd

    2012-01-01

    Among gene families it is the Hox genes and among metazoan animals it is the insects (Hexapoda) that have attracted particular attention for studying the evolution of development. Surprisingly though, no Hox genes have been isolated from 26 out of 35 insect orders yet, and the existing sequences derive mainly from only two orders (61% from Hymenoptera and 22% from Diptera). We have designed insect specific primers and isolated 37 new partial homeobox sequences of Hox cluster genes (lab, pb, Hox3, ftz, Antp, Scr, abd-a, Abd-B, Dfd, and Ubx) from six insect orders, which are crucial to insect phylogenetics. These new gene sequences provide a first step towards comparative Hox gene studies in insects. Furthermore, comparative distance analyses of homeobox sequences reveal a correlation between gene divergence rate and species radiation success with insects showing the highest rate of homeobox sequence evolution. PMID:22685537

  10. Genome-wide Generation and Systematic Phenotyping of Knockout Mice Reveals New Roles for Many Genes

    PubMed Central

    White, Jacqueline K.; Gerdin, Anna-Karin; Karp, Natasha A.; Ryder, Ed; Buljan, Marija; Bussell, James N.; Salisbury, Jennifer; Clare, Simon; Ingham, Neil J.; Podrini, Christine; Houghton, Richard; Estabel, Jeanne; Bottomley, Joanna R.; Melvin, David G.; Sunter, David; Adams, Niels C.; Baker, Lauren; Barnes, Caroline; Beveridge, Ryan; Cambridge, Emma; Carragher, Damian; Chana, Prabhjoat; Clarke, Kay; Hooks, Yvette; Igosheva, Natalia; Ismail, Ozama; Jackson, Hannah; Kane, Leanne; Lacey, Rosalind; Lafont, David Tino; Lucas, Mark; Maguire, Simon; McGill, Katherine; McIntyre, Rebecca E.; Messager, Sophie; Mottram, Lynda; Mulderrig, Lee; Pearson, Selina; Protheroe, Hayley J.; Roberson, Laura-Anne; Salsbury, Grace; Sanderson, Mark; Sanger, Daniel; Shannon, Carl; Thompson, Paul C.; Tuck, Elizabeth; Vancollie, Valerie E.; Brackenbury, Lisa; Bushell, Wendy; Cook, Ross; Dalvi, Priya; Gleeson, Diane; Habib, Bishoy; Hardy, Matt; Liakath-Ali, Kifayathullah; Miklejewska, Evelina; Price, Stacey; Sethi, Debarati; Trenchard, Elizabeth; von Schiller, Dominique; Vyas, Sapna; West, Anthony P.; Woodward, John; Wynn, Elizabeth; Evans, Arthur; Gannon, David; Griffiths, Mark; Holroyd, Simon; Iyer, Vivek; Kipp, Christian; Lewis, Morag; Li, Wei; Oakley, Darren; Richardson, David; Smedley, Damian; Agu, Chukwuma; Bryant, Jackie; Delaney, Liz; Gueorguieva, Nadia I.; Tharagonnet, Helen; Townsend, Anne J.; Biggs, Daniel; Brown, Ellen; Collinson, Adam; Dumeau, Charles-Etienne; Grau, Evelyn; Harrison, Sarah; Harrison, James; Ingle, Catherine E.; Kundi, Helen; Madich, Alla; Mayhew, Danielle; Metcalf, Tom; Newman, Stuart; Pass, Johanna; Pearson, Laila; Reynolds, Helen; Sinclair, Caroline; Wardle-Jones, Hannah; Woods, Michael; Alexander, Liam; Brown, Terry; Flack, Francesca; Frost, Carole; Griggs, Nicola; Hrnciarova, Silvia; Kirton, Andrea; McDermott, Jordan; Rogerson, Claire; White, Gemma; Zielezinski, Pawel; DiTommaso, Tia; Edwards, Andrew; Heath, Emma; Mahajan, Mary Ann; Yalcin, Binnaz; Tannahill, David; Logan, Darren W.; MacArthur, Daniel G.; Flint, Jonathan; Mahajan, Vinit B.; Tsang, Stephen H.; Smyth, Ian; Watt, Fiona M.; Skarnes, William C.; Dougan, Gordon; Adams, David J.; Ramirez-Solis, Ramiro; Bradley, Allan; Steel, Karen P.

    2013-01-01

    Summary Mutations in whole organisms are powerful ways of interrogating gene function in a realistic context. We describe a program, the Sanger Institute Mouse Genetics Project, that provides a step toward the aim of knocking out all genes and screening each line for a broad range of traits. We found that hitherto unpublished genes were as likely to reveal phenotypes as known genes, suggesting that novel genes represent a rich resource for investigating the molecular basis of disease. We found many unexpected phenotypes detected only because we screened for them, emphasizing the value of screening all mutants for a wide range of traits. Haploinsufficiency and pleiotropy were both surprisingly common. Forty-two percent of genes were essential for viability, and these were less likely to have a paralog and more likely to contribute to a protein complex than other genes. Phenotypic data and more than 900 mutants are openly available for further analysis. PaperClip PMID:23870131

  11. Conditional Gene Targeting in Mouse High Endothelial Venules

    PubMed Central

    Kawashima, Hiroto; Hirakawa, Jotaro; Tobisawa, Yuki; Fukuda, Minoru; Saga, Yumiko

    2009-01-01

    High endothelial venules (HEVs) are specialized blood vessels of secondary lymphoid organs composed of endothelial cells with a characteristic cuboidal morphology. Lymphocytes selectively adhere to and migrate across HEVs to initiate immune responses. In this study, we established a novel transgenic mouse line expressing Cre recombinase under the transcriptional control of the gene encoding HEV-expressed sulfotransferase, N-acetylglucosamine-6-O-sulfotransferase 2 (GlcNAc6ST-2), using bacterial artificial chromosome recombineering. Crossing these transgenic mice with the ROSA26 reporter strain, which expresses lacZ following Cre-mediated recombination, and staining the resulting progeny with 5-bromo-4-chloro-5-indolyl-β-D-galactoside indicated that Cre recombinase was specifically expressed in mAb MECA79-reactive HEVs in secondary lymphoid organs but not in any other blood vessels of the transgenic mice. The expression of Cre recombinase correlated with a developmental switch, from immature, mAb MECA367-reactive HEVs to mature, mAb MECA79-reactive HEVs in neonatal lymph nodes. In addition to the HEVs, Cre recombinase was also strongly expressed in the colonic villi, which recapitulated the intrinsic expression of GlcNAc6ST-2 as confirmed in GlcNAc6ST-2GFP/GFP knock-in mice and by RT-PCR. Furthermore, treatment with an antimicrobial agent revealed that the colonic expression of Cre recombinase in the transgenic mice was regulated by commensal bacteria in the colon. In addition, Cre recombinase was expressed in a small subset of cells in the brain, testis, stomach, small intestine, and lung. In view of the restricted expression of Cre recombinase, this transgenic mouse line should be useful for elucidating tissue-specific gene functions using the Cre/loxP system. PMID:19380794

  12. Using PCR to Target Misconceptions about Gene Expression †

    PubMed Central

    Wright, Leslie K.; Newman, Dina L.

    2013-01-01

    We present a PCR-based laboratory exercise that can be used with first- or second-year biology students to help overcome common misconceptions about gene expression. Biology students typically do not have a clear understanding of the difference between genes (DNA) and gene expression (mRNA/protein) and often believe that genes exist in an organism or cell only when they are expressed. This laboratory exercise allows students to carry out a PCR-based experiment designed to challenge their misunderstanding of the difference between genes and gene expression. Students first transform E. coli with an inducible GFP gene containing plasmid and observe induced and un-induced colonies. The following exercise creates cognitive dissonance when actual PCR results contradict their initial (incorrect) predictions of the presence of the GFP gene in transformed cells. Field testing of this laboratory exercise resulted in learning gains on both knowledge and application questions on concepts related to genes and gene expression. PMID:23858358

  13. Network analysis of microRNAs, transcription factors, target genes and host genes in nasopharyngeal carcinoma

    PubMed Central

    WANG, HAO; XU, ZHIWEN; MA, MENGYAO; WANG, NING; WANG, KUNHAO

    2016-01-01

    Numerous studies on the morbidity of nasopharyngeal carcinoma (NPC) have identified several genes, microRNAs (miRNAs or miRs) and transcription factors (TFs) that influence the pathogenesis of NPC. However, summarizing all the regulatory networks involved in NPC is challenging. In the present study, the genes, miRNAs and TFs involved in NPC were considered as the nodes of the so-called regulatory network, and the associations between them were investigated. To clearly represent these associations, three regulatory networks were built seperately, namely, the differentially expressed network, the associated network and the global network. The differentially expressed network is the most important one of these three networks, since its nodes are differentially expressed genes whose mutations may lead to the development of NPC. Therefore, by modifying the aberrant expression of those genes that are differentially expressed in this network, their dysregulation may be corrected and the tumorigenesis of NPC may thus be prevented. Analysis of the aforementioned three networks highlighted the importance of certain pathways, such as self-adaptation pathways, in the development of NPC. For example, cyclin D1 (CCND1) was observed to regulate Homo sapiens-miR-20a, which in turn targeted CCND1. The present study conducted a systematic analysis of the pathogenesis of NPC through the three aforementioned regulatory networks, and provided a theoretical model for biologists. Future studies are required to evaluate the influence of the highlighted pathways in NPC. PMID:27313701

  14. Gene targeting, genome editing: from Dolly to editors.

    PubMed

    Tan, Wenfang; Proudfoot, Chris; Lillico, Simon G; Whitelaw, C Bruce A

    2016-06-01

    One of the most powerful strategies to investigate biology we have as scientists, is the ability to transfer genetic material in a controlled and deliberate manner between organisms. When applied to livestock, applications worthy of commercial venture can be devised. Although initial methods used to generate transgenic livestock resulted in random transgene insertion, the development of SCNT technology enabled homologous recombination gene targeting strategies to be used in livestock. Much has been accomplished using this approach. However, now we have the ability to change a specific base in the genome without leaving any other DNA mark, with no need for a transgene. With the advent of the genome editors this is now possible and like other significant technological leaps, the result is an even greater diversity of possible applications. Indeed, in merely 5 years, these 'molecular scissors' have enabled the production of more than 300 differently edited pigs, cattle, sheep and goats. The advent of genome editors has brought genetic engineering of livestock to a position where industry, the public and politicians are all eager to see real use of genetically engineered livestock to address societal needs. Since the first transgenic livestock reported just over three decades ago the field of livestock biotechnology has come a long way-but the most exciting period is just starting. PMID:26847670

  15. Cytogenomic profiling of breast cancer brain metastases reveals potential for repurposing targeted therapeutics

    PubMed Central

    Bollig-Fischer, Aliccia; Michelhaugh, Sharon K.; Wijesinghe, Priyanga; Dyson, Greg; Kruger, Adele; Palanisamy, Nallasivam; Choi, Lydia; Alosh, Baraa; Ali-Fehmi, Rouba; Mittal, Sandeep

    2015-01-01

    Breast cancer brain metastases remain a significant clinical problem. Chemotherapy is ineffective and a lack of treatment options result in poor patient outcomes. Targeted therapeutics have proven to be highly effective in primary breast cancer, but lack of molecular genomic characterization of metastatic brain tumors is hindering the development of new treatment regimens. Here we contribute to fill this void by reporting on gene copy number variation (CNV) in 10 breast cancer metastatic brain tumors, assayed by array comparative genomic hybridization (aCGH). Results were compared to a list of cancer genes verified by others to influence cancer. Cancer gene aberrations were identified in all specimens and pathway-level analysis was applied to aggregate data, which identified stem cell pluripotency pathway enrichment and highlighted recurring, significant amplification of SOX2, PIK3CA, NTRK1, GNAS, CTNNB1, and FGFR1. For a subset of the metastatic brain tumor samples (n=4) we compared patient-matched primary breast cancer specimens. The results of our CGH analysis and validation by alternative methods indicate that oncogenic signals driving growth of metastatic tumors exist in the original cancer. This report contributes support for more rapid development of new treatments of metastatic brain tumors, the use of genomic-based diagnostic tools and repurposed drug treatments. PMID:25970776

  16. A genome-wide survey reveals abundant rice blast R genes in resistant cultivars.

    PubMed

    Zhang, Xiaohui; Yang, Sihai; Wang, Jiao; Jia, Yanxiao; Huang, Ju; Tan, Shengjun; Zhong, Yan; Wang, Ling; Gu, Longjiang; Chen, Jian-Qun; Pan, Qinghua; Bergelson, Joy; Tian, Dacheng

    2015-10-01

    Plant resistance genes (R genes) harbor tremendous allelic diversity, constituting a robust immune system effective against microbial pathogens. Nevertheless, few functional R genes have been identified for even the best-studied pathosystems. Does this limited repertoire reflect specificity, with most R genes having been defeated by former pests, or do plants harbor a rich diversity of functional R genes, the composite behavior of which is yet to be characterized? Here, we survey 332 NBS-LRR genes cloned from five resistant Oryza sativa (rice) cultivars for their ability to confer recognition of 12 rice blast isolates when transformed into susceptible cultivars. Our survey reveals that 48.5% of the 132 NBS-LRR loci tested contain functional rice blast R genes, with most R genes deriving from multi-copy clades containing especially diversified loci. Each R gene recognized, on average, 2.42 of the 12 isolates screened. The abundant R genes identified in resistant genomes provide extraordinary redundancy in the ability of host genotypes to recognize particular isolates. If the same is true for other pathogens, many extant NBS-LRR genes retain functionality. Our success at identifying rice blast R genes also validates a highly efficient cloning and screening strategy. PMID:26248689

  17. Targeted Ablation of the Abcc6 Gene Results in Ectopic Mineralization of Connective Tissues

    PubMed Central

    Klement, John F.; Matsuzaki, Yasushi; Jiang, Qiu-Jie; Terlizzi, Joseph; Choi, Hae Young; Fujimoto, Norihiro; Li, Kehua; Pulkkinen, Leena; Birk, David E.; Sundberg, John P.; Uitto, Jouni

    2005-01-01

    Pseudoxanthoma elasticum (PXE), characterized by connective tissue mineralization of the skin, eyes, and cardiovascular system, is caused by mutations in the ABCC6 gene. ABCC6 encodes multidrug resistance-associated protein 6 (MRP6), which is expressed primarily in the liver and kidneys. Mechanisms producing ectopic mineralization as a result of these mutations remain unclear. To elucidate this complex disease, a transgenic mouse was generated by targeted ablation of the mouse Abcc6 gene. Abcc6 null mice were negative for Mrp6 expression in the liver, and complete necropsies revealed profound mineralization of several tissues, including skin, arterial blood vessels, and retina, while heterozygous animals were indistinguishable from the wild-type mice. Particularly striking was the mineralization of vibrissae, as confirmed by von Kossa and alizarin red stains. Electron microscopy revealed mineralization affecting both elastic structures and collagen fibers. Mineralization of vibrissae was noted as early as 5 weeks of age and was progressive with age in Abcc6−/− mice but was not observed in Abcc6+/− or Abcc6+/+ mice up to 2 years of age. A total body computerized tomography scan of Abcc6−/− mice revealed mineralization in skin and subcutaneous tissue as well as in the kidneys. These data demonstrate aberrant mineralization of soft tissues in PXE-affected organs, and, consequently, these mice recapitulate features of this complex disease. PMID:16135817

  18. TFIIS.h, a new target of p53, regulates transcription efficiency of pro-apoptotic bax gene

    PubMed Central

    Liao, Jun-Ming; Cao, Bo; Deng, Jun; Zhou, Xiang; Strong, Michael; Zeng, Shelya; Xiong, Jianping; Flemington, Erik; Lu, Hua

    2016-01-01

    Tumor suppressor p53 transcriptionally regulates hundreds of genes involved in various cellular functions. However, the detailed mechanisms underlying the selection of p53 targets in response to different stresses are still elusive. Here, we identify TFIIS.h, a transcription elongation factor, as a new transcriptional target of p53, and also show that it can enhance the efficiency of transcription elongation of apoptosis-associated bax gene, but not cell cycle-associated p21 (CDKN1A) gene. TFIIS.h is revealed as a p53 target through microarray analysis of RNAs extracted from cells treated with or without inauhzin (INZ), a p53 activator, and further confirmed by RT-q-PCR, western blot, luciferase reporter, and ChIP assays. Interestingly, knocking down TFIIS.h impairs, but overexpressing TFIIS.h promotes, induction of bax, but not other p53 targets including p21, by p53 activation. In addition, overexpression of TFIIS.h induces cell death in a bax- dependent fashion. These findings reveal a mechanism by which p53 utilizes TFIIS.h to selectively promote the transcriptional elongation of the bax gene, upsurging cell death in response to severe DNA damage. PMID:27005522

  19. Response to Nodal morphogen gradient is determined by the kinetics of target gene induction

    PubMed Central

    Dubrulle, Julien; Jordan, Benjamin M; Akhmetova, Laila; Farrell, Jeffrey A; Kim, Seok-Hyung; Solnica-Krezel, Lilianna; Schier, Alexander F

    2015-01-01

    Morphogen gradients expose cells to different signal concentrations and induce target genes with different ranges of expression. To determine how the Nodal morphogen gradient induces distinct gene expression patterns during zebrafish embryogenesis, we measured the activation dynamics of the signal transducer Smad2 and the expression kinetics of long- and short-range target genes. We found that threshold models based on ligand concentration are insufficient to predict the response of target genes. Instead, morphogen interpretation is shaped by the kinetics of target gene induction: the higher the rate of transcription and the earlier the onset of induction, the greater the spatial range of expression. Thus, the timing and magnitude of target gene expression can be used to modulate the range of expression and diversify the response to morphogen gradients. DOI: http://dx.doi.org/10.7554/eLife.05042.001 PMID:25869585

  20. Chitinase genes revealed and compared in bacterial isolates, DNA extracts and a metagenomic library from a phytopathogen suppressive soil

    SciTech Connect

    Hjort, K.; Bergstrom, M.; Adesina, M.F.; Jansson, J.K.; Smalla, K.; Sjoling, S.

    2009-09-01

    Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3) from bacterial isolates with antifungal and chitinase activities. Terminal-restriction fragment length polymorphism (T-RFLP) of chitinase genes revealed differences in amplified chitinase genes from the metagenomic library and the directly extracted DNA, but approximately 40% of the identified chitinase terminal-restriction fragments (TRFs) were found in both sources. All of the chitinase TRFs from the isolates were matched to TRFs in the directly extracted DNA and the metagenomic library. The most abundant chitinase TRF in the soil DNA and the metagenomic library corresponded to the TRF{sup 103} of the isolate, Streptomyces mutomycini and/or Streptomyces clavifer. There were good matches between T-RFLP profiles of chitinase gene fragments obtained from different sources of DNA. However, there were also differences in both the chitinase and the 16S rRNA gene T-RFLP patterns depending on the source of DNA, emphasizing the lack of complete coverage of the gene diversity by any of the approaches used.

  1. Reiterated Targeting Peptides on the Nanoparticle Surface Significantly Promote Targeted Vascular Endothelial Growth Factor Gene Delivery to Stem Cells.

    PubMed

    Wang, Dong-Dong; Yang, Mingying; Zhu, Ye; Mao, Chuanbin

    2015-12-14

    Nonviral gene delivery vectors hold great promise for gene therapy due to the safety concerns with viral vectors. However, the application of nonviral vectors is hindered by their low transfection efficiency. Herein, in order to tackle this challenge, we developed a nonviral vector integrating lipids, sleeping beauty transposon system and 8-mer stem cell targeting peptides for safe and efficient gene delivery to hard-to-transfect mesenchymal stem cells (MSCs). The 8-mer MSC-targeting peptides, when synthetically reiterated in three folds and chemically presented on the surface, significantly promoted the resultant lipid-based nanoparticles (LBNs) to deliver VEGF gene into MSCs with a high transfection efficiency (∼52%) and long-lasting gene expression (for longer than 170 h) when compared to nonreiterated peptides. However, the reiterated stem cell targeting peptides do not enable the highly efficient gene transfer to other control cells. This work suggests that the surface presentation of the reiterated stem cell-targeting peptides on the nonviral vectors is a promising method for improving the efficiency of cell-specific nonviral gene transfection in stem cells. PMID:26588028

  2. Correlation Analysis between SNP and Expression Arrays in Gliomas Identify Potentially Relevant Targets Genes1

    PubMed Central

    Kotliarov, Yuri; Kotliarova, Svetlana; Charong, Nurdina; Li, Aiguo; Walling, Jennifer; Aquilanti, Elisa; Ahn, Susie; Steed, Mary Ellen; Su, Qin; Center, Angela; Zenklusen, Jean C; Fine, Howard A.

    2008-01-01

    Primary brain tumors are a major cause of cancer mortality in the United States. Therapy for gliomas, the most common type of primary brain tumors, remains suboptimal. The development of improved therapeutics will require greater knowledge of the biology of gliomas at both the genomic and transcriptional levels. We have previously reported whole genome profiling of chromosome copy number alterations (CNA) in gliomas, and now present our findings on how those changes may affect transcription of genes that may be involved in tumor induction and progression. By calculating correlation values of mRNA expression vs. DNA copy number average in a moving window around a given RNA probeset, biologically relevant information can be gained that is obscured by the analysis of a single data type. Correlation coefficients ranged from −0.6 to 0.7; highly significant when compared to previously studies. Most correlated genes are located on chromosomes 1, 7, 9, 10, 13, 14, 19, 20 and 22, chromosomes known to have genomic alterations in gliomas. Additionally, we were able to identify CNAs whose gene expression correlation suggests possible epigenetic regulation. This analysis revealed a number of interesting candidates such as CXCL12, PTER, LRRN6C, among others. The results have been verified using real-time PCR and methylation sequencing assays. These data will further help differentiate genes involved in the induction and/or maintenance of the tumorigenic process from those that are mere passenger mutations, thereby enriching for a population of potentially new therapeutic molecular targets. PMID:19190341

  3. LPTS: A Novel Tumor Suppressor Gene and a Promising Drug Target for Cancer Intervention.

    PubMed

    Baichuan, Li; Cao, Songshen; Liu, Yunlai

    2015-01-01

    Liver-related putative tumor suppressor (lpts) is a liver-related tumor suppressor candidate gene initially isolated by positional candidate cloning method. Three translation products of lpts gene are found, that are LPTS-L, LPTS-S and LPTS-M respectively. The gene highly expresses in normal tissues but lowly in cancer tissues. The LPTS proteins can suppress the activity of telomerase and trigger apoptosis for tumor cells in vivo and in vitro, despite that the detailed anti-cancer mechanism remains undefined. This review successively describes the lpts genomic assembly, transcriptional regulation and structure-activity evaluation of different LPTS isoforms; then it represents the LPTS binding partners, for example Pin2/TRF1 and MCRS2, which play important roles in decreasing telomerase activity, which benefits to reveal the anticancer mechanism; subsequently, it surveys several patents of recombinant LPTS proteins such as TAT-LPTS-LC, PinX1/C-G4S-9R-G4S-mBAFF and PinX1/C-9R-mBAF that can inhibit the growth of tumor cells. Lpts gene is becoming a promising drug target for cancer intervention owing to its powerful inhibition efficacy on telomerase activity, and recombinant LPTS proteins claimed by a couple of patents seem to be potential anti-cancer agents. PMID:25479038

  4. Magnetic nanoparticles for targeted therapeutic gene delivery and magnetic-inducing heating on hepatoma

    NASA Astrophysics Data System (ADS)

    Yuan, Chenyan; An, Yanli; Zhang, Jia; Li, Hongbo; Zhang, Hao; Wang, Ling; Zhang, Dongsheng

    2014-08-01

    Gene therapy holds great promise for treating cancers, but their clinical applications are being hampered due to uncontrolled gene delivery and expression. To develop a targeted, safe and efficient tumor therapy system, we constructed a tissue-specific suicide gene delivery system by using magnetic nanoparticles (MNPs) as carriers for the combination of gene therapy and hyperthermia on hepatoma. The suicide gene was hepatoma-targeted and hypoxia-enhanced, and the MNPs possessed the ability to elevate temperature to the effective range for tumor hyperthermia as imposed on an alternating magnetic field (AMF). The tumoricidal effects of targeted gene therapy associated with hyperthermia were evaluated in vitro and in vivo. The experiment demonstrated that hyperthermia combined with a targeted gene therapy system proffer an effective tool for tumor therapy with high selectivity and the synergistic effect of hepatoma suppression.

  5. A protocol for construction of gene targeting vectors and generation of homologous recombinant ES cells

    PubMed Central

    Bouabe, Hicham; Okkenhaug, Klaus

    2015-01-01

    Summary The completion of human and mouse genome sequencing has confronted us with huge amount of data sequences that certainly need decades and many generations of scientists to be reasonably interpreted and assigned to physiological functions, and subsequently fruitfully translated into medical application. A means to assess the function of genes provides gene targeting in mouse embryonic stem (ES) cells that enables to introduce site-specific modifications in the mouse genome, and analyze their physiological consequences. Gene targeting enables almost any type of genetic modifications of interest, ranging from gene insertion (e.g. insertion of human-specific genes or reporter genes), gene disruption, point mutations, short and long range deletions, inversions. Site-specific modification into the genome of ES cells can be reached by homologous recombination using targeting vectors. Here, we describe a protocol to generate targeting constructs and homologous recombinant ES cells. PMID:23996269

  6. Global Analysis of Fission Yeast Mating Genes Reveals New Autophagy Factors

    PubMed Central

    Sun, Ling-Ling; Shen, En-Zhi; Yang, Bing; Dong, Meng-Qiu; He, Wan-Zhong; Du, Li-Lin

    2013-01-01

    Macroautophagy (autophagy) is crucial for cell survival during starvation and plays important roles in animal development and human diseases. Molecular understanding of autophagy has mainly come from the budding yeast Saccharomyces cerevisiae, and it remains unclear to what extent the mechanisms are the same in other organisms. Here, through screening the mating phenotype of a genome-wide deletion collection of the fission yeast Schizosaccharomyces pombe, we obtained a comprehensive catalog of autophagy genes in this highly tractable organism, including genes encoding three heretofore unidentified core Atg proteins, Atg10, Atg14, and Atg16, and two novel factors, Ctl1 and Fsc1. We systematically examined the subcellular localization of fission yeast autophagy factors for the first time and characterized the phenotypes of their mutants, thereby uncovering both similarities and differences between the two yeasts. Unlike budding yeast, all three Atg18/WIPI proteins in fission yeast are essential for autophagy, and we found that they play different roles, with Atg18a uniquely required for the targeting of the Atg12–Atg5·Atg16 complex. Our investigation of the two novel factors revealed unforeseen autophagy mechanisms. The choline transporter-like protein Ctl1 interacts with Atg9 and is required for autophagosome formation. The fasciclin domain protein Fsc1 localizes to the vacuole membrane and is required for autophagosome-vacuole fusion but not other vacuolar fusion events. Our study sheds new light on the evolutionary diversity of the autophagy machinery and establishes the fission yeast as a useful model for dissecting the mechanisms of autophagy. PMID:23950735

  7. Expression of a novel P22 ORFan gene reveals the phage carrier state in Salmonella typhimurium.

    PubMed

    Cenens, William; Mebrhatu, Mehari T; Makumi, Angella; Ceyssens, Pieter-Jan; Lavigne, Rob; Van Houdt, Rob; Taddei, François; Aertsen, Abram

    2013-01-01

    We discovered a novel interaction between phage P22 and its host Salmonella Typhimurium LT2 that is characterized by a phage mediated and targeted derepression of the host dgo operon. Upon further investigation, this interaction was found to be instigated by an ORFan gene (designated pid for phage P22 encoded instigator of dgo expression) located on a previously unannotated moron locus in the late region of the P22 genome, and encoding an 86 amino acid protein of 9.3 kDa. Surprisingly, the Pid/dgo interaction was not observed during strict lytic or lysogenic proliferation of P22, and expression of pid was instead found to arise in cells that upon infection stably maintained an unintegrated phage chromosome that segregated asymmetrically upon subsequent cell divisions. Interestingly, among the emerging siblings, the feature of pid expression remained tightly linked to the cell inheriting this phage carrier state and became quenched in the other. As such, this study is the first to reveal molecular and genetic markers authenticating pseudolysogenic development, thereby exposing a novel mechanism, timing, and populational distribution in the realm of phage-host interactions. PMID:23483857

  8. Chemopreventive agents alters global gene expression pattern: predicting their mode of action and targets.

    PubMed

    Narayanan, Bhagavathi A

    2006-12-01

    Chemoprevention has the potential to be a major component of colon, breast, prostate and lung cancer control. Epidemiological, experimental, and clinical studies provide evidence that antioxidants, anti-inflammatory agents, n-3 polyunsaturated fatty acids and several other phytochemicals possess unique modes of action against cancer growth. However, the mode of action of several of these agents at the gene transcription level is not completely understood. Completion of the human genome sequence and the advent of DNA microarrays using cDNAs enhanced the detection and identification of hundreds of differentially expressed genes in response to anticancer drugs or chemopreventive agents. In this review, we are presenting an extensive analysis of the key findings from studies using potential chemopreventive agents on global gene expression patterns, which lead to the identification of cancer drug targets. The summary of the study reports discussed in this review explains the extent of gene alterations mediated by more than 20 compounds including antioxidants, fatty acids, NSAIDs, phytochemicals, retinoids, selenium, vitamins, aromatase inhibitor, lovastatin, oltipraz, salvicine, and zinc. The findings from these studies further reveal the utility of DNA microarray in characterizing and quantifying the differentially expressed genes that are possibly reprogrammed by the above agents against colon, breast, prostate, lung, liver, pancreatic and other cancer types. Phenolic antioxidant resveratrol found in berries and grapes inhibits the formation of prostate tumors by acting on the regulatory genes such as p53 while activating a cascade of genes involved in cell cycle and apoptosis including p300, Apaf-1, cdk inhibitor p21, p57 (KIP2), p53 induced Pig 7, Pig 8, Pig 10, cyclin D, DNA fragmentation factor 45. The group of genes significantly altered by selenium includes cyclin D1, cdk5, cdk4, cdk2, cdc25A and GADD 153. Vitamine D shows impact on p21(Waf1/Cip1) p27 cyclin B

  9. Cross-species transcriptomic approach reveals genes in hamster implantation sites.

    PubMed

    Lei, Wei; Herington, Jennifer; Galindo, Cristi L; Ding, Tianbing; Brown, Naoko; Reese, Jeff; Paria, Bibhash C

    2014-12-01

    The mouse model has greatly contributed to understanding molecular mechanisms involved in the regulation of progesterone (P4) plus estrogen (E)-dependent blastocyst implantation process. However, little is known about contributory molecular mechanisms of the P4-only-dependent blastocyst implantation process that occurs in species such as hamsters, guineapigs, rabbits, pigs, rhesus monkeys, and perhaps humans. We used the hamster as a model of P4-only-dependent blastocyst implantation and carried out cross-species microarray (CSM) analyses to reveal differentially expressed genes at the blastocyst implantation site (BIS), in order to advance the understanding of molecular mechanisms of implantation. Upregulation of 112 genes and downregulation of 77 genes at the BIS were identified using a mouse microarray platform, while use of the human microarray revealed 62 up- and 38 down-regulated genes at the BIS. Excitingly, a sizable number of genes (30 up- and 11 down-regulated genes) were identified as a shared pool by both CSMs. Real-time RT-PCR and in situ hybridization validated the expression patterns of several up- and down-regulated genes identified by both CSMs at the hamster and mouse BIS to demonstrate the merit of CSM findings across species, in addition to revealing genes specific to hamsters. Functional annotation analysis found that genes involved in the spliceosome, proteasome, and ubiquination pathways are enriched at the hamster BIS, while genes associated with tight junction, SAPK/JNK signaling, and PPARα/RXRα signalings are repressed at the BIS. Overall, this study provides a pool of genes and evidence of their participation in up- and down-regulated cellular functions/pathways at the hamster BIS. PMID:25252651

  10. A Genetic Mosaic Screen Reveals Ecdysone-Responsive Genes Regulating Drosophila Oogenesis.

    PubMed

    Ables, Elizabeth T; Hwang, Grace H; Finger, Danielle S; Hinnant, Taylor D; Drummond-Barbosa, Daniela

    2016-01-01

    Multiple aspects of Drosophila oogenesis, including germline stem cell activity, germ cell differentiation, and follicle survival, are regulated by the steroid hormone ecdysone. While the transcriptional targets of ecdysone signaling during development have been studied extensively, targets in the ovary remain largely unknown. Early studies of salivary gland polytene chromosomes led to a model in which ecdysone stimulates a hierarchical transcriptional cascade, wherein a core group of ecdysone-sensitive transcription factors induce tissue-specific responses by activating secondary branches of transcriptional targets. More recently, genome-wide approaches have identified hundreds of putative ecdysone-responsive targets. Determining whether these putative targets represent bona fide targets in vivo, however, requires that they be tested via traditional mutant analysis in a cell-type specific fashion. To investigate the molecular mechanisms whereby ecdysone signaling regulates oogenesis, we used genetic mosaic analysis to screen putative ecdysone-responsive genes for novel roles in the control of the earliest steps of oogenesis. We identified a cohort of genes required for stem cell maintenance, stem and progenitor cell proliferation, and follicle encapsulation, growth, and survival. These genes encode transcription factors, chromatin modulators, and factors required for RNA transport, stability, and ribosome biogenesis, suggesting that ecdysone might control a wide range of molecular processes during oogenesis. Our results suggest that, although ecdysone target genes are known to have cell type-specific roles, many ecdysone response genes that control larval or pupal cell types at developmental transitions are used reiteratively in the adult ovary. These results provide novel insights into the molecular mechanisms by which ecdysone signaling controls oogenesis, laying new ground for future studies. PMID:27226164

  11. A Genetic Mosaic Screen Reveals Ecdysone-Responsive Genes Regulating Drosophila Oogenesis

    PubMed Central

    Ables, Elizabeth T.; Hwang, Grace H.; Finger, Danielle S.; Hinnant, Taylor D.; Drummond-Barbosa, Daniela

    2016-01-01

    Multiple aspects of Drosophila oogenesis, including germline stem cell activity, germ cell differentiation, and follicle survival, are regulated by the steroid hormone ecdysone. While the transcriptional targets of ecdysone signaling during development have been studied extensively, targets in the ovary remain largely unknown. Early studies of salivary gland polytene chromosomes led to a model in which ecdysone stimulates a hierarchical transcriptional cascade, wherein a core group of ecdysone-sensitive transcription factors induce tissue-specific responses by activating secondary branches of transcriptional targets. More recently, genome-wide approaches have identified hundreds of putative ecdysone-responsive targets. Determining whether these putative targets represent bona fide targets in vivo, however, requires that they be tested via traditional mutant analysis in a cell-type specific fashion. To investigate the molecular mechanisms whereby ecdysone signaling regulates oogenesis, we used genetic mosaic analysis to screen putative ecdysone-responsive genes for novel roles in the control of the earliest steps of oogenesis. We identified a cohort of genes required for stem cell maintenance, stem and progenitor cell proliferation, and follicle encapsulation, growth, and survival. These genes encode transcription factors, chromatin modulators, and factors required for RNA transport, stability, and ribosome biogenesis, suggesting that ecdysone might control a wide range of molecular processes during oogenesis. Our results suggest that, although ecdysone target genes are known to have cell type-specific roles, many ecdysone response genes that control larval or pupal cell types at developmental transitions are used reiteratively in the adult ovary. These results provide novel insights into the molecular mechanisms by which ecdysone signaling controls oogenesis, laying new ground for future studies. PMID:27226164

  12. Complete genome-wide screening and subtractive genomic approach revealed new virulence factors, potential drug targets against bio-war pathogen Brucella melitensis 16M

    PubMed Central

    Pradeepkiran, Jangampalli Adi; Sainath, Sri Bhashyam; Kumar, Konidala Kranthi; Bhaskar, Matcha

    2015-01-01

    Brucella melitensis 16M is a Gram-negative coccobacillus that infects both animals and humans. It causes a disease known as brucellosis, which is characterized by acute febrile illness in humans and causes abortions in livestock. To prevent and control brucellosis, identification of putative drug targets is crucial. The present study aimed to identify drug targets in B. melitensis 16M by using a subtractive genomic approach. We used available database repositories (Database of Essential Genes, Kyoto Encyclopedia of Genes and Genomes Automatic Annotation Server, and Kyoto Encyclopedia of Genes and Genomes) to identify putative genes that are nonhomologous to humans and essential for pathogen B. melitensis 16M. The results revealed that among 3 Mb genome size of pathogen, 53 putative characterized and 13 uncharacterized hypothetical genes were identified; further, from Basic Local Alignment Search Tool protein analysis, one hypothetical protein showed a close resemblance (50%) to Silicibacter pomeroyi DUF1285 family protein (2RE3). A further homology model of the target was constructed using MODELLER 9.12 and optimized through variable target function method by molecular dynamics optimization with simulating annealing. The stereochemical quality of the restrained model was evaluated by PROCHECK, VERIFY-3D, ERRAT, and WHATIF servers. Furthermore, structure-based virtual screening was carried out against the predicted active site of the respective protein using the glycerol structural analogs from the PubChem database. We identified five best inhibitors with strong affinities, stable interactions, and also with reliable drug-like properties. Hence, these leads might be used as the most effective inhibitors of modeled protein. The outcome of the present work of virtual screening of putative gene targets might facilitate design of potential drugs for better treatment against brucellosis. PMID:25834405

  13. A gene locus for targeted ectopic gene integration in Zymoseptoria tritici.

    PubMed

    Kilaru, S; Schuster, M; Latz, M; Das Gupta, S; Steinberg, N; Fones, H; Gurr, S J; Talbot, N J; Steinberg, G

    2015-06-01

    Understanding the cellular organization and biology of fungal pathogens requires accurate methods for genomic integration of mutant alleles or fluorescent fusion-protein constructs. In Zymoseptoria tritici, this can be achieved by integrating of plasmid DNA randomly into the genome of this wheat pathogen. However, untargeted ectopic integration carries the risk of unwanted side effects, such as altered gene expression, due to targeting regulatory elements, or gene disruption following integration into protein-coding regions of the genome. Here, we establish the succinate dehydrogenase (sdi1) locus as a single "soft-landing" site for targeted ectopic integration of genetic constructs by using a carboxin-resistant sdi1(R) allele, carrying the point-mutation H267L. We use various green and red fluorescent fusion constructs and show that 97% of all transformants integrate correctly into the sdi1 locus as single copies. We also demonstrate that such integration does not affect the pathogenicity of Z. tritici, and thus the sdi1 locus is a useful tool for virulence analysis in genetically modified Z. tritici strains. Furthermore, we have developed a vector which facilitates yeast recombination cloning and thus allows assembly of multiple overlapping DNA fragments in a single cloning step for high throughput vector and strain generation. PMID:26092798

  14. In vivo phosphoproteomics analysis reveals the cardiac targets of β-adrenergic receptor signaling.

    PubMed

    Lundby, Alicia; Andersen, Martin N; Steffensen, Annette B; Horn, Heiko; Kelstrup, Christian D; Francavilla, Chiara; Jensen, Lars J; Schmitt, Nicole; Thomsen, Morten B; Olsen, Jesper V

    2013-06-01

    β-Blockers are widely used to prevent cardiac arrhythmias and to treat hypertension by inhibiting β-adrenergic receptors (βARs) and thus decreasing contractility and heart rate. βARs initiate phosphorylation-dependent signaling cascades, but only a small number of the target proteins are known. We used quantitative in vivo phosphoproteomics to identify 670 site-specific phosphorylation changes in murine hearts in response to acute treatment with specific βAR agonists. The residues adjacent to the regulated phosphorylation sites exhibited a sequence-specific preference (R-X-X-pS/T), and integrative analysis of sequence motifs and interaction networks suggested that the kinases AMPK (adenosine 5'-monophosphate-activated protein kinase), Akt, and mTOR (mammalian target of rapamycin) mediate βAR signaling, in addition to the well-established pathways mediated by PKA (cyclic adenosine monophosphate-dependent protein kinase) and CaMKII (calcium/calmodulin-dependent protein kinase type II). We found specific regulation of phosphorylation sites on six ion channels and transporters that mediate increased ion fluxes at higher heart rates, and we showed that phosphorylation of one of these, Ser(92) of the potassium channel KV7.1, increased current amplitude. Our data set represents a quantitative analysis of phosphorylated proteins regulated in vivo upon stimulation of seven-transmembrane receptors, and our findings reveal previously unknown phosphorylation sites that regulate myocardial contractility, suggesting new potential targets for the treatment of heart disease and hypertension. PMID:23737553

  15. Genome-Wide Mapping of Targets of Maize Histone Deacetylase HDA101 Reveals Its Function and Regulatory Mechanism during Seed Development[OPEN

    PubMed Central

    Yang, Hua; Liu, Xinye; Xin, Mingming; Du, Jinkun; Hu, Zhaorong; Peng, HuiRu; Sun, Qixin; Ni, Zhongfu; Yao, Yingyin

    2016-01-01

    Histone deacetylases (HDACs) regulate histone acetylation levels by removing the acetyl group from lysine residues. The maize (Zea mays) HDAC HDA101 influences several aspects of development, including kernel size; however, the molecular mechanism by which HDA101 affects kernel development remains unknown. In this study, we find that HDA101 regulates the expression of transfer cell-specific genes, suggesting that their misregulation may be associated with the defects in differentiation of endosperm transfer cells and smaller kernels observed in hda101 mutants. To investigate HDA101 function during the early stages of seed development, we performed genome-wide mapping of HDA101 binding sites. We observed that, like mammalian HDACs, HDA101 mainly targets highly and intermediately expressed genes. Although loss of HDA101 can induce histone hyperacetylation of its direct targets, this often does not involve variation in transcript levels. A small subset of inactive genes that must be negatively regulated during kernel development is also targeted by HDA101 and its loss leads to hyperacetylation and increased expression of these inactive genes. Finally, we report that HDA101 interacts with members of different chromatin remodeling complexes, such as NFC103/MSI1 and SNL1/SIN3-like protein corepressors. Taken together, our results reveal a complex genetic network regulated by HDA101 during seed development and provide insight into the different mechanisms of HDA101-mediated regulation of transcriptionally active and inactive genes. PMID:26908760

  16. Genome-Wide Mapping of Targets of Maize Histone Deacetylase HDA101 Reveals Its Function and Regulatory Mechanism during Seed Development.

    PubMed

    Yang, Hua; Liu, Xinye; Xin, Mingming; Du, Jinkun; Hu, Zhaorong; Peng, HuiRu; Rossi, Vincenzo; Sun, Qixin; Ni, Zhongfu; Yao, Yingyin

    2016-03-01

    Histone deacetylases (HDACs) regulate histone acetylation levels by removing the acetyl group from lysine residues. The maize (Zea mays)HDACHDA101 influences several aspects of development, including kernel size; however, the molecular mechanism by which HDA101 affects kernel development remains unknown. In this study, we find that HDA101 regulates the expression of transfer cell-specific genes, suggesting that their misregulation may be associated with the defects in differentiation of endosperm transfer cells and smaller kernels observed inhda101mutants. To investigate HDA101 function during the early stages of seed development, we performed genome-wide mapping of HDA101 binding sites. We observed that, like mammalianHDACs, HDA101 mainly targets highly and intermediately expressed genes. Although loss of HDA101 can induce histone hyperacetylation of its direct targets, this often does not involve variation in transcript levels. A small subset of inactive genes that must be negatively regulated during kernel development is also targeted by HDA101 and its loss leads to hyperacetylation and increased expression of these inactive genes. Finally, we report that HDA101 interacts with members of different chromatin remodeling complexes, such as NFC103/MSI1 and SNL1/SIN3-like protein corepressors. Taken together, our results reveal a complex genetic network regulated by HDA101 during seed development and provide insight into the different mechanisms of HDA101-mediated regulation of transcriptionally active and inactive genes. PMID:26908760

  17. Population and Functional Genomics of Neisseria Revealed with Gene-by-Gene Approaches

    PubMed Central

    Harrison, Odile B.

    2016-01-01

    Rapid low-cost whole-genome sequencing (WGS) is revolutionizing microbiology; however, complementary advances in accessible, reproducible, and rapid analysis techniques are required to realize the potential of these data. Here, investigations of the genus Neisseria illustrated the gene-by-gene conceptual approach to the organization and analysis of WGS data. Using the gene and its link to phenotype as a starting point, the BIGSdb database, which powers the PubMLST databases, enables the assembly of large open-access collections of annotated genomes that provide insight into the evolution of the Neisseria, the epidemiology of meningococcal and gonococcal disease, and mechanisms of Neisseria pathogenicity. PMID:27098959

  18. Population and Functional Genomics of Neisseria Revealed with Gene-by-Gene Approaches.

    PubMed

    Maiden, Martin C J; Harrison, Odile B

    2016-08-01

    Rapid low-cost whole-genome sequencing (WGS) is revolutionizing microbiology; however, complementary advances in accessible, reproducible, and rapid analysis techniques are required to realize the potential of these data. Here, investigations of the genus Neisseria illustrated the gene-by-gene conceptual approach to the organization and analysis of WGS data. Using the gene and its link to phenotype as a starting point, the BIGSdb database, which powers the PubMLST databases, enables the assembly of large open-access collections of annotated genomes that provide insight into the evolution of the Neisseria, the epidemiology of meningococcal and gonococcal disease, and mechanisms of Neisseria pathogenicity. PMID:27098959

  19. Strategies for Profiling Single Mouse Intestinal Epithelial Cells by Targeted Gene Expression

    PubMed Central

    McDowell, W.; Box, A.; Staehling, K.; Wang, F.; Li, L.; Zueckert-Gaudenz, K.

    2014-01-01

    Targeted gene expression profiling of single cells permits the study of heterogeneity in cell populations. Here, a pool of mouse intestinal crypt-base CD44+/GRP78- cells was collected by fluorescence activated cell sorting. Aliquots were either loaded onto Fluidigm's C1 System for microfluidic cell capture and cDNA synthesis in nanoliter volumes, or flow-sorted directly into individual PCR plate wells for cDNA synthesis in microliter volumes. The pre-amplified cDNAs were transferred to the BioMark System for EvaGreen real-time PCR. The two sample preparation methods were compared by expression analysis of 86 genes, using Fluidigm's SINGuLAR R-scripts. After outlier identification, gene expression values from 42% of the “C1” and 92% of the “flow” wells were retained. For 55 of the genes, expression was measured in both the “C1” and “flow” cells. Genes with a high variance in expression likely stemming from the sample preparation method and/or unspecific amplification were removed. Hierarchical clustering on the remaining data revealed gene clusters that contributed to the expected Lgr5hi and Lgr5lo intestinal stem cell (ISC) populations as well as a small population of differentiated cells. The subpopulations could be defined by either method. However, as ISCs quickly undergo apoptosis at room temperature, the use of the C1 System provided no clear advantage over the direct sorting of the fragile cells into lysis/RT reaction buffer. Specifically, the C1 quality control step to verify the number of captured cells and cell viability was omitted to accelerate processing.

  20. Genomic Analysis Reveals Contrasting PIFq Contribution to Diurnal Rhythmic Gene Expression in PIF-Induced and -Repressed Genes.

    PubMed

    Martin, Guiomar; Soy, Judit; Monte, Elena

    2016-01-01

    Members of the PIF quartet (PIFq; PIF1, PIF3, PIF4, and PIF5) collectively contribute to induce growth in Arabidopsis seedlings under short day (SD) conditions, specifically promoting elongation at dawn. Their action involves the direct regulation of growth-related and hormone-associated genes. However, a comprehensive definition of the PIFq-regulated transcriptome under SD is still lacking. We have recently shown that SD and free-running (LL) conditions correspond to "growth" and "no growth" conditions, respectively, correlating with greater abundance of PIF protein in SD. Here, we present a genomic analysis whereby we first define SD-regulated genes at dawn compared to LL in the wild type, followed by identification of those SD-regulated genes whose expression depends on the presence of PIFq. By using this sequential strategy, we have identified 349 PIF/SD-regulated genes, approximately 55% induced and 42% repressed by both SD and PIFq. Comparison with available databases indicates that PIF/SD-induced and PIF/SD-repressed sets are differently phased at dawn and mid-morning, respectively. In addition, we found that whereas rhythmicity of the PIF/SD-induced gene set is lost in LL, most PIF/SD-repressed genes keep their rhythmicity in LL, suggesting differential regulation of both gene sets by the circadian clock. Moreover, we also uncovered distinct overrepresented functions in the induced and repressed gene sets, in accord with previous studies in other examined PIF-regulated processes. Interestingly, promoter analyses showed that, whereas PIF/SD-induced genes are enriched in direct PIF targets, PIF/SD-repressed genes are mostly indirectly regulated by the PIFs and might be more enriched in ABA-regulated genes. PMID:27458465

  1. Genomic Analysis Reveals Contrasting PIFq Contribution to Diurnal Rhythmic Gene Expression in PIF-Induced and -Repressed Genes

    PubMed Central

    Martin, Guiomar; Soy, Judit; Monte, Elena

    2016-01-01

    Members of the PIF quartet (PIFq; PIF1, PIF3, PIF4, and PIF5) collectively contribute to induce growth in Arabidopsis seedlings under short day (SD) conditions, specifically promoting elongation at dawn. Their action involves the direct regulation of growth-related and hormone-associated genes. However, a comprehensive definition of the PIFq-regulated transcriptome under SD is still lacking. We have recently shown that SD and free-running (LL) conditions correspond to “growth” and “no growth” conditions, respectively, correlating with greater abundance of PIF protein in SD. Here, we present a genomic analysis whereby we first define SD-regulated genes at dawn compared to LL in the wild type, followed by identification of those SD-regulated genes whose expression depends on the presence of PIFq. By using this sequential strategy, we have identified 349 PIF/SD-regulated genes, approximately 55% induced and 42% repressed by both SD and PIFq. Comparison with available databases indicates that PIF/SD-induced and PIF/SD-repressed sets are differently phased at dawn and mid-morning, respectively. In addition, we found that whereas rhythmicity of the PIF/SD-induced gene set is lost in LL, most PIF/SD-repressed genes keep their rhythmicity in LL, suggesting differential regulation of both gene sets by the circadian clock. Moreover, we also uncovered distinct overrepresented functions in the induced and repressed gene sets, in accord with previous studies in other examined PIF-regulated processes. Interestingly, promoter analyses showed that, whereas PIF/SD-induced genes are enriched in direct PIF targets, PIF/SD-repressed genes are mostly indirectly regulated by the PIFs and might be more enriched in ABA-regulated genes. PMID:27458465

  2. Genome-wide analysis of murine renal distal convoluted tubular cells for the target genes of mineralocorticoid receptor

    SciTech Connect

    Ueda, Kohei; Fujiki, Katsunori; Shirahige, Katsuhiko; Gomez-Sanchez, Celso E.; Fujita, Toshiro; Nangaku, Masaomi; Nagase, Miki

    2014-02-28

    Highlights: • We define a target gene of MR as that with MR-binding to the adjacent region of DNA. • We use ChIP-seq analysis in combination with microarray. • We, for the first time, explore the genome-wide binding profile of MR. • We reveal 5 genes as the direct target genes of MR in the renal epithelial cell-line. - Abstract: Background and objective: Mineralocorticoid receptor (MR) is a member of nuclear receptor family proteins and contributes to fluid homeostasis in the kidney. Although aldosterone-MR pathway induces several gene expressions in the kidney, it is often unclear whether the gene expressions are accompanied by direct regulations of MR through its binding to the regulatory region of each gene. The purpose of this study is to identify the direct target genes of MR in a murine distal convoluted tubular epithelial cell-line (mDCT). Methods: We analyzed the DNA samples of mDCT cells overexpressing 3xFLAG-hMR after treatment with 10{sup −7} M aldosterone for 1 h by chromatin immunoprecipitation with deep-sequence (ChIP-seq) and mRNA of the cell-line with treatment of 10{sup −7} M aldosterone for 3 h by microarray. Results: 3xFLAG-hMR overexpressed in mDCT cells accumulated in the nucleus in response to 10{sup −9} M aldosterone. Twenty-five genes were indicated as the candidate target genes of MR by ChIP-seq and microarray analyses. Five genes, Sgk1, Fkbp5, Rasl12, Tns1 and Tsc22d3 (Gilz), were validated as the direct target genes of MR by quantitative RT-qPCR and ChIP-qPCR. MR binding regions adjacent to Ctgf and Serpine1 were also validated. Conclusions: We, for the first time, captured the genome-wide distribution of MR in mDCT cells and, furthermore, identified five MR target genes in the cell-line. These results will contribute to further studies on the mechanisms of kidney diseases.

  3. Computational gene expression profiling under salt stress reveals patterns of co-expression.

    PubMed

    Sanchita; Sharma, Ashok

    2016-03-01

    Plants respond differently to environmental conditions. Among various abiotic stresses, salt stress is a condition where excess salt in soil causes inhibition of plant growth. To understand the response of plants to the stress conditions, identification of the responsible genes is required. Clustering is a data mining technique used to group the genes with similar expression. The genes of a cluster show similar expression and function. We applied clustering algorithms on gene expression data of Solanum tuberosum showing differential expression in Capsicum annuum under salt stress. The clusters, which were common in multiple algorithms were taken further for analysis. Principal component analysis (PCA) further validated the findings of other cluster algorithms by visualizing their clusters in three-dimensional space. Functional annotation results revealed that most of the genes were involved in stress related responses. Our findings suggest that these algorithms may be helpful in the prediction of the function of co-expressed genes. PMID:26981411

  4. Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes

    EPA Science Inventory

    Aims: Compare specificity and sensitivity of quantitative PCR (qPCR) assays targeting single and multi-copy gene regions of Escherichia coli. Methods and Results: A previously reported assay targeting the uidA gene (uidA405) was used as the basis for comparing the taxono...

  5. Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA.

    PubMed

    Svitashev, Sergei; Young, Joshua K; Schwartz, Christine; Gao, Huirong; Falco, S Carl; Cigan, A Mark

    2015-10-01

    Targeted mutagenesis, editing of endogenous maize (Zea mays) genes, and site-specific insertion of a trait gene using clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)-guide RNA technology are reported in maize. DNA vectors expressing maize codon-optimized Streptococcus pyogenes Cas9 endonuclease and single guide RNAs were cointroduced with or without DNA repair templates into maize immature embryos by biolistic transformation targeting five different genomic regions: upstream of the liguleless1 (LIG1) gene, male fertility genes (Ms26 and Ms45), and acetolactate synthase (ALS) genes (ALS1 and ALS2). Mutations were subsequently identified at all sites targeted, and plants containing biallelic multiplex mutations at LIG1, Ms26, and Ms45 were recovered. Biolistic delivery of guide RNAs (as RNA molecules) directly into immature embryo cells containing preintegrated Cas9 also resulted in targeted mutations. Editing the ALS2 gene using either single-stranded oligonucleotides or double-stranded DNA vectors as repair templates yielded chlorsulfuron-resistant plants. Double-strand breaks generated by RNA-guided Cas9 endonuclease also stimulated insertion of a trait gene at a site near LIG1 by homology-directed repair. Progeny showed expected Mendelian segregation of mutations, edits, and targeted gene insertions. The examples reported in this study demonstrate the utility of Cas9-guide RNA technology as a plant genome editing tool to enhance plant breeding and crop research needed to meet growing agriculture demands of the future. PMID:26269544

  6. Reverse Chemical Genetics: Comprehensive Fitness Profiling Reveals the Spectrum of Drug Target Interactions.

    PubMed

    Wong, Lai H; Sinha, Sunita; Bergeron, Julien R; Mellor, Joseph C; Giaever, Guri; Flaherty, Patrick; Nislow, Corey

    2016-09-01

    The emergence and prevalence of drug resistance demands streamlined strategies to identify drug resistant variants in a fast, systematic and cost-effective way. Methods commonly used to understand and predict drug resistance rely on limited clinical studies from patients who are refractory to drugs or on laborious evolution experiments with poor coverage of the gene variants. Here, we report an integrative functional variomics methodology combining deep sequencing and a Bayesian statistical model to provide a comprehensive list of drug resistance alleles from complex variant populations. Dihydrofolate reductase, the target of methotrexate chemotherapy drug, was used as a model to identify functional mutant alleles correlated with methotrexate resistance. This systematic approach identified previously reported resistance mutations, as well as novel point mutations that were validated in vivo. Use of this systematic strategy as a routine diagnostics tool widens the scope of successful drug research and development. PMID:27588687

  7. Identification of the human ApoAV gene as a novel ROR{alpha} target gene

    SciTech Connect

    Lind, Ulrika; Nilsson, Tina; McPheat, Jane; Stroemstedt, Per-Erik; Bamberg, Krister; Balendran, Clare; Kang, Daiwu . E-mail: Daiwu.Kang@astrazeneca.com

    2005-04-29

    Retinoic acid receptor-related orphan receptor-{alpha} (ROR{alpha}) (NR1F1) is an orphan nuclear receptor with a potential role in metabolism. Previous studies have shown that ROR{alpha} regulates transcription of the murine Apolipoprotein AI gene and human Apolipoprotein CIII genes. In the present study, we present evidence that ROR{alpha} also induces transcription of the human Apolipoprotein AV gene, a recently identified apolipoprotein associated with triglyceride levels. Adenovirus-mediated overexpression of ROR{alpha} increased the endogenous expression of ApoAV in HepG2 cells and ROR{alpha} also enhanced the activity of an ApoAV promoter construct in transiently transfected HepG2 cells. Deletion and mutation studies identified three AGGTCA motifs in the ApoAV promoter that mediate ROR{alpha} transactivation, one of which overlaps with a previously identified binding site for PPAR{alpha}. Together, these results suggest a novel mechanism whereby ROR{alpha} modulates lipid metabolism and implies ROR{alpha} as a potential target for the treatment of dyslipidemia and atherosclerosis.

  8. Targeted gene walking by low stringency polymerase chain reaction: Assignment of a putative human brain sodium channel gene (SCN3A) to chromosome 2q24-31

    SciTech Connect

    Malo, M.S.; Srivastava, K.; Andresen, J.M.; Ingram, V.M.; Chen, X.N.; Korenberg, J.R.

    1994-04-12

    The authors have developed a low stringency polymerase chain reaction (LSPCR) to isolate the unknown neighboring region around a known DNA sequence, thus allowing efficient targeted gene walking. The method involves the polymerase chain reaction (PCR) with a single primer under conditions of low stringency for primer annealing (40{degrees}C) for the first few cycles followed by more cycles at high stringency (55{degrees}C). Nested PCRs with end-labeled primers are then used to generate a ladder of radioactive bands, which accurately identifies the targeted fragment(s). They performed LSPCR on human placental DNA using a highly conserved sodium channel-specific primer for 5 cycles at 40{degrees}C followed by 27 cycles at 55{degrees}C for primer annealing. Subsequently, using higher stringency (55{degrees}C) PCR with radiolabeled nested primers for 8 cycles, they have isolated a 0.66-kb fragment of a putative human sodium channel gene. Partial sequence (325 bp) of this fragment revealed a 270-bp region (exon) with homology to the rat brain sodium channel III{alpha} (RBIII) gene at the nucleotide (87%) and amino acid (92%) levels. Therefore, the authors putatively assign this sequence as a part of a gene coding the {alpha}-subunit of a human brain type III sodium channel (SCN3A). Using PCR on two human/rodent somatic cell hybrid panels with primers specific to this putative SCN3A gene, they have localized this gene to chromosome 2. Fluorescence in situ hybridization to human metaphase chromosomes was used to sublocalize the SCN3A gene to chromosome at 2q24-31. In conclusion, LSPCR is an efficient and sensitive method for targeted gene walking and is also useful for the isolation of homologous genes in related species.

  9. Applications of CRISPR/Cas9 technology for targeted mutagenesis, gene replacement and stacking of genes in higher plants.

    PubMed

    Luo, Ming; Gilbert, Brian; Ayliffe, Michael

    2016-07-01

    Mutagenesis continues to play an essential role for understanding plant gene function and, in some instances, provides an opportunity for plant improvement. The development of gene editing technologies such as TALENs and zinc fingers has revolutionised the targeted mutation specificity that can now be achieved. The CRISPR/Cas9 system is the most recent addition to gene editing technologies and arguably the simplest requiring only two components; a small guide RNA molecule (sgRNA) and Cas9 endonuclease protein which complex to recognise and cleave a specific 20 bp target site present in a genome. Target specificity is determined by complementary base pairing between the sgRNA and target site sequence enabling highly specific, targeted mutation to be readily engineered. Upon target site cleavage, error-prone endogenous repair mechanisms produce small insertion/deletions at the target site usually resulting in loss of gene function. CRISPR/Cas9 gene editing has been rapidly adopted in plants and successfully undertaken in numerous species including major crop species. Its applications are not restricted to mutagenesis and target site cleavage can be exploited to promote sequence insertion or replacement by recombination. The multiple applications of this technology in plants are described. PMID:27146973

  10. Sequence analysis of the ERCC2 gene regions in human, mouse, and hamster reveals three linked genes.

    PubMed

    Lamerdin, J E; Stilwagen, S A; Ramirez, M H; Stubbs, L; Carrano, A V

    1996-06-15

    The ERCC2 (excision repair cross-complementing rodent repair group 2) gene product is involved in transcription-coupled repair as an integral member of the basal transcription factor BTF2/TFIIH complex. Defects in this gene can result in three distinct human disorders, namely the cancer-prone syndrome xeroderma pigmentosum complementation group D, trichothiodystrophy, and Cockayne syndrome. We report the comparative analysis of 91.6 kb of new sequence including 54.3 kb encompassing the human ERCC2 locus, the syntenic region in the mouse (32.6 kb), and a further 4.7 kb of sequence 3' of the previously reported ERCC2 region in the hamster. In addition to ERCC2, our analysis revealed the presence of two previously undescribed genes in all three species. The first is centromeric (in the human) to ERCC2 and is most similar to the kinesin light chain gene in sea urchin. The second gene is telomeric (in the human) to ERCC2 and contains a motif found in ankyrins, some cell cycle proteins, and transcription factors. Multiple EST matches to this putative new gene indicate that it is expressed in several human tissues, including breast. The identification and description of two new genes provides potential candidate genes for disorders mapping to this region of 19q13.2. PMID:8786141

  11. Sequence analysis of the ERCC2 gene regions in human, mouse, and hamster reveals three linked genes

    SciTech Connect

    Lamerdin, J.E.; Stilwagen, S.A.; Ramirez, M.H.

    1996-06-15

    The ERCC2 (excision repair cross-complementing rodent repair group 2) gene product is involved in transcription-coupled repair as an integral member of the basal transcription factor BTF2/TFIIH complex. Defects in this gene can result in three distinct human disorders, namely the cancer-prone syndrome xeroderma pigmentosum complementation group D, trichothiodystrophy, and Cockayne syndrome. We report the comparative analysis of 91.6 kb of new sequence including 54.3 kb encompassing the human ERCC2 locus, the syntenic region in the mouse (32.6 kb), and a further 4.7 kb of sequence 3{prime} of the previously reported ERCC2 region in the hamster. In addition to ERCC2, our analysis revealed the presence of two previously undescribed genes in all three species. The first is centromeric (in the human) to ERCC2 and is most similar to the kinesin light chain gene in sea urchin. The second gene is telomeric (in the human) to ERCC2 and contains a motif found in ankyrins, some cell proteins, and transcription factors. Multiple EST matches to this putative new gene indicate that it is expressed in several human tissues, including breast. The identification and description of two new genes provides potential candidate genes for disorders mapping to this region of 19q13.2. 42 refs., 6 figs., 3 tabs.

  12. Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases

    PubMed Central

    Santiago, Yolanda; Chan, Edmond; Liu, Pei-Qi; Orlando, Salvatore; Zhang, Lin; Urnov, Fyodor D.; Holmes, Michael C.; Guschin, Dmitry; Waite, Adam; Miller, Jeffrey C.; Rebar, Edward J.; Gregory, Philip D.; Klug, Aaron; Collingwood, Trevor N.

    2008-01-01

    Gene knockout is the most powerful tool for determining gene function or permanently modifying the phenotypic characteristics of a cell. Existing methods for gene disruption are limited by their efficiency, time to completion, and/or the potential for confounding off-target effects. Here, we demonstrate a rapid single-step approach to targeted gene knockout in mammalian cells, using engineered zinc-finger nucleases (ZFNs). ZFNs can be designed to target a chosen locus with high specificity. Upon transient expression of these nucleases the target gene is first cleaved by the ZFNs and then repaired by a natural—but imperfect—DNA repair process, nonhomologous end joining. This often results in the generation of mutant (null) alleles. As proof of concept for this approach we designed ZFNs to target the dihydrofolate reductase (DHFR) gene in a Chinese hamster ovary (CHO) cell line. We observed biallelic gene disruption at frequencies >1%, thus obviating the need for selection markers. Three new genetically distinct DHFR−/− cell lines were generated. Each new line exhibited growth and functional properties consistent with the specific knockout of the DHFR gene. Importantly, target gene disruption is complete within 2–3 days of transient ZFN delivery, thus enabling the isolation of the resultant DHFR−/− cell lines within 1 month. These data demonstrate further the utility of ZFNs for rapid mammalian cell line engineering and establish a new method for gene knockout with application to reverse genetics, functional genomics, drug discovery, and therapeutic recombinant protein production. PMID:18359850

  13. Quantitative Phosphoproteomics Reveals Wee1 Kinase as a Therapeutic Target in a Model of Proneural Glioblastoma.

    PubMed

    Lescarbeau, Rebecca S; Lei, Liang; Bakken, Katrina K; Sims, Peter A; Sarkaria, Jann N; Canoll, Peter; White, Forest M

    2016-06-01

    Glioblastoma (GBM) is the most common malignant primary brain cancer. With a median survival of about a year, new approaches to treating this disease are necessary. To identify signaling molecules regulating GBM progression in a genetically engineered murine model of proneural GBM, we quantified phosphotyrosine-mediated signaling using mass spectrometry. Oncogenic signals, including phosphorylated ERK MAPK, PI3K, and PDGFR, were found to be increased in the murine tumors relative to brain. Phosphorylation of CDK1 pY15, associated with the G2 arrest checkpoint, was identified as the most differentially phosphorylated site, with a 14-fold increase in phosphorylation in the tumors. To assess the role of this checkpoint as a potential therapeutic target, syngeneic primary cell lines derived from these tumors were treated with MK-1775, an inhibitor of Wee1, the kinase responsible for CDK1 Y15 phosphorylation. MK-1775 treatment led to mitotic catastrophe, as defined by increased DNA damage and cell death by apoptosis. To assess the extensibility of targeting Wee1/CDK1 in GBM, patient-derived xenograft (PDX) cell lines were also treated with MK-1775. Although the response was more heterogeneous, on-target Wee1 inhibition led to decreased CDK1 Y15 phosphorylation and increased DNA damage and apoptosis in each line. These results were also validated in vivo, where single-agent MK-1775 demonstrated an antitumor effect on a flank PDX tumor model, increasing mouse survival by 1.74-fold. This study highlights the ability of unbiased quantitative phosphoproteomics to reveal therapeutic targets in tumor models, and the potential for Wee1 inhibition as a treatment approach in preclinical models of GBM. Mol Cancer Ther; 15(6); 1332-43. ©2016 AACR. PMID:27196784

  14. Identification of C/EBPβ Target Genes in ALK+ Anaplastic Large Cell Lymphoma (ALCL) by Gene Expression Profiling and Chromatin Immunoprecipitation

    PubMed Central

    Bonzheim, Irina; Irmler, Martin; Klier-Richter, Margit; Steinhilber, Julia; Anastasov, Nataša; Schäfer, Sabine; Adam, Patrick; Beckers, Johannes; Raffeld, Mark; Fend, Falko; Quintanilla-Martinez, Leticia

    2013-01-01

    C/EBPβ (CCAAT enhancer binding protein) is a transcription factor that plays a crucial role in survival and transformation of ALK+ anaplastic large cell lymphoma (ALCL). The aim of this study was to identify the downstream targets of C/EBPβ responsible for ALK-mediated oncogenesis. C/EBPβ was knocked down in ALK+ ALCL cell lines with a C/EBPβ-shRNA, followed by gene expression profiling (GEP). GEP analysis revealed a reproducible signature of genes that were significantly regulated by C/EBPβ. Classification into biological categories revealed overrepresentation of genes involved in the immune response, apoptosis and cell proliferation. Transcriptional regulation by C/EBPβ was found in 6 of 11 (BCL2A1, G0S2, TRIB1, S100A9, DDX21 and DDIT4) genes investigated by chromatin immunoprecipitation. We demonstrated that BCL2A1, G0S2 and DDX21 play a crucial role in survival and proliferation of ALK+ ALCL cells. DDX21, a gene involved in rRNA biogenesis, was found differentially overexpressed in primary ALK+ ALCL cases. All three candidate genes were validated in primary ALCL cases by either immunohistochemistry or RT-qPCR. In conclusion, we identified and validated several key C/EBPβ-regulated genes with major impact on survival and cell growth in ALK+ ALCL, supporting the central role of C/EBPβ in ALK-mediated oncogenesis. PMID:23741337

  15. Seamless Genome Editing in Rice via Gene Targeting and Precise Marker Elimination.

    PubMed

    Nishizawa-Yokoi, Ayako; Saika, Hiroaki; Toki, Seiichi

    2016-01-01

    Positive-negative selection using hygromycin phosphotransferase (hpt) and diphtheria toxin A-fragment (DT-A) as positive and negative selection markers, respectively, allows enrichment of cells harboring target genes modified via gene targeting (GT). We have developed a successful GT system employing positive-negative selection and subsequent precise marker excision via the piggyBac transposon derived from the cabbage looper moth to introduce desired modifications into target genes in the rice genome. This approach could be applied to the precision genome editing of almost all endogenous genes throughout the genome, at least in rice. PMID:27557691

  16. Identification of TTP mRNA targets in human dendritic cells reveals TTP as a critical regulator of dendritic cell maturation.

    PubMed

    Emmons, Jillian; Townley-Tilson, W H Davin; Deleault, Kristen M; Skinner, Stephen J; Gross, Robert H; Whitfield, Michael L; Brooks, Seth A

    2008-05-01

    Dendritic cells provide a critical link between innate and adaptive immunity and are essential to prime a naive T-cell response. The transition from immature dendritic cells to mature dendritic cells involves numerous changes in gene expression; however, the role of post-transcriptional changes in this process has been largely ignored. Tristetraprolin is an AU-rich element mRNA-binding protein that has been shown to regulate the stability of a number of cytokines and chemokines of mRNAs. Using TTP immunoprecipitations and Affymetrix GeneChips, we identified 393 messages as putative TTP mRNA targets in human dendritic cells. Gene ontology analysis revealed that approximately 25% of the identified mRNAs are associated with protein synthesis. We also identified six MHC Class I alleles, five MHC Class II alleles, seven chemokine and chemokine receptor genes, indoleamine 2,3 dioxygenase, and CD86 as putative TTP ligands. Real-time PCR was used to validate the GeneChip data for 15 putative target genes and functional studies performed for six target genes. These data establish that TTP regulates the expression of DUSP1, IDO, SOD2, CD86, and MHC Class I-B and F via the 3'-untranslated region of each gene. A novel finding is the demonstration that TTP can interact with and regulate the expression of non-AU-rich element-containing messages. The data implicate TTP as having a broader role in regulating and limiting the immune response than previously suspected. PMID:18367721

  17. Genome-wide analysis reveals gene expression and metabolic network dynamics during embryo development in Arabidopsis.

    PubMed

    Xiang, Daoquan; Venglat, Prakash; Tibiche, Chabane; Yang, Hui; Risseeuw, Eddy; Cao, Yongguo; Babic, Vivijan; Cloutier, Mathieu; Keller, Wilf; Wang, Edwin; Selvaraj, Gopalan; Datla, Raju

    2011-05-01

    Embryogenesis is central to the life cycle of most plant species. Despite its importance, because of the difficulty associated with embryo isolation, global gene expression programs involved in plant embryogenesis, especially the early events following fertilization, are largely unknown. To address this gap, we have developed methods to isolate whole live Arabidopsis (Arabidopsis thaliana) embryos as young as zygote and performed genome-wide profiling of gene expression. These studies revealed insights into patterns of gene expression relating to: maternal and paternal contributions to zygote development, chromosomal level clustering of temporal expression in embryogenesis, and embryo-specific functions. Functional analysis of some of the modulated transcription factor encoding genes from our data sets confirmed that they are critical for embryogenesis. Furthermore, we constructed stage-specific metabolic networks mapped with differentially regulated genes by combining the microarray data with the available Kyoto Encyclopedia of Genes and Genomes metabolic data sets. Comparative analysis of these networks revealed the network-associated structural and topological features, pathway interactions, and gene expression with reference to the metabolic activities during embryogenesis. Together, these studies have generated comprehensive gene expression data sets for embryo development in Arabidopsis and may serve as an important foundational resource for other seed plants. PMID:21402797

  18. Network and pathway analysis of microRNAs, transcription factors, target genes and host genes in human glioma

    PubMed Central

    ZHANG, YING; ZHAO, SHISHUN; XU, ZHIWEN

    2016-01-01

    To date, there has been rapid development with regard to gene and microRNA (miR/miRNA) research in gliomas. However, the regulatory mechanisms of the associated genes and miRNAs remain unclear. In the present study, the genes, miRNAs and transcription factors (TFs) were considered as elements in the regulatory network, and focus was placed on the associations between TFs and miRNAs, miRNAs and target genes, and miRNAs and host genes. In order to show the regulatory correlation clearly, all the elements were investigated and three regulatory networks, namely the differentially-expressed, related and global networks, were constructed. Certain important pathways were highlighted, with analysis of the similarities and differences among the networks. Next, the upstream and downstream elements of differentially-expressed genes, miRNAs and predicted TFs were listed. The most notable aspect of the present study was the three levels of network, particularly the differentially-expressed network, since the differentially-expressed associations that these networks provide appear at the initial stages of cancers such as glioma. If the states of the differentially-expressed associations can be adjusted to the normal state via alterations in regulatory associations, which were also recorded in the study networks and tables, it is likely that cancer can be regulated or even avoided. In the present study, the differentially-expressed network illuminated the pathogenesis of glioma; for example, a TF can regulate one or more miRNAs, and a target gene can be targeted by one or more miRNAs. Therefore, the host genes and target genes, the host genes and TFs, and the target genes and TFs indirectly affect each other through miRNAs. The association also exists between TFs and TFs, target genes and target genes, and host genes and host genes. The present study also demonstrated self-adaption associations and circle-regulations. The related network further described the regulatory mechanism

  19. Cell-by-Cell Dissection of Gene Expression and Chromosomal Interactions Reveals Consequences of Nuclear Reorganization

    PubMed Central

    2005-01-01

    The functional consequences of long-range nuclear reorganization were studied in a cell-by-cell analysis of gene expression and long-range chromosomal interactions in the Drosophila eye and eye imaginal disk. Position-effect variegation was used to stochastically perturb gene expression and probe nuclear reorganization. Variegating genes on rearrangements of Chromosomes X, 2, and 3 were probed for long-range interactions with heterochromatin. Studies were conducted only in tissues known to express the variegating genes. Nuclear structure was revealed by fluorescence in situ hybridization with probes to the variegating gene and heterochromatin. Gene expression was determined alternately by immunofluorescence against specific proteins and by eye pigment autofluorescence. This allowed cell-by-cell comparisons of nuclear architecture between cells in which the variegating gene was either expressed or silenced. Very strong correlations between heterochromatic association and silencing were found. Expressing cells showed a broad distribution of distances between variegating genes and their own centromeric heterochromatin, while silenced cells showed a very tight distribution centered around very short distances, consistent with interaction between the silenced genes and heterochromatin. Spatial and temporal analysis of interactions with heterochromatin indicated that variegating genes primarily associate with heterochromatin in cells that have exited the cell cycle. Differentiation was not a requirement for association, and no differences in association were observed between cell types. Thus, long-range interactions between distal chromosome regions and their own heterochromatin have functional consequences for the organism. PMID:15737020

  20. Whole blood hypoxia-related gene expression reveals novel pathways to obstructive sleep apnea in humans.

    PubMed

    Perry, Juliana C; Guindalini, Camila; Bittencourt, Lia; Garbuio, Silverio; Mazzotti, Diego R; Tufik, Sergio

    2013-12-01

    In this study, our goal was to identify the key genes that are associated with obstructive sleep apnea (OSA). Thirty-five volunteers underwent full in-lab polysomnography and, according to the sleep apnea hypopnea index (AHI), were classified into control, mild-to-moderate OSA and severe OSA groups. Severe OSA patients were assigned to participate in a continuous positive airway pressure (CPAP) protocol for 6 months. Blood was collected and the expression of 84 genes analyzed using the RT(2) Profiler™ PCR array. Mild-to-moderate OSA patients demonstrated down-regulation of 2 genes associated with induction of apoptosis, while a total of 13 genes were identified in severe OSA patients. After controlling for body mass index, PRPF40A and PLOD3 gene expressions were strongly and independently associated with AHI scores. This research protocol highlights a number of molecular targets that might help the development of novel therapeutic strategies. PMID:23994550

  1. Real-time imaging of Huntingtin aggregates diverting target search and gene transcription

    PubMed Central

    Li, Li; Liu, Hui; Dong, Peng; Li, Dong; Legant, Wesley R; Grimm, Jonathan B; Lavis, Luke D; Betzig, Eric; Tjian, Robert; Liu, Zhe

    2016-01-01

    The presumptive altered dynamics of transient molecular interactions in vivo contributing to neurodegenerative diseases have remained elusive. Here, using single-molecule localization microscopy, we show that disease-inducing Huntingtin (mHtt) protein fragments display three distinct dynamic states in living cells – 1) fast diffusion, 2) dynamic clustering and 3) stable aggregation. Large, stable aggregates of mHtt exclude chromatin and form 'sticky' decoy traps that impede target search processes of key regulators involved in neurological disorders. Functional domain mapping based on super-resolution imaging reveals an unexpected role of aromatic amino acids in promoting protein-mHtt aggregate interactions. Genome-wide expression analysis and numerical simulation experiments suggest mHtt aggregates reduce transcription factor target site sampling frequency and impair critical gene expression programs in striatal neurons. Together, our results provide insights into how mHtt dynamically forms aggregates and disrupts the finely-balanced gene control mechanisms in neuronal cells. DOI: http://dx.doi.org/10.7554/eLife.17056.001 PMID:27484239

  2. Transcriptome network analysis reveals potential candidate genes for squamous lung cancer.

    PubMed

    Bai, Jing; Hu, Sheng

    2012-01-01

    Squamous lung cancer is a common type of lung cancer; however, its mechanism of oncogenesis is still unknown. The aim of this study was to screen candidate genes of squamous lung cancer using a bioinformatics strategy and elucidate the mechanism of squamous lung cancer. Published microarray data of the GSE3268 series was obtained from Gene Expression Omnibus (GEO). Significance analysis of microarrays was performed using the software R, and differentially expressed genes by R analysis were harvested. The relationship between transcription factors and target genes in cancer were collected from the Transcriptional regulatory element database. A transcriptome network analysis method was used to construct gene regulation networks and select the candidate genes for squamous lung cancer. SPI1, FLI1, FOS, ETS2, EGR1 and PPARG were defined as candidate genes for squamous lung cancer by the transcriptome network analysis method. Among them, 5 genes had been reported to be involved in lung cancer, except SPI1 and FLI1. Effective recall on previous knowledge conferred strong confidence in these methods. It is demonstrated that transcriptome network analysis is useful in the identification of candidate genes in disease. PMID:21922129

  3. Essential Gene Discovery in the Basidiomycete Cryptococcus neoformans for Antifungal Drug Target Prioritization

    PubMed Central

    Ianiri, Giuseppe

    2015-01-01

    ABSTRACT Fungal diseases represent a major burden to health care globally. As with other pathogenic microbes, there is a limited number of agents suitable for use in treating fungal diseases, and resistance to these agents can develop rapidly. Cryptococcus neoformans is a basidiomycete fungus that causes cryptococcosis worldwide in both immunocompromised and healthy individuals. As a basidiomycete, it diverged from other common pathogenic or model ascomycete fungi more than 500 million years ago. Here, we report C. neoformans genes that are essential for viability as identified through forward and reverse genetic approaches, using an engineered diploid strain and genetic segregation after meiosis. The forward genetic approach generated random insertional mutants in the diploid strain, the induction of meiosis and sporulation, and selection for haploid cells with counterselection of the insertion event. More than 2,500 mutants were analyzed, and transfer DNA (T-DNA) insertions in several genes required for viability were identified. The genes include those encoding the thioredoxin reductase (Trr1), a ribosome assembly factor (Rsa4), an mRNA-capping component (Cet1), and others. For targeted gene replacement, the C. neoformans homologs of 35 genes required for viability in ascomycete fungi were disrupted, meiosis and sporulation were induced, and haploid progeny were evaluated for their ability to grow on selective media. Twenty-one (60%) were found to be required for viability in C. neoformans. These genes are involved in mitochondrial translation, ergosterol biosynthesis, and RNA-related functions. The heterozygous diploid mutants were evaluated for haploinsufficiency on a number of perturbing agents and drugs, revealing phenotypes due to the loss of one copy of an essential gene in C. neoformans. This study expands the knowledge of the essential genes in fungi using a basidiomycete as a model organism. Genes that have no mammalian homologs and are essential

  4. RNA Sequencing Revealed Numerous Polyketide Synthase Genes in the Harmful Dinoflagellate Karenia mikimotoi

    PubMed Central

    Kimura, Kei; Okuda, Shujiro; Nakayama, Kei; Shikata, Tomoyuki; Takahashi, Fumio; Yamaguchi, Haruo; Skamoto, Setsuko; Yamaguchi, Mineo; Tomaru, Yuji

    2015-01-01

    The dinoflagellate Karenia mikimotoi forms blooms in the coastal waters of temperate regions and occasionally causes massive fish and invertebrate mortality. This study aimed to elucidate the toxic effect of K. mikimotoi on marine organisms by using the genomics approach; RNA-sequence libraries were constructed, and data were analyzed to identify toxin-related genes. Next-generation sequencing produced 153,406 transcript contigs from the axenic culture of K. mikimotoi. BLASTX analysis against all assembled contigs revealed that 208 contigs were polyketide synthase (PKS) sequences. Thus, K. mikimotoi was thought to have several genes encoding PKS metabolites and to likely produce toxin-like polyketide molecules. Of all the sequences, approximately 30 encoded eight PKS genes, which were remarkably similar to those of Karenia brevis. Our phylogenetic analyses showed that these genes belonged to a new group of PKS type-I genes. Phylogenetic and active domain analyses showed that the amino acid sequence of four among eight Karenia PKS genes was not similar to any of the reported PKS genes. These PKS genes might possibly be associated with the synthesis of polyketide toxins produced by Karenia species. Further, a homology search revealed 10 contigs that were similar to a toxin gene responsible for the synthesis of saxitoxin (sxtA) in the toxic dinoflagellate Alexandrium fundyense. These contigs encoded A1–A3 domains of sxtA genes. Thus, this study identified some transcripts in K. mikimotoi that might be associated with several putative toxin-related genes. The findings of this study might help understand the mechanism of toxicity of K. mikimotoi and other dinoflagellates. PMID:26561394

  5. Cas9-Assisted Targeting of CHromosome segments CATCH enables one-step targeted cloning of large gene clusters

    PubMed Central

    Jiang, Wenjun; Zhao, Xuejin; Gabrieli, Tslil; Lou, Chunbo; Ebenstein, Yuval; Zhu, Ting F.

    2015-01-01

    The cloning of long DNA segments, especially those containing large gene clusters, is of particular importance to synthetic and chemical biology efforts for engineering organisms. While cloning has been a defining tool in molecular biology, the cloning of long genome segments has been challenging. Here we describe a technique that allows the targeted cloning of near-arbitrary, long bacterial genomic sequences of up to 100 kb to be accomplished in a single step. The target genome segment is excised from bacterial chromosomes in vitro by the RNA-guided Cas9 nuclease at two designated loci, and ligated to the cloning vector by Gibson assembly. This technique can be an effective molecular tool for the targeted cloning of large gene clusters that are often expensive to synthesize by gene synthesis or difficult to obtain directly by traditional PCR and restriction-enzyme-based methods. PMID:26323354

  6. Applications of Gene Targeting Technology to Mental Retardation and Developmental Disability Research

    ERIC Educational Resources Information Center

    Pimenta, Aurea F.; Levitt, Pat

    2005-01-01

    The human and mouse genome projects elucidated the sequence and position map of innumerous genes expressed in the central nervous system (CNS), advancing our ability to manipulate these sequences and create models to investigate regulation of gene expression and function. In this article, we reviewed gene targeting methodologies with emphasis on…

  7. Global analyses of endonucleolytic cleavage in mammals reveal expanded repertoires of cleavage-inducing small RNAs and their targets

    PubMed Central

    Cass, Ashley A.; Bahn, Jae Hoon; Lee, Jae-Hyung; Greer, Christopher; Lin, Xianzhi; Kim, Yong; Hsiao, Yun-Hua Esther; Xiao, Xinshu

    2016-01-01

    In mammals, small RNAs are important players in post-transcriptional gene regulation. While their roles in mRNA destabilization and translational repression are well appreciated, their involvement in endonucleolytic cleavage of target RNAs is poorly understood. Very few microRNAs are known to guide RNA cleavage. Endogenous small interfering RNAs are expected to induce target cleavage, but their target genes remain largely unknown. We report a systematic study of small RNA-mediated endonucleolytic cleavage in mouse through integrative analysis of small RNA and degradome sequencing data without imposing any bias toward known small RNAs. Hundreds of small cleavage-inducing RNAs and their cognate target genes were identified, significantly expanding the repertoire of known small RNA-guided cleavage events. Strikingly, both small RNAs and their target sites demonstrated significant overlap with retrotransposons, providing evidence for the long-standing speculation that retrotransposable elements in mRNAs are leveraged as signals for gene targeting. Furthermore, our analysis showed that the RNA cleavage pathway is also present in human cells but affecting a different repertoire of retrotransposons. These results show that small