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Sample records for gene deletion mutants

  1. Functional profiling in Streptococcus mutans: construction and examination of a genomic collection of gene deletion mutants.

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    Quivey, R G; Grayhack, E J; Faustoferri, R C; Hubbard, C J; Baldeck, J D; Wolf, A S; MacGilvray, M E; Rosalen, P L; Scott-Anne, K; Santiago, B; Gopal, S; Payne, J; Marquis, R E

    2015-12-01

    A collection of tagged deletion mutant strains was created in Streptococcus mutans UA159 to facilitate investigation of the aciduric capability of this oral pathogen. Gene-specific barcoded deletions were attempted in 1432 open reading frames (representing 73% of the genome), and resulted in the isolation of 1112 strains (56% coverage) carrying deletions in distinct non-essential genes. As S. mutans virulence is predicated upon the ability of the organism to survive an acidic pH environment, form biofilms on tooth surfaces, and out-compete other oral microflora, we assayed individual mutant strains for the relative fitness of the deletion strain, compared with the parent strain, under acidic and oxidative stress conditions, as well as for their ability to form biofilms in glucose- or sucrose-containing medium. Our studies revealed a total of 51 deletion strains with defects in both aciduricity and biofilm formation. We have also identified 49 strains whose gene deletion confers sensitivity to oxidative damage and deficiencies in biofilm formation. We demonstrate the ability to examine competitive fitness of mutant organisms using the barcode tags incorporated into each deletion strain to examine the representation of a particular strain in a population. Co-cultures of deletion strains were grown either in vitro in a chemostat to steady-state values of pH 7 and pH 5 or in vivo in an animal model for oral infection. Taken together, these data represent a mechanism for assessing the virulence capacity of this pathogenic microorganism and a resource for identifying future targets for drug intervention to promote healthy oral microflora.

  2. Parallel analysis of tagged deletion mutants efficiently identifies genes involved in endoplasmic reticulum biogenesis.

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    Wright, Robin; Parrish, Mark L; Cadera, Emily; Larson, Lynnelle; Matson, Clinton K; Garrett-Engele, Philip; Armour, Chris; Lum, Pek Yee; Shoemaker, Daniel D

    2003-07-30

    Increased levels of HMG-CoA reductase induce cell type- and isozyme-specific proliferation of the endoplasmic reticulum. In yeast, the ER proliferations induced by Hmg1p consist of nuclear-associated stacks of smooth ER membranes known as karmellae. To identify genes required for karmellae assembly, we compared the composition of populations of homozygous diploid S. cerevisiae deletion mutants following 20 generations of growth with and without karmellae. Using an initial population of 1,557 deletion mutants, 120 potential mutants were identified as a result of three independent experiments. Each experiment produced a largely non-overlapping set of potential mutants, suggesting that differences in specific growth conditions could be used to maximize the comprehensiveness of similar parallel analysis screens. Only two genes, UBC7 and YAL011W, were identified in all three experiments. Subsequent analysis of individual mutant strains confirmed that each experiment was identifying valid mutations, based on the mutant's sensitivity to elevated HMG-CoA reductase and inability to assemble normal karmellae. The largest class of HMG-CoA reductase-sensitive mutations was a subset of genes that are involved in chromatin structure and transcriptional regulation, suggesting that karmellae assembly requires changes in transcription or that the presence of karmellae may interfere with normal transcriptional regulation.

  3. Phenotype profiling of single gene deletion mutants of E. coli using Biolog technology.

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    Tohsato, Yukako; Mori, Hirotada

    2008-01-01

    Phenotype MicroArray (PM) technology is high-throughput phenotyping system and is directly applicable to assay the effects of genetic changes in cells. In this study, we performed comprehensive PM analysis using single gene deletion mutants of central metabolic pathway and related genes. To elucidate the structure of central metabolic networks in Escherichia coli K-12, we focused 288 different PM conditions of carbon and nitrogen sources and performed bioinformatic analysis. For data processing, we employed noise reduction procedures. The distance between each of the mutants was defined by Manhattan distance and agglomerative Ward's hierarchical method was applied for clustering analysis. As a result, five clusters were revealed which represented to activate or repress cellular respiratory activities. Furthermore, the results might suggest that Glyceraldehyde-3P plays a key role as a molecular switch of central metabolic network.

  4. [Repression of the enzyme inducible syntheses in Escherichia coli K12 mutant with a deleted ptsH gene].

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    Gershanovich, V N; Il'ina, T S; Rusina, O Iu; Iurovitskaia, N V; Bol'shakova, T N

    1977-01-01

    The genome of lambda phage with thermosensitive repressor was integrated into the pts region of the E. coli chromosome. Such a lysogenic culture behaves as a pts mutant at 30 degrees. Heating of cells of this strain leads to the induction of lambda prophage and formation of deletions in the pts region. A mutant with a deletion covering ptsH gene was isolated after prophage induction. The deletion nature of pts mutation was confirmed in genetic and biochemical experiments. It was shown that the deletion is small and does not involve ptsI and lig genes. The isolated deltaptsH mutant possesses all characteristics of pts mutants: pleiotropic impairment of transport and utilization of a number of carbohydrates, repression of the enzyme inducible synthesis and resistance to catabolite repression with glucose. These data (together with earlier ones) allow us to conclude that the phosphorylated form of HPr is involved (in direct of indirect manner/ in activation of DNA transcription.

  5. Functional dissection of regulatory models using gene expression data of deletion mutants.

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    Jin'e Li

    Full Text Available Genome-wide gene expression profiles accumulate at an alarming rate, how to integrate these expression profiles generated by different laboratories to reverse engineer the cellular regulatory network has been a major challenge. To automatically infer gene regulatory pathways from genome-wide mRNA expression profiles before and after genetic perturbations, we introduced a new Bayesian network algorithm: Deletion Mutant Bayesian Network (DM_BN. We applied DM_BN to the expression profiles of 544 yeast single or double deletion mutants of transcription factors, chromatin remodeling machinery components, protein kinases and phosphatases in S. cerevisiae. The network inferred by this method identified causal regulatory and non-causal concurrent interactions among these regulators (genetically perturbed genes that are strongly supported by the experimental evidence, and generated many new testable hypotheses. Compared to networks reconstructed by routine similarity measures or by alternative Bayesian network algorithms, the network inferred by DM_BN excels in both precision and recall. To facilitate its application in other systems, we packaged the algorithm into a user-friendly analysis tool that can be downloaded at http://www.picb.ac.cn/hanlab/DM_BN.html.

  6. Generation of stable mutants and targeted gene deletion strains in Cryptococcus neoformans through electroporation.

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    Lin, Xiaorong; Chacko, Nadia; Wang, Linqi; Pavuluri, Yashwant

    2015-04-01

    Cryptococcus neoformans is the etiologic agent of cryptococcal meningitis that causes more than half a million deaths worldwide each year. This capsulated basidiomycetous yeast also serves as a model for micropathogenic studies. The ability to make stable mutants, either via ectopic integration or homologous recombination, has been accomplished using biolistic transformation. This technical advance has greatly facilitated the research on the basic biology and pathogenic mechanisms of this pathogen in the past two decades. However, biolistic transformation is costly, and its reproducibility varies widely. Here we found that stable ectopic integration or targeted gene deletion via homologous replacement could be accomplished through electroporative transformation. The stability of the transformants obtained through electroporation and the frequency of homologous replacement is highly dependent on the selective marker. A frequency of homologous recombination among the stable transformants obtained by electroporation is comparable to those obtained by biolistic transformation (∼10%) when dominant drug selection markers are used, which is much higher than what has been previously reported for electroporation when auxotrophic markers were used (0.001% to 0.1%). Furthermore, disruption of the KU80 gene or generation of gene deletion constructs using the split marker strategy, two approaches known to increase homologous replacement among transformants obtained through biolistic transformation, also increase the frequency of homologous replacement among transformants obtained through electroporation. Therefore, electroporation provides a low cost alternative for mutagenesis in Cryptococcus.

  7. Analysis of pools of targeted Salmonella deletion mutants identifies novel genes affecting fitness during competitive infection in mice.

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    Carlos A Santiviago

    2009-07-01

    Full Text Available Pools of mutants of minimal complexity but maximal coverage of genes of interest facilitate screening for genes under selection in a particular environment. We constructed individual deletion mutants in 1,023 Salmonella enterica serovar Typhimurium genes, including almost all genes found in Salmonella but not in related genera. All mutations were confirmed simultaneously using a novel amplification strategy to produce labeled RNA from a T7 RNA polymerase promoter, introduced during the construction of each mutant, followed by hybridization of this labeled RNA to a Typhimurium genome tiling array. To demonstrate the ability to identify fitness phenotypes using our pool of mutants, the pool was subjected to selection by intraperitoneal injection into BALB/c mice and subsequent recovery from spleens. Changes in the representation of each mutant were monitored using T7 transcripts hybridized to a novel inexpensive minimal microarray. Among the top 120 statistically significant spleen colonization phenotypes, more than 40 were mutations in genes with no previously known role in this model. Fifteen phenotypes were tested using individual mutants in competitive assays of intraperitoneal infection in mice and eleven were confirmed, including the first two examples of attenuation for sRNA mutants in Salmonella. We refer to the method as Array-based analysis of cistrons under selection (ABACUS.

  8. Analysis of pools of targeted Salmonella deletion mutants identifies novel genes affecting fitness during competitive infection in mice.

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    Carlos A Santiviago

    2009-07-01

    Full Text Available Pools of mutants of minimal complexity but maximal coverage of genes of interest facilitate screening for genes under selection in a particular environment. We constructed individual deletion mutants in 1,023 Salmonella enterica serovar Typhimurium genes, including almost all genes found in Salmonella but not in related genera. All mutations were confirmed simultaneously using a novel amplification strategy to produce labeled RNA from a T7 RNA polymerase promoter, introduced during the construction of each mutant, followed by hybridization of this labeled RNA to a Typhimurium genome tiling array. To demonstrate the ability to identify fitness phenotypes using our pool of mutants, the pool was subjected to selection by intraperitoneal injection into BALB/c mice and subsequent recovery from spleens. Changes in the representation of each mutant were monitored using T7 transcripts hybridized to a novel inexpensive minimal microarray. Among the top 120 statistically significant spleen colonization phenotypes, more than 40 were mutations in genes with no previously known role in this model. Fifteen phenotypes were tested using individual mutants in competitive assays of intraperitoneal infection in mice and eleven were confirmed, including the first two examples of attenuation for sRNA mutants in Salmonella. We refer to the method as Array-based analysis of cistrons under selection (ABACUS.

  9. The einkorn wheat (Triticum monococcum) mutant, maintained vegetative phase, is caused by a deletion in the VRN1 gene.

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    Shitsukawa, Naoki; Ikari, Chihiro; Shimada, Sanae; Kitagawa, Satoshi; Sakamoto, Koichi; Saito, Hiroyuki; Ryuto, Hiromichi; Fukunishi, Nobuhisa; Abe, Tomoko; Takumi, Shigeo; Nasuda, Shuhei; Murai, Koji

    2007-04-01

    The einkorn wheat (Triticum monococcum) mutant, maintained vegetative phase (mvp), was induced by nitrogen ion-beam treatment and was identified by its inability to transit from the vegetative to reproductive phase. In our previous study, we showed that WAP1 (wheat APETALA1) is a key gene in the regulatory pathway that controls phase transition from vegetative to reproductive growth in common wheat. WAP1 is an ortholog of the VRN1 gene that is responsible for vernalization insensitivity in einkorn wheat. The mvp mutation resulted from deletion of the VRN1 coding and promoter regions, demonstrating that WAP1/VRN1 is an indispensable gene for phase transition in wheat. Expression analysis of flowering-related genes in mvp plants indicated that wheat GIGANTIA (GI), CONSTANS (CO) and SUPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) genes either act upstream of or in a different pathway to WAP1/VRN1.

  10. Analysis of a large cluster of nonessential genes deleted from a vaccinia virus terminal transposition mutant.

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    Kotwal, G J; Moss, B

    1988-12-01

    The principal objectives of this study were to analyze the structure and coding potential of a long segment of DNA missing from a previously isolated (B. Moss, E. Winters, and J. A. Cooper (1981) J. Virol. 40, 387-395) attenuated variant of vaccinia virus strain WR and to examine the precise changes in the genome accompanying the deletion. The sequences of a 14.5-kbp region located at the left end of the standard vaccinia virus genome, extending from within the inverted terminal repetition (ITR) of the HindIII C fragment to the end of the HindIII N fragment, and of a 3-kbp segment from a corresponding region of the variant genome were determined. A comparison of these sequences revealed that the variant contained a deletion of 12 kbp and an insertion of 2.1 kbp. The origin of the inserted DNA was traced to the HindIII B region by using oligonucleotide probes indicating that a transposition of unique DNA located adjacent to the right ITR had occurred. Structural analysis indicated no extensive homologies, nucleotide substitutions, additions, or deletions at the boundaries of the transposed DNA. Examination of the right end of the variant genome indicated that a copy of the transposed DNA was still present and, therefore, the length of the ITR had been increased by 2.1 kbp. The variant genome could have formed by a mechanism that resulted in the replacement of a 22-kbp left-terminal fragment with a 12-kbp right-terminal fragment. The DNA missing from the variant and contained within the standard vaccinia virus WR genome contains 17 contiguous open reading frames (ORFs), all of which are directed leftward and apparently not required for replication in cultured cells. One deleted ORF has a 60% sequence similarity to another gene encoding a 42,000-Da protein present within the ITR suggesting that duplications have previously occurred during the evolution of vaccinia virus. Another deleted ORF has a 39% sequence similarity to a complement 4b binding protein. The

  11. Construction and characterization of pta gene-deleted mutant of Clostridium tyrobutyricum for enhanced butyric acid fermentation.

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    Zhu, Ying; Liu, Xiaoguang; Yang, Shang-Tian

    2005-04-20

    Clostridium tyrobutyricum ATCC 25755 is an acidogenic bacterium, producing butyrate and acetate as its main fermentation products. In order to decrease acetate and increase butyrate production, integrational mutagenesis was used to disrupt the gene associated with the acetate formation pathway in C. tyrobutyricum. A nonreplicative integrational plasmid containing the phosphotransacetylase gene (pta) fragment cloned from C. tyrobutyricum by using degenerate primers and an erythromycin resistance cassette were constructed and introduced into C. tyrobutyricum by electroporation. Integration of the plasmid into the homologous region on the chromosome inactivated the target pta gene and produced the pta-deleted mutant (PTA-Em), which was confirmed by Southern hybridization. SDS-PAGE and two-dimensional protein electrophoresis results indicated that protein expression was changed in the mutant. Enzyme activity assays using the cell lysate showed that the activities of PTA and acetate kinase (AK) in the mutant were reduced by more than 60% for PTA and 80% for AK. The mutant grew more slowly in batch fermentation with glucose as the substrate but produced 15% more butyrate and 14% less acetate as compared to the wild-type strain. Its butyrate productivity was approximately 2-fold higher than the wild-type strain. Moreover, the mutant showed much higher tolerance to butyrate inhibition, and the final butyrate concentration was improved by 68%. However, inactivation of pta gene did not completely eliminate acetate production in the fermentation, suggesting the existence of other enzymes (or pathways) also leading to acetate formation. This is the first-reported genetic engineering study demonstrating the feasibility of using a gene-inactivation technique to manipulate the acetic acid formation pathway in C. tyrobutyricum in order to improve butyric acid production from glucose.

  12. Construction of brewing-wine Aspergillus oryzae pyrG- mutant by pyrG gene deletion and its application in homology transformation.

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    Du, Yu; Xie, Guizhen; Yang, Chunfa; Fang, Baishan; Chen, Hongwen

    2014-06-01

    pyrG(-) host cells are indispensable for pyrG(-) based transformation system. Isolations of pyrG(-) host cells by random mutations are limited by time-consuming, unclear genetic background and potential interferences of homogenous recombination. The purpose of this study was to construct brewing-wine Aspergillus oryzae pyrG(-) mutant by site-directed mutation of pyrG gene deletion which would be used as a host for further transformation. pMD-pyrGAB, a vector carrying pyrG deletion cassette, was used to construct pyrG(-) mutant of A. oryzae. Three stable pyrG deletion mutants of A. oryzae were isolated by resistant to 5-fluoroorotic acid and confirmed by polymerase chain reaction analysis, indicating that pyrG was completely excised. The ΔpyrG mutants were applied as pyrG(-) host cells to disrupt xdh gene encoding xylitol dehydrogenase, which involves in xylitol production of A. oryzae. The xdh disruption mutants were efficiently constructed by transforming a pMD-pyrG-xdh disruption plasmid carrying pyrG, and the produced xylitol concentration of the Δxdh mutant was three times as much as that of the ΔpyrG recipient. Site-directed pyrG gene deletion is thus an effective way for the isolation of pyrG(-) host cells, and the established host-vector system could be applied in further functional genomics analysis and molecular breeding of A. oryzae.

  13. Analysis of Two Complementary Single-Gene Deletion Mutant Libraries of Salmonella Typhimurium in Intraperitoneal Infection of BALB/c Mice

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    Silva-Valenzuela, Cecilia A.; Molina-Quiroz, Roberto C.; Desai, Prerak; Valenzuela, Camila; Porwollik, Steffen; Zhao, Ming; Hoffman, Robert M.; Andrews-Polymenis, Helene; Contreras, Inés; Santiviago, Carlos A.; McClelland, Michael

    2016-01-01

    Two pools of individual single gene deletion (SGD) mutants of S. Typhimurium 14028s encompassing deletions of 3,923 annotated non-essential ORFs and sRNAs were screened by intraperitoneal (IP) injection in BALB/c mice followed by recovery from spleen and liver 2 days post infection. The relative abundance of each mutant was measured by microarray hybridization. The two mutant libraries differed in the orientation of the antibiotic resistance cassettes (either sense-oriented KanR, SGD-K, or antisense-oriented CamR, SGD-C). Consistent systemic colonization defects were observed in both libraries and both organs for hundreds of mutants of genes previously reported to be important after IP injection in this animal model, and for about 100 new candidate genes required for systemic colonization. Four mutants with a range of apparent fitness defects were confirmed using competitive infections with the wild-type parental strain: ΔSTM0286, ΔSTM0551, ΔSTM2363, and ΔSTM3356. Two mutants, ΔSTM0286 and ΔSTM2363, were then complemented in trans with a plasmid encoding an intact copy of the corresponding wild-type gene, and regained the ability to fully colonize BALB/c mice systemically. These results suggest the presence of many more undiscovered Salmonella genes with phenotypes in IP infection of BALB/c mice, and validate the libraries for application to other systems. PMID:26779130

  14. Transcriptome Analysis of a Ustilago maydis ust1 Deletion Mutant Uncovers Involvement of Laccase and Polyketide Synthase Genes in Spore Development.

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    Islamovic, Emir; García-Pedrajas, María D; Chacko, Nadia; Andrews, David L; Covert, Sarah F; Gold, Scott E

    2015-01-01

    Ustilago maydis, causal agent of corn smut disease, is a dimorphic fungus alternating between a saprobic budding haploid and an obligate pathogenic filamentous dikaryon. Maize responds to U. maydis colonization by producing tumorous structures, and only within these does the fungus sporulate, producing melanized sexual teliospores. Previously we identified Ust1, an APSES (Asm1p, Phd1p, Sok2p, Efg1p, and StuAp) transcription factor, whose deletion led to filamentous haploid growth and the production of highly pigmented teliospore-like structures in culture. In this study, we analyzed the transcriptome of a ust1 deletion mutant and functionally characterized two highly upregulated genes with potential roles in melanin biosynthesis: um05361, encoding a putative laccase (lac1), and um06414, encoding a polyketide synthase (pks1). The Δlac1 mutant strains showed dramatically reduced virulence on maize seedlings and fewer, less-pigmented teliospores in adult plants. The Δpks1 mutant was unaffected in seedling virulence but adult plant tumors generated hyaline, nonmelanized teliospores. Thus, whereas pks1 appeared to be restricted to the synthesis of melanin, lac1 showed a broader role in virulence. In conclusion, the ust1 deletion mutant provided an in vitro model for sporulation in U. maydis, and functional analysis supports the efficacy of this in vitro mutant analysis for identification of genes involved in in planta teliosporogenesis.

  15. Comparative studies of genome-wide maps of nucleosomes between deletion mutants of elp3 and hos2 genes of Saccharomyces cerevisiae.

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    Takashi Matsumoto

    Full Text Available In order to elucidate the influence of histone acetylation upon nucleosomal DNA length and nucleosome position, we compared nucleosome maps of the following three yeast strains; strain BY4741 (control, the elp3 (one of histone acetyltransferase genes deletion mutant, and the hos2 (one of histone deactylase genes deletion mutant of Saccharomyces cerevisiae. We sequenced mononucleosomal DNA fragments after treatment with micrococcal nuclease. After mapping the DNA fragments to the genome, we identified the nucleosome positions. We showed that the distributions of the nucleosomal DNA lengths of the control and the hos2 disruptant were similar. On the other hand, the distribution of the nucleosomal DNA lengths of the elp3 disruptant shifted toward shorter than that of the control. It strongly suggests that inhibition of Elp3-induced histone acetylation causes the nucleosomal DNA length reduction. Next, we compared the profiles of nucleosome mapping numbers in gene promoter regions between the control and the disruptant. We detected 24 genes with low conservation level of nucleosome positions in promoters between the control and the elp3 disruptant as well as between the control and the hos2 disruptant. It indicates that both Elp3-induced acetylation and Hos2-induced deacetylation influence the nucleosome positions in the promoters of those 24 genes. Interestingly, in 19 of the 24 genes, the profiles of nucleosome mapping numbers were similar between the two disruptants.

  16. A suitable streptomycin-resistant mutant for constructing unmarked in-frame gene deletions using rpsL as a counter-selection marker.

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    Yu-Kuo Tsai

    Full Text Available The streptomycin counter-selection system is a useful tool for constructing unmarked in-frame gene deletions, which is a fundamental approach to study bacteria and their pathogenicity at the molecular level. A prerequisite for this system is acquiring a streptomycin-resistant strain due to rpsL mutations, which encodes the ribosomal protein S12. However, in this study no streptomycin resistance was found to be caused by rpsL mutations in all 127 clinical strains of Klebsiella pneumoniae isolated from liver abscess patients. By screening 107 spontaneous mutants of streptomycin resistance from a clinical strain of K. pneumoniae, nucleotide substitution or insertion located within the rpsL was detected in each of these strains. Thirteen different mutants with varied S12 proteins were obtained, including nine streptomycin-dependent mutants. The virulence of all four streptomycin-resistant mutants was further evaluated. Compared with the parental strain, the K42N, K42T and K87R mutants showed a reduction in growth rate, and the K42N and K42T mutants became susceptible to normal human serum. In the mice LD50 (the bacterial dose that caused 50% death assay, the K42N and K42T mutants were ∼ 1,000-fold less lethal (∼ 2 × 10(5 CFU and the K87R mutant was ∼ 50-fold less lethal (∼ 1 × 10(4 CFU than the parental strain (∼ 2 × 10(2 CFU. A K42R mutant showed non-observable effects on the above assays, while this mutant exhibited a small cost (P < 0.01 in an in vitro growth competition experiment. In summary, most of the K. pneumoniae strains with streptomycin resistance caused by rpsL mutations are less virulent than their parental strain in the absence of streptomycin. The K42R mutant showed similar pathogenicity to its parental strain and should be one of the best choices when using rpsL as a counter-selection marker.

  17. Expression of brown-midrib in a spontaneous sorghum mutant is linked to a 5'-UTR deletion in lignin biosynthesis gene SbCAD2.

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    Li, Huang; Huang, Yinghua

    2017-09-15

    Brown midrib (bmr) mutants in sorghum (Sorghum bicolor (L.) Moench) and several other C4 grasses are associated with reduced lignin concentration, altered lignin composition and improved cell wall digestibility, which are desirable properties in biomass development for the emerging lignocellulosic biofuel industry. Studying bmr mutants has considerably expanded our understanding of the molecular basis underlying lignin biosynthesis and perturbation in grasses. In this study, we performed quantitative trait locus (QTL) analysis, identified and cloned a novel cinnamyl alcohol dehydrogenase allele (SbCAD2) that has an 8-bp deletion in its 5'-untranslated region (UTR), conferring the spontaneous brown midrib trait and lignin reduction in the sorghum germplasm line PI 595743. Complementation test and gene expression analysis revealed that this non-coding region alteration is associated with the significantly reduced expression of the SbCAD2 in PI 595743 throughout its growth stages. Moreover, a promoter-GUS fusion study with transgenic Arabidopsis thaliana plants found that SbCAD2 promoter is functionally conserved, driving a specific expression pattern in lignifying vascular tissues. Taken together, our results revealed the genetic basis of bmr occurrence in this spontaneous sorghum mutant and suggested the regulatory region of the SbCAD2 can be a target site for optimizing lignin modification in sorghum and other bioenergy crops.

  18. The Immature Fiber Mutant Phenotype of Cotton (Gossypium hirsutum Is Linked to a 22-bp Frame-Shift Deletion in a Mitochondria Targeted Pentatricopeptide Repeat Gene

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    Gregory N. Thyssen

    2016-06-01

    Full Text Available Cotton seed trichomes are the most important source of natural fibers globally. The major fiber thickness properties influence the price of the raw material, and the quality of the finished product. The recessive immature fiber (im gene reduces the degree of fiber cell wall thickening by a process that was previously shown to involve mitochondrial function in allotetraploid Gossypium hirsutum. Here, we present the fine genetic mapping of the im locus, gene expression analysis of annotated proteins near the locus, and association analysis of the linked markers. Mapping-by-sequencing identified a 22-bp deletion in a pentatricopeptide repeat (PPR gene that is completely linked to the immature fiber phenotype in 2837 F2 plants, and is absent from all 163 cultivated varieties tested, although other closely linked marker polymorphisms are prevalent in the diversity panel. This frame-shift mutation results in a transcript with two long open reading frames: one containing the N-terminal transit peptide that targets mitochondria, the other containing only the RNA-binding PPR domains, suggesting that a functional PPR protein cannot be targeted to mitochondria in the im mutant. Taken together, these results suggest that PPR gene Gh_A03G0489 is involved in the cotton fiber wall thickening process, and is a promising candidate gene at the im locus. Our findings expand our understanding of the molecular mechanisms that modulate cotton fiber fineness and maturity, and may facilitate the development of cotton varieties with superior fiber attributes.

  19. Identification of genes involved in the toxic response of Saccharomyces cerevisiae against iron and copper overload by parallel analysis of deletion mutants.

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    Jo, William J; Loguinov, Alex; Chang, Michelle; Wintz, Henri; Nislow, Corey; Arkin, Adam P; Giaever, Guri; Vulpe, Chris D

    2008-01-01

    Iron and copper are essential nutrients for life as they are required for the function of many proteins but can be toxic if present in excess. Accumulation of these metals in the human body as a consequence of overload disorders and/or high environmental exposures has detrimental effects on health. The budding yeast Saccharomyces cerevisiae is an accepted cellular model for iron and copper metabolism in humans primarily because of the high degree of conservation between pathways and proteins involved. Here we report a systematic screen using yeast deletion mutants to identify genes involved in the toxic response to growth-inhibitory concentrations of iron and copper sulfate. We aimed to understand the cellular responses to toxic concentrations of these two metals by analyzing the different subnetworks and biological processes significantly enriched with these genes. Our results indicate the presence of two different detoxification pathways for iron and copper that converge toward the vacuole. The product of several of the identified genes in these pathways form molecular complexes that are conserved in mammals and include the retromer, endosomal sorting complex required for transport (ESCRT) and AP-3 complexes, suggesting that the mechanisms involved can be extrapolated to humans. Our data also suggest a disruption in ion homeostasis and, in particular, of iron after copper exposure. Moreover, the identification of treatment-specific genes associated with biological processes such as DNA double-strand break repair for iron and tryptophan biosynthesis for copper suggests differences in the mechanisms by which these two metals are toxic at high concentrations.

  20. A population of deletion mutants and an integrated mapping and Exome-seq pipeline for gene discovery in maize

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    To better understand maize endosperm filling and maturation, we developed a novel functional genomics platform that combined Bulked Segregant RNA and Exome sequencing (BSREx-seq) to map causative mutations and identify candidate genes within mapping intervals. Using gamma-irradiation of B73 maize to...

  1. [Ferric iron absorption in deltar p f F xoo, a gene deletion mutant of Xanthomonas oryzae pv. oryzae, assayed using atomic absorption spectrophotometry].

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    Sun, Lei; Wu, Mao-Sen; He, Chen-Yang

    2010-04-01

    The ferric iron absorption is one of the most important limiting factors of bacterial growth of Xanthomonas oryzae pv. oryzae. It has been previously speculated that r p f F xoo might be involved in the ferric iron metabolism of the pathogen. In the present study, deltar p f F xoo, a gene deletion mutant, was generated from the wild-type strain PXO99A of Xoo through the homologous recombination, and Fe content was assayed using flame atomic absorption in PXO99A and deltar p f F xoo. The results indicated that the recovery was 99.7% and the relative standard deviation was 1.89 under optimized AAS operating conditions. The increase in Fe absorption in PXO99A and deltar p f F xoo was observed with the increasing time. However, the ferric content of deltar p f F xoo was significantly lower than that of PXO99A (P < 0.05). It is suggested that r p f F xoo is involved in iron metabolism in Xanthomonas oryzae pv. oryzae.

  2. Spontaneous asj-2J mutant mouse as a model for generalized arterial calcification of infancy: a large deletion/insertion mutation in the Enpp1 gene.

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    Qiaoli Li

    Full Text Available Generalized arterial calcification of infancy (GACI, an autosomal recessive disorder caused by mutations in the ENPP1 gene, manifests with extensive mineralization of the cardiovascular system. The affected individuals in most cases die within the first year of life, and there is currently no effective treatment for this disorder. In this study, we characterized a spontaneous mutant mouse, asj-2J, as a model for GACI. These mice were identified as part of a phenotypic deviant search in a large-scale production colony of BALB/cJ mice at The Jackson Laboratory. They demonstrated a characteristic gait due to stiffening of the joints, with phenotypic similarity to a previously characterized asj ("ages with stiffened joints" mouse, caused by a missense mutation in the Enpp1 gene. Complementation testing indicated that asj-2J and asj were allelic. PCR-based mutation detection strategy revealed in asj-2J mice a large, 40,035 bp, deletion spanning from intron 1 to the 3'-untranslated region of the Enpp1 gene, coupled with a 74 bp insertion. This was accompanied with a significant reduction in the plasma PPi concentration and reduced PPi/Pi ratio. As a consequence, extensive aberrant mineralization affecting the arterial vasculature, a number of internal organs, and the dermal sheath of vibrissae, a progressive biomarker of the ectopic mineralization process, was demonstrated by a combination of micro computed tomography, histopathology with calcium-specific stains, and direct chemical assay of calcium. Comparison of the asj and asj-2J mice demonstrated that the latter ones, particularly when placed on an acceleration diet high in phosphate and low in magnesium, had more extensive mineralization. Thus, the asj-2J mouse serves as a novel model for GACI, a currently intractable disorder.

  3. Polypeptone induces dramatic cell lysis in ura4 deletion mutants of fission yeast.

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    Yuzy Matsuo

    Full Text Available Polypeptone is widely excluded from Schizosaccharomyces pombe growth medium. However, the reasons why polypeptone should be avoided have not been documented. Polypeptone dramatically induced cell lysis in the ura4 deletion mutant when cells approached the stationary growth phase, and this phenotype was suppressed by supplementation of uracil. To determine the specificity of this cell lysis phenotype, we created deletion mutants of other genes involved in de novo biosynthesis of uridine monophosphate (ura1, ura2, ura3, and ura5. Cell lysis was not observed in these gene deletion mutants. In addition, concomitant disruption of ura1, ura2, ura3, or ura5 in the ura4 deletion mutant suppressed cell lysis, indicating that cell lysis induced by polypeptone is specific to the ura4 deletion mutant. Furthermore, cell lysis was also suppressed when the gene involved in coenzyme Q biosynthesis was deleted. This is likely because Ura3 requires coenzyme Q for its activity. The ura4 deletion mutant was sensitive to zymolyase, which mainly degrades (1,3-beta-D glucan, when grown in the presence of polypeptone, and cell lysis was suppressed by the osmotic stabiliser, sorbitol. Finally, the induction of cell lysis in the ura4 deletion mutant was due to the accumulation of orotidine-5-monophosphate. Cell wall integrity was dramatically impaired in the ura4 deletion mutant when grown in the presence of polypeptone. Because ura4 is widely used as a selection marker in S. pombe, caution needs to be taken when evaluating phenotypes of ura4 mutants.

  4. Complete androgen insensitivity syndrome due to a new frameshift deletion in exon 4 of the androgen receptor gene: Functional analysis of the mutant receptor

    OpenAIRE

    Lobaccaro, J.M.; Lumbroso, S.; Poujol, Nicolas; Georget, V.; Brinkmann, Albert; Malpuech, Georges; Sultan, C.

    1995-01-01

    textabstractWe studied the androgen receptor gene in a large kindred with complete androgen insensitivity syndrome and negative receptor-binding activity, single-strand conformation polymorphism (SSCP) analysis and sequencing identified a 13 base pair deletion within exon 4. This was responsible for a predictive frameshift in the open reading frame and introduction of a premature stop codon at position 783 instead of 919. The deletion was reproduced in androgen receptor wildtype cDNA and tran...

  5. Antibodies with higher bactericidal activity induced by a Neisseria gonorrhoeae Rmp deletion mutant strain.

    Directory of Open Access Journals (Sweden)

    Guocai Li

    Full Text Available Neisseria gonorrhoeae (N. gonorrhoeae outer membrane protein reduction modifiable protein (Rmp has strong immunogenicity. However, anti-Rmp antibodies block rather than preserve the antibacterial effects of protective antibodies, which hampers the development of vaccines for gonococcal infections. We herein constructed an Rmp deletion mutant strain of N. gonorrhoeae by gene homologous recombination. The 261-460 nucleotide residues of Rmp gene amplified from N. gonorrhoeae WHO-A strain were replaced with a kanamycin-resistant Kan gene amplified from pET-28a. The resultant hybridized DNA was transformed into N. gonorrhoeae WHO-A strain. PCR was used to screen the colonies in which wild-type Rmp gene was replaced with a mutant gene fragment. Western blotting revealed that the Rmp deletion mutant strain did not express Rmp protein. Rmp deletion did not alter the morphological and Gram staining properties of the mutant strain that grew slightly more slowly than the wild-type one. Rmp gene mutated stably throughout 25 generations of passage. Antibody-mediated complement-dependent cytotoxicity assay indicated that the antibodies induced by the mutant strain had evidently higher bactericidal activities than those induced by the wild-type strain. Further modification of the Rmp deletion mutant strain is still required in the development of novel live attenuated vaccines for gonorrhea by Opa genes deletion or screening of phenotypic variant strains that do not express Opa proteins.

  6. Antibodies with higher bactericidal activity induced by a Neisseria gonorrhoeae Rmp deletion mutant strain.

    Science.gov (United States)

    Li, Guocai; Xie, Rushan; Zhu, Xiaoping; Mao, Yanli; Liu, Shuangxi; Jiao, Hongmei; Yan, Hua; Xiong, Kun; Ji, Mingchun

    2014-01-01

    Neisseria gonorrhoeae (N. gonorrhoeae) outer membrane protein reduction modifiable protein (Rmp) has strong immunogenicity. However, anti-Rmp antibodies block rather than preserve the antibacterial effects of protective antibodies, which hampers the development of vaccines for gonococcal infections. We herein constructed an Rmp deletion mutant strain of N. gonorrhoeae by gene homologous recombination. The 261-460 nucleotide residues of Rmp gene amplified from N. gonorrhoeae WHO-A strain were replaced with a kanamycin-resistant Kan gene amplified from pET-28a. The resultant hybridized DNA was transformed into N. gonorrhoeae WHO-A strain. PCR was used to screen the colonies in which wild-type Rmp gene was replaced with a mutant gene fragment. Western blotting revealed that the Rmp deletion mutant strain did not express Rmp protein. Rmp deletion did not alter the morphological and Gram staining properties of the mutant strain that grew slightly more slowly than the wild-type one. Rmp gene mutated stably throughout 25 generations of passage. Antibody-mediated complement-dependent cytotoxicity assay indicated that the antibodies induced by the mutant strain had evidently higher bactericidal activities than those induced by the wild-type strain. Further modification of the Rmp deletion mutant strain is still required in the development of novel live attenuated vaccines for gonorrhea by Opa genes deletion or screening of phenotypic variant strains that do not express Opa proteins.

  7. A rice mutant displaying a heterochronically elongated internode carries a 100 kb deletion

    Institute of Scientific and Technical Information of China (English)

    Mika Hayashi-Tsugane; Masahiko Maekawa; Qian Qian; Hirokazu Kobayashi; Shigeru Iida; Kazuo Tsugane

    2011-01-01

    We have isolated a recessive rice mutant,designated as indeterminate growth(ing),which displays creeping and apparent heterochronic phenotypes in the vegetative period with lanky and winding culms.Rough mapping and subsequent molecular characterization revealed that the ing mutant carries a large deletion,which corresponds to a 103 kb region in the Nipponbare genome,containing nine annotated genes on chromosome 3.Of these annotated genes,the SLRI gene encoding a DELLA protein is the only one that is well characterized in its function,and its null mutation,which is caused by a single base deletion in the middle of the intronless SLR1 gene,confers a slender phenotype that bears close resemblance to the ing mutant phenotype.The primary cause of the ing mutant phenotype is the deletion of the SLR1 gene,and the ing mutant appears to be the first characterized mutant having the entire SLRI sequence deleted.Our results also suggest that the deleted region of 103 kb does not contain an indispensable gene,whose dysfunction must result in a lethal phenotype.

  8. Construction of sufC gene deleted mutant in salmonella enterica serovar typhi%伤寒沙门菌sufC基因缺陷变异株的制备

    Institute of Scientific and Technical Information of China (English)

    李美芬; 朱超望

    2011-01-01

    目的 为深入研究sufC基因在伤寒沙门菌中的功能作用,制备sufC基因缺陷变异株.方法 根据GeneBank公布的伤寒沙门菌sufC基因序列,设计sufC缺失用PCR特异性引物,制备缺失sufC基因的同源性核苷酸片段,连接自杀质粒后导入伤寒沙门菌野生株,诱导同源重组,重组菌经PCR观察及序列分析鉴定,将完全重组稳定的相应菌株作为伤寒沙门菌sufC基因缺陷变异株,并经测序分析加以确定.结果 PCR及序列分析证实,缺陷变异株的sufC基因缺失495个碱基.结论 伤寒沙门菌sufC基因缺陷株构建成功,为进一步研究其在伤寒沙门菌中的功能作用奠定了基础.%Objective For investigating the funcation of sufC gene, the deletion mutant of the sufC gene was constructed in Salmonella enterica serovar Typhi (S. Typhi). Methods According to sufC gene sequence from the Gene Bank, designed sufC deletions using PCR specific primer. Constructed deletions of the sufC gene homology in nucleotide fragment, and connected the suicide plasmid. The suicide plasmid including sufC gene homology in nucleotide fragment which was transferred into S. Typhi wild strains. It was induction of homologous recombination. The recombination was valided by PCR and sequencing analysis. The completely stable recombinant strains were sufC gene defective mutant of S. Typhi. Results PCR and sequencing analysis confirmed that the sufC gene mutant was deleted 495 bp. Conclusions The sufC gene deleted mutant of S. Enterica serovar Typhi was generated successfully which was a foundation to study the function of the sufC gene in S. Enterica serovar Typhi.

  9. Complete androgen insensitivity syndrome due to a new frameshift deletion in exon 4 of the androgen receptor gene: Functional analysis of the mutant receptor

    NARCIS (Netherlands)

    J.M. Lobaccaro; S. Lumbroso; N. Poujol (Nicolas); V. Georget; A.O. Brinkmann (Albert); G. Malpuech (Georges); C. Sultan

    1995-01-01

    textabstractWe studied the androgen receptor gene in a large kindred with complete androgen insensitivity syndrome and negative receptor-binding activity, single-strand conformation polymorphism (SSCP) analysis and sequencing identified a 13 base pair deletion within exon 4. This was responsible for

  10. Construction and characterization of a bovine herpesvirus 5 mutant with a deletion of the gI, gE and US9 genes Construção e caracterização de um mutante herpesvírus bovino 5 com uma deleção nos genes gI, gE e US9

    Directory of Open Access Journals (Sweden)

    Ana Cláudia Franco

    2007-12-01

    Full Text Available Bovine herpesvirus 5 (BoHV-5 is a important cause of viral encephalitis in cattle in South America. Within the framework of developing a differential vaccine against BoHV-5, a deletion mutant was constructed based on a Brazilian BoHV-5 isolate. The target of the deletions were genes that code proteins implicated in the neurovirulence of BoHV-5, the glycoprotein I (gI, glycoprotein E (gE and membrane protein US9. To construct the deletion mutant of BoHV-5, the flanking regions of all three genes were cloned in a prokaryotic plasmid. This deletion fragment was co-transfected with the viral DNA into bovine cells. Identification of deletion mutants was performed by immunostaining with an anti-gE monoclonal antibody. One of the gE negative viral populations found was purified, amplified and further examined by restriction endonuclesase analysis of its genomic DNA. The plaque sizes and penetration kinetics of the deletion mutant and wild type viruses were compared. The plaque sizes of the deletion mutant were significantly smaller than those of the parental strain (p O herpesvírus bovino 5 (BoHV-5 é uma causa importante de encefalite viral em bovinos na América do Sul. Buscando o desenvolvimento de uma vacina diferencial contra o BoHV-5, um mutante deletado foi construído com base em um isolado brasileiro deste vírus. O alvo das deleções foram genes que codificam proteínas implicadas na neurovirulência do BoHV-5, a glicoproteína I (gI, a glicoproteína E (gE e a proteína de membrana US9. Para construir o mutante deletado de BoHV-5, as regiões flanqueadoras dos três genes foram clonadas em um plasmídeo procarioto. Este fragmento de deleção foi co-transfectado com o DNA viral em células de bovinos. A identificação dos mutantes deletados foi feita por meio da técnica de imunoperoxidase com um anticorpo monoclonal anti-gE. Uma das populacões virais gE negativas encontradas foi purificada, amplificada e seu genoma foi examinado por an

  11. The mitochondrial genome of the fission yeast Schizosaccharomyces pombe : 5. Characterization of mitochondrial deletion mutants.

    Science.gov (United States)

    Ahne, F; Merlos-Lange, A M; Lang, B F; Wolf, K

    1984-09-01

    The three mutator strains ana (r)-8, ana (r)-14, and diu (r)-301 were shown to produce respiratory deficient mutants at different rates. The frequency of respiratory deficient mutants in a culture could be increased by adding ethidium bromide. According to their cytochrome spectra and enzymatic activities they form three classes, namely mutants defective in cytochrome oxidase, in cytochrome b, and in both cytochromes. By restriction enzyme analysis of mitochondrial DNA from about 100 mutants, 22 deletion mutants were identified. The deletions, ranging from 50 to 1,500 base pairs were physically mapped. Deletions were localized in the genes coding for subunit 1 of cytochrome oxidase with its two introns, within the cytochrome b gene and its intron, and within the genes for subunits 2 and 3 of cytochrome oxidase. In several cases, where the physical mapping yielded ambiguous results, pairwise genetic crosses ruled out an overlap between two neighbouring deletions.Using these mitochondrial deletion mutants as tester strains, it was shown that only tetrad analysis and chemical haploidization, but not mitotic segregation analysis, allows a decision between chromosomal and mitochondrial inheritance of respiratory deficiency in Schizosaccharomyces pombe.

  12. A Mutant with Expression Deletion of Gene Sec-1 in a 1RS.1BL Line and Its Effect on Production Quality of Wheat.

    Directory of Open Access Journals (Sweden)

    Zhi Li

    Full Text Available The chromosome arm 1RS of rye (Secale cereal L. has been used worldwide as a source of genes for agronomic and resistant improvement. However, the 1RS arm in wheat has end-use quality defects that are partially attributable to the presence of ω-secalins, which are encoded by genes at the Sec-1 locus. Various attempts in removing the Sec-1 genes from the 1RS.1BL translocation chromosome have been made. In the present study, two new primary 1RS.1BL translocation lines, T917-26 and T917-15, were developed from a cross between wheat variety "A42912" and Chinese local rye "Weining." The lines T917-15 and T917-26 carried a pair of intact and homogeneous 1RS.1BL chromosomes. The line T917-26 also harbored an expression deletion of some genes at the Sec-1 locus, which originated from a mutation that occurred simultaneously with wheat-rye chromosome translocations. These results suggest that the accompanying mutations of the evolutionarily significant translocations are remarkable resources for plant improvement. Comparison of translocation lines with its wheat parent showed improvements in the end-use quality parameters, which included protein content (PC, water absorption (WA, sodium dodecyl sulfate sedimentation (SDSS, wet gluten (WG, dry gluten (DG and dough stickiness (DS, whereas significant reduction in gluten index (GI and stability time (ST were observed. These findings indicate that 1RS in wheat has produced a higher amount of protein, although these comprised worse compositions. However, in the T917-26 line that harbored an expression deletion mutation in the Sec-1 genes, the quality parameters were markedly improved relative to its sister line, T917-15, especially for GI and DS (P < 0.05. These results indicated that expression deletion of Sec-1 genes significantly improves the end-use quality of wheat cultivars harboring the 1RS.1BL translocation. Strategies to remove the Sec-1 genes from the 1RS.1BL translocation in wheat improvement are

  13. 替代失活法构建变形链球菌LuxS基因缺陷株%Construction of Streptococcus mutans LuxS gene deletion mutants by long flanking homology polymerase chain reaction

    Institute of Scientific and Technical Information of China (English)

    李月恒; 陈惠珍; 周智; 陈娇; 李锐; 尹一兵; 张雪梅

    2011-01-01

    目的 LuxS基因是变形链球菌生物膜早期形成过程中的关键基因,构建该基因的缺陷菌.方法 采用长臂同源多聚酶链反应(LFH-PCR)方法构建含红霉素耐药基因片段的LuxS基因上、下游同源序列的连接片段,转化到变形链球菌中,在红霉素的平板上筛选缺陷菌株,并采用PCR鉴定.结果 对变形链球菌LuxS基因缺陷菌株进行PCR和DNA序列测定分析证实构建成功.结论 成功构建出变形链球菌LuxS基因的缺陷菌株,为后期针对变形链球菌LuxS基因的相关研究奠定基础.%Objective To construct LuxS deletion mutant of Streptococcus mutans and get the capsule deficient strain. Method Long flanking homology polymerase chain reaction(LFH-PCR) was introduced to generate a gene disruption construct consisting of Emr cassette with long flanking homology regions to the target gene. The electroporation competence of Streptococcus mutans was then transformed with this PCR product. Then the positive transformants were counted on selective agar containing erythromycin and identified by PCR. Result Identification by PCR and sequencing confirmed the validity of the LuxS deletion mutant of Streptococcus mutans. Conclusion The successful construction of the LuxS deletion mutant can be used in further functional genome research.

  14. Further characterization of a highly attenuated Yersinia pestis CO92 mutant deleted for the genes encoding Braun lipoprotein and plasminogen activator protease in murine alveolar and primary human macrophages.

    Science.gov (United States)

    van Lier, Christina J; Tiner, Bethany L; Chauhan, Sadhana; Motin, Vladimir L; Fitts, Eric C; Huante, Matthew B; Endsley, Janice J; Ponnusamy, Duraisamy; Sha, Jian; Chopra, Ashok K

    2015-03-01

    We recently characterized the Δlpp Δpla double in-frame deletion mutant of Yersinia pestis CO92 molecularly, biologically, and immunologically. While Braun lipoprotein (Lpp) activates toll-like receptor-2 to initiate an inflammatory cascade, plasminogen activator (Pla) protease facilitates bacterial dissemination in the host. The Δlpp Δpla double mutant was highly attenuated in evoking bubonic and pneumonic plague, was rapidly cleared from mouse organs, and generated humoral and cell-mediated immune responses to provide subsequent protection to mice against a lethal challenge dose of wild-type (WT) CO92. Here, we further characterized the Δlpp Δpla double mutant in two murine macrophage cell lines as well as in primary human monocyte-derived macrophages to gauge its potential as a live-attenuated vaccine candidate. We first demonstrated that the Δpla single and the Δlpp Δpla double mutant were unable to survive efficiently in murine and human macrophages, unlike WT CO92. We observed that the levels of Pla and its associated protease activity were not affected in the Δlpp single mutant, and, likewise, deletion of the pla gene from WT CO92 did not alter Lpp levels. Further, our study revealed that both Lpp and Pla contributed to the intracellular survival of WT CO92 via different mechanisms. Importantly, the ability of the Δlpp Δpla double mutant to be phagocytized by macrophages, to stimulate production of tumor necrosis factor-α and interleukin-6, and to activate the nitric oxide killing pathways of the host cells remained unaltered when compared to the WT CO92-infected macrophages. Finally, macrophages infected with either the WT CO92 or the Δlpp Δpla double mutant were equally efficient in their uptake of zymosan particles as determined by flow cytometric analysis. Overall, our data indicated that although the Δlpp Δpla double mutant of Y. pestis CO92 was highly attenuated, it retained the ability to elicit innate and subsequent acquired immune

  15. Impaired long-term memory retention and working memory in sdy mutant mice with a deletion in Dtnbp1, a susceptibility gene for schizophrenia

    Directory of Open Access Journals (Sweden)

    Takao Keizo

    2008-10-01

    Full Text Available Abstract Background Schizophrenia is a complex genetic disorder caused by multiple genetic and environmental factors. The dystrobrevin-binding protein 1 (DTNBP1: dysbindin-1 gene is a major susceptibility gene for schizophrenia. Genetic variations in DTNBP1 are associated with cognitive functions, general cognitive ability and memory function, and clinical features of patients with schizophrenia including negative symptoms and cognitive decline. Since reduced expression of dysbindin-1 has been observed in postmortem brains of patients with schizophrenia, the sandy (sdy mouse, which has a deletion in the Dtnbp1 gene and expresses no dysbindin-1 protein, could be an animal model of schizophrenia. To address this issue, we have carried out a comprehensive behavioral analysis of the sdy mouse in this study. Results In a rotarod test, sdy mice did not exhibit motor learning whilst the wild type mice did. In a Barnes circular maze test both sdy mice and wild type mice learned to selectively locate the escape hole during the course of the training period and in the probe trial conducted 24 hours after last training. However, sdy mice did not locate the correct hole in the retention probe tests 7 days after the last training trial, whereas wild type mice did, indicating impaired long-term memory retention. A T-maze forced alternation task, a task of working memory, revealed no effect of training in sdy mice despite the obvious effect of training in wild type mice, suggesting a working memory deficit. Conclusion Sdy mouse showed impaired long-term memory retention and working memory. Since genetic variation in DTNBP1 is associated with both schizophrenia and memory function, and memory function is compromised in patients with schizophrenia, the sdy mouse may represent a useful animal model to investigate the mechanisms of memory dysfunction in the disorder.

  16. Widely Used Herpes Simplex Virus 1 ICP0 Deletion Mutant Strain dl1403 and Its Derivative Viruses Do Not Express Glycoprotein C Due to a Secondary Mutation in the gC Gene.

    Directory of Open Access Journals (Sweden)

    Cristina W Cunha

    Full Text Available Herpes simplex virus 1 (HSV-1 ICP0 is a multi-functional phosphoprotein expressed with immediate early kinetics. An ICP0 deletion mutant, HSV-1 dl1403, has been widely used to study the roles of ICP0 in the HSV-1 replication cycle including gene expression, latency, entry and assembly. We show that HSV-1 dl1403 virions lack detectable levels of envelope protein gC, and that gC is not synthesized in infected cells. Sequencing of the gC gene from HSV-1 dl1403 revealed a single amino acid deletion that results in a frameshift mutation. The HSV-1 dl1403 gC gene is predicted to encode a polypeptide consisting of the original 62 N-terminal amino acids of the gC protein followed by 112 irrelevant, non-gC residues. The mutation was also present in a rescuant virus and in two dl1403-derived viruses, D8 and FXE, but absent from the parental 17+, suggesting that the mutation was introduced during the construction of the dl1403 virus, and not as a result of passage in culture.

  17. NADPH-dependent reductive biotransformation with Escherichia coli and its pfkA deletion mutant: influence on global gene expression and role of oxygen supply.

    Science.gov (United States)

    Siedler, Solvej; Bringer, Stephanie; Polen, Tino; Bott, Michael

    2014-10-01

    An Escherichia coli ΔpfkA mutant lacking the major phosphofructokinase possesses a partially cyclized pentose phosphate pathway leading to an increased NADPH per glucose ratio. This effect decreases the amount of glucose required for NADPH regeneration in reductive biotransformations, such as the conversion of methyl acetoacetate (MAA) to (R)-methyl 3-hydroxybutyrate (MHB) by an alcohol dehydrogenase from Lactobacillus brevis. Here, global transcriptional analyses were performed to study regulatory responses during reductive biotransformation. DNA microarray analysis revealed amongst other things increased expression of soxS, supporting previous results indicating that a high NADPH demand contributes to the activation of SoxR, the transcriptional activator of soxS. Furthermore, several target genes of the ArcAB two-component system showed a lower mRNA level in the reference strain than in the ΔpfkA mutant, pointing to an increased QH2 /Q ratio in the reference strain. This prompted us to analyze yields and productivities of MAA reduction to MHB under different oxygen regimes in a bioreactor. Under anaerobic conditions, the specific MHB production rates of both strains were comparable (7.4 ± 0.2 mmolMHB  h(-1)  gcdw (-1) ) and lower than under conditions of 15% dissolved oxygen, where those of the reference strain (12.8 mmol h(-1)  gcdw (-1) ) and of the ΔpfkA mutant (11.0 mmol h(-1)  gcdw (-1) ) were 73% and 49% higher. While the oxygen transfer rate (OTR) of the reference strain increased after the addition of MAA, presumably due to the oxidation of the acetate accumulated before MAA addition, the OTR of the ΔpfkA strain strongly decreased, indicating a very low respiration rate despite sufficient oxygen supply. The latter effect can likely be attributed to a restricted conversion of NADPH into NADH via the soluble transhydrogenase SthA, as the enzyme is outcompeted in the presence of MAA by the recombinant NADPH-dependent alcohol

  18. Biological characteristics of rpoS gene deleted mutant in Salmonella typhi%伤寒沙门菌rpoS基因缺陷变异株的生物学特性研究

    Institute of Scientific and Technical Information of China (English)

    杜鸿; 周慧琴; 朱雪明

    2010-01-01

    Objective To investigate the biological characteristics of rpoS gene deleted mutation in Salmonella typhi under different stress conditions,so as to explore the target gene for the prevention and treament of Salmonella typhi infection.Methods rpoS gene deleted mutant of Salmonella typhi was prepared by homologious recombination.rpoS mutant and parental strains were incubated under iso-osmia and various stress conditions:acid stress(pH 4.2),high osmolarity stress(NaCl 300 mmol/L),bile stress (1.5 mmol/L sodiumdeoxycbolate)and oxidative stress(1 mmol/L H2O2).The growth curves were compared between mutant and parental strains under different incubation conditions(t test).Results rpoS gene deleted mutant of Salmonella typhi Was successfully generated.Compared with the parental strain,the survival ability of rpoS mutant was significantly compromised under the acid stress,high osmolarity stress and oxidative stress(t values at4 h were 12.864,3.594 and 12.979;t values at 14 h were6.497,3.039 and 10.440,P<0.05 or<0.01).Conclusion rpoS is important for Salmonella typhi to overcome the acid,high osmolarity and oxidative stresses,and it may be a target gene for the prevention and treatment of Salmonella typhi infection.%目的 研究rpoS基因在应激环境下的生物学作用,为临床预防和治疗伤寒沙门菌感染提供可能的靶基因.方法 利用原核生物基因同源重组技术制备伤寒沙门菌rpoS基因缺陷变异株.绘制生长曲线,对比rpoS基因缺陷变异株与野生株在等渗条件和酸应激(pH 4.2)、高渗应激(NaCl 300 mmoL/L)、胆汁应激(脱氧胆酸钠终浓度为1.5 mmoL/L)及氧应激(1 mmol/L H2O2)条件下的生长能力,采用t检验进行统计学分析.结果 成功制备伤寒沙门菌rpoS基因缺陷变异株.与野生株相比,rpoS基因缺陷变异株在酸应激、高渗应激和氧应激条件下的生存能力明显降低(4 h时的t值分别为12.864、3.594和12.979,14 h时的t值分别为6.497、3.039和10.440,P

  19. Deletion and interallelic complementation analysis of Steel mutant mice

    Energy Technology Data Exchange (ETDEWEB)

    Bedell, M.A.; Cleveland, L.S.; Copeland, N.G. [NCI-Frederick Cancer Research and Development Center, MD (United States)] [and others

    1996-03-01

    Mutations at the Steel (Sl) locus produce pleiotropic effects on viability as well as hematopoiesis, pigmentation and fertility. Several homozygous viable Sl alleles have previously have been shown to contain either structural alterations in mast cell growth factor (Mgf) or regulatory mutations that affect expression of the Mgf gene. More severe Sl alleles cause lethality to homozygous embryos and all lethal Sl alleles examined to date contain deletions that remove the entire Mgf coding region. As the timing of the lethality varies from early to late in gestation, it is possible that some deletions may affect other closely linked genes in addition to Mgf. We have analyzed the extent of deleted sequences in seven homozygous lethal Sl alleles. The results of this analysis suggests that late gestation lethality represents the Sl null phenotype and that peri-implantation lethality results from the deletion of at least one essential gene that maps proximal to Sl. We have also examined gene dosage effects of Sl comparing the phenotypes of mice homozygous and hemizygous for each of four viable Sl alleles. Lastly, we show that certain combinations of the viable Sl alleles exhibit interallelic complementation. Possible mechanisms by which such complementation could occur are discussed. 39 refs., 3 figs., 3 tabs.

  20. A genome-wide screen for Schizosaccharomyces pombe deletion mutants that affect telomere length

    Institute of Scientific and Technical Information of China (English)

    Ning-Ning Liu; Tian Xu Han; Li-Lin Du; Jin-Qiu Zhou

    2010-01-01

    @@ Dear Editor, Both the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae are popular model organisms, and studies using these models have provided many informative clues for solving fundamental biological questions [1], such as DNA replication,cell cycle regulation and gene transcription. Since the completion of genome sequencing of these fungi [2, 3],systematic genetic modification, e.g. gene deletion, has become possible, and genome-wide phenotypic screening for gene function has been widely carried out. For example, Askree et al. and Gatbonton et al. examined the telomere-length change in about 4 800 non-essential gene deletion mutants of S. cerevisiae, and found that about 250 genes are involved in telomere-length regulation.

  1. Growth, physicochemical properties, and morphogenesis of Chinese wild-type PRV Fa and its gene-deleted mutant strain PRV SA215

    Directory of Open Access Journals (Sweden)

    Tang Shanhu

    2011-06-01

    Full Text Available Abstract Background PRV Fa is common in China and causes most of the pseudorabies in the pig industry. A PRV SA215 strain with deleted gE, gI, and TK genes was constructed to develop a commercial attenuated live vaccine. However, the physicochemical properties, growth pattern, penetration kinetics, and morphogenesis of the PRV SA215 and its parental PRV Fa strain are unclear. Results A series of experiments were conducted to characterize both strains and provide more information. PRV Fa and PRV SA215 were found to have similar penetration patterns, with about 5 min half-time of penetration. The SA215 strain exhibited a slight delay in entry compared with PRV Fa. In the one-step growth test, the titers of the SA215 strain were first detected at 8 h, rapidly increased, and peaked at 12 h. A plateau was formed between 12-36 h of culturing. PRV SA215 showed delayed replication and approximately 10-30-fold lower titers during 0-16 h of culturing compared with the PRV-Fa strain. After 16 h, the PRV Fa titers dramatically decreased, whereas those of PRV SA215 were prolonged to 36 h and reached a titer value equal to that of PRV Fa and then decreased. Both strains were sensitive to both heat and acid-alkali treatments; however, PRV Fa was relatively more stable to heat treatment than PRV SA215. Both strains could propagate in the cultures with pH values from 5.0 to 9.0. Cultures with pH below 3.0 or above 11.0 were fatal to both strains. Both strains had considerable resistance to freeze-thawing treatments. Morphogenetic investigations showed that typical phases in the maturation pathway were observed in the PRV Fa-infected PK15 cells, whereas secondary envelopment was not observed in the PRV SA215 strain. Instead, capsid aggregations with concomitants of electrodense materials were observed. Conclusions These results suggest that PRV SA215 is a promising strain for vaccine development

  2. 耐甲氧西林表皮葡萄球菌 psm-mec 缺失突变株的构建%Construction of mutant strains of methicillin resistant Staphylococcus epidermidis with psm-mec gene deletion

    Institute of Scientific and Technical Information of China (English)

    杨永长; 胡洪华; 陈亮; 刘华; 喻华; 黄文芳

    2015-01-01

    Objective To construct mutant strains of methicillin resistant Staphylococcus epidermi-dis (MRSE) with psm-mec gene deletion and to investigate the function of psm-mec gene.Methods The drug sensitivity test and DNA sequence analysis were performed to screen out the tetracycline and chloram -phenicol sensitive clinical strains of MRSE , whose upstream and downstream sequences of psm-mec gene were identical to those of the Staphylococcus epidermidis reference strain RP62A.The recombinant plasmid pBT2-Δpsm-mec was constructed by using the fusion PCR and a temperature sensitive shuttle plasmid .After being identified , the plasmid was transformed into the Staphylococcus aureus RN4220 strain by electropora-tion, and then transformed into the selected clinical isolates of MRSE .The mutant strains of MRSE with psm-mec deletion were screened out and identified after homologous recombination .The differences in biofilm formation between the mutant and wild-type strains were analyzed for further elucidation the relationships be-tween the psm-mec gene and biofilm formation in MRSE strains .Results Three clinical MRSE isolates for the construction of mutant strains with psm-mec gene deletion were screened out and identified by using drug sensitivity test and sequence alignment analysis .The mutants constructed via homogenous recombination were screened out and identified .Compared with the corresponding wild-type strains, the three mutants with psm-mec gene deletion showed significantly decreased ability of biofilm formation , demonstrating that the psm-mec genes strains induced the biofilm formation of MRSE .Conclusion The Δpsm-mec mutant strains were successfully constructed .The psm-mec gene played an important role in the biofilm formation of Staphy-lococcus epidermdis.%目的:构建耐甲氧西林表皮葡萄球菌( MRSE)的psm-mec缺失突变株,并对psm-mec的功能进行初步研究。方法运用药敏试验、DNA序列分析技术筛选psm-mec上下游序

  3. X-ray survival characteristics and genetic analysis for nine saccharomyces deletion mutants that show altered radiation sensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Game, John C.; Williamson, Marsha S.; Baccari, Clelia

    2004-01-07

    The availability of a genome-wide set of Saccharomyces deletion mutants provides a chance to identify all the yeast genes involved in DNA repair. Using X-rays, we are screening these mutants to identify additional genes that show increased sensitivity to the lethal effects of ionizing radiation. For each mutant identified as sensitive, we are confirming that the sensitivity phenotype co-segregates with the deletion allele and are obtaining multipoint survival-versus-dose assays in at least two haploid and one homozygous diploid strains. We present data for deletion mutants involving the genes DOT1, MDM20, NAT3, SPT7, SPT20, GCN5, HFI1, DCC1 and VID21/EAF1, and discuss their potential roles in repair. Eight of these genes have a clear radiation-sensitive phenotype when deleted, but the ninth, GCN5, has at most a borderline phenotype. None of the deletions confer substantial sensitivity to ultra-violet radiation, although one or two may confer marginal sensitivity. The DOT1 gene is of interest because its only known function is to methylate one lysine residue in the core of the histone H3 protein. We find that histone H3 mutants (supplied by K. Struhl) in which this residue is replaced by other amino-acids are also X-ray sensitive, seeming to confirm that methylation of the lysine-79 residue is required for effective repair of radiation damage.

  4. Construction and characterization of partially ntrC-deleted mutants in Alcaligenes faecalis

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To study the effect of ntrC gene product on the expression and regulation of other important nitrogen-fixing genes in Alcaligenes faecalis, partially ntrC-deleted mutants of A. faecalis have been generated. To start with, the ntrC gene of A. faecalis was cloned into a suicide plasmid pSUP202 to create a recombinant plasmid pSUM1. The ntrC gene in pSUM1 was then replaced by a lacZ-Kmr fragment resulted in the generation of a plasmid pSUM2. The lacZ fragment in pSUM2 was further removed and a plasmid pSUM3 produced. As a second step, the plasmid pSUM2 or pSUM3 was introduced into the wild type of A. faecalis A1501 by conjugation and two partially ntrC-deleted mutants A15CM1 (ntrC∷lacZ) and A15CM2 (ntrC-) were obtained. To understand the regulatory effect of the NtrC on the expression of nifH and nifA, a nifH-lacZ gene or a nifA-lacZ gene was introduced into the ntrC- mutant by conjugation. The results indicated that: (ⅰ) although the ntrC- mutant was nif + , its nitrogen fixation activity was only 20% that of the wild type; (ⅱ) the ntrC- mutant failed to grow on the medium containing nitrate as a sole nitrogen source; (ⅲ) the regulation of ntrC gene expression did not require its own product; (ⅳ) the expression of nifH in A . faecalis was positively regulated by the ntrC. Deletion of the ntrC resulted in the reduction of nifH expression or even totally inactivated nitrogen fixation; (ⅴ) there was no obvious influence on the expression of nifA in A. faecalis if the ntrC gene was deleted.

  5. Catalytic properties of ADAM12 and its domain deletion mutants

    DEFF Research Database (Denmark)

    Jacobsen, Jonas; Visse, Robert; Sørensen, Hans Peter

    2008-01-01

    Human ADAM12 (a disintegrin and metalloproteinase) is a multidomain zinc metalloproteinase expressed at high levels during development and in human tumors. ADAM12 exists as two splice variants: a classical type 1 membrane-anchored form (ADAM12-L) and a secreted splice variant (ADAM12-S) consisting...... of pro, catalytic, disintegrin, cysteine-rich, and EGF domains. Here we present a novel activity of recombinant ADAM12-S and its domain deletion mutants on S-carboxymethylated transferrin (Cm-Tf). Cleavage of Cm-Tf occurred at multiple sites, and N-terminal sequencing showed that the enzyme exhibits...... restricted specificity but a consensus sequence could not be defined as its subsite requirements are promiscuous. Kinetic analysis revealed that the noncatalytic C-terminal domains are important regulators of Cm-Tf activity and that ADAM12-PC consisting of the pro domain and catalytic domain is the most...

  6. Selection of Mycoplasma hominis PG21 deletion mutants by cultivation in the presence of monoclonal antibody 552

    DEFF Research Database (Denmark)

    Jensen, Lise Torp; Ladefoged, Søren; Birkelund, Svend

    1995-01-01

    Three mutants of Mycoplasma hominis PG21 were isolated and shown to contain alterations in the size of a repeat-containing gene encoding a surface-localized 135-kDa antigen designated Lmp1. The mutants were isolated by cultivating M. hominis for a 3-month period in the presence of Lmp1-specific...... characterized. The mutants showed deletions of a various number of repeats. The deletions were accompanied by a decrease in size of the proteins. With increasing size of deletions, agglutination and growth inhibition by MAb 552 became less pronounced. Spontaneous aggregation of the mutant M. hominis cells...... in culture medium was, however, increased, indicating that the repeated elements may be of importance for repulsion of the cells....

  7. The ARABIDOPSIS accession Pna-10 is a naturally occurring sng1 deletion mutant.

    Science.gov (United States)

    Li, Xu; Bergelson, Joy; Chapple, Clint

    2010-01-01

    Sinapoylmalate is the major sinapate ester found in leaves of Arabidopsis thaliana, where it plays an important role in UV-B protection. Metabolic profiling of rosette leaves from 96 Arabidopsis accessions revealed that the Pna-10 accession accumulates sinapoylglucose instead of sinapoylmalate. This unique leaf sinapate ester profile is similar to that of the previously characterized sinapoylglucose accumulator1 (sng1) mutants. SNG1 encodes sinapoylglucose:malate sinapoyltransferase (SMT), a serine carboxypeptidase-like (SCPL) enzyme that catalyzes the conversion of sinapoylglucose to sinapoylmalate. In the reference Columbia genome, the SNG1 gene is located in a cluster of five SCPL genes on Chromosome II. PCR and sequencing analysis of the same genomic region in the Pna-10 accession revealed a 13-kb deletion that eliminates the SNG1 gene (At2g22990) and the gene encoding sinapoylglucose:anthocyanin sinapoyltransferase (SAT) (At2g23000). In addition to its sinapoylmalate-deficient phenotype, and consistent with the loss of SAT, Pna-10 is unable to accumulate sinapoylated anthocyanins. Interestingly, the Pna-17 accession, collected from the same location as Pna-10, has no such deletion. Further analysis of 135 lines collected from the same location as Pna-10 and Pna-17 revealed that four more lines contain the deletion found in Pna-10 accession, suggesting that either the deletion found in Pna-10 is a recent event that has not yet been eliminated through selection or that sinapoylmalate is dispensable for the growth of Arabidopsis under field conditions.

  8. Whole genome HBV deletion profiles and the accumulation of preS deletion mutant during antiviral treatment

    Directory of Open Access Journals (Sweden)

    Zhang Dake

    2012-12-01

    Full Text Available Abstract Background Hepatitis B virus (HBV, because of its error-prone viral polymerase, has a high mutation rate leading to widespread substitutions, deletions, and insertions in the HBV genome. Deletions may significantly change viral biological features complicating the progression of liver diseases. However, the clinical conditions correlating to the accumulation of deleted mutants remain unclear. In this study, we explored HBV deletion patterns and their association with disease status and antiviral treatment by performing whole genome sequencing on samples from 51 hepatitis B patients and by monitoring changes in deletion variants during treatment. Clone sequencing was used to analyze preS regions in another cohort of 52 patients. Results Among the core, preS, and basic core promoter (BCP deletion hotspots, we identified preS to have the highest frequency and the most complex deletion pattern using whole genome sequencing. Further clone sequencing analysis on preS identified 70 deletions which were classified into 4 types, the most common being preS2. Also, in contrast to the core and BCP regions, most preS deletions were in-frame. Most deletions interrupted viral surface epitopes, and are possibly involved in evading immuno-surveillance. Among various clinical factors examined, logistic regression showed that antiviral medication affected the accumulation of deletion mutants (OR = 6.81, 95% CI = 1.296 ~ 35.817, P = 0.023. In chronic carriers of the virus, and individuals with chronic hepatitis, the deletion rate was significantly higher in the antiviral treatment group (Fisher exact test, P = 0.007. Particularly, preS2 deletions were associated with the usage of nucleos(tide analog therapy (Fisher exact test, P = 0.023. Dynamic increases in preS1 or preS2 deletions were also observed in quasispecies from samples taken from patients before and after three months of ADV therapy. In vitro experiments demonstrated that

  9. The immature fiber mutant phenotype of cotton (Gossypium hirsutum) is linked to a 22-bp frame-shift deletion in a mitochondria targeted pentatricopeptide repeat gene

    Science.gov (United States)

    Cotton seed trichomes are the globally most important source of natural fibers. The major fiber thickness properties influence the price of the raw material and the quality of the finished product. The recessive immature fiber (im) gene reduces the degree of fiber cell wall thickening by a process...

  10. Tripeptidase Gene (pepT) of Lactococcus lactis : Molecular Cloning and Nucleotide Sequencing of pepT and Construction of a Chromosomal Deletion Mutant

    NARCIS (Netherlands)

    Mierau, Igor; Haandrikman, Alfred J.; Velterop, Odilia; Tan, Paris S.T.; Leenhouts, Kees L.; Konings, Wilhelmus; Venema, Gerhardus; Kok, Jan

    1994-01-01

    The gene encoding a tripeptidase (pepT) of lactococcus lactis subsp. cremoris (formerly subsp. lactis) MG1363 was cloned from a genomic library in pUC19 and subsequently sequenced. The tripeptidase of L. lactis was shown to be homologous to PepT of Salmonella typhimurium with 47.4% identity in the d

  11. Seven gene deletions in seven days

    DEFF Research Database (Denmark)

    Ingemann Jensen, Sheila; Lennen, Rebecca; Herrgard, Markus;

    2015-01-01

    enables growth at 37 °C, thereby facilitating removal of integrated antibiotic cassettes and deletion of additional genes in the same day. Phosphorothioated primers were demonstrated to enable simultaneous deletions during one round of electroporation. Utilizing these methods, we constructed strains...

  12. Correlation between transcript profiles and fitness of deletion mutants in anaerobic chemostat cultures of Saccharomyces cerevisiae

    Science.gov (United States)

    Tai, Siew Leng; Snoek, Ishtar; Luttik, Marijke A. H.; Almering, Marinka J. H.; Walsh, Michael C.; Pronk, Jack T.; Daran, Jean-Marc

    2007-01-01

    The applicability of transcriptomics for functional genome analysis rests on the assumption that global information on gene function can be inferred from transcriptional regulation patterns. This study investigated whether Saccharomyces cerevisiae genes that show a consistently higher transcript level under anaerobic than aerobic conditions do indeed contribute to fitness in the absence of oxygen. Tagged deletion mutants were constructed in 27 S. cerevisiae genes that showed a strong and consistent transcriptional upregulation under anaerobic conditions, irrespective of the nature of the growth-limiting nutrient (glucose, ammonia, sulfate or phosphate). Competitive anaerobic chemostat cultivation showed that only five out of the 27 mutants (eug1Δ, izh2Δ, plb2Δ, ylr413wΔ and yor012wΔ) conferred a significant disadvantage relative to a tagged reference strain. The implications of this study are that: (i) transcriptome analysis has a very limited predictive value for the contribution of individual genes to fitness under specific environmental conditions, and (ii) competitive chemostat cultivation of tagged deletion strains offers an efficient approach to select relevant leads for functional analysis studies. PMID:17322208

  13. Physiology of deletion mutants in the anaerobic β-myrcene degradation pathway in Castellaniella defragrans

    Directory of Open Access Journals (Sweden)

    Lüddeke Frauke

    2012-09-01

    Full Text Available Abstract Background Monoterpenes present a large and versatile group of unsaturated hydrocarbons of plant origin with widespread use in the fragrance as well as food industry. The anaerobic β-myrcene degradation pathway in Castellaniella defragrans strain 65Phen differs from well known aerobic, monooxygenase-containing pathways. The initial enzyme linalool dehydratase-isomerase ldi/LDI catalyzes the hydration of β-myrcene to (S-(+-linalool and its isomerization to geraniol. A high-affinity geraniol dehydrogenase geoA/GeDH and a geranial dehydrogenase geoB/GaDH contribute to the formation of geranic acid. A genetic system was for the first time applied for the betaproteobacterium to prove in vivo the relevance of the linalool dehydratase-isomerase and the geraniol dehydrogenase. In-frame deletion cassettes were introduced by conjugation and two homologous recombination events. Results Polar effects were absent in the in-frame deletion mutants C. defragrans Δldi and C. defragrans ΔgeoA. The physiological characterization of the strains demonstrated a requirement of the linalool dehydratase-isomerase for growth on acyclic monoterpenes, but not on cyclic monoterpenes. The deletion of geoA resulted in a phenotype with hampered growth rate on monoterpenes as sole carbon and energy source as well as reduced biomass yields. Enzyme assays revealed the presence of a second geraniol dehydrogenase. The deletion mutants were in trans complemented with the broad-host range expression vector pBBR1MCS-4ldi and pBBR1MCS-2geoA, restoring in both cases the wild type phenotype. Conclusions In-frame deletion mutants of genes in the anaerobic β-myrcene degradation revealed novel insights in the in vivo function. The deletion of a high-affinity geraniol dehydrogenase hampered, but did not preclude growth on monoterpenes. A second geraniol dehydrogenase activity was present that contributes to the β-myrcene degradation pathway. Growth on cyclic monoterpenes

  14. Huntington's disease and mitochondrial DNA deletions: event or regular mechanism for mutant huntingtin protein and CAG repeats expansion?!

    Science.gov (United States)

    Banoei, Mohammad Mehdi; Houshmand, Massoud; Panahi, Mehdi Shafa Shariat; Shariati, Parvin; Rostami, Maryam; Manshadi, Masoumeh Dehghan; Majidizadeh, Tayebeh

    2007-11-01

    The mitochondrial DNA (mtDNA) may play an essential role in the pathogenesis of the respiratory chain complex activities in neurodegenerative disorders such as Huntington's disease (HD). Research studies were conducted to determine the possible levels of mitochondrial defect (deletion) in HD patients and consideration of interaction between the expanded Huntingtin gene as a nuclear gene and mitochondria as a cytoplasmic organelle. To determine mtDNA damage, we investigated deletions based in four areas of mitochondrial DNA, in a group of 60 Iranian patients clinically diagnosed with HD and 70 healthy controls. A total of 41 patients out of 60 had CAG expansion (group A). About 19 patients did not show expansion but had the clinical symptoms of HD (group B). MtDNA deletions were classified into four groups according to size; 9 kb, 7.5 kb, 7 kb, and 5 kb. We found one of the four-mtDNA deletions in at least 90% of samples. Multiple deletions have also been observed in 63% of HD patients. None of the normal control (group C) showed mtDNA deletions. The sizes or locations of the deletions did not show a clear correlation with expanded CAG repeat and age in our samples. The study presented evidence that HD patients had higher frequencies of mtDNA deletions in lymphocytes in comparison to the controls. It is thus proposed that CAG repeats instability and mutant Htt are causative factor in mtDNA damage.

  15. Changes in photosynthesis and pigmentation in an agp deletion mutant of the cyanobacterium Synechocystis sp.

    Science.gov (United States)

    Miao, Xiaoling; Wu, Qingyu; Wu, Guifang; Zhao, Nanming

    2003-03-01

    The agp gene encoding ADP-glucose pyrophosphorylase is involved in cyanobacterial glycogen synthesis. By in vitro DNA recombination technology, agp deletion mutant (agp-) of cyanobacterium Synechocystis sp. PCC 6803 was constructed. This mutation led to a complete absence of glycogen biosynthesis. As compared with WT (wild type), a 60% decrease in ratio of the c-phycocyanine/chlorophyll a and no significant change in the carotenoid/chlorophyll a were observed in agp- cells. The agp- mutant had 38% less photosynthetic capacity when grown in light over 600 micromol m(-2) s(-1). Under lower light intensity, the final biomass of the mutant strain was only 1.1 times of that of the WT strain under mixotrophic condition after 6 d culture. Under higher light intensity, however, the final biomass of the WT strain under mixotrophic conditions was 3 times that of the mutant strain after 6 d culture and 1.5 times under photoautotrophic conditions. The results indicate that there is a minimum requirement for glycogen synthesis for normal growth and development in cyanobacteria.

  16. Phenotype MicroArray Analysis of Escherichia coli K-12 Mutants with Deletions of All Two-Component Systems

    Science.gov (United States)

    Zhou, Lu; Lei, Xiang-He; Bochner, Barry R.; Wanner, Barry L.

    2003-01-01

    Two-component systems are the most common mechanism of transmembrane signal transduction in bacteria. A typical system consists of a histidine kinase and a partner response regulator. The histidine kinase senses an environmental signal, which it transmits to its partner response regulator via a series of autophosphorylation, phosphotransfer, and dephosphorylation reactions. Much work has been done on particular systems, including several systems with regulatory roles in cellular physiology, communication, development, and, in the case of bacterial pathogens, the expression of genes important for virulence. We used two methods to investigate two-component regulatory systems in Escherichia coli K-12. First, we systematically constructed mutants with deletions of all two-component systems by using a now-standard technique of gene disruption (K. A. Datsenko and B. L. Wanner, Proc. Natl. Acad. Sci. USA 97:6640-6645, 2000). We then analyzed these deletion mutants with a new technology called Phenotype MicroArrays, which permits assays of nearly 2,000 growth phenotypes simultaneously. In this study we tested 100 mutants, including mutants with individual deletions of all two-component systems and several related genes, including creBC-regulated genes (cbrA and cbrBC), phoBR-regulated genes (phoA, phoH, phnCDEFGHIJKLMNOP, psiE, and ugpBAECQ), csgD, luxS, and rpoS. The results of this battery of nearly 200,000 tests provided a wealth of new information concerning many of these systems. Of 37 different two-component mutants, 22 showed altered phenotypes. Many phenotypes were expected, and several new phenotypes were also revealed. The results are discussed in terms of the biological roles and other information concerning these systems, including DNA microarray data for a large number of the same mutants. Other mutational effects are also discussed. PMID:12897016

  17. Somatic mosaicism for a DMD gene deletion

    Energy Technology Data Exchange (ETDEWEB)

    Saito, Kayoko; Ikeya, Kiyoko; Kondo, Eri [Tokyo Women`s Medical College (Japan)] [and others

    1995-03-13

    Mosaicism is a mixed state, with two cell populations of different genetic origins caused by a cell mutation occurring after fertilization. In the present case, DNA analysis of lymphocytes led to a DMD diagnosis before death. Postmortem immunocytochemical and DNA analysis showed somatic mosaicism. At age 18 years, blood lymphocyte DNA analysis showed a DMD gene deletion, upstream from exon 7 to the 5{prime} end containing both muscle and brain promoters. As the patient`s mother and elder sister had no deletions, he was considered to have a new mutation. Immunocytochemical studies of postmortem tissues showed that dystrophin was absent from the tongue, deltoid, intercostal, psoas and rectus femoris muscles, but there was a mix of dystrophin-positive and negative fibers in the rectus abdominis, cardiac, temporalis and sternocleidomastoid muscles. All diaphragm cells were dystrophin positive. Polymerase chain reaction (PCR) amplification from all tissues except the temporalis and sternocleidomastoid muscles, diaphragm and kidney, in which no deletion was found, showed the deletion from at least exon 6 to the 5{prime} end containing both muscle and brain promoters. In this case, a genomic deletion of the DMD gene contributed to the formation of tissues derived from both ectoderm and endoderm, and cells of mesodermal origin showed genotypic and phenotypic heterogeneity. Our results indicate a mutation of the present case may have occurred just before the period of germ layer formation. 34 refs., 7 figs.

  18. Transcriptome profiling of TDC cluster deletion mutant of Enterococcus faecalis V583

    Directory of Open Access Journals (Sweden)

    Marta Perez

    2016-09-01

    Full Text Available The species Enterococcus faecalis is able to catabolise the amino acid tyrosine into the biogenic amine tyramine by the tyrosine decarboxilase (TDC pathway Ladero et al. (2012 [1]. The TDC cluster comprises four genes: tyrS, an aminoacyl-tRNA synthetase-like gene; tdcA, which encodes the tyrosine decarboxylase; tyrP, a tyrosine/tyramine exchanger gene and nhaC-2, which encodes an Na+/H+ antiporter and whose role in the tyramine biosynthesis remains unknown [2]. In E. faecalis V583 the last three genes are co-transcribed as a single polycistronic mRNA forming the catabolic operon, while tyrS is transcribed independently of the catabolic genes as a monocistronic mRNA [2]. The catabolic operon is transcriptionally induced by tyrosine and acidic pH. On the opposite, the tyrS expression is repressed by tyrosine concentrations [2]. In this work we report the transcriptional profiling of the TDC cluster deletion mutant (E. faecalis V583 ΔTDC [2] compared to the wild-type strain, both grown in M17 medium supplemented with tyrosine. The transcriptional profile data of TDC cluster-regulated genes were deposited in the Gene Expression Omnibus (GEO database under accession no. GSE77864.

  19. Transcriptome profiling of TDC cluster deletion mutant of Enterococcus faecalis V583.

    Science.gov (United States)

    Perez, Marta; Ladero, Victor; Del Rio, Beatriz; Redruello, Begoña; de Jong, Anne; Kuipers, Oscar P; Kok, Jan; Martin, M Cruz; Fernandez, Maria; Alvarez, Miguel A

    2016-09-01

    The species Enterococcus faecalis is able to catabolise the amino acid tyrosine into the biogenic amine tyramine by the tyrosine decarboxilase (TDC) pathway Ladero et al. (2012) [1]. The TDC cluster comprises four genes: tyrS, an aminoacyl-tRNA synthetase-like gene; tdcA, which encodes the tyrosine decarboxylase; tyrP, a tyrosine/tyramine exchanger gene and nhaC-2, which encodes an Na(+)/H(+) antiporter and whose role in the tyramine biosynthesis remains unknown [2]. In E. faecalis V583 the last three genes are co-transcribed as a single polycistronic mRNA forming the catabolic operon, while tyrS is transcribed independently of the catabolic genes as a monocistronic mRNA [2]. The catabolic operon is transcriptionally induced by tyrosine and acidic pH. On the opposite, the tyrS expression is repressed by tyrosine concentrations [2]. In this work we report the transcriptional profiling of the TDC cluster deletion mutant (E. faecalis V583 ΔTDC) [2] compared to the wild-type strain, both grown in M17 medium supplemented with tyrosine. The transcriptional profile data of TDC cluster-regulated genes were deposited in the Gene Expression Omnibus (GEO) database under accession no. GSE77864.

  20. Cloning and characterization of a novel deletion mutant of heterogeneous nuclear ribonucleoprotein M4 from human dendritic cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To identify differentially expressed genes from antigen-stimulated human dendritic cells (DC), subtractive cloning was adopted and more than ten novel genes differentially expressed were cloned. One is a deletion mutant of heterogeneous nuclear ribonucleoprotein (hnRNP) M4 in which the residues from 159 to 197 of hnRNP M4 have been absent. The deletion mutant was shown to be co-expressed with hnRNP M4 in cell lines. The mutant was expressed in antigen-stimulated DC but not in normal DC. Northern blot analysis revealed the presence of a major hnRNP M4 deletion mutant Mrna transcript of 2.4 kilobase with the highest levels in peripheral lymphocytes, lung, liver and spleen. It was also expressed in bone marrow-derived stromal cells (BMSC), BMSC treated with several cytokines but not in BMSC treated with TNF-a. The results revealed a new member of hnRNP family and suggested that hnRNP would participate in antigen process and presentation.

  1. Cloning and characterization of a novel deletion mutant of heterogeneous nuclear ribonucleoprotein M4 from human dendritic cells

    Institute of Scientific and Technical Information of China (English)

    黄欣; 赵忠良; 袁正隆; 张明徽; 朱学军; 陈国友; 曹雪涛

    2000-01-01

    To identify differentially expressed genes from antigen-stimulated human dendritic cells (DC), subtractive cloning was adopted and more than ten novel genes differentially expressed were cloned. One is a deletion mutant of heterogeneous nuclear ribonucleoprotein (hnRNP) M4 in which the residues from 159 to 197 of hnRNP M4 have been absent. The deletion mutant was shown to- be co-expressed with hnRNP M4 in cell lines. The mutant was expressed in antigen-stimulated DC but not in normal DC. Northern blot analysis revealed the presence of a major hnRNP M4 deletion mutant mRNA transcript of 2.4 kilobase with the highest levels in peripheral lymphocytes, lung, liver and spleen. It was also expressed in bone marrow-derived stromal cells (BMSC), BMSC treated with several cytokines but not in BMSC treated with TNF-a. The results revealed a new member of hnRNP family and suggested that hnRNP would participate in antigen process and presentation.

  2. Cloning and characterization of a novel deletion mutant of heterogeneous nuclear ribonucleoprotein M4 from human dendritic cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To identify differentially expressed genes from antigen-stimulated human dendritic cells (DC), subtractive cloning was adopted and more than ten novel genes differentially expressed were cloned. One is a deletion mutant of heterogeneous nuclear ribonucleoprotein (hnRNP) M4 in which the residues from 159 to 197 of hnRNP M4 have been absent. The deletion mutant was shown to be co-expressed with hnRNP M4 in cell lines. The mutant was expressed in antigen-stimulated DC but not in normal DC. Northern blot analysis revealed the presence of a major hnRNP M4 deletion mutant mRNA transcript of 2.4 kilobase with the highest levels in peripheral lymphocytes, lung, liver and spleen. It was also expressed in bone marrow-derived stromal cells (BMSC), BMSC treated with several cytokines but not in BMSC treated with TNF-a. The results revealed a new member of hnRNP family and suggested that hnRNP would participate in antigen process and presentation.

  3. Selection of Mycoplasma hominis PG21 deletion mutants by cultivation in the presence of monoclonal antibody 552

    DEFF Research Database (Denmark)

    Jensen, L T; Ladefoged, S; Birkelund, S;

    1995-01-01

    characterized. The mutants showed deletions of a various number of repeats. The deletions were accompanied by a decrease in size of the proteins. With increasing size of deletions, agglutination and growth inhibition by MAb 552 became less pronounced. Spontaneous aggregation of the mutant M. hominis cells...

  4. Deletion and deletion/insertion mutations in the juxtamembrane domain of the FLT3 gene in adult acute myeloid leukemia

    Directory of Open Access Journals (Sweden)

    Kristin K. Deeb

    2014-01-01

    Full Text Available In contrast to FLT3 ITD mutations, in-frame deletions in the FLT3 gene have rarely been described in adult acute leukemia. We report two cases of AML with uncommon in-frame mutations in the juxtamembrane domain of the FLT3 gene: a 3-bp (c.1770_1774delCTACGinsGT; p.F590_V592delinsLF deletion/insertion and a 12-bp (c.1780_1791delTTCAGAGAATAT; p.F594_Y597del deletion. We verified by sequencing that the reading frame of the FLT3 gene was preserved and by cDNA analysis that the mRNA of the mutant allele was expressed in both cases. Given the recent development of FLT3 inhibitors, our findings may be of therapeutic value for AML patients harboring similar FLT3 mutations.

  5. Transcriptomic profile of aguR deletion mutant of Lactococcus lactis subsp. cremoris CECT 8666

    Directory of Open Access Journals (Sweden)

    Beatriz del Rio

    2015-12-01

    Full Text Available Lactococcus lactis subsp. cremoris CECT 8666 (formerly GE2-14 is a dairy strain that catabolizes agmatine (a decarboxylated derivative of arginine into the biogenic amine putrescine by the agmatine deiminase (AGDI pathway [1]. The AGDI cluster of L. lactis is composed by five genes aguR, aguB, aguD, aguA and aguC. The last four genes are responsible for the deamination of agmatine to putrescine and are co-transcribed as a single policistronic mRNA forming the catabolic operon aguBDAC [1]. aguR encodes a transmembrane protein that functions as a one-component signal transduction system that senses the agmatine concentration of the medium and accordingly regulates the transcription of aguBDAC [2], which is also transcriptionally regulated by carbon catabolic repression (CCR via glucose, but not by other sugars such as lactose and galactose [1,3]. Here we report the transcriptional profiling of the aguR gene deletion mutant (L. lactis subsp. cremoris CECT 8666 ∆aguR [2] compared to the wild type strain, both grown in M17 medium with galactose as carbon source and supplemented with agmatine. The transcriptional profiling data of AguR-regulated genes were deposited in the Gene Expression Omnibus (GEO database under accession no. GSE59514.

  6. Transcriptomic profile of aguR deletion mutant of Lactococcus lactis subsp. cremoris CECT 8666.

    Science.gov (United States)

    Del Rio, Beatriz; Linares, Daniel M; Redruello, Begoña; Martin, Maria Cruz; Fernandez, Maria; de Jong, Anne; Kuipers, Oscar P; Ladero, Victor; Alvarez, Miguel A

    2015-12-01

    Lactococcus lactis subsp. cremoris CECT 8666 (formerly GE2-14) is a dairy strain that catabolizes agmatine (a decarboxylated derivative of arginine) into the biogenic amine putrescine by the agmatine deiminase (AGDI) pathway [1]. The AGDI cluster of L. lactis is composed by five genes aguR, aguB, aguD, aguA and aguC. The last four genes are responsible for the deamination of agmatine to putrescine and are co-transcribed as a single policistronic mRNA forming the catabolic operon aguBDAC[1]. aguR encodes a transmembrane protein that functions as a one-component signal transduction system that senses the agmatine concentration of the medium and accordingly regulates the transcription of aguBDAC[2], which is also transcriptionally regulated by carbon catabolic repression (CCR) via glucose, but not by other sugars such as lactose and galactose [1], [3]. Here we report the transcriptional profiling of the aguR gene deletion mutant (L. lactis subsp. cremoris CECT 8666 ∆aguR) [2] compared to the wild type strain, both grown in M17 medium with galactose as carbon source and supplemented with agmatine. The transcriptional profiling data of AguR-regulated genes were deposited in the Gene Expression Omnibus (GEO) database under accession no. GSE59514.

  7. 鼠伤寒沙门菌spvB基因缺陷变异株的制备及其抗酸能力检测%Construction of a spvB Gene-deleted Mutant of Salmonella Enterica Serovar Typhi and Its Survival Ability in Acid Condition

    Institute of Scientific and Technical Information of China (English)

    吴春雪; 陈强; 李红; 余晓君; 朱春晖; 李岚; 何美娟; 刘晓艳

    2014-01-01

    Objective To investigate the function of spvB gene,a toxicity gene of Salmonella enterica serovar Typhi,by constructing the spvB gene-deleted mutant and examinimg its survival ability in acid condition.Methods According to the se-quences of spvB gene of Salmonella enterica serovar Typhi,specific primers were designed for PCR.The homologous DNA fragments with spvB gene deleted were constructed,which was cloned into the suicide plasmid pCVD442 and then transferred into the target cells of Salmonella enterica serovar Typhi.The recombination was visualized by PCR,and the complete recombi-nant strain was selected as the spvB gene-deleted mutant strain and confirmed by the corresponding sequencing analysis.Under the acid condition,the survival ability of the spvB mutant and parent was compared by using the growth curve.Results A dele-tion of 1 748 bp of the spvB gene was confirmed by PCR and sequencing analysis in spvB gene-deleted mutants.The number of live wild-type strains were significantly greater than that of spvB gene-deleted mutants under the acid condition for 1 and 2 h, and the difference was statistically significant(P<0.05).The survival rate of the wild-type strains was 85.6% and 74.9% at 1 and 2 h,significantly higher than that of the spvB gene-deleted mutants,which was 68.0% and 42.3%.Conclusion The spvB gene-deleted mutant of Salmonella enterica serovar Typhi was successfully generated and its survival ability was significantly compromised under the acid condition,which lays a foundation for studying the function of the spvB gene in Salmonella enteri-ca serovar Typhi.%目的:为深入研究鼠伤寒沙门菌毒力基因 spvB的功能,制备鼠伤寒沙门菌 spvB基因完全缺陷变异株,观察spvB基因缺陷株在体外酸性环境中的生存能力。方法根据鼠伤寒沙门菌spvB基因序列,设计PCR特异性引物,制备spvB基因缺陷性同源性核苷酸片段,导入自杀质粒 pCVD442后再导入鼠伤寒沙门菌野生

  8. Construction and characterization of the cAMP receptor protein gene deletion mutant of Salmonella typhimurium SL1344 strain%鼠伤寒沙门菌SL1344株cAMP受体蛋白基因缺失株的构建及其生物学特性

    Institute of Scientific and Technical Information of China (English)

    廖成水; 程相朝; 赵战勤; 张春杰; 李银聚; 吴庭才; 郁川; 王晓利; 胡阿勇

    2011-01-01

    The cAMP receptor protein gene(crp) deletion mutant of Salmonella typhimurium SL1344 strain was constructed by the allelic exchange introduced by the transduction of suicide plasmid.In addition,the biological characteristics of the mutant were determined.Firstly,the upstream and downstream fragments of crp gene were amplified from SL1344 strain genome.The two fragments were successively cloned into the suicide pRE112 vector to construct the recombinant suicide vector pREΔcrp harboring the 321 bp-deleted crp fragment.The recombination suicide vector was conjugated with SL1344 and the unmarked crp deleted strain without resistance was selected by two-step method and crp deletion on the genome was determined by PCR.The serotype of the mutant was 1,4,5,12:i:1,2,identical to the parent SL1344.The mutant was stable with the recombinant Δcrp gene in vitro.However,the carbohydrate fermentation or utilization assays of the mutant were differed from the parent SL1344 strain,obviously.The growth velocity of the mutant was more slowly compared with SL1344.The chicken lethal test showed that the virulence of the SL1344 Δcrp mutant strain with LD50 of 7.40×109 CFU was 32 456 times lower than the parent SL1344 strain with LD50 of 2.28×105 CFU.These results showed that the SL1344 Δcrp mutant was constructed successfully.It is likely that this Δcrp mutant could be adapted to develop attenuated Salmonella vaccine.%通过自杀性质粒介导的细菌同源重组技术,构建鼠伤寒沙门菌SL1344株的crp基因缺失疫苗候选菌株,并对其生物学特性进行初步研究。首先构建含缺失321bp crp基因的重组自杀性质粒pREΔcrp,然后利用重组自杀性质粒介导的等位基因交换技术,两步法筛选SL1344的Δcrp缺失株,用PCR鉴定结果表明该缺失株构建成功。生物学特性研究发现,缺失株ΔcrpSL1344保留了亲本菌株SL1344的血清型1,4,5,12:i:1,2,且能够稳定遗传缺失321bp的crp

  9. Residual virulence and immunogenicity of CGV26 and CGV2631 B. melitensis Rev. 1 deletion mutant strains in sheep after subcutaneous or conjunctival vaccination.

    Science.gov (United States)

    Guilloteau, Laurence A; Laroucau, Karine; Olivier, Michel; Grillo, Maria Jesus; Marin, Clara M; Verger, Jean-Michel; Blasco, Jose-Maria

    2006-04-24

    The CGV26 and CGV2631 strains are novel engineered Brucella melitensis Rev.1 mutant strains deleted for the bp26 gene or for both bp26 and omp31 genes, respectively, coding for proteins of diagnostic significance. The residual virulence and immunogenicity of both mutants were compared to the parental Rev.1 strain in sheep after subcutaneous or conjunctival vaccination. The deletion of the bp26 gene or both bp26 and omp31 genes had no significant effect on the intracellular survival of the Rev.1 strain in ovine macrophage cultures. The kinetics of infection induced by both mutants in sheep was similar to the Rev.1 strain, and inoculation by the subcutaneous route produced wider and more generalized infections than the conjunctival route. All strains were cleared from lymph nodes and organs within 3 months after inoculation. The CGV26 and CGV2631 mutants induced both specific systemic antibody response and lymphoproliferation in sheep. The kinetics of the responses induced by the mutants was quite similar to that of the parental Rev.1 strain, except for the intensity of the lymphoproliferative response, which was attenuated for the CGV2631 mutant. In conclusion, the residual virulence of both CGV26 and CGV2631 mutants in sheep was similar to that of the parental Rev.1 vaccine strain. These mutants induced also significant specific antibody and cell-mediated immunity in sheep and are suitable to be evaluated as potential vaccine candidates against B. melitensis and B. ovis infections in sheep.

  10. Construction of LuxS gene deletion of Streptococcus mutans and evaluation of the acid tolerance of the mutant%变异链球菌LuxS基因缺陷株的建立及其耐酸能力的研究

    Institute of Scientific and Technical Information of China (English)

    韩福胜; 韩玉植; 刘宇霞

    2010-01-01

    Objective To construct Streptococcus mutans UA159 mutants with deletion of LuxS gene related to quorum-sensing pathway and evaluate the aciduricity of the mutants. Methods Using S. mutans UA159 as materials, the PCR fragments of the upstream and downstream regions of LuxS and erythromycin resistance(Eymr) gene of PJT10 were cloned into plasmid PUC19. The resulting constructs were integrated into the chromosome of S. mutans. LuxS gene deletion mutant was then constructed in S. mutans by means of allelic exchange and selected for resistance to erythromycin. The aciduric ability of the mutant under different pH was measured and S. mutans UA159 was used as control. Results The LuxS-deleted status of S. mutans mutants were confirmed by various PCR and DNA sequencing. The results showed that Eymr gene take the place of LuxS gene, while the mutant can not induce bioluminescenece. The LuxS mutant strain displayed a decreased growth ability with the decreasing pH values compared to those of the wild-type strain UA159. Conclusion A LuxS-negative mutants of S. mutans is constructed. The LuxS quorum sensing system is involved in the regulation of aciduricity of S. mutans UA159.%目的 建立LuxS基因缺失的变异链球菌突变菌株,并对突变株的耐酸能力进行研究.方法 以变异链球菌UA159为材料,运用基因重组方法将红霉素抗性基因(Eymr)与LuxS基因上下游区域的2个基因片段按一定顺序重组到质粒载体PUC19的多克隆位点中,获得了具有红霉素抗性的重组质粒,将载体质粒转化到含完整LuxS基因的变异链球菌UA159中,利用红霉素抗性筛选出LuxS基因缺失的突变株.检测变异链球菌LuxS基因突变菌株在不同pH环境下生长情况,并以正常菌株为对照.结果 PCR基因扩增结果显示,突变株LuxS基因已被Eymr基因完全替换,不能再编码合成AI-2(autoinducer 2)信号分子,扩增产物经DNA测序证实筛选得到了LuxS基因缺失的突变株,并

  11. Directed construction and analysis of a Sinorhizobium meliloti pSymA deletion mutant library.

    Science.gov (United States)

    Yurgel, Svetlana N; Mortimer, Michael W; Rice, Jennifer T; Humann, Jodi L; Kahn, Michael L

    2013-03-01

    Resources from the Sinorhizobium meliloti Rm1021 open reading frame (ORF) plasmid libraries were used in a medium-throughput method to construct a set of 50 overlapping deletion mutants covering all of the Rm1021 pSymA megaplasmid except the replicon region. Each resulting pSymA derivative carried a defined deletion of approximately 25 ORFs. Various phenotypes, including cytochrome c respiration activity, the ability of the mutants to grow on various carbon and nitrogen sources, and the symbiotic effectiveness of the mutants with alfalfa, were analyzed. This approach allowed us to systematically evaluate the potential impact of regions of Rm1021 pSymA for their free-living and symbiotic phenotypes.

  12. Deletion mutants of region E1 a of AD12 E1 plasmids: Effect on oncogenic transformation

    NARCIS (Netherlands)

    Bos, J.L.; Jochemsen, A.G.; Bernards, R.A.; Schrier, P.I.; Ormondt, H. van; Eb, A.J. van der

    1983-01-01

    Plasmids containing the El region of Ad12 DNA can transform certain rodent cells into oncogenic cells. To study the role of the Ela subregion in the process of oncogenic transformation, Ad12 region El mutants carrying deletions in the Ela region were constructed. Deletion mutants pR7 and pR8 affect

  13. RDNA cloning vector pVE1, deletion and hybrid mutants and recombinant derivatives thereof products and processes

    Energy Technology Data Exchange (ETDEWEB)

    MacNeil, T.; Gibbons, P.H.

    1987-10-27

    This patent describes novel plasmid pVE1, deletion mutants thereof, recombinant derivatives thereof, which is the same as the genome or nucleic acid of such plasmids and derivatives of such genome, which are useful as recombinant DNA cloning vectors into host organisms, such as bacteria, for example, Streptomyces avermitilis. Portions of such plasmid genome are additionally useful as adjuncts in recombinant DNA cloning procedures, for examples: 1. to permit the maintenance of cloned DNA in the host, either in an integrated state or as an autonomous element; 2. to serve as promoters for increasing expression of endogenous or foreign genes wherein the promoters are ligated to such genes or otherwise serve as promoters; and 3. to serve as regulatory elements for achieving control over endogenous and foreign gene expression. As cloning vectors, pVE1 its deletion mutants, and other derivatives serve for the amplification and transfer of DNA sequences (genes) coding for useful functions. Such modified cloning vectors are introduced into the recipient organism by conjugation or transformation; wherein the hybrid DNA functions in an integrated mode and/or in plasmid mode.

  14. Construction and characterization of ack deleted mutant of Clostridium tyrobutyricum for enhanced butyric acid and hydrogen production.

    Science.gov (United States)

    Liu, Xiaoguang; Zhu, Ying; Yang, Shang-Tian

    2006-01-01

    Clostridium tyrobutyricum produces butyrate, acetate, H(2), and CO(2) as its main fermentation products from glucose and xylose. To improve butyric acid and hydrogen production, integrational mutagenesis was used to create a metabolically engineered mutant with inactivated ack gene, encoding acetate kinase (AK) associated with the acetate formation pathway. A non-replicative plasmid containing the acetate kinase gene (ack) fragment was constructed and introduced into C. tyrobutyricum by electroporation. Integration of the plasmid into the homologous region on the chromosome should inactivate the target ack gene and produce ack-deleted mutant, PAK-Em. Enzyme activity assays showed that the AK activity in PAK-Em decreased by approximately 50%; meanwhile, phosphotransacetylase (PTA) and hydrogenase activities each increased by approximately 40%. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the expression of protein with approximately 32 kDa molecular mass was reduced significantly in the mutant. Compared to the wild type, the mutant grew more slowly at pH 6.0 and 37 degrees C, with a lower specific growth rate of 0.14 h(-1) (vs 0.21 h(-1) for the wild type), likely due to the partially impaired PTA-AK pathway. However, the mutant produced 23.5% more butyrate (0.42 vs 0.34 g/g glucose) at a higher final concentration of 41.7 g/L (vs 19.98 g/L) as a result of its higher butyrate tolerance as indicated in the growth kinetics study using various intial concentrations of butyrate in the media. The mutant also produced 50% more hydrogen (0.024 g/g) from glucose than the wild type. Immobilized-cell fermentation of PAK-Em in a fibrous-bed bioreactor (FBB) further increased the final butyric acid concentration (50.1 g/L) and the butyrate yield (0.45 g/g glucose). Furthermore, in the FBB fermentation at pH 5.0 with xylose as the substrate, only butyric acid was produced by the mutant, whereas the wild type produced large amounts

  15. Group II intron-anchored gene deletion in Clostridium.

    Directory of Open Access Journals (Sweden)

    Kaizhi Jia

    Full Text Available Clostridium plays an important role in commercial and medical use, for which targeted gene deletion is difficult. We proposed an intron-anchored gene deletion approach for Clostridium, which combines the advantage of the group II intron "ClosTron" system and homologous recombination. In this approach, an intron carrying a fragment homologous to upstream or downstream of the target site was first inserted into the genome by retrotransposition, followed by homologous recombination, resulting in gene deletion. A functional unknown operon CAC1493-1494 located in the chromosome, and an operon ctfAB located in the megaplasmid of C. acetobutylicum DSM1731 were successfully deleted by using this approach, without leaving antibiotic marker in the genome. We therefore propose this approach can be used for targeted gene deletion in Clostridium. This approach might also be applicable for gene deletion in other bacterial species if group II intron retrotransposition system is established.

  16. Large contiguous gene deletions in Sjögren-Larsson syndrome.

    Science.gov (United States)

    Engelstad, Holly; Carney, Gael; S'aulis, Dana; Rise, Janae; Sanger, Warren G; Rudd, M Katharine; Richard, Gabriele; Carr, Christopher W; Abdul-Rahman, Omar A; Rizzo, William B

    2011-11-01

    Sjögren-Larsson syndrome (SLS) is an autosomal recessive disorder characterized by ichthyosis, mental retardation, spasticity and mutations in the ALDH3A2 gene for fatty aldehyde dehydrogenase, an enzyme that catalyzes the oxidation of fatty aldehyde to fatty acid. More than 70 mutations have been identified in SLS patients, including small deletions or insertions, missense mutations, splicing defects and complex nucleotide changes. We now describe 2 SLS patients whose disease is caused by large contiguous gene deletions of the ALDH3A2 locus on 17p11.2. The deletions were defined using long distance inverse PCR and microarray-based comparative genomic hybridization. A 24-year-old SLS female was homozygous for a 352-kb deletion involving ALDH3A2 and 4 contiguous genes including ALDH3A1, which codes for the major soluble protein in cornea. Although lacking corneal disease, she showed severe symptoms of SLS with uncommon deterioration in oral motor function and loss of ambulation. The other 19-month-old female patient was a compound heterozygote for a 1.44-Mb contiguous gene deletion and a missense mutation (c.407C>T, P136L) in ALDH3A2. These studies suggest that large gene deletions may account for up to 5% of the mutant alleles in SLS. Geneticists should consider the possibility of compound heterozygosity for large deletions in patients with SLS and other inborn errors of metabolism, which has implications for carrier testing and prenatal diagnosis.

  17. Characterization of a spontaneous nonmagnetic mutant of Magnetospirillum gryphiswaldense reveals a large deletion comprising a putative magnetosome island.

    Science.gov (United States)

    Schübbe, Sabrina; Kube, Michael; Scheffel, André; Wawer, Cathrin; Heyen, Udo; Meyerdierks, Anke; Madkour, Mohamed H; Mayer, Frank; Reinhardt, Richard; Schüler, Dirk

    2003-10-01

    Frequent spontaneous loss of the magnetic phenotype was observed in stationary-phase cultures of the magnetotactic bacterium Magnetospirillum gryphiswaldense MSR-1. A nonmagnetic mutant, designated strain MSR-1B, was isolated and characterized. The mutant lacked any structures resembling magnetosome crystals as well as internal membrane vesicles. The growth of strain MSR-1B was impaired under all growth conditions tested, and the uptake and accumulation of iron were drastically reduced under iron-replete conditions. A large chromosomal deletion of approximately 80 kb was identified in strain MSR-1B, which comprised both the entire mamAB and mamDC clusters as well as further putative operons encoding a number of magnetosome-associated proteins. A bacterial artificial chromosome clone partially covering the deleted region was isolated from the genomic library of wild-type M. gryphiswaldense. Sequence analysis of this fragment revealed that all previously identified mam genes were closely linked with genes encoding other magnetosome-associated proteins within less than 35 kb. In addition, this region was remarkably rich in insertion elements and harbored a considerable number of unknown gene families which appeared to be specific for magnetotactic bacteria. Overall, these findings suggest the existence of a putative large magnetosome island in M. gryphiswaldense and other magnetotactic bacteria.

  18. Construction and characterization of a glycoprotein E deletion mutant of bovine herpesvirus type 1.2 strain isolated in Brazil Construção e caracterização de uma amostra de BoHV-1.2 isolada no Brasil com uma deleção no gene da glicoproteína E

    Directory of Open Access Journals (Sweden)

    Ana C. Franco

    2002-09-01

    Full Text Available This paper describes the construction and characterization of a Brazilian strain of bovine herpesvirus type 1.2a (BoHV-1.2a with a deletion of the glycoprotein E (gE gene. The deletion was introduced by co-transfection of a deletion fragment containing the 5´and 3´gE flanking regions and genomic DNA of wild type BoHV-1 into bovine cells. Isolation of gE deletion mutant was performed by immunoperoxidase staining with an anti-gE monoclonal antibody. Viral clones were plaque purified and further examined by restriction endonuclesase digestion and Southern blot hybridization. This gE deletion mutant will be evaluated as a vaccinal virus, in order to determine its potential use for a differential vaccine.Este artigo descreve a construção e caracterização de uma amostra de um herpesvírus bovino tipo 1.2a (BoHV-1.2a que apresenta uma deleção na região genômica que codifica a glicoproteína E (gE. A deleção gênica foi induzida através da co-transfecção de um fragmento de deleção, contendo as regiões 5´e 3´flanqueadoras da gE, com o DNA viral intacto de uma amostra viral isolada de um animal que apresentava doença respiratória. O isolamento do vírus gE negativo (gE- foi realizado com auxílio da técnica de imunoperoxidase em que foi utilizado como anticorpo primário um anticorpo monoclonal anti-gE. O vírus gE- foi purificado e o DNA isolado desta amostra foi examinado através das técnicas de análise por enzimas de restrição e "Southern blot". Esta amostra gE- será avaliada como candidata para compor uma vacina diferencial contra a rinotraqueíte infecciosa dos bovinos.

  19. Construction of a mutant strain of Streptococcus mutans with clpC-deletion to study the role of clpC ;gene in genetic competence%clpC 基因对变异链球菌感受态形成的影响

    Institute of Scientific and Technical Information of China (English)

    徐巧丽; 饶慧华; 马晓波; 黄朝阳; 郑港森; 张加勤; 宋秀宇

    2015-01-01

    目的:构建变异链球菌clpC缺陷突变株,检测该基因对变异链球菌感受态形成的影响。方法分别以变异链球菌UA159基因组和pIB107质粒为模板,PCR扩增clpC基因片段和卡那霉素基因盒(lox71-KMR-lox66);将clpC基因片段插入pMD-19T simple载体,经ClaⅠ/EcoRⅠ酶切、补平后连入卡那霉素基因盒,构建clpC缺陷突变同源重组载体pCKX2;SalⅠ线性化pCKX2并转化变异链球菌,卡那霉素筛选阳性菌落;质粒pCrePA转化阳性菌株,30℃培养剔除卡那霉素基因盒;37℃培养去除pCrePA,获得clpC缺陷突变株,PCR和测序鉴定;提取细菌总RNA并逆转录成cDNA,用RT-PCR法扩增clpC缺失序列的核苷酸片段并进行产物的电泳分析;pDL276分别转化变异链球菌和clpC缺陷突变株,观察感受态细胞形成变化。结果 PCR和测序结果证实成功构建同源重组载体pCKX2及变异链球菌clpC缺陷突变株;RT-PCR结果显示,△clpC缺失的核苷酸片段PCR产物电泳结果并无相应的条带出现;clpC缺陷突变株的感受态形成期延迟并延长维持期。结论 clpC基因具有负调控变异链球菌晚期感受态细胞形成的作用。%Objective To construct a mutant strain of Streptococcus mutans ( S.mutans ) with clpC-deletion and to investigate the role of clpC gene in genetic competence.Methods The fragment of clpC gene and the kanamycin resistant cassette flanked by two loxP sites were amplified by PCR.The purified fragment of clpC gene was cloned into pMD-19T simple vector to construct pCKX1.The pCKX1 vector was digested with ClaⅠ/EcoRⅠ, then blunted and introduced into lox71-KMR-lox66 to obtain pCKX2 vector via homologous recombination.The pCKX2 vector was linearized with SalⅠ and transformed into S.mutans UA159 strain.The positive strains constructed via homologous recombination were screened with kanamycin and transformed with the thermosensitive plasmid pCrePA.The KMR

  20. Deletion analysis of oligomycin PKS genes (olmA) in Streptomyces avermitilis

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xiaolin; CHEN Zhi; ZHAO Jinlei; SONG Yuan; WEN Ying; LI Jilun

    2004-01-01

    Gene deletion vector pXL05(pKC1139∷△olmA1 +△olmA4) was used to disrupt oligomycin PKS encoding genes (olmA) in Streptomyces avermitilis CZ8-73, the producer of anthelmintic avermectins B and the cell growth inhibitor oligomycin. olmA gene cluster in the chromosome was displaced by deletion allele on the plasmid via double crossover. Four of disruptants were confirmed by Southern blotting. Shaking flask experiments and HPLC analyses showed that the four mutants no longer produced the toxic oligomycin, but only made four components of avermectins B, which were avermectin B1a, B1b, B2a, B2b. The yields of avermectins B in these mutants were separately equal to those in CZ8-73. This revealed that olmA genes deletion did not affect the biosynthesis of avermectins. The deletion mutants were proved to be genetically stable, and thus might be promising strains in industrial production of avermectins B.

  1. Deletion of ku homologs increases gene targeting frequency in Streptomyces avermitilis.

    Science.gov (United States)

    Zhang, Xiaojuan; Chen, Wei; Zhang, Yang; Jiang, Libin; Chen, Zhi; Wen, Ying; Li, Jilun

    2012-06-01

    Streptomyces avermitilis is an industrially important soil bacterium known for production of avermectins, which are antiparasitic agents useful in animal health care, agriculture, and treatment of human infections. ku genes play a key role in the non-homologous end-joining pathway for repair of DNA double strand breaks. We identified homologs of eukaryotic ku70 and ku80 genes, termed ku1 and ku2, in S. avermitilis. Mutants with deletion of ku1, ku2, and both genes were constructed and their phenotypic changes were characterized. Deletion of ku genes had no apparent adverse effects on growth, spore formation, or avermectin production. The ku mutants, in comparison to wild-type strain, were slightly more sensitive to the DNA-damaging agent ethyl methanesulfonate, but not to UV exposure or to bleomycin. Gene targeting frequencies by homologous recombination were higher in the ku mutants than in wild-type strain. We conclude that ku-deleted strains will be useful hosts for efficient gene targeting and will facilitate functional analysis of genes in S. avermitilis and other industrially important bacterial strains.

  2. Deletions of the elastin gene in Williams Syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Greenberg, F.; Nickerson, E.; McCaskill, C. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1994-09-01

    To investigate deletions in the elastin gene in patients with Williams Syndrome (WS), we screened 37 patients and their parents for deletions in the elastin gene by both fluorescence in situ hybridization (FISH) using cosmid cELN272 containing the 5{prime} end of the elastin gene and by polymerase chain reaction (PCR) using a primer pair which amplifies intron 17 in the elastin gene, producing a polymorphic amplification product. Thirty-two patients have been investigated by both the FISH and PCR techniques, one patient was studied only by PCR, and 4 patients were studied only by FISH. Overall, 34 of 37 patients (92%) were deleted for the elastin gene. Using the PCR marker, 14 patients were informative and 12 were shown to be deleted [maternal (n=5) and paternal (n=7)]. Using cosmid cELN272, 33 of 36 patients demonstrated a deletion of chromosome 7q11.23. In one family, both the mother and daughter were deleted due to an apparently de novo deletion arising in the mother. Three patients were not deleted using the elastin cosmid; 2 of these patients have classic WS. Another non-deleted patient has the typical facial features and hypercalcemia but normal intelligence. These three patients will be important in delineating the critical region(s) responsible for the facial features, hypercalcemia, mental retardation and supravalvular aortic stenosis (SVAS). There was not an absolute correlation between deletions in elastin and SVAS, although these individuals may be at risk for other cardiovascular complications such as hypertention. Since the majority of WS patients are deleted for a portion of the elastin gene, most likely this marker will be an important diagnostic tool, although more patients will need to be studied. Those patients who are not deleted but clinically have WS will be missed using only this one marker. Expansion of the critical region to other loci and identification of additional markers will be essential for identifying all patients with WS.

  3. Multi-exon deletions of the FBN1 gene in Marfan syndrome

    Directory of Open Access Journals (Sweden)

    Schrijver Iris

    2001-10-01

    Full Text Available Abstract Background Mutations in the fibrillin -1 gene (FBN1 cause Marfan syndrome (MFS, an autosomal dominant multi-system connective tissue disorder. The 200 different mutations reported in the 235 kb, 65 exon-containing gene include only one family with a genomic multi-exon deletion. Methods We used long-range RT-PCR for mutation detection and long-range genomic PCR and DNA sequencing for identification of deletion breakpoints, allele-specific transcript analyses to determine stability of the mutant RNA, and pulse-chase studies to quantitate fibrillin synthesis and extracellular matrix deposition in cultured fibroblasts. Southern blots of genomic DNA were probed with three overlapping fragments covering the FBN1 coding exons Results Two novel multi-exon FBN1 deletions were discovered. Identical nucleotide pentamers were found at or near the intronic breakpoints. In a Case with classic MFS, an in-frame deletion of exons 42 and 43 removed the C-terminal 24 amino acids of the 5th LTBP (8-cysteine domain and the adjacent 25th calcium-binding EGF-like (6-cysteine domain. The mutant mRNA was stable, but fibrillin synthesis and matrix deposition were significantly reduced. A Case with severe childhood-onset MFS has a de novo deletion of exons 44–46 that removed three EGF-like domains. Fibrillin protein synthesis was normal, but matrix deposition was strikingly reduced. No genomic rearrangements were detected by Southern analysis of 18 unrelated MFS samples negative for FBN1 mutation screening. Conclusions Two novel deletion cases expand knowledge of mutational mechanisms and genotype/phenotype correlations of fibrillinopathies. Deletions or mutations affecting an LTBP domain may result in unstable mutant protein cleavage products that interfere with microfibril assembly.

  4. The Changed Rule of Infected Piglets by a Mutant of Porcine Reproductive and Respiratory Syndrome Virus with Nsp2 Gene Deletion%感染PRRSV Nsp2基因部分缺失变异株的仔猪抗体变化规律

    Institute of Scientific and Technical Information of China (English)

    张凤华; 卢晓艳; 徐红运; 赵琳; 刘玉松; 范旭; 夏平安; 崔保安

    2011-01-01

    为了解PRRSV Nsp2基因缺失变异株的致病性,采用2个Nsp2基因分别连续缺失3个和89个碱基的PRRSV变异株Hn-2(GenBank:FJ237419)和Hn-4(GenBank:FJ237421)的病毒液,人工滴鼻感染断奶仔猪,同时用Marc-145健康仔猪细胞培养液滴鼻作对照,在感染后0,7,14,24,32,41,50,60和70 d无菌采血,分离血清,用RTPCR方法检测病毒、用N-ELISA方法和免疫荧光抑制试验分别检测抗PRRSV N蛋白抗体和抗PRRSV中和抗体,比较分析不同时期病毒复制和抗体产生的变化规律.结果表明,仔猪感染Hn-2和Hn-4毒株后7~14 d均能检测到病毒,第7天能检测到抗PRRSV N蛋白抗体,并一直维持较高水平,第41和50天检测到较低水平的中和抗体,随后中和抗体效价逐渐升高.试验猪均未出现死亡,表明PRRSV Nsp2基因缺失变异株不一定导致感染猪的死亡.%In order to understand the pathogenicity of PRRSV about Nsp2 gene deletion mutant, The PRRS negative healthy piglets of 20 days-old were inoculated by intranasal with a Mutant of PRRSV Hn-2 ( GenBank; FJ237419)and Hn-4 (GenBank; FJ237421) strains respectively, which delete 3 base pairs and 89 base pairs in the Nsp2 gene respectively, while healthy piglets were used for control, the sera were separated aseptically on the 0,7, 14,24,32,41,50,60,70 d respectively, using RT-PCR to detect the virus, using ELISA method and hnmunofluo-rescence inhibition test for detection of anti-PRRSV N protein antibody and neutralizing antibodies, variation of the virus and antibody were comparied in different periods of time. The results showed that the PRRSV could be detected in 7 -14 d after infected with the Mutant Hn-2, and Hn-4 strain, anti-PRRSV N protein antibodies could be detected on the 7th day and has maintained a high level, respectively, the low level of neutralizing antibodies could be detected respectively on 41 d and 50 d, and the neutralization antibody increased gradually. The test pigs were not of death

  5. Characterization of a lymphoblastoid line deleted for lambda immunoglobulin genes

    Energy Technology Data Exchange (ETDEWEB)

    Hough, C.A., White, B.N., Holden, J.A. [Queen`s Univ., Ontario (Canada)

    1995-04-01

    While characterizing the cat eye syndrome (CES) supernumerary chromosome for the presence of {lambda} immunoglobulin gene region sequences, a lymphoblastoid cell line from one CES patient was identified in which there was selection of cells deleted from some IGLC and IGLV genes. Two distinct deletions, one on each chromosome 22, were identified, presumably arising from independent somatic recombination events occurring during B-lymphocyte differentiation. The extent of the deleted regions was determined using probes from the various IGLV subgroups and they each covered at least 82 kilobases. The precise definition of the deletions was not possible because of conservation of some restriction sites in the IGLV region. The cell line was used to map putative IGLV genes within the recombinant phage {lambda}V{lambda}135 to the distal part of the IGLV gene region. 35 refs., 4 figs.

  6. Bilateral hand amyotrophy with PMP-22 gene deletion.

    Science.gov (United States)

    Gochard, A; Guennoc, A M; Praline, J; Malinge, M C; de Toffol, B; Corcia, P

    2007-01-01

    Hereditary neuropathy with liability to pressure palsies (HNPP) phenotypes are heterogeneous. We report the case of a 52-year-old woman without medical history, who complained of bilateral hand weakness suggestive first of a motor neuron disorder. The presence of a diffuse predominant distal demyelinating neuropathy suggested a deletion of PMP-22 gene, which was confirmed by genetic analysis. This case report underlines a novel phenotype related to the deletion of PMP-22 gene.

  7. Characterization of a cold-active lipase from Psychrobacter cryohalolentis K5(T) and its deletion mutants.

    Science.gov (United States)

    Novototskaya-Vlasova, K A; Petrovskaya, L E; Rivkina, E M; Dolgikh, D A; Kirpichnikov, M P

    2013-04-01

    A gene coding for cold-active lipase from the psychrotrophic Gram-negative bacterium Psychrobacter cryohalolentis K5(T) isolated from a Siberian cryopeg has been cloned and expressed in Escherichia coli. The recombinant protein Lip1Pc with a 6× histidine tag at its C-terminus was purified by nickel affinity chromatography. With p-nitrophenyl dodecanoate (C12) as a substrate, the purified recombinant protein displayed maximum lipolytic activity at 25°C and pH 8.0. Increasing the temperature above 40°C and addition of various metal ions and organic solvents inhibited the enzymatic activity of Lip1Pc. Most nonionic detergents, such as Triton X-100 and Tween 20, slightly increased the lipase activity, while SDS completely inhibited it. To investigate the functional significance of the Lip1Pc N-terminal domain, we constructed five deletion mutants of this protein. The ND1 and ND2 mutants displayed specific activity reduced by 30-35%, while other truncated proteins were completely inactive. Both mutants demonstrated increased activity towards p-nitrophenyl decanoate (C10) and impaired utilization of C16 substrate. Although optimum reaction temperature of ND2 lowered to 20°C, it displayed enhanced stability by 44% after incubation at 40°C. The results prove that the N-terminal domain of Lip1Pc has a fundamental impact on the activity and stability of the protein.

  8. Gene deletion strategy to examine the involvement of the two chondroitin lyases in Flavobacterium columnare virulence.

    Science.gov (United States)

    Li, Nan; Qin, Ting; Zhang, Xiao Lin; Huang, Bei; Liu, Zhi Xin; Xie, Hai Xia; Zhang, Jin; McBride, Mark J; Nie, Pin

    2015-11-01

    Flavobacterium columnare is an important bacterial pathogen of freshwater fish that causes high mortality of infected fish and heavy economic losses in aquaculture. The pathogenesis of this bacterium is poorly understood, in part due to the lack of efficient methods for genetic manipulation. In this study, a gene deletion strategy was developed and used to determine the relationship between the production of chondroitin lyases and virulence. The F. johnsoniae ompA promoter (PompA) was fused to sacB to construct a counterselectable marker for F. columnare. F. columnare carrying PompA-sacB failed to grow on media containing 10% sucrose. A suicide vector carrying PompA-sacB was constructed, and a gene deletion strategy was developed. Using this approach, the chondroitin lyase-encoding genes, cslA and cslB, were deleted. The ΔcslA and ΔcslB mutants were both partially deficient in digestion of chondroitin sulfate A, whereas a double mutant (ΔcslA ΔcslB) was completely deficient in chondroitin lyase activity. Cells of F. columnare wild-type strain G4 and of the chondroitin lyase-deficient ΔcslA ΔcslB mutant exhibited similar levels of virulence toward grass carp in single-strain infections. Coinfections, however, revealed a competitive advantage for the wild type over the chondroitin lyase mutant. The results indicate that chondroitin lyases are not essential virulence factors of F. columnare but may contribute to the ability of the pathogen to compete and cause disease in natural infections. The gene deletion method developed in this study may be employed to investigate the virulence factors of this bacterium and may have wide application in many other members of the phylum Bacteroidetes.

  9. Systematic deletion of homeobox genes in Podospora anserina uncovers their roles in shaping the fruiting body.

    Directory of Open Access Journals (Sweden)

    Evelyne Coppin

    Full Text Available Higher fungi, which comprise ascomycetes and basidiomycetes, play major roles in the biosphere. Their evolutionary success may be due to the extended dikaryotic stage of their life cycle, which is the basis for their scientific name: the Dikarya. Dikaryosis is maintained by similar structures, the clamp in basidiomycetes and the crozier in ascomycetes. Homeodomain transcription factors are required for clamp formation in all basidiomycetes studied. We identified all the homeobox genes in the filamentous ascomycete fungus Podospora anserina and constructed deletion mutants for each of these genes and for a number of gene combinations. Croziers developed normally in these mutants, including those with up to six deleted homeogenes. However, some mutants had defects in maturation of the fruiting body, an effect that could be rescued by providing wild-type maternal hyphae. Analysis of mutants deficient in multiple homeogenes revealed interactions between the genes, suggesting that they operate as a complex network. Similar to their role in animals and plants, homeodomain transcription factors in ascomycetes are involved in shaping multicellular structures.

  10. Systematic deletion of homeobox genes in Podospora anserina uncovers their roles in shaping the fruiting body.

    Science.gov (United States)

    Coppin, Evelyne; Berteaux-Lecellier, Véronique; Bidard, Frédérique; Brun, Sylvain; Ruprich-Robert, Gwenaël; Espagne, Eric; Aït-Benkhali, Jinane; Goarin, Anne; Nesseir, Audrey; Planamente, Sara; Debuchy, Robert; Silar, Philippe

    2012-01-01

    Higher fungi, which comprise ascomycetes and basidiomycetes, play major roles in the biosphere. Their evolutionary success may be due to the extended dikaryotic stage of their life cycle, which is the basis for their scientific name: the Dikarya. Dikaryosis is maintained by similar structures, the clamp in basidiomycetes and the crozier in ascomycetes. Homeodomain transcription factors are required for clamp formation in all basidiomycetes studied. We identified all the homeobox genes in the filamentous ascomycete fungus Podospora anserina and constructed deletion mutants for each of these genes and for a number of gene combinations. Croziers developed normally in these mutants, including those with up to six deleted homeogenes. However, some mutants had defects in maturation of the fruiting body, an effect that could be rescued by providing wild-type maternal hyphae. Analysis of mutants deficient in multiple homeogenes revealed interactions between the genes, suggesting that they operate as a complex network. Similar to their role in animals and plants, homeodomain transcription factors in ascomycetes are involved in shaping multicellular structures.

  11. Altered murine tissue colonization by Borrelia burgdorferi following targeted deletion of linear plasmid 17-carried genes.

    Science.gov (United States)

    Casselli, Timothy; Tourand, Yvonne; Bankhead, Troy

    2012-05-01

    The causative agent of Lyme disease, Borrelia burgdorferi, possesses a segmented genome comprised of a single linear chromosome and upwards of 23 linear and circular plasmids. Much of what is known about plasmid-borne genes comes from studying laboratory clones that have spontaneously lost one or more plasmids during in vitro passage. Some plasmids, including the linear plasmid lp17, are never or rarely reported to be lost during routine culture; therefore, little is known about the requirement of these conserved plasmids for infectivity. In this study, the effects of deleting regions of lp17 were examined both in vitro and in vivo. A mutant strain lacking the genes bbd16 to bbd25 showed no deficiency in the ability to establish infection or disseminate to the bloodstream of mice; however, colonization of peripheral tissues was delayed. Despite the ability to colonize ear, heart, and joint tissues, this mutant exhibited a defect in bladder tissue colonization for up to 56 days postinfection. This phenotype was not observed in immunodeficient mice, suggesting that bladder colonization by the mutant strain was inhibited by an adaptive immune-based mechanism. Moreover, the mutant displayed increased expression of outer surface protein C in vitro, which was correlated with the absence of the gene bbd18. To our knowledge, this is the first report involving genetic manipulation of lp17 in an infectious clone of B. burgdorferi and reveals for the first time the effects of lp17 gene deletion during murine infection by the Lyme disease spirochete.

  12. Isolation and analysis of two Escherichia coli K-12 ilv attenuator deletion mutants with high-level constitutive expression of an ilv-lac fusion operon.

    OpenAIRE

    Bennett, D. C.; Umbarger, H E

    1984-01-01

    A lysogenizing lambda phage, lambda dilv-lac11, was constructed to carry an ilvD-lac operon fusion. Expression from the phage of the ilvE and lacZ genes is controlled by an intact ilv control region also carried by this phage. Two spontaneous mutants of lambda dilv-lac11 that have high-level constitutive expression of the ilv-lac fusion operon were isolated by growth on a beta-chloroalanine selective medium. The mutants were shown by nucleotide sequence determination to contain large deletion...

  13. Exon Deletions of Parkin Gene in Patients with Parkinson Disease

    Institute of Scientific and Technical Information of China (English)

    王涛; 梁直厚; 孙圣刚; 曹学兵; 彭海; 刘红进; 童萼塘

    2004-01-01

    Summary: Mutations in the parkin gene have recently been identified in familial and isolated patients with early-onset Parkinson disease (PD) and that subregions between exon 2 and 4 of the parkin gene are hot spots of deletive mutations. To study the distribution of deletions in the parkin gene among variant subset patients with PD in China, and to explore the role of parkin gene in the pathogenesis of PD, 63 patients were divided into early onset and later onset groups. Exons 1-12 were amplified by PCR, templated by the genomic DNA of patients, and then the deletion distribution detected by agarose electrophoresis. Four patients were found to be carrier of exon deletions in 63 patients with PD. The location of the deletion was on exon 2 (1 case), exon 3 (2 cases) and exon 4 (1 case). All patients were belong to the group of early onset PD. The results showed that parkin gene deletion on exon 2, exon 3 and exon 4 found in Chinese population contributes partly to early onset PD.

  14. Suicide Vector Construction of Haemophilus parasuis hhd B Gene Marker-free Deleted

    Institute of Scientific and Technical Information of China (English)

    Song Shuai; Li Miao; Li Yan; Jiang Zhiyong; Cai Rujian; Yang Dongxia; Li Chunling

    2015-01-01

    To construct the suicide vector of hhd B gene marker-free mutant in Haemophilus parasuis( HPS),two pairs of specific primers were designed and synthesized according to the hhd B gene upstream and downstream sequences of HPS published in Gen Bank. The hhd B gene upstream and downstream sequences were amplified by PCR,which were further ligated( hhd B-up + down) through overlapping PCR method. NotⅠand SalⅠrestriction enzyme sites were introduced on both ends of the ligated sequence. After the corresponding digestion,the hhd B-up + down sequence was directionally cloned to the suicide plasmid vector p EMOC2. Results showed that the suicide vector of hhd B gene marker-free deleted( p EMOC2Δhhd B) with stable inheritance in E. coli β2155 strain was successfully obtained,thereby laying the foundation for construction of HPS-hhd B gene marker-free mutant strain.

  15. A genome-wide deletion mutant screen identifies pathways affected by nickel sulfate in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Dai Wei

    2009-11-01

    Full Text Available Abstract Background The understanding of the biological function, regulation, and cellular interactions of the yeast genome and proteome, along with the high conservation in gene function found between yeast genes and their human homologues, has allowed for Saccharomyces cerevisiae to be used as a model organism to deduce biological processes in human cells. Here, we have completed a systematic screen of the entire set of 4,733 haploid S. cerevisiae gene deletion strains (the entire set of nonessential genes for this organism to identify gene products that modulate cellular toxicity to nickel sulfate (NiSO4. Results We have identified 149 genes whose gene deletion causes sensitivity to NiSO4 and 119 genes whose gene deletion confers resistance. Pathways analysis with proteins whose absence renders cells sensitive and resistant to nickel identified a wide range of cellular processes engaged in the toxicity of S. cerevisiae to NiSO4. Functional categories overrepresented with proteins whose absence renders cells sensitive to NiSO4 include homeostasis of protons, cation transport, transport ATPases, endocytosis, siderophore-iron transport, homeostasis of metal ions, and the diphthamide biosynthesis pathway. Functional categories overrepresented with proteins whose absence renders cells resistant to nickel include functioning and transport of the vacuole and lysosome, protein targeting, sorting, and translocation, intra-Golgi transport, regulation of C-compound and carbohydrate metabolism, transcriptional repression, and chromosome segregation/division. Interactome analysis mapped seven nickel toxicity modulating and ten nickel-resistance networks. Additionally, we studied the degree of sensitivity or resistance of the 111 nickel-sensitive and 72 -resistant strains whose gene deletion product has a similar protein in human cells. Conclusion We have undertaken a whole genome approach in order to further understand the mechanism(s regulating the cell

  16. Targeted gene deletions in C. elegans using transposon excision

    Science.gov (United States)

    Frøkjær-Jensen, Christian; Davis, M. Wayne; Hollopeter, Gunther; Taylor, Jon; Harris, Todd; Nix, Paola; Lofgren, Rachel; Prestgard-Duke, Michael; Bastiani, Michael; Moerman, Donald G.; Jorgensen, Erik M.

    2010-01-01

    We have developed a method, MosDel, to generate targeted knock-outs of genes in C. elegans. We make use of the Mos1 transposase to excise a Mos1 transposon adjacent to the region to be deleted. The double-strand break is repaired using injected DNA as a template. Repair can delete up to 25 kb of DNA and simultaneously insert a positive selection marker. PMID:20418868

  17. 布鲁菌Ⅳ型分泌系统效应蛋白 DK63-887基因缺失株的构建及鉴定%Construction and Identification of Ⅳ Secretion System Effector Protein DK63-887 Gene Deletion Mutant of Brucella

    Institute of Scientific and Technical Information of China (English)

    江雅丽; 陈创夫; 李志强; 李默; 李爽; 王震; 张欢; 张辉; 郭飞

    2016-01-01

    To construct the DK63-887 gene deletion mutant (16MΔDK63-887)of Brucella melitensis 16M (referred to as 16M),and explore the relationship between the gene and 16M-mediated autophagy,the methods of homologous recombination and replacement were applied,and the kanamycin gene was used to replace DK63-887 gene obtaining mutant 16MΔDK63-887.Under the same conditions of shaking culture, we observed growth of the parental strain 16M,vaccine strain M5-90,mutant 16MΔDK63-887,and then, each strain was placed in different environments,the survival rate of which was observed.Mouse macropha-ges were infected with parental and mutant strains,then we compared their ability of surviving in host cells and detected autophagy-related genes using RT-qPCR.The 16MΔDK63-887 mutant strain was successfully obtained.The mutants were genetically stable within 20 passages.Compared with the parental strain,the growing trends of mutant and parent strains were coincident in vitro ,but there were some differences in the concentration of bacteria.The mutants had decreased survival rate under the stress conditions.After 4h post-infection,the numbers of the mutant strains in host cells significantly decreased.ULK1 and beclin1 ex-pression levels of the mutants significantly reduced which were detected by RT-qPCR(P <0.01).The re-sults of this study showed that Brucella type Ⅳ secretion system effector proteins associated with 16M me-diated-cell autophagy,the research laid the foundation for the study on 16M intracellular parasitism mecha-nisms.%为了构建羊布鲁菌16M(简称16M)的 DK63-887基因缺失株(16MΔDK63-887),探讨该基因与16M 介导自噬的关系。利用同源重组和抗性替换的方法,以卡那基因替换 DK63-887基因,获得突变株16MΔDK63-887。将亲本株16M、疫苗株 M5-90、突变株16MΔDK63-887在相同条件下振荡培养,观察其生长趋势变化;将各菌株置于不同外界环境中,观察其生存率;将各菌株侵染小

  18. Construction of deletion mutants in the phosphotransferase transport system and adenosine triphosphate-binding cassette transporters in Listeria monocytogenes and analysis of their growth under different stress conditions

    Directory of Open Access Journals (Sweden)

    Marina Ceruso

    2013-10-01

    Full Text Available Functional genomics approaches enable us to investigate the biochemical, cellular, and physiological properties of each gene product and are nowadays applied to enhance food safety by understanding microbial stress responses in food and host-pathogen interactions. Listeria monocytogenes is a food-borne pathogen that causes listeriosis and is difficult to eliminate this pathogen since it can survive under multiple stress conditions such as low pH and low temperature. Detailed studies are needed to determine its mode of action and to understand the mechanisms that protect the pathogen when it is subjected to stress. In this study, deletion mutants of phosphotransferase transport system genes (PTS and adenosine triphosphate(ATP-binding cassette transporters (ABC of Listeria monocytogenes F2365 were created using molecular techniques. These mutants and the wild-type were tested under different stress conditions, such as in solutions with different NaCl concentration, pH value and for nisin resistance. Results demonstrate that the behaviour of these deletion mutants is different from the wild type. In particular, deleted genes may be involved in L. monocytogenes resistance to nisin and to acid and salt concentrations. Functional genomics research on L. monocytogenes allows a better understanding of the genes related to stress responses and this knowledge may help in intervention strategies to control this food-borne pathogen. Furthermore, specific gene markers can be used to identify and subtype L. monocytogenes. Thus, future development of this study will focus on additional functional analyses of important stress response-related genes, as well as on methods for rapid and sensitive detection of L. monocytogenes such as using DNA microarrays.

  19. Deletion of luxS further attenuates the virulence of the avian pathogenic Escherichia coli aroA mutant.

    Science.gov (United States)

    Han, Xiangan; Bai, Hao; Tu, Jian; Yang, Lijun; Xu, Da; Wang, Shaohui; Qi, Kezong; Fan, Guobo; Zhang, Yuxi; Zuo, Jiakun; Tian, Mingxing; Ding, Chan; Yu, Shengqing

    2015-11-01

    In this study, an aroA-deletion avian pathogenic Escherichia coli (APEC) mutant (strain DE17ΔaroA) and aroA and luxS double deletion APEC mutant (strain DE17ΔluxSΔaroA) were constructed from the APEC DE17 strain. The results showed that as compared to DE17ΔaroA, the virulence of DE17ΔluxSΔaroA was further attenuated by 200- and 31.7-fold, respectively, in ducklings based on the 50% lethal dose. The adherence and invasion abilities of DE17ΔluxSΔaroA and DE17ΔaroA were reduced by 36.5%/42.5% and 25.8%/29.3%, respectively, as compared to the wild-type strain DE17 (p < 0.05 and 0.01, respectively). Furthermore, in vivo studies showed that the bacterial loads of DE17ΔluxSΔaroA were reduced by 8400- and 11,333-fold in the spleen and blood of infected birds, respectively, while those of DE17ΔaroA were reduced by 743- and 1000-fold, respectively, as compared to the wild-type strain DE17. Histopathological analysis showed both that the mutants were associated with reduced pathological changes in the liver, spleen, and kidney of ducklings, and changes in DE17ΔluxSΔaroA-infected ducklings were reduced to a greater degree than those infected with DE17ΔaroA. Real-time polymerase chain reaction analysis further demonstrated that the mRNA levels of virulence-related genes (i.e., tsh, ompA, vat, iucD, pfs, fyuA, and fimC) were significantly decreased in DE17ΔaroA, especially in DE17ΔluxSΔaroA, as compared to DE17 (p < 0.05). In addition, the deletion of aroA or the double deletion of aroA and luxS reduced bacterial motility. To evaluate the potential use of DE17ΔluxSΔaroA as a vaccine candidate, 50 7-day-old ducklings were divided randomly into five groups of ten each for the experiment. The results showed that the ducklings immunized with inactivated DE17, DE17ΔluxS, DE17ΔaroA, and DE17ΔluxSΔaroA were 70.0%, 70.0%, 70.0, and 80.0% protected, respectively, after challenge with strain APEC DE17. The results of this study suggest that the double deletion of

  20. Generation of a Pseudomonas aeruginosa elastase gene targeted deletion mutant by Red recombination system%采用Red重组系统敲除铜绿假单胞菌弹性蛋白酶基因

    Institute of Scientific and Technical Information of China (English)

    余华; 熊浚智; 何晓梅; 盛哈蕾; 蔡文强; 谢玮; 张克斌

    2013-01-01

    The aim of this study is to obtain the elastase activity negative strain by knocking out the elastase gene in Pseudomonas aeruginosa PAO1. Three genes of Red recombination system from λ phage were amplified and cloned into Esche-richia-Pseudomonas shuttle vector pUCP, and the pUCP-Red vector was transformed into PAO1 competent cells by electropo-ration. Then the recombinant DNA fragment which contains gentamycin antibiotic cassette flanked by two 80-bp homology sequences of elastase gene upstream and downstream locuses respectively was obtained by conventional cloning methods. And the fragment was electroporated into PAOl/pUCP-Red competent cells and screened on LB plate containing gentamycin and carben-icillin. The elastase gene knocked-out strain was verified by the methods of PCR, RT-PCR and enzyme activity assays. The elastase activity negative strain was successfully obtained in this study by using the Red recombination system. The elastase gene knocked-out strain obtained in this study provides the basis and materials for systemic study of pathogenicity mechanism of elastase in Pseudomonas aeruginosa.%目的 敲除铜绿假单胞菌弹性蛋白酶基因,获得无弹性蛋白酶活性的铜绿假单胞菌菌株.方法 采用PCR从pKD46质粒上扩增λ噬菌体的Red重组酶基因,并将其克隆到大肠杆菌和铜绿假单胞菌穿梭质粒pUCP多克隆位点上,电击转化铜绿假单胞菌PAO1感受态细胞,构建PAO1/pUCP-Red基因敲除体系.常规基因操作构建两端与弹性蛋白酶基因上、下游同源,中间为庆大霉素抗性基因的线性打靶片段;并将其电击转化pUCP-Red/PAO1感受态;采用庆大霉素和羧苄青霉素抗性平板初步筛选阳性重组菌;通过PCR、RT-PCR及弹性蛋白酶活性检测方法,鉴定菌株弹性蛋白酶基因的敲除情况.结果 本研究通过构建Red重组系统,获得了无弹性蛋白酶活性的铜绿假单胞菌菌株.结论 本研究成功敲除了铜绿假单胞菌弹性蛋

  1. Pathogenicity and immunogenicity of recombinant Tiantan Vaccinia Virus with deleted C12L and A53R genes.

    Science.gov (United States)

    Dai, Kaifan; Liu, Ying; Liu, Mingjie; Xu, Jianqing; Huang, Wei; Huang, Xianggang; Liu, Lianxing; Wan, Yanmin; Hao, Yanling; Shao, Yiming

    2008-09-15

    Interest is increasing regarding replicating poxvirus as HIV vaccine vector. In China, the Tiantan Vaccinia Virus (TV) has been used most extensively in the battle of eradicating smallpox. Recently, TV was developing as vaccine vector to fight against infectious diseases such as human immunodeficiency virus (HIV). However, replicating vaccinia virus sometimes may pose serious post-vaccination complications, especially in immunosuppressed individuals. To develop a safer and more effective TV-based vector, we constructed C12L (vIL-18 binding protein) and A53R (vTNF receptor homolog) gene-deleted mutants which are based on parental TV and VTKgpe (TV expressing HIV gagpol and env gene), respectively. The pathogenicity and immunogenicity were also evaluated. Deleting these two immunomodulatory genes lessened the virulence of the parental virus in both mice and rabbit models. Notably, C12L deletion mutant attenuated the skin virulence of parental virus by as high as approximate 2 logs. Furthermore, VTKgpe with A53R and C12L gene deletion retains the high immunogenicity of the parental virus to elicit strong humoral and cellular responses to the HIV target genes despite the remarkable attenuation. These data suggest that deletion of the cytokine viroceptor gene is feasible to obtain a safer and replication-competent TV vector for vaccination and immunotherapy.

  2. A Yeast Mutant Deleted of GPH1 Bears Defects in Lipid Metabolism.

    Directory of Open Access Journals (Sweden)

    Martina Gsell

    Full Text Available In a previous study we demonstrated up-regulation of the yeast GPH1 gene under conditions of phosphatidylethanolamine (PE depletion caused by deletion of the mitochondrial (M phosphatidylserine decarboxylase 1 (PSD1 (Gsell et al., 2013, PLoS One. 8(10:e77380. doi: 10.1371/journal.pone.0077380. Gph1p has originally been identified as a glycogen phosphorylase catalyzing degradation of glycogen to glucose in the stationary growth phase of the yeast. Here we show that deletion of this gene also causes decreased levels of phosphatidylcholine (PC, triacylglycerols and steryl esters. Depletion of the two non-polar lipids in a Δgph1 strain leads to lack of lipid droplets, and decrease of the PC level results in instability of the plasma membrane. In vivo labeling experiments revealed that formation of PC via both pathways of biosynthesis, the cytidine diphosphate (CDP-choline and the methylation route, is negatively affected by a Δgph1 mutation, although expression of genes involved is not down regulated. Altogether, Gph1p besides its function as a glycogen mobilizing enzyme appears to play a regulatory role in yeast lipid metabolism.

  3. Construction and characterization of a glycoprotein E deletion mutant of bovine herpesvirus type 1.2 strain isolated in Brazil

    NARCIS (Netherlands)

    Franco, A.C.; Rijsewijk, F.A.M.; Flores, E.F.; Weiblen, R.; Roehe, P.M.

    2002-01-01

    This paper describes the construction and characterization of a Brazilian strain of bovine herpesvirus type 1.2a (BoHV-1.2a) with a deletion of the glycoprotein E (gE) gene. The deletion was introduced by co-transfection of a deletion fragment containing the 5´and 3´gE flanking regions and genomic D

  4. Gene Deletion by Fluorescence-Reported Allelic Exchange Mutagenesis in Chlamydia trachomatis

    Directory of Open Access Journals (Sweden)

    Konrad E. Mueller

    2016-01-01

    Full Text Available Although progress in Chlamydia genetics has been rapid, genomic modification has previously been limited to point mutations and group II intron insertions which truncate protein products. The bacterium has thus far been intractable to gene deletion or more-complex genomic integrations such as allelic exchange. Herein, we present a novel suicide vector dependent on inducible expression of a chlamydial gene that renders Chlamydia trachomatis fully genetically tractable and permits rapid reverse genetics by fluorescence-reported allelic exchange mutagenesis (FRAEM. We describe the first available system of targeting chlamydial genes for deletion or allelic exchange as well as curing plasmids from C. trachomatis serovar L2. Furthermore, this approach permits the monitoring of mutagenesis by fluorescence microscopy without disturbing bacterial growth, a significant asset when manipulating obligate intracellular organisms. As proof of principle, trpA was successfully deleted and replaced with a sequence encoding both green fluorescent protein (GFP and β-lactamase. The trpA-deficient strain was unable to grow in indole-containing medium, and this phenotype was reversed by complementation with trpA expressed in trans. To assess reproducibility at alternate sites, FRAEM was repeated for genes encoding type III secretion effectors CTL0063, CTL0064, and CTL0065. In all four cases, stable mutants were recovered one passage after the observation of transformants, and allelic exchange was limited to the specific target gene, as confirmed by whole-genome sequencing. Deleted sequences were not detected by quantitative real-time PCR (qPCR from isogenic mutant populations. We demonstrate that utilization of the chlamydial suicide vector with FRAEM renders C. trachomatis highly amenable to versatile and efficient genetic manipulation.

  5. A Haemophilus ducreyi CpxR deletion mutant is virulent in human volunteers.

    Science.gov (United States)

    Labandeira-Rey, Maria; Dodd, Dana; Fortney, Kate R; Zwickl, Beth; Katz, Barry P; Janowicz, Diane M; Spinola, Stanley M; Hansen, Eric J

    2011-06-15

    Haemophilus ducreyi 35000HP contains a homolog of the CpxRA 2-component signal transduction system, which controls the cell envelope stress response system in other gram-negative bacteria and regulates some important H. ducreyi virulence factors. A H. ducreyi cpxR mutant was compared with its parent for virulence in the human challenge model of experimental chancroid. The pustule formation rate in 5 volunteers was 33% (95% confidence interval [CI], 1.3%-65.3%) at 15 parent sites and 40% (95% CI, 18.1%-61.9%) at 15 mutant sites (P = .35). Thus, the cpxR mutant was not attenuated for virulence. Inactivation of the H. ducreyi cpxR gene did not reduce the ability of this mutant to express certain proven virulence factors, including the DsrA serum resistance protein and the LspA2 protein, which inhibits phagocytosis. These results expand our understanding of the involvement of the CpxRA system in regulating virulence expression in H. ducreyi.

  6. Gene deletion in an Italian haemophilia B subject.

    Science.gov (United States)

    Bernardi, F; del Senno, L; Barbieri, R; Buzzoni, D; Gambari, R; Marchetti, G; Conconi, F; Panicucci, F; Positano, M; Pitruzzello, S

    1985-01-01

    DNA from 20 Italian haemophilia B patients was analysed by the Southern blotting technique and hybridisation to a factor IX cDNA probe. A large deletion of factor IX gene was detected in one patient with antibodies to the infused factor; the EcoRI pattern of the other 19 subjects examined was normal. Images PMID:4045960

  7. Gene deletion in an Italian haemophilia B subject.

    OpenAIRE

    De Bernardi, F.; del Senno, L; Barbieri, R.; Buzzoni, D; Gambari, R.; Marchetti, G.; Conconi, F; Panicucci, F; Positano, M; Pitruzzello, S

    1985-01-01

    DNA from 20 Italian haemophilia B patients was analysed by the Southern blotting technique and hybridisation to a factor IX cDNA probe. A large deletion of factor IX gene was detected in one patient with antibodies to the infused factor; the EcoRI pattern of the other 19 subjects examined was normal.

  8. Analysis of a naturally-occurring deletion mutant of Spodoptera frugiperda multiple nucleopolyhedrovirus reveals sf58 as a new per os infectivity factor of lepidopteran-infecting baculoviruses.

    Science.gov (United States)

    Simón, Oihane; Palma, Leopoldo; Williams, Trevor; López-Ferber, Miguel; Caballero, Primitivo

    2012-01-01

    The Nicaraguan population of Spodoptera frugiperda multiple nucleopolyhedrovirus, SfMNPV-NIC, is structured as a mixture of nine genotypes (A-I). Occlusion bodies (OBs) of SfMNPV-C, -D and -G pure genotypes are incapable of oral transmission; a phenotype which in SfMNPV-C and -D is due to the absence of pif1 and pif2 genes. The complete sequence of the SfMNPV-G genome was determined to identify possible factors involved in this phenotype. Deletions of 4860 bp (22,366-27,225) and 60 bp (119,759-119,818) were observed in SfMNPV-G genome compared with that of the predominant complete genotype SfMNPV-B (132,954 bp). However no genes homologous to previously described per os infectivity factors were located within the deleted sequences. Significant differences were detected in the nucleotide sequence in sf58 gene (unknown function) that produced changes in the amino acid sequence and the predicted secondary structure of the corresponding protein. This gene is conserved only in lepidopteran baculoviruses (alpha- and betabaculoviruses). To determine the role of sf58 in peroral infectivity a deletion mutant was constructed using bacmid technology. OBs of the deletion mutant (Sf58null) were not orally infectious for S. frugiperda larvae, whereas Sf58null rescue virus OBs recovered oral infectivity. Sf58null DNA and occlusion derived virions (ODVs) were as infective as SfMNPV bacmid DNA and ODVs in intrahemocelically infected larvae or cell culture, indicating that defects in ODV or OB morphogenesis were not involved in the loss of peroral infectivity. Addition of optical brightener or the presence of the orally infectious SfMNPV-B OBs in mixtures with SfMNPV-G OBs did not recover Sf58null OB infectivity. According to these results sf58 is a new per os infectivity factor present only in lepidopteran baculoviruses.

  9. PARK2 deletions occur frequently in sporadic colorectal cancer and accelerate adenoma development in Apc mutant mice.

    Science.gov (United States)

    Poulogiannis, George; McIntyre, Rebecca E; Dimitriadi, Maria; Apps, John R; Wilson, Catherine H; Ichimura, Koichi; Luo, Feijun; Cantley, Lewis C; Wyllie, Andrew H; Adams, David J; Arends, Mark J

    2010-08-24

    In 100 primary colorectal carcinomas, we demonstrate by array comparative genomic hybridization (aCGH) that 33% show DNA copy number (DCN) loss involving PARK2, the gene encoding PARKIN, the E3 ubiquitin ligase whose deficiency is responsible for a form of autosomal recessive juvenile parkinsonism. PARK2 is located on chromosome 6 (at 6q25-27), a chromosome with one of the lowest overall frequencies of DNA copy number alterations recorded in colorectal cancers. The PARK2 deletions are mostly focal (31% approximately 0.5 Mb on average), heterozygous, and show maximum incidence in exons 3 and 4. As PARK2 lies within FRA6E, a large common fragile site, it has been argued that the observed DCN losses in PARK2 in cancer may represent merely the result of enforced replication of locally vulnerable DNA. However, we show that deficiency in expression of PARK2 is significantly associated with adenomatous polyposis coli (APC) deficiency in human colorectal cancer. Evidence of some PARK2 mutations and promoter hypermethylation is described. PARK2 overexpression inhibits cell proliferation in vitro. Moreover, interbreeding of Park2 heterozygous knockout mice with Apc(Min) mice resulted in a dramatic acceleration of intestinal adenoma development and increased polyp multiplicity. We conclude that PARK2 is a tumor suppressor gene whose haploinsufficiency cooperates with mutant APC in colorectal carcinogenesis.

  10. Engineering validamycin production by tandem deletion of γ-butyrolactone receptor genes in Streptomyces hygroscopicus 5008.

    Science.gov (United States)

    Tan, Gao-Yi; Peng, Yao; Lu, Chenyang; Bai, Linquan; Zhong, Jian-Jiang

    2015-03-01

    Paired homologs of γ-butyrolactone (GBL) biosynthesis gene afsA and GBL receptor gene arpA are located at different positions in genome of Streptomyces hygroscopicus 5008. Inactivation of afsA homologs dramatically decreased biosynthesis of validamycin, an important anti-fungal antibiotic and a critical substrate for antidiabetic drug synthesis, and the deletion of arpA homologs increased validamycin production by 26% (ΔshbR1) and 20% (ΔshbR3). By double deletion, the ΔshbR1/R3 mutant showed higher transcriptional levels of adpA-H (the S. hygroscopicus ortholog of the global regulatory gene adpA) and validamycin biosynthetic genes, and validamycin production increased by 55%. Furthermore, by engineering a high-producing industrial strain via tandem deletion of GBL receptor genes, validamycin production and productivity were enhanced from 19 to 24 g/L (by 26%) and from 6.7 to 9.7 g/L(-1) d(-1) (by 45%), respectively, which was the highest ever reported. The strategy demonstrated here may be useful to engineering other Streptomyces spp. with multiple pairs of afsA-arpA homologs.

  11. Glucocerebrosidase 2 gene deletion rescues type 1 Gaucher disease

    OpenAIRE

    Mistry, Pramod K.; LIU, Jun; Sun, Li; Chuang, Wei-Lien; Yuen, Tony; Yang, Ruhua; Lu, Ping; Zhang, Kate; Li, Jianhua; Keutzer, Joan; Stachnik, Agnes; Mennone, Albert; Boyer, James L; Jain, Dhanpat; Brady, Roscoe O

    2014-01-01

    Type 1 Gaucher disease (GD1) is a rare autosomal recessive disorder caused by inherited mutations in the glucocerebrosidase (GBA1) gene. This disease results in a marked accumulation of glycosphingolipid substrates, causing visceromegaly, cytopenia, and osteopenia. Here, we have rescued this clinical phenotype in GD1 mice by genetically deleting Gba2, a gene encoding a downstream extralysosomal enzyme, GBA2. We also report that sphingosine production in GD1 patients may contribute to the low-...

  12. Increased frequency of DNA deletions in pink-eyed unstable mice carrying a mutation in the Werner syndrome gene homologue.

    Science.gov (United States)

    Lebel, Michel

    2002-01-01

    Werner syndrome (WS) is a rare autosomal recessive disorder characterized by genomic instability and the premature onset of a number of age-related diseases, including cancers. Accumulating evidence indicates that the WS gene product is involved in resolving aberrant DNA structures that may arise during the process of DNA replication and/or transcription. To estimate the frequency of DNA deletions directly in the skin of mouse embryos, mice with a deletion of part of the murine WRN helicase domain were created. These mutant mice were then crossed to the pink-eyed unstable animals, which have a 70 kb internal duplication at the pink-eyed dilution (p) gene. This report indicates that the frequency of deletion of the duplicated sequence at the p locus is elevated in mice with a mutation in the WRN allele when compared with wild-type mice. In addition, the inhibitor of topoisomerase I camptothecin also increases the frequency of deletion at the p locus. This frequency is even more elevated in WRN mutant mice treated with camptothecin. In contrast, while the inhibition of poly(ADP-ribose) polymerase (PARP) activity by 3-aminobenzamide increases the frequency of DNA deletion, mutant WRN mice are not significantly more sensitive to the inhibition of PARP activity than wild-type animals.

  13. Poliovirus temperature-sensitive mutant containing a single nucleotide deletion in the 5'-noncoding region of the viral RNA.

    Science.gov (United States)

    Racaniello, V R; Meriam, C

    1986-12-01

    The effect on viral replication of deleting nucleotide 10 of the poliovirus RNA genome was determined. This deletion, which removes a base pair from a predicted hairpin structure in the viral RNA, was introduced into full-length cDNA. Virus recovered after transfection of HeLa cells with the mutated cDNA contained the expected deletion and was temperature sensitive for plaque formation. Analysis of viral replication by one-step growth experiments indicated that mutant virus production at the nonpermissive temperature was at least 100 times less than that of wild type virus, and release of virus from mutant-infected cells was delayed. The synthesis of positive- and negative-strand viral RNA in mutant virus-infected cells was temperature sensitive. Virus-specific protein synthesis in mutant virus-infected cells was not temperature sensitive but occurred at a slower rate than that of wild type virus at permissive and nonpermissive temperatures. Replication of the mutant virus was sensitive to actinomycin D, in contrast to the wild type parent virus, which was resistant to the drug. Mutant virus stocks contained a small percentage of ts+ viruses that were able to form plaques at the nonpermissive temperature. Nucleotide sequence analysis of genomic RNA from these ts+ viruses revealed a single base change at position 34 from a G to U. In the positive RNA strand, the effect of this mutation is to restore to the hairpin structure the single base pair whose formation was prevented by the original deletion. The ts+ pseudorevertants replicated to similar titers as wild type virus at 33 and 38.5 degrees and were partially sensitive to actinomycin D.

  14. Biological and virulence characteristics of Salmonella enterica serovar Typhimurium following deletion of glucose-inhibited division (gidA) gene.

    Science.gov (United States)

    Shippy, Daniel C; Eakley, Nicholas M; Bochsler, Philip N; Chopra, Ashok K; Fadl, Amin A

    2011-06-01

    Salmonella enterica serovar Typhimurium is a frequent cause of enteric disease due to the consumption of contaminated food. Identification and characterization of bacterial factors involved in Salmonella pathogenesis would help develop effective strategies for controlling salmonellosis. To investigate the role of glucose-inhibited division gene (gidA) in Salmonella virulence, we constructed a Salmonella mutant strain in which gidA was deleted. Deletion of gidA rendered Salmonella deficient in the invasion of intestinal epithelial cells, bacterial motility, intracellular survival, and induction of cytotoxicity in host cells. Deletion of gidA rendered the organism to display a filamentous morphology compared to the normal rod-shaped nature of Salmonella. Furthermore, a significant attenuation in the induction of inflammatory cytokines and chemokines, histopathological lesions, and systemic infection was observed in mice infected with the gidA mutant. Most importantly, a significant increase in LD(50) was observed in mice infected with the gidA mutant, and mice immunized with the gidA mutant were able to survive a lethal dose of wild-type Salmonella. Additionally, deletion of gidA significantly altered the expression of several bacterial factors associated with pathogenesis as indicated by global transcriptional and proteomic profiling. Taken together, our data indicate GidA as a potential regulator of Salmonella virulence genes.

  15. Functional analysis of the PsbX protein by deletion of the corresponding gene in Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Funk, C

    2000-12-01

    The psbX gene (sml0002) coding for a 4.1 kDa protein in Photosystem II of plants and cyanobacteria was deleted in both wild type and in a Photosystem I-less mutant of the cyanobacterium Synechocystis sp. PCC 6803. Polymerase chain reaction and sequencing analysis showed that the mutants had completely segregated. Deletion of the PsbX protein does not seem to influence growth rate, electron transport or water oxidation ability. Whereas a high light induction of the psbX mRNA could be observed in wild type, deletion of the gene did not lead to high light sensibility. Light saturation measurements and 77K fluorescence measurements indicated a minor disconnection of the antenna in the deletion mutant. Furthermore, fluorescence induction measurements as well as immuno-staining of the D1 protein showed that the amount of Photosystem II complexes in the mutants was reduced by 30%. Therefore, PsbX does not seem to be necessary for the Photosystem II electron transport, but directly or indirectly involved in the regulation of the amount of functionally active Photosystem II centres in Synechocystis sp. PCC 6803.

  16. Functional Importance of Deletion Mutant Genotypes in an Insect Nucleopolyhedrovirus Population

    Science.gov (United States)

    Simón, Oihane; Williams, Trevor; López-Ferber, Miguel; Caballero, Primitivo

    2005-01-01

    A Nicaraguan isolate of a nucleopolyhedrovirus (SfNIC) that attacks the fall armyworm, Spodoptera frugiperda, survives as a mixture of nine genotypes (SfNIC A to I) that all present genomic deletions, except variant B (complete genotype). Sequencing of cloned restriction fragments revealed that genotypic variants lack between 5 and 16 of the open reading frames present in a contiguous sequence of 18 kb of the SfNIC genome. The absence of oral infectivity of SfNIC-C and -D variants is related to the deletion of the pif and/or pif-2 gene, while that of SfNIC-G remains unexplained. The presence of open reading frame 10, homolog of Se030, also appeared to influence pathogenicity in certain variants. Previous studies demonstrated a significant positive interaction between genotypes B and C. We compared the median lethal concentration of single genotypes (A, B, C, D, and F) and co-occluded genotype mixtures (B+A, B+D, B+F, A+C, and F+C in a 3:1 ratio). Mixtures B+A and B+D showed increased pathogenicity, although only B+D restored the activity of the mixture to that of the natural population. Mixtures of two deletion variants (A+C and F+C) did not show interactions in pathogenicity. We conclude that minority genotypes have an important influence on the overall pathogenicity of the population. These results clearly demonstrate the value of retaining genotypic diversity in virus-based bioinsecticides. PMID:16085811

  17. Expression of a Mutant kcnj2 Gene Transcript in Zebrafish.

    Science.gov (United States)

    Leong, Ivone U S; Skinner, Jonathan R; Shelling, Andrew N; Love, Donald R

    2013-01-01

    Long QT 7 syndrome (LQT7, also known as Andersen-Tawil syndrome) is a rare autosomal-dominant disorder that causes cardiac arrhythmias, periodic paralysis, and dysmorphic features. Mutations in the human KCNJ2 gene, which encodes for the subunit of the potassium inwardly-rectifying channel (IK1), have been associated with the disorder. The majority of mutations are considered to be dominant-negative as mutant proteins interact to limit the function of wild type KCNJ2 proteins. Several LQT7 syndrome mouse models have been created that vary in the physiological similarity to the human disease. To complement the LQT7 mouse models, we investigated the usefulness of the zebrafish as an alternative model via a transient approach. Initial bioinformatic analysis identified the zebrafish orthologue of the human KCNJ2 gene, together with a spatial expression profile that was similar to that of human. The expression of a kcnj2-12 transcript carrying an in-frame deletion of critical amino acids identified in human studies resulted in embryos that exhibited defects in muscle development, thereby affecting movement, a decrease in jaw size, pupil-pupil distance, and signs of scoliosis. These defects correspond to some phenotypes expressed by human LQT7 patients.

  18. Absence of mutation at the 5'-upstream promoter region of the TPM4 gene from cardiac mutant axolotl (Ambystoma mexicanum).

    Science.gov (United States)

    Denz, Christopher R; Zhang, Chi; Jia, Pingping; Du, Jianfeng; Huang, Xupei; Dube, Syamalima; Thomas, Anish; Poiesz, Bernard J; Dube, Dipak K

    2011-09-01

    Tropomyosins are a family of actin-binding proteins that show cell-specific diversity by a combination of multiple genes and alternative RNA splicing. Of the 4 different tropomyosin genes, TPM4 plays a pivotal role in myofibrillogenesis as well as cardiac contractility in amphibians. In this study, we amplified and sequenced the upstream regulatory region of the TPM4 gene from both normal and mutant axolotl hearts. To identify the cis-elements that are essential for the expression of the TPM4, we created various deletion mutants of the TPM4 promoter DNA, inserted the deleted segments into PGL3 vector, and performed promoter-reporter assay using luciferase as the reporter gene. Comparison of sequences of the promoter region of the TPM4 gene from normal and mutant axolotl revealed no mutations in the promoter sequence of the mutant TPM4 gene. CArG box elements that are generally involved in controlling the expression of several other muscle-specific gene promoters were not found in the upstream regulatory region of the TPM4 gene. In deletion experiments, loss of activity of the reporter gene was noted upon deletion which was then restored upon further deletion suggesting the presence of both positive and negative cis-elements in the upstream regulatory region of the TPM4 gene. We believe that this is the first axolotl promoter that has ever been cloned and studied with clear evidence that it functions in mammalian cell lines. Although striated muscle-specific cis-acting elements are absent from the promoter region of TPM4 gene, our results suggest the presence of positive and negative cis-elements in the promoter region, which in conjunction with positive and negative trans-elements may be involved in regulating the expression of TPM4 gene in a tissue-specific manner.

  19. Global gene expression of a murein (Braun) lipoprotein mutant of Salmonella enterica serovar Typhimurium by microarray analysis.

    Science.gov (United States)

    Fadl, A A; Galindo, C L; Sha, J; Klimpel, G R; Popov, V L; Chopra, A K

    2006-06-01

    Braun/murein lipoprotein (Lpp) is one of the major outer membrane components of gram-negative enteric bacteria involved in inflammatory responses and septic shock. In previous studies, we reported that two copies of the lipoprotein (lpp) gene (designated as lppA and lppB) existed on the chromosome of Salmonella enterica serovar Typhimurium. Deletion of both lppA and lppB genes rendered Salmonella defective in invasion, motility, induction of cytotoxicity, and production of inflammatory cytokines/chemokines. The lppAB double-knockout (DKO) mutant was attenuated in mice, and animals immunized with this mutant were protected against subsequent challenge with lethal doses of wild-type (wt) S. Typhimurium. To better understand how deletion of the lpp gene might affect Salmonella virulence, we performed global transcriptional profiling of the genes in the wt and the lppAB DKO mutant of S. Typhimurium using microarrays. Our data revealed alterations in the expression of flagellar genes, invasion-associated type III secretion system genes, and transcriptional virulence gene regulators in the lppAB DKO mutant compared to wt S. Typhimurium. These data correlated with the lppAB DKO mutant phenotype and provided possible mechanism(s) of Lpp-associated attenuation in S. Typhimurium. Although these studies were performed in in vitro grown bacteria, our future research will be targeted at global transcriptional profiling of the genes in in vivo grown wt S. Typhimurium and its Lpp mutant.

  20. Population stratification of a common APOBEC gene deletion polymorphism.

    Directory of Open Access Journals (Sweden)

    Jeffrey M Kidd

    2007-04-01

    Full Text Available The APOBEC3 gene family plays a role in innate cellular immunity inhibiting retroviral infection, hepatitis B virus propagation, and the retrotransposition of endogenous elements. We present a detailed sequence and population genetic analysis of a 29.5-kb common human deletion polymorphism that removes the APOBEC3B gene. We developed a PCR-based genotyping assay, characterized 1,277 human diversity samples, and found that the frequency of the deletion allele varies significantly among major continental groups (global FST = 0.2843. The deletion is rare in Africans and Europeans (frequency of 0.9% and 6%, more common in East Asians and Amerindians (36.9% and 57.7%, and almost fixed in Oceanic populations (92.9%. Despite a worldwide frequency of 22.5%, analysis of data from the International HapMap Project reveals that no single existing tag single nucleotide polymorphism may serve as a surrogate for the deletion variant, emphasizing that without careful analysis its phenotypic impact may be overlooked in association studies. Application of haplotype-based tests for selection revealed potential pitfalls in the direct application of existing methods to the analysis of genomic structural variation. These data emphasize the importance of directly genotyping structural variation in association studies and of accurately resolving variant breakpoints before proceeding with more detailed population-genetic analysis.

  1. Role of the RuvAB protein in avoiding spontaneous formation of deletion mutations in the Escherichia coli K-12 endogenous tonB gene.

    Science.gov (United States)

    Mashimo, Kazumi; Nagata, Yuki; Kawata, Masakado; Iwasaki, Hiroshi; Yamamoto, Kazuo

    2004-10-08

    The endogenous tonB gene of Escherichia coli was used as a target for spontaneous deletion mutations which were isolated from ruvAB-, recG-, and ruvC- cells. The rates of tonB mutation were essentially the same in ruv+, ruvAB-, recG-, and ruvC- cells. We analyzed tonB mutants by sequencing. In the ruv+, recG-, and ruvC- strains, the spectra were different from those obtained from the ruvAB- cells, where deletions dominated followed by IS insertions, base substitutions, and frameshifts, in that order. We then analyzed the tonB-trp large deletion, due to simultaneous mutations of the trp operon, and found that the frequency in ruvAB- was higher than those in ruv+, recG-, and ruvC- cells. To characterize deletion formation further, we analyzed all the tonB mutants from one colicin plate. Seven deletions were identified at five sites from the 45 tonB mutants of ruv+ cells and 24 deletions at 11 sites from the 43 tonB mutants of ruvAB- cells. Thus, the ruvAB- strain is a deletion mutator. We discuss the role of RuvAB in avoiding deletions. Copyright 2004 Elsevier Inc.

  2. Effects of Chlorophyll Availability on Fluorescence Components of Photosystems in the ORF469-Deletion Mutant of Cyanobacterium

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    PCR-amplified ORF469 fragment from Synechocystis sp.PCC 6803 was cloned into pUC118 and a construct was made in which part of ORF469 was deleted and replaced by erythromycin resistance cassette.Transformation of wild type strain of Synechocystis sp.PCC 6803 with this construct yielded a mutant in which ORF469 was deleted.In the resulting mutant, the light-independent pathway of chlorophyll biosynthesis was inactivated and availability of chlorophyll was fully dependent on light.When propagated the mutant in dark, the chlorophyll was non-detectable and protochlorophyllide with 645 nm fluorescence emission peak was accumulated.Meanwhile, the fluorescence emission peaks (excited at 435 nm) of thylakoids at 685 nm, 695 nm and 725 nm, which represented relative chlorophyll-binding proteins, disappeared.Upon return of dark-grown ORF469 mutant to the light, greening occurred and chlorophyll was synthesized to assembly fluorescence emission components in photosystems.Newly synthesized chlorophyll combined the fluorescence component of 685 nm at first, then 725 nm and 695 nm at last, which indicates a pecking order for biogenesis of chlorophyll-binding proteins when availability of chlorophyll is limited.The mutant lacking ORF469 in Synechocystis sp.PCC 6803 was suggested as an excellent cyanobacterial system for studies on the interactions between chlorophyll and chlorophyll-binding proteins in photosystems.

  3. Large deletion in the NF1 gene associated with dysmorphism

    Energy Technology Data Exchange (ETDEWEB)

    Hughes, H.E.; Maynard, J.; Sourour, E. [University Hospital of Wales, Cardiff (United Kingdom)] [and others

    1994-09-01

    Neurofibromatosis type 1 is an autosomal dominant disorder with a prevalence of 1 in 3000. The major clinical features of the disease include cafe-au-lait spots, neurofibromas, Lisch nodules and auxillary freckling. Six sporadic NF1 patients with dysmorphism and intellectual impairment have been described to have a large deletion extending beyond the NF1 gene. We report another spordiac NF1 patient with severe developmental delay, early growth failure and dysmorphism (not Noonan-like) associated with a large deletion involving the NF1 gene. A panel of 12 polymorphic DNA markers within 4 cM of the NF1 gene were used to screen for the NF1 gene rearrangements. With all the polymorphic markers, only a single band was ever observed in this affected individual. However, with DNA probe EW301 which maps to 17p, a biparental inheritance was observed. Analysis with several microsatellite markers indicated that this patient had not inherited an allele from the father. A reduction in the hybridization signal was also observed when DNA from this patient was screened with cDNAs AE25, P5, B3A, and an extragenic marker EW206, clearly indicating hemizygosity at these loci. The combined evidence of dosage reduction and biparental inheritance with DNA marker EW301 indictates that this patient has a deletion of paternal origin rather than uniparental disomy. Pulsed-field gel electrophoresis has not, so far, revealed any evidence of an altered band pattern; however, studies are continuing. FISH analysis is currently in progress using YACs and cosmids to define the extent of this deletion.

  4. Research Progress on Pseudorabies Gene-deleted Vaccine

    Institute of Scientific and Technical Information of China (English)

    JIN Sheng-zao; CHEN Huang-chun; XIONG Fu

    2002-01-01

    Pseudorabies is caused by pseudorabies virus (PrV), which is a member of family Herpesviridae, subfamily Alphaherpesvirinae and is the agent of acute infectious disease in many domestic and wild animals. Swine was the natural host and reservior of PRV, which inflicts major economic loss in pig industries world wide. Immunization with safe, effective vaccine is main measurements to prevent the disease.In this assay, research progress on PRV gene-deleted vaccine used extensively today was discussed.

  5. Trapping of normal EB1 ligands in aggresomes formed by an EB1 deletion mutant

    Directory of Open Access Journals (Sweden)

    Askham Jon M

    2005-04-01

    Full Text Available Abstract Background EB1 is a microtubule tip-associated protein that interacts with the APC tumour suppressor protein and the p150glued subunit of dynactin. We previously reported that an EB1 deletion mutant that retains both of these interactions but does not directly associate with microtubules (EB1-ΔN2-GFP spontaneously formed perinuclear aggregates when expressed in COS-7 cells. Results In the present study live imaging indicated that EB1-ΔN2-GFP aggregates underwent dynamic microtubule-dependent changes in morphology and appeared to be internally cohesive. EB1-ΔN2-GFP aggregates were phase-dense structures that displayed microtubule-dependent accumulation around the centrosome, were immunoreactive for both the 20s subunit of the proteasome and ubiquitin, and induced the collapse of the vimentin cytoskeleton. Fractionation studies revealed that a proportion of EB1-ΔN2-GFP was detergent-insoluble and ubiquitylated, indicating that EB1-ΔN2-GFP aggregates are aggresomes. Immunostaining also revealed that APC and p150glued were present in EB1-ΔN2-GFP aggregates, whereas EB3 was not. Furthermore, evidence for p150glued degradation was found in the insoluble fraction of EB1-ΔN2-GFP transfected cultures. Conclusion Our data indicate that aggresomes can be internally cohesive and may not represent a simple "aggregate of aggregates" assembled around the centrosome. Our observations also indicate that a partially misfolded protein may retain the ability to interact with its normal physiological ligands, leading to their co-assembly into aggresomes. This supports the idea that the trapping and degradation of co-aggregated proteins might contribute to human pathologies characterised by aggresome formation.

  6. Modeling of gap gene expression in Drosophila Kruppel mutants.

    Directory of Open Access Journals (Sweden)

    Konstantin Kozlov

    Full Text Available The segmentation gene network in Drosophila embryo solves the fundamental problem of embryonic patterning: how to establish a periodic pattern of gene expression, which determines both the positions and the identities of body segments. The gap gene network constitutes the first zygotic regulatory tier in this process. Here we have applied the systems-level approach to investigate the regulatory effect of gap gene Kruppel (Kr on segmentation gene expression. We acquired a large dataset on the expression of gap genes in Kr null mutants and demonstrated that the expression levels of these genes are significantly reduced in the second half of cycle 14A. To explain this novel biological result we applied the gene circuit method which extracts regulatory information from spatial gene expression data. Previous attempts to use this formalism to correctly and quantitatively reproduce gap gene expression in mutants for a trunk gap gene failed, therefore here we constructed a revised model and showed that it correctly reproduces the expression patterns of gap genes in Kr null mutants. We found that the remarkable alteration of gap gene expression patterns in Kr mutants can be explained by the dynamic decrease of activating effect of Cad on a target gene and exclusion of Kr gene from the complex network of gap gene interactions, that makes it possible for other interactions, in particular, between hb and gt, to come into effect. The successful modeling of the quantitative aspects of gap gene expression in mutant for the trunk gap gene Kr is a significant achievement of this work. This result also clearly indicates that the oversimplified representation of transcriptional regulation in the previous models is one of the reasons for unsuccessful attempts of mutant simulations.

  7. Emergence of porcine reproductive and respiratory syndrome virus deletion mutants: Correlation with the porcine antibody response to a hypervariable site in the ORF 3 structural glycoprotein

    DEFF Research Database (Denmark)

    Oleksiewicz, M.B.; Bøtner, Anette; Toft, P.

    2000-01-01

    reading frames, the same PRRSV genetic locus codes for the ORF 3 "RKASLSTS" sequence, and a previously described ORF 4 epitope (Meulenherg, J. J. M., Van Nieuwstadt, A. P,, Van Essen-Zandbergen, A., and Langeveld, J. P. M., 1997, J. Virol. 71, 6061-6067). Sequence analysis identified naturally occurring...... deletion mutants at this ORF 3/4 site. Phylogenetic analysis showed the presence of a highly accurate ORF 3 molecular clock, according to which deletion mutants and nondeleted viruses evolved at differing speeds. Furthermore, deletion mutants and nondeleted viruses evolved as separate lineages...

  8. Deletion of gene encoding methyltransferase (gidB) confers high-level antimicrobial resistance in Salmonella.

    Science.gov (United States)

    Mikheil, Dareen M; Shippy, Daniel C; Eakley, Nicholas M; Okwumabua, Ogi E; Fadl, Amin A

    2012-04-01

    The glucose-inhibited division gene (gid)B, which resides in the gid operon, was thought to have a role in the modulation of genes similar to that of gidA. Recent studies have indicated that GidB is a methyltransferase enzyme that is involved in the methylation of the 16S ribosomal RNA (rRNA) in Escherichia coli. In this study, we investigated the role of GidB in susceptibility to antibiotics and the overall biology of Salmonella. A gidB isogenic mutant of Salmonella was constructed and subsequently characterized under different conditions. Our data indicated that growth and invasion characteristics of the gidB mutant were similar to those of the wild type (WT). The gidB mutant was outgrown by the WT in a competitive growth assay, indicating a compromised overall bacterial fitness. Under the stress of nalidixic acid, the gidB mutant's motility was significantly reduced. Similarly, the mutant showed a filamentous morphology and smaller colony size compared with the rod-shaped and large colonies of the WT in the presence of nalidixic acid. Most importantly, deletion of gidB conferred high-level resistance to the aminoglycoside antibiotics streptomycin and neomycin. A primer extension assay determined the methylation site for the WT to be at G527 of the 16S rRNA. A lack of methylation in the mutant indicated that GidB is required for this methylation. Taken together, these data indicate that the GidB enzyme has a significant role in the alteration of antibiotic susceptibility and the modulation of growth and morphology under stress conditions in Salmonella.

  9. Microhomology-mediated deletion and gene conversion in African trypanosomes.

    Science.gov (United States)

    Glover, Lucy; Jun, Junho; Horn, David

    2011-03-01

    Antigenic variation in African trypanosomes is induced by DNA double-strand breaks (DSBs). In these protozoan parasites, DSB repair (DSBR) is dominated by homologous recombination (HR) and microhomology-mediated end joining (MMEJ), while non-homologous end joining (NHEJ) has not been reported. To facilitate the analysis of chromosomal end-joining, we established a system whereby inter-allelic repair by HR is lethal due to loss of an essential gene. Analysis of intrachromosomal end joining in individual DSBR survivors exclusively revealed MMEJ-based deletions but no NHEJ. A survey of microhomologies typically revealed sequences of between 5 and 20 bp in length with several mismatches tolerated in longer stretches. Mean deletions were of 54 bp on the side closest to the break and 284 bp in total. Break proximity, microhomology length and GC-content all favored repair and the pattern of MMEJ described above was similar at several different loci across the genome. We also identified interchromosomal gene conversion involving HR and MMEJ at different ends of a duplicated sequence. While MMEJ-based deletions were RAD51-independent, one-sided MMEJ was RAD51 dependent. Thus, we describe the features of MMEJ in Trypanosoma brucei, which is analogous to micro single-strand annealing; and RAD51 dependent, one-sided MMEJ. We discuss the contribution of MMEJ pathways to genome evolution, subtelomere recombination and antigenic variation.

  10. A deletion in the gene encoding sphingomyelin phosphodiesterase 3 (Smpd3) results in osteogenesis and dentinogenesis imperfecta in the mouse.

    Science.gov (United States)

    Aubin, Isabelle; Adams, Carolyn P; Opsahl, Sibylle; Septier, Dominique; Bishop, Colin E; Auge, Nathalie; Salvayre, Robert; Negre-Salvayre, Anne; Goldberg, Michel; Guénet, Jean-Louis; Poirier, Christophe

    2005-08-01

    The mouse mutation fragilitas ossium (fro) leads to a syndrome of severe osteogenesis and dentinogenesis imperfecta with no detectable collagen defect. Positional cloning of the locus identified a deletion in the gene encoding neutral sphingomyelin phosphodiesterase 3 (Smpd3) that led to complete loss of enzymatic activity. Our knowledge of SMPD3 function is consistent with the pathology observed in mutant mice and provides new insight into human pathologies.

  11. Deletion of cagA gene of Helicobacter pylori by PCR products

    Institute of Scientific and Technical Information of China (English)

    Xun Zeng; Li-Hua He; Yan Yin; Mao-Jun Zhang; Jian-Zhong Zhang

    2005-01-01

    AIM: Cytotoxin-associated protein (antigen) A (CagA)plays an important role in Helicobacter pylori(H pylori)pathogenesis. Our aim was to obtain cagA mutant strains by a new mutation method so as to better understand the mechanism of CagA in epithelial cells. METHODS: In contrast with the traditional method using suicide plasmid, we constructed cagA- mutant strains directly with PCR products. The constructed mutant clones grew on selective media and allelic exchange was confirmed by Southern blot. Furthermore, two different transformation methods, electroporation, and natural transformation, were compared with regard to the efficiency of recombination.RESULTS: The mutation by PCR products could be completed within 3-5 d, and the recombination rate by electroporation and natural transformation was 4.02×10-8 and 1.03x 10-9 respectively. Mutation rate by electroporation (4.02× 10-8) was far higher than by natural transformation (1.03× 10-9) (P = 0.000<0.005).CONCLUSION: cagA- mutant strains have been constructed,which is important for further study on the function of CagA in epithelial cells. A mutation method by directly using PCR products has been proved successful with a much higher mutation rate, and is easier, especially when in combination with electroporation. This method could be widely used in gene deletion of H pylori.

  12. Identification of Mutant Genes and Introgressed Tiger Salamander DNA in the Laboratory Axolotl, Ambystoma mexicanum.

    Science.gov (United States)

    Woodcock, M Ryan; Vaughn-Wolfe, Jennifer; Elias, Alexandra; Kump, D Kevin; Kendall, Katharina Denise; Timoshevskaya, Nataliya; Timoshevskiy, Vladimir; Perry, Dustin W; Smith, Jeramiah J; Spiewak, Jessica E; Parichy, David M; Voss, S Randal

    2017-12-01

    The molecular genetic toolkit of the Mexican axolotl, a classic model organism, has matured to the point where it is now possible to identify genes for mutant phenotypes. We used a positional cloning-candidate gene approach to identify molecular bases for two historic axolotl pigment phenotypes: white and albino. White (d/d) mutants have defects in pigment cell morphogenesis and differentiation, whereas albino (a/a) mutants lack melanin. We identified in white mutants a transcriptional defect in endothelin 3 (edn3), encoding a peptide factor that promotes pigment cell migration and differentiation in other vertebrates. Transgenic restoration of Edn3 expression rescued the homozygous white mutant phenotype. We mapped the albino locus to tyrosinase (tyr) and identified polymorphisms shared between the albino allele (tyr (a) ) and tyr alleles in a Minnesota population of tiger salamanders from which the albino trait was introgressed. tyr (a) has a 142 bp deletion and similar engineered alleles recapitulated the albino phenotype. Finally, we show that historical introgression of tyr (a) significantly altered genomic composition of the laboratory axolotl, yielding a distinct, hybrid strain of ambystomatid salamander. Our results demonstrate the feasibility of identifying genes for traits in the laboratory Mexican axolotl.

  13. Deletion of cdvB paralogous genes of Sulfolobus acidocaldarius impairs cell division.

    Science.gov (United States)

    Yang, Nuan; Driessen, Arnold J M

    2014-03-01

    The majority of Crenarchaeota utilize the cell division system (Cdv) to divide. This system consists of three highly conserved genes, cdvA, cdvB and cdvC that are organized in an operon. CdvC is homologous to the AAA-type ATPase Vps4, involved in multivesicular body biogenesis in eukaryotes. CdvA is a unique archaeal protein that interacts with the membrane, while CdvB is homologous to the eukaryal Vps24 and forms helical filaments. Most Crenarcheota contain additional CdvB paralogs. In Sulfolobus acidocaldarius these are termed CdvB1-3. We have used a gene inactivation approach to determine the impact of these additional cdvB genes on cell division. Independent deletion mutants of these genes were analyzed for growth and protein localization. One of the deletion strains (ΔcdvB3) showed a severe growth defect on plates and delayed growth on liquid medium. It showed the formation of enlarged cells and a defect in DNA segregation. Since these defects are accompanied with an aberrant localization of CdvA and CdvB, we conclude that CdvB3 fulfills an important accessory role in cell division.

  14. Amplified and homozygously deleted genes in glioblastoma: impact on gene expression levels.

    Directory of Open Access Journals (Sweden)

    Inês Crespo

    Full Text Available BACKGROUND: Glioblastoma multiforme (GBM displays multiple amplicons and homozygous deletions that involve relevant pathogenic genes and other genes whose role remains unknown. METHODOLOGY: Single-nucleotide polymorphism (SNP-arrays were used to determine the frequency of recurrent amplicons and homozygous deletions in GBM (n = 46, and to evaluate the impact of copy number alterations (CNA on mRNA levels of the genes involved. PRINCIPAL FINDINGS: Recurrent amplicons were detected for chromosomes 7 (50%, 12 (22%, 1 (11%, 4 (9%, 11 (4%, and 17 (4%, whereas homozygous deletions involved chromosomes 9p21 (52% and 10q (22%. Most genes that displayed a high correlation between DNA CNA and mRNA levels were coded in the amplified chromosomes. For some amplicons the impact of DNA CNA on mRNA expression was restricted to a single gene (e.g., EGFR at 7p11.2, while for others it involved multiple genes (e.g., 11 and 5 genes at 12q14.1-q15 and 4q12, respectively. Despite homozygous del(9p21 and del(10q23.31 included multiple genes, association between these DNA CNA and RNA expression was restricted to the MTAP gene. CONCLUSIONS: Overall, our results showed a high frequency of amplicons and homozygous deletions in GBM with variable impact on the expression of the genes involved, and they contributed to the identification of other potentially relevant genes.

  15. Deletion of a malaria invasion gene reduces death and anemia, in model hosts.

    Directory of Open Access Journals (Sweden)

    Noé D Gómez

    Full Text Available Malaria parasites induce complex cellular and clinical phenotypes, including anemia, cerebral malaria and death in a wide range of mammalian hosts. Host genes and parasite 'toxins' have been implicated in malarial disease, but the contribution of parasite genes remains to be fully defined. Here we assess disease in BALB/c mice and Wistar rats infected by the rodent malaria parasite Plasmodium berghei with a gene knock out for merozoite surface protein (MSP 7. MSP7 is not essential for infection but in P. falciparum, it enhances erythrocyte invasion by 20%. In vivo, as compared to wild type, the P. berghei Δmsp7 mutant is associated with an abrogation of death and a decrease from 3% to 2% in peak, circulating parasitemia. The Δmsp7 mutant is also associated with less anemia and modest increase in the size of follicles in the spleen. Together these data show that deletion of a single parasite invasion ligand modulates blood stage disease, as measured by death and anemia. This work is the first to assess the contribution of a gene present in all plasmodial species in severe disease.

  16. Transcriptional Responses of the Bdtf1-Deletion Mutant to the Phytoalexin Brassinin in the Necrotrophic Fungus Alternaria brassicicola

    Directory of Open Access Journals (Sweden)

    Yangrae Cho

    2014-07-01

    Full Text Available Brassica species produce the antifungal indolyl compounds brassinin and its derivatives, during microbial infection. The fungal pathogen Alternaria brassicicola detoxifies brassinin and possibly its derivatives. This ability is an important property for the successful infection of brassicaceous plants. Previously, we identified a transcription factor, Bdtf1, essential for the detoxification of brassinin and full virulence. To discover genes that encode putative brassinin-digesting enzymes, we compared gene expression profiles between a mutant strain of the transcription factor and wild-type A. brassicicola under two different experimental conditions. A total of 170 and 388 genes were expressed at higher levels in the mutants than the wild type during the infection of host plants and saprophytic growth in the presence of brassinin, respectively. In contrast, 93 and 560 genes were expressed, respectively, at lower levels in the mutant than the wild type under the two conditions. Fifteen of these genes were expressed at lower levels in the mutant than in the wild type under both conditions. These genes were assumed to be important for the detoxification of brassinin and included Bdtf1 and 10 putative enzymes. This list of genes provides a resource for the discovery of enzyme-coding genes important in the chemical modification of brassinin.

  17. Enhancement or attenuation of disease by deletion of genes from Citrus tristeza virus.

    Science.gov (United States)

    Tatineni, Satyanarayana; Dawson, William O

    2012-08-01

    Stem pitting is a common virus-induced disease of perennial woody plants induced by a range of different viruses. The phenotype results from sporadic areas of the stem in which normal xylem and phloem development is prevented during growth of stems. These alterations interfere with carbohydrate transport, resulting in reduced plant growth and yield. Citrus tristeza virus (CTV), a phloem-limited closterovirus, induces economically important stem-pitting diseases of citrus. CTV has three nonconserved genes (p33, p18, and p13) that are not related to genes of other viruses and that are not required for systemic infection of some species of citrus, which allowed us to examine the effect of deletions of these genes on symptom phenotypes. In the most susceptible experimental host, Citrus macrophylla, the full-length virus causes only very mild stem-pitting symptoms. Surprisingly, we found that certain deletion combinations (p33 and p18 and/or p13) induced greatly increased stem-pitting symptoms, while other combinations (p13 or p13 plus p18) resulted in reduced stem pitting. These results suggest that the stem-pitting phenotype, which is one of more economically important disease phenotypes, can result not from a specific sequence or protein but from a balance between the expression of different viral genes. Unexpectedly, using green fluorescent protein-tagged full-length virus and deletion mutants (CTV9Δp33 and CTV9Δp33Δp18Δp13), the virus was found at pitted areas in abnormal locations outside the normal ring of phloem. Thus, increased stem pitting was associated not only with a prevention of xylem production but also with a proliferation of cells that supported viral replication, suggesting that at random areas of stems the virus can elicit changes in cellular differentiation and development.

  18. Fine Mapping and Cloning of Leafy Head Mutant Gene pla1-5 in Rice

    Directory of Open Access Journals (Sweden)

    Gong-neng FENG

    2013-09-01

    Full Text Available We identified a leafy head mutant pla1-5 (plastochron 1-5 from the progeny of japonica rice cultivar Taipei 309 treated with 60Co-γ ray irradiation. The pla1-5 mutant has a dwarf phenotype and small leaves. Compared with its wild type, pla1-5 has more leaves and fewer tillers, and it fails to produce normal panicles at the maturity stage. Genetic analysis showed that the pla1-5 phenotype is controlled by a single recessive nuclear gene. Using the map-based cloning strategy, we narrowed down the location of the target gene to a 58-kb region between simple sequence repeat markers CHR1027 and CHR1030 on the long arm of chromosome 10. The target gene cosegregated with molecular markers CHR1028 and CHR1029. There were five predicted genes in the mapped region. The results from sequencing analysis revealed that there was one base deletion in the first exon of LOC_Os10g26340 encoding cytochrome P450 CYP78A11 in the pla1-5 mutant, which might result in a downstream frame shift and premature termination. These results suggest that the P450 CYP78A11 gene is the candidate gene of PLA1-5.

  19. Construction of fbpA-deletion Mutant of Listeria Monocytogenes%单核细胞增生性李斯特菌fbpa基因敲除菌株的构建

    Institute of Scientific and Technical Information of China (English)

    李胜军; 阎雪晶; 王舰

    2013-01-01

    Objective To construct an fbpA-deletion mutant of Listeria monocytogenes. Methods The fbpA gene and its upstream, downstream genes of Listeria monocytogenes were cloned into plasmid pCR Ⅱ. The upstream and downstream fragments were ligated into the pAULA using restriction enzyme as pAULA-ΔfbpA. To achieve allelic exchange, pAULA-ΔfbpA was introduced into Listeria monocytogenes by electroporation. The mutant was confirmed by PCR and Western blot. Results The fbpA gene was not detected in genome of fbpA-deletion mutant of Listeria monocytogenes,and FbpA was not expressed in fbpA-deletion mutant of Listeria monocytogenes. Conclusion The fbpA-deletion mutant of Listeria monocytogenes was constructed successfully.%目的 构建单核细胞增生性李斯特菌fbpa基因敲除菌株.方法 克隆fbpa及其上、下游基因,构建其载体质粒;通过酶切反应将上、下游基因分别重组到载体质粒中,形成同源重组质粒;同源重组质粒电转入细菌内,进行同源重组;采用PCR、Western blot鉴定敲除菌株.结果 单核细胞增生性李斯特菌fbpa基因敲除菌株基因组DNA无fbpa基因片段,且无FbpA蛋白表达.结论 成功构建单核细胞增生性李斯特菌fbpa基因敲除菌株.

  20. VIP Gene Deletion in Mice Causes Cardiomyopathy Associated with Upregulation of Heart Failure Genes

    Energy Technology Data Exchange (ETDEWEB)

    Szema, Anthony M.; Hamidi, Sayyed A.; Smith, S. David; Benveniste, Helene; Katare, Rajesh Gopalrao

    2013-05-20

    Vasoactive Intestinal Peptide (VIP), a pulmonary vasodilator and inhibitor of vascular smooth muscle proliferation, is absent in pulmonary arteries of patients with idiopathic pulmonary arterial hypertension (PAH). We previously determined that targeted deletion of the VIP gene in mice leads to PAH with pulmonary vascular remodeling and right ventricular (RV) dilatation. Whether the left ventricle is also affected by VIP gene deletion is unknown. In the current study, we examined if VIP knockout mice (VIP-/-) develop both right (RV) and left ventricular (LV) cardiomyopathy, manifested by LV dilatation and systolic dysfunction, as well as overexpression of genes conducive to heart failure.

  1. Differentially expressed genes in the ovary of the sixth day of pupal "Ming" lethal egg mutant of silkworm, Bombyx mori.

    Science.gov (United States)

    Gao, Peng; Chen, An-Li; Zhao, Qiao-Ling; Shen, Xing-Jia; Qiu, Zhi-Yong; Xia, Ding-Guo; Tang, Shun-Ming; Zhang, Guo-Zheng

    2013-09-15

    The "Ming" lethal egg mutant (l-em) is a vitelline membrane mutant in silkworm, Bombyx mori. The eggs laid by the l-em mutant lose water, ultimately causing death within an hour. Previous studies have shown that the deletion of BmEP80 is responsible for the l-em mutation in silkworm, B. mori. In the current study, digital gene expression (DGE) was performed to investigate the difference of gene expression in ovaries between wild type and l-em mutant on the sixth day of the pupal stage to obtain a global view of gene expression profiles using the ovaries of three l-em mutants and three wild types. The results showed a total of 3,463,495 and 3,607,936 clean tags in the wild type and the l-em mutant libraries, respectively. Compared with those of wild type, 239 differentially expressed genes were detected in the l-em mutant, wherein 181 genes are up-regulated and 58 genes are down-regulated in the mutant strain. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis results showed that no pathway was significantly enriched and three pathways are tightly related to protein synthesis among the five leading pathways. Moreover, the expression profiles of eight important differentially expressed genes related to oogenesis changed. These results provide a comprehensive gene expression analysis of oogenesis and vitellogenesis in B. mori which facilitates understanding of both the specific molecular mechanism of the 1-em mutant and Lepidopteran oogenesis in general.

  2. STUDY OF DELETION OF P16 GENE IN THE PROGRESSION OF BRAIN ASTROCYTOMAS

    Institute of Scientific and Technical Information of China (English)

    Zhai Guang; Yuan Xianhou

    1998-01-01

    Objective:To study the relationship between deletion of P16 gene and occurrence and progression of astrocytomas. Methods: The techniques of polymerase chain reaction (PCR) and immunohistochemistry were used to detect the deletion of exon2 of P16 gene and expression of P16 gene in 52 cases of Brain astrocytoma.Results: The deletion rate of exon2 of P16 gene in the tumors analyzed was 34.6%. Most of them with deletion of exon2 of p16 gene were high grade astrocytomas (grade Ⅲ 42%, grade Ⅳ 50%). 61.5% of the tumors were absent from expression of p16 and the deletion rate of p16 protein increased with the grade of astrocytoma (X2=10.83, P<0.005). Conclusion: Deletion of p16 gene and protein may correlate with the malignant progression of astrocytoma.

  3. Effects of deletion of mutant huntingtin in steroidogenic factor 1 neurons on the psychiatric and metabolic phenotype in the BACHD mouse model of Huntington disease.

    Directory of Open Access Journals (Sweden)

    Barbara Baldo

    Full Text Available Psychiatric and metabolic features appear several years before motor disturbances in the neurodegenerative Huntington's disease (HD, caused by an expanded CAG repeat in the huntingtin (HTT gene. Although the mechanisms leading to these aspects are unknown, dysfunction in the hypothalamus, a brain region controlling emotion and metabolism, has been suggested. A direct link between the expression of the disease causing protein, huntingtin (HTT, in the hypothalamus and the development of metabolic and psychiatric-like features have been shown in the BACHD mouse model of HD. However, precisely which circuitry in the hypothalamus is critical for these features is not known. We hypothesized that expression of mutant HTT in the ventromedial hypothalamus, an area involved in the regulation of metabolism and emotion would be important for the development of these non-motor aspects. Therefore, we inactivated mutant HTT in a specific neuronal population of the ventromedial hypothalamus expressing the transcription factor steroidogenic factor 1 (SF1 in the BACHD mouse using cross-breeding based on a Cre-loxP system. Effects on anxiety-like behavior were assessed using the elevated plus maze and novelty-induced suppressed feeding test. Depressive-like behavior was assessed using the Porsolt forced swim test. Effects on the metabolic phenotype were analyzed using measurements of body weight and body fat, as well as serum insulin and leptin levels. Interestingly, the inactivation of mutant HTT in SF1-expressing neurons exerted a partial positive effect on the depressive-like behavior in female BACHD mice at 4 months of age. In this cohort of mice, no anxiety-like behavior was detected. The deletion of mutant HTT in SF1 neurons did not have any effect on the development of metabolic features in BACHD mice. Taken together, our results indicate that mutant HTT regulates metabolic networks by affecting hypothalamic circuitries that do not involve the SF1 neurons

  4. Enhanced gene replacements in Ku80 disruption mutants of the dermatophyte, Trichophyton mentagrophytes.

    Science.gov (United States)

    Yamada, Tsuyoshi; Makimura, Koichi; Hisajima, Tatsuya; Ishihara, Yumiko; Umeda, Yoshiko; Abe, Shigeru

    2009-09-01

    The frequency of targeted gene disruption via homologous recombination is low in the clinically important dermatophyte, Trichophyton mentagrophytes. The Ku genes, Ku70 and Ku80, encode key components of the nonhomologous end-joining pathway involved in DNA double-strand break repair. Their deletion increases the homologous recombination frequency, facilitating targeted gene disruption. To improve the homologous recombination frequency in T. mentagrophytes, the Ku80 ortholog was inactivated. The nucleotide sequence of the Ku80 locus containing a 2788-bp ORF encoding a predicted product of 728 amino acids was identified, and designated as TmKu80. The predicted TmKu80 product showed a high degree of amino acid sequence similarity to known fungal Ku80 proteins. Ku80 disruption mutant strains of T. mentagrophytes were constructed by Agrobacterium tumefaciens-mediated genetic transformation. The average homologous recombination frequency was 73.3 +/- 25.2% for the areA/nit-2-like nitrogen regulatory gene (tnr) in Ku80(-) mutants, about 33-fold higher than that in wild-type controls. A high frequency (c. 67%) was also obtained for the Tri m4 gene encoding a putative serine protease. Ku80(-) mutant strains will be useful for large-scale reverse genetics studies of dermatophytes, including T. mentagrophytes, providing valuable information on the basic mechanisms of host invasion.

  5. Bivalent vaccination against pneumonic pasteurellosis in domestic sheep and goats with modified-live in-frame lktA deletion mutants of Mannheimia haemolytica.

    Science.gov (United States)

    Briggs, Robert E; Hauglund, Melissa J; Maheswaran, Samuel K; Tatum, Fred M

    2013-11-01

    A temperature-sensitive shuttle vector, pBB80C, was utilized to generate in-frame deletion mutants of the leukotoxin structural gene (lktA) of Mannheimia haemolytica serotypes 1, 2, 5, 6, 7, 8, 9, and 12. Culture supernatants from the mutants contained a truncated protein with an approximate molecular weight of 66 kDa which was reactive to anti-leukotoxin monoclonal antibody. No protein reactive to anti-LktA monoclonal antibody was detected at the molecular weight 100-105 kDa of native LktA. Sheep and goats vaccinated intramuscularly with a mixture of serotypes 5 and 6 mutants were resistant to virulent challenge with a mixture of the wild-type parent strains. These vaccinates responded serologically to both vaccine serotypes and exhibited markedly-reduced lung lesion volume and pulmonary infectious load compared to control animals. Control animals yielded a mixture of serotypes from lung lobes, but the proportion even within an individual animal varied widely from 95% serotype 5-95% serotype 6. Cultures recovered from liver were homogeneous, but two animals yielded serotype 5 and the other two yielded serotype 6 in pure culture. Published by Elsevier Ltd.

  6. Deletion of the Braun lipoprotein-encoding gene and altering the function of lipopolysaccharide attenuate the plague bacterium.

    Science.gov (United States)

    Sha, Jian; Kirtley, Michelle L; van Lier, Christina J; Wang, Shaofei; Erova, Tatiana E; Kozlova, Elena V; Cao, Anthony; Cong, Yingzi; Fitts, Eric C; Rosenzweig, Jason A; Chopra, Ashok K

    2013-03-01

    Braun (murein) lipoprotein (Lpp) and lipopolysaccharide (LPS) are major components of the outer membranes of Enterobacteriaceae family members that are capable of triggering inflammatory immune responses by activating Toll-like receptors 2 and 4, respectively. Expanding on earlier studies that demonstrated a role played by Lpp in Yersinia pestis virulence in mouse models of bubonic and pneumonic plague, we characterized an msbB in-frame deletion mutant incapable of producing an acyltransferase that is responsible for the addition of lauric acid to the lipid A moiety of LPS, as well as a Δlpp ΔmsbB double mutant of the highly virulent Y. pestis CO92 strain. Although the ΔmsbB single mutant was minimally attenuated, the Δlpp single mutant and the Δlpp ΔmsbB double mutant were significantly more attenuated than the isogenic wild-type (WT) bacterium in bubonic and pneumonic animal models (mouse and rat) of plague. These data correlated with greatly reduced survivability of the aforementioned mutants in murine macrophages. Furthermore, the Δlpp ΔmsbB double mutant was grossly compromised in its ability to disseminate to distal organs in mice and in evoking cytokines/chemokines in infected animal tissues. Importantly, mice that survived challenge with the Δlpp ΔmsbB double mutant, but not the Δlpp or ΔmsbB single mutant, in a pneumonic plague model were significantly protected against a subsequent lethal WT CO92 rechallenge. These data were substantiated by the fact that the Δlpp ΔmsbB double mutant maintained an immunogenicity comparable to that of the WT strain and induced long-lasting T-cell responses against heat-killed WT CO92 antigens. Taken together, the data indicate that deletion of the msbB gene augmented the attenuation of the Δlpp mutant by crippling the spread of the double mutant to the peripheral organs of animals and by inducing cytokine/chemokine responses. Thus, the Δlpp ΔmsbB double mutant could provide a new live-attenuated background

  7. Deletion of PLCB1 gene in schizophrenia-affected patients.

    Science.gov (United States)

    Lo Vasco, Vincenza Rita; Cardinale, Giuseppina; Polonia, Patrizia

    2012-04-01

    A prevalence of 1% in the general population and approximately 50% concordance rate in monozygotic twins was reported for schizophrenia, suggesting that genetic predisposition affecting neurodevelopmental processes might combine with environmental risk factors. A multitude of pathways seems to be involved in the aetiology and/or pathogenesis of schizophrenia, including dopaminergic, serotoninergic, muscarinic and glutamatergic signalling. The phosphoinositide signal transduction system and related phosphoinositide-specific phospholipase C (PI-PLC) enzymes seem to represent a point of convergence in these networking pathways during the development of selected brain regions. The existence of a susceptibility locus on the short arm of chromosome 20 moved us to analyse PLCB1, the gene codifying for PI-PLC β1 enzyme, which maps on 20p12. By using interphase fluorescent in situ hybridization methodology, we found deletions of PLCB1 in orbito-frontal cortex samples of schizophrenia-affected patients.

  8. [THE EFFECT OF waaL LIGASE GENES DELETION ON MOTILITY AND STRESS ADAPTATION REACTIONS OF YERSINIA ENTEROCOLITICA 6471/76].

    Science.gov (United States)

    Shevchenko, J I; Shilina, J V; Pozur, V K; Skurnik, M

    2015-01-01

    The aim of current study was to estimate the influence of waaL(OS) and waaL(PS) genes deletion on lipopolysaccharide (LPS) synthesis, bacterial motility and stress resistance of bacteria Yersinia enterocolitica 6471/76. Single and double waaL mutants were created by replacing the wild-type alleles in bacterial chromosome for mutant ones. The phenotypes of mutants were visualized by DOC-PAGE gels stained with silver and immunoblot with specific to O-polysaccharide and outer core monoclonal antibodies. Bacterial motility was evaluated by the diameter of the migration zone. Wild type bacteria and mutants were analyzed by bacterial growth curves in a hypertonic medium. Participation of WaaL ligases in resistance to osmotic pressure was found only in case of both ligese genes deletion. Also the YeO3_os_ps mutants showed motility decreasing, which recovered after adding a functionally active gene. Thus, deletion of both waaL ligase genes lead to a drastic reduction in bacterial motility and increase their sensitivity to hypertonic medium that can indirectly characterize biological role of WaaL ligases.

  9. Rice mutant resources for gene discovery

    NARCIS (Netherlands)

    Hirochika, H.; Guiderdoni, E.; An, G.; Hsing, Y.I.; Eun, M.Y.; Han, C.D.; Upadhyaya, N.; Ramachandran, S.; Zhang, Q.F.; Pereira, A.B.; Sundaresan, V.; Leung, H.

    2004-01-01

    With the completion of genomic sequencing of rice, rice has been firmly established as a model organism for both basic and applied research. The next challenge is to uncover the functions of genes predicted by sequence analysis. Considering the amount of effort and the diversity of disciplines requi

  10. Contribution of Scaffoldins to Biomass Degradation by Clostridium Thermocellum: The Effect of Scaffoldin-Deletions on Expression of Other Genes

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Qi; Podkaminer, Kara; Resch, Michael G.; Donohoe, Bryon; Olson, Daniel G.; Baker, John O.; Klingeman, Dawn M.; Syed, Mustafa; Wilson, Charlotte M.; Brown, Steven D.; Yang, Shihui; Magnusson, Lauren; Maness, Pin-Ching; Decker, Steve R.; Lynd, Lee R.; Bomble, Yannick J.; Himmel, Michael E.

    2014-04-28

    The cellulosome system contributes greatly to the extreme efficiency of C. thermocellum cellulose degradation. In order to further understand the cellulosome working mechanism, we have knocked out C. thermocellum scaffoldin genes to generate a variety of deletion mutants. The knockout most detrimental to enzymatic hydrolysis by the secretome is that of the primary scaffoldin CipA. Deletion of multiple secondary scaffoldins results in secretome activities intermediate between those of the parent strain and the CipA-knockout mutants. The order of relative secretome activities is the same, whether the cellulosic substrate is microcrystalline cellulose (Avicel) or deacetylated acid-pretreated corn stover (DACS), but the relative magnitudes of the deletion effects are strongly substrate-dependent. Similar trends are observed in fermentation studies of the abilities of the parent and knockout strains themselves to utilize Avicel and DACS. Data from transcriptomic and proteomic studies of these strains when grown on both substrates are used to relate the activity and growth effects of the deletions to their effects on the overall expression of lignocellulose-degrading enzymes by C. thermocellum.

  11. Metabolic flux balance analysis and the in silico analysis of Escherichia coli K-12 gene deletions

    Directory of Open Access Journals (Sweden)

    Edwards Jeremy S

    2000-07-01

    Full Text Available Abstract Background Genome sequencing and bioinformatics are producing detailed lists of the molecular components contained in many prokaryotic organisms. From this 'parts catalogue' of a microbial cell, in silico representations of integrated metabolic functions can be constructed and analyzed using flux balance analysis (FBA. FBA is particularly well-suited to study metabolic networks based on genomic, biochemical, and strain specific information. Results Herein, we have utilized FBA to interpret and analyze the metabolic capabilities of Escherichia coli. We have computationally mapped the metabolic capabilities of E. coli using FBA and examined the optimal utilization of the E. coli metabolic pathways as a function of environmental variables. We have used an in silico analysis to identify seven gene products of central metabolism (glycolysis, pentose phosphate pathway, TCA cycle, electron transport system essential for aerobic growth of E. coli on glucose minimal media, and 15 gene products essential for anaerobic growth on glucose minimal media. The in silico tpi-, zwf, and pta- mutant strains were examined in more detail by mapping the capabilities of these in silico isogenic strains. Conclusions We found that computational models of E. coli metabolism based on physicochemical constraints can be used to interpret mutant behavior. These in silica results lead to a further understanding of the complex genotype-phenotype relation. Supplementary information: http://gcrg.ucsd.edu/supplementary_data/DeletionAnalysis/main.htm

  12. 肠炎沙门氏菌鸡源株ompR基因缺失株的构建及生物学特性与亲本株的比较%Construction and characterization of an ompR gene deletion mutant from Salmonella enteritidis

    Institute of Scientific and Technical Information of China (English)

    董洪燕; 彭大新; 焦新安; 张小荣; 陈素娟; 卢艳; 耿士忠; 刘秀梵

    2011-01-01

    [目的]为了探讨ompR基因在肠炎沙门氏菌生物被膜形成及毒力中的作用.[方法]以肠炎沙门氏菌作为母本,运用自杀性载体pGMB151构建了ompR基因缺失株,结晶紫染色法和扫描电镜观察测定缺失株的生物被膜形成能力,细胞的吸附和侵入及小鼠攻毒试验测定缺失株的毒力.[结果]RT-PCR和蛋白表达证明了ompR基因缺失株构建成功;该缺失株不表达纤维素和菌毛,不形成生物被膜;上皮细胞吸附和侵入试验表明缺失株与野生株具有相同的吸附和侵入率;BALB/c鼠腹腔感染性试验表明,缺失株的半数致死量为10 667CFU,而野生株的半数致死量小于2 CFU.[结论]ompR基因既是肠炎沙门氏菌生物膜形成的调控基因,又是重要的毒力基因.%[Objective] To investigate the role of ompR gene from Salmonella enteritidis in biofilm formation and virulence. [Methods] We constructed an ompR mutant of Salmonella enteritidis by suicide plasmid pCMBlSl. Biofilm forming ability of the mutant was detected by crystal violet assay and scanning electron micrography. Virulence of the mutant was determined by assay of adherence to and invasion of epithelial cells, and mouse challenge experiments. [Results] The ompR mutant was confirmed by RT-PCR and the pattern of outer membrane protein. The mutant did not produce cellulose, curli, and biofilm, and showed similar adherence percentage to and invasion percentage of epithelial cells as wild type strain. In addition, intraperitoneal challenge of bacteria in BALB/c mice revealed that LD50 of the mutant strain was 106.67 CFU, while that of the wild type strain was less than 2 CFU. [ Conclusion] These data indicate that the ompR gene is involved in both biofilm formation and virulence in Salmonella enteritidis.

  13. Pathogenicity and protective activity in pregnant goats of a Brucella melitensis Deltaomp25 deletion mutant.

    Science.gov (United States)

    Edmonds, M D; Cloeckaert, A; Hagius, S D; Samartino, L E; Fulton, W T; Walker, J V; Enright, F M; Booth, N J; Elzer, P H

    2002-06-01

    The Brucella melitensis mutant BM 25, which lacks the major 25 kDa outer membrane protein Omp25, has previously been found to be attenuated in the murine brucellosis model. In the present study, the capacity of the Deltaomp25 mutant to colonise and cause abortions in the caprine host was evaluated. The vaccine potential of BM 25 was also investigated in goats. Inoculation of nine pregnant goats in late gestation with the B. melitensis mutant resulted in 0/9 abortions, while the virulent parental strain, B. melitensis 16M, induced 6/6 dams to abort (Pgoats for two weeks post-infection. Owing to the ability of BM 25 to colonise both non-pregnant and pregnant adults without inducing abortions, a vaccine efficacy study was performed. Vaccination of goats prior to breeding with either BM 25 or the current caprine vaccine B. melitensis strain Rev. 1 resulted in 100 per cent protection against abortion following challenge in late gestation with virulent strain 16M (Pmelitensis Deltaomp25 mutant, BM 25, may be a safe and efficacious alternative to strain Rev. 1 when dealing with goat herds of mixed age and pregnancy status.

  14. Deletion of Osr2 Partially Rescues Tooth Development in Runx2 Mutant Mice

    Science.gov (United States)

    Kwon, H.J.E.; Park, E.K.; Jia, S.; Liu, H.; Lan, Y.

    2015-01-01

    Tooth organogenesis depends on genetically programmed sequential and reciprocal inductive interactions between the dental epithelium and neural crest–derived mesenchyme. Previous studies showed that the Msx1 and Runx2 transcription factors are required for activation of odontogenic signals, including Bmp4 and Fgf3, in the early tooth mesenchyme to drive tooth morphogenesis through the bud-to-cap transition and that Runx2 acts downstream of Msx1 to activate Fgf3 expression. Recent studies identified Osr2 as a repressor of tooth development and showed that inactivation of Osr2 rescued molar tooth morphogenesis in the Msx1-/- mutant mice as well as in mice with neural crest–specific inactivation of Bmp4. Here we show that Runx2 expression is expanded in the tooth bud mesenchyme in Osr2-/- mutant mouse embryos and is partially restored in the tooth mesenchyme in Msx1-/-Osr2-/- mutants in comparison with Msx1-/- and wild-type embryos. Whereas mandibular molar development arrested at the bud stage and maxillary molar development arrested at the bud-to-cap transition in Runx2-/- mutant mice, both mandibular and maxillary molar tooth germs progressed to the early bell stage, with rescued expression of Msx1 and Bmp4 in the dental papilla as well as expression of Bmp4, p21, and Shh in the primary enamel knot in the Osr2-/-Runx2-/- compound mutants. In contrast to the Msx1-/-Osr2-/- compound mutants, which exhibit nearly normal first molar morphogenesis, the Osr2-/-Runx2-/- compound mutant embryos failed to activate the expression of Fgf3 and Fgf10 in the dental papilla and exhibited significant deficit in cell proliferation in both the dental epithelium and mesenchyme in comparison with the control embryos. These data indicate that Runx2 synergizes with Msx1 to drive tooth morphogenesis through the bud-to-cap transition and that Runx2 controls continued tooth growth and morphogenesis beyond the cap stage through activation of Fgf3 and Fgf10 expression in the dental

  15. Deletion of Osr2 Partially Rescues Tooth Development in Runx2 Mutant Mice.

    Science.gov (United States)

    Kwon, H J E; Park, E K; Jia, S; Liu, H; Lan, Y; Jiang, R

    2015-08-01

    Tooth organogenesis depends on genetically programmed sequential and reciprocal inductive interactions between the dental epithelium and neural crest-derived mesenchyme. Previous studies showed that the Msx1 and Runx2 transcription factors are required for activation of odontogenic signals, including Bmp4 and Fgf3, in the early tooth mesenchyme to drive tooth morphogenesis through the bud-to-cap transition and that Runx2 acts downstream of Msx1 to activate Fgf3 expression. Recent studies identified Osr2 as a repressor of tooth development and showed that inactivation of Osr2 rescued molar tooth morphogenesis in the Msx1(-/-) mutant mice as well as in mice with neural crest-specific inactivation of Bmp4. Here we show that Runx2 expression is expanded in the tooth bud mesenchyme in Osr2(-/-) mutant mouse embryos and is partially restored in the tooth mesenchyme in Msx1(-/-)Osr2(-/-) mutants in comparison with Msx1(-/-) and wild-type embryos. Whereas mandibular molar development arrested at the bud stage and maxillary molar development arrested at the bud-to-cap transition in Runx2(-/-) mutant mice, both mandibular and maxillary molar tooth germs progressed to the early bell stage, with rescued expression of Msx1 and Bmp4 in the dental papilla as well as expression of Bmp4, p21, and Shh in the primary enamel knot in the Osr2(-/-)Runx2(-/-) compound mutants. In contrast to the Msx1(-/-)Osr2(-/-) compound mutants, which exhibit nearly normal first molar morphogenesis, the Osr2(-/-)Runx2(-/-) compound mutant embryos failed to activate the expression of Fgf3 and Fgf10 in the dental papilla and exhibited significant deficit in cell proliferation in both the dental epithelium and mesenchyme in comparison with the control embryos. These data indicate that Runx2 synergizes with Msx1 to drive tooth morphogenesis through the bud-to-cap transition and that Runx2 controls continued tooth growth and morphogenesis beyond the cap stage through activation of Fgf3 and Fgf10 expression

  16. [From gene to disease; deletion of the DAZ-gene from the Y-chromosome in oligo- or azoospermia

    NARCIS (Netherlands)

    Tuerlings, J.H.A.M.; Hoefsloot, L.H.; Kremer, J.A.M.

    2001-01-01

    DAZ gene deletions at the azoospermia factor (AZF) locus on the Y chromosome, have been implicated as one of the major causes of idiopathic male subfertility. Deletions of the entire DAZ gene have been reported in azoospermia as well as in oligozoospermia. The DAZ gene encodes a RNA binding protein

  17. Hepatitis B virus S gene escape mutants

    Directory of Open Access Journals (Sweden)

    Purdy Michael

    2007-01-01

    Full Text Available Hepatitis B virus (HBV can be classified into nine immunological subtypes or eight genotypes. The most prevalent genotypes in Asia are genotypes B and C. HBV is transmitted parenteraly and can produce either asymptomatic or symptomatic disease. Although the consequences of acute hepatitis B can be severe, serious sequelae are associated with chronic infections. HBV seroprevalence ranges from intermediate (2%-7% to high (≥8% levels in Asia. Several strategies for the control and prevention of HBV infection have been found to be efficacious. They include vaccination and the administration of HBIG, interferon-a and nucleoside/nucleotide analogues. However, these procedures also apply selective pressures on HBV in infected individuals leading to the generation and accumulation of mutations in the S gene. Most of these mutations occur in the major hydrophilic region (MHR of the S gene. These mutations create public health concerns as they can be responsible for reactivation of hepatitis B and occult hepatitis B infection. The inability to detect occult infections means that these individuals may become blood donors. This suggests that new strategies for donor evaluation and selection may need to be developed to protect the blood supply.

  18. Glucocerebrosidase 2 gene deletion rescues type 1 Gaucher disease

    Science.gov (United States)

    Mistry, Pramod K.; Liu, Jun; Sun, Li; Chuang, Wei-Lien; Yuen, Tony; Yang, Ruhua; Lu, Ping; Zhang, Kate; Li, Jianhua; Keutzer, Joan; Stachnik, Agnes; Mennone, Albert; Boyer, James L.; Jain, Dhanpat; Brady, Roscoe O.; New, Maria I.; Zaidi, Mone

    2014-01-01

    The inherited deficiency of the lysosomal glucocerebrosidase (GBA) due to mutations in the GBA gene results in Gaucher disease (GD). A vast majority of patients present with nonneuronopathic, type 1 GD (GD1). GBA deficiency causes the accumulation of two key sphingolipids, glucosylceramide (GL-1) and glucosylsphingosine (LysoGL-1), classically noted within the lysosomes of mononuclear phagocytes. How metabolites of GL-1 or LysoGL-1 produced by extralysosomal glucocerebrosidase GBA2 contribute to the GD1 pathophysiology is not known. We recently recapitulated hepatosplenomegaly, cytopenia, hypercytokinemia, and the bone-formation defect of human GD1 through conditional deletion of Gba in Mx1–Cre+:GD1 mice. Here we show that the deletion of Gba2 significantly rescues the GD1 clinical phenotype, despite enhanced elevations in GL-1 and LysoGL-1. Most notably, the reduced bone volume and bone formation rate are normalized. These results suggest that metabolism of GL-1 or LysoGL-1 into downstream bioactive lipids is a major contributor to the bone-formation defect. Direct testing revealed a strong inhibition of osteoblast viability by nanomolar concentrations of sphingosine, but not of ceramide. These findings are consistent with toxicity of high circulating sphingosine levels in GD1 patients, which decline upon enzyme-replacement therapy; serum ceramide levels remain unchanged. Together, complementary results from mice and humans affected with GD1 not only pinpoint sphingosine as being an osteoblast toxin, but also set forth Gba2 as a viable therapeutic target for the development of inhibitors to ameliorate certain disabling consequences of GD1. PMID:24639522

  19. Effects of the deletion and over-expression of Fusarium graminearum gene FgHal2 on host response to mycovirus Fusarium graminearum virus 1.

    Science.gov (United States)

    Yu, Jisuk; Lee, Kyung-Mi; Son, Moonil; Kim, Kook-Hyung

    2015-09-01

    The mycovirus Fusarium graminearum virus 1 (FgV1) is associated with reduced virulence (hypovirulence) of Fusarium graminearum. Transcriptomic and proteomic expression profiling have shown that many F. graminearum genes are differentially expressed as a consequence of FgV1 infection. Several of these genes may be related to the maintenance of the virus life cycle. The host gene, FgHal2, which has a highly conserved 3'-phosphoadenosine 5'-phosphatase (PAP phosphatase-like) domain or inositol monophosphatase (IMPase) superfamily domain, shows reduced expression in response to FgV1 infection. We generated targeted gene deletion and over-expression mutants to clarify the possible function(s) of FgHal2 and its relationship to FgV1. The gene deletion mutant showed retarded growth, reduced aerial mycelia formation and reduced pigmentation, whereas over-expression mutants were morphologically similar to the wild-type (WT). Furthermore, compared with the WT, the gene deletion mutant produced fewer conidia and these showed abnormal morphology. The FgHal2 expression level was decreased by FgV1 infection at 120 h post-inoculation (hpi), whereas the levels were nine-fold greater for both the virus-free and virus-infected over-expression mutant than for the WT. FgV1 RNA accumulation was decreased in the deletion mutant at 48, 72 and 120 hpi. FgV1 RNA accumulation in the over-expression mutant was reduced relative to that of the WT at 48 and 120 hpi, but was similar to that of the WT at 72 hpi. The vertical transmission rate of FgV1 in the gene deletion mutant was low, suggesting that FgHal2 may be required for the maintenance of FgV1 in the host cell. Together, these results indicate that the putative 3'(2'),5'-bisphosphate nucleotidase gene, FgHal2, has diverse biological functions in the host fungus and may affect the viral RNA accumulation and transmission of FgV1.

  20. Use of the meganuclease I-SceI of Saccharomyces cerevisiae to select for gene deletions in actinomycetes.

    Science.gov (United States)

    Fernández-Martínez, Lorena T; Bibb, Mervyn J

    2014-11-18

    The search for new natural products is leading to the isolation of novel actinomycete species, many of which will ultimately require genetic analysis. Some of these isolates will likely exhibit low intrinsic frequencies of homologous recombination and fail to sporulate under laboratory conditions, exacerbating the construction of targeted gene deletions and replacements in genetically uncharacterised strains. To facilitate the genetic manipulation of such species, we have developed an efficient method to generate gene or gene cluster deletions in actinomycetes by homologous recombination that does not introduce any other changes to the targeted organism's genome. We have synthesised a codon optimised I-SceI gene for expression in actinomycetes that results in the production of the yeast I-SceI homing endonuclease which produces double strand breaks at a unique introduced 18 base pair recognition sequence. Only those genomes that undergo homologous recombination survive, providing a powerful selection for recombinants, approximately half of which possess the desired mutant genotype. To demonstrate the efficacy and efficiency of the system, we deleted part of the gene cluster for the red-pigmented undecylprodiginine complex of compounds in Streptomyces coelicolor M1141. We believe that the system we have developed will be broadly applicable across a wide range of actinomycetes.

  1. Susceptibility genes for schizophrenia: mutant models, endophenotypes and psychobiology.

    Science.gov (United States)

    O'Tuathaigh, Colm M P; Desbonnet, Lieve; Moran, Paula M; Waddington, John L

    2012-01-01

    Schizophrenia is characterised by a multifactorial aetiology that involves genetic liability interacting with epigenetic and environmental factors to increase risk for developing the disorder. A consensus view is that the genetic component involves several common risk alleles of small effect and/or rare but penetrant copy number variations. Furthermore, there is increasing evidence for broader, overlapping genetic-phenotypic relationships in psychosis; for example, the same susceptibility genes also confer risk for bipolar disorder. Phenotypic characterisation of genetic models of candidate risk genes and/or putative pathophysiological processes implicated in schizophrenia, as well as examination of epidemiologically relevant gene × environment interactions in these models, can illuminate molecular and pathobiological mechanisms involved in schizophrenia. The present chapter outlines both the evidence from phenotypic studies in mutant mouse models related to schizophrenia and recently described mutant models addressing such gene × environment interactions. Emphasis is placed on evaluating the extent to which mutant phenotypes recapitulate the totality of the disease phenotype or model selective endophenotypes. We also discuss new developments and trends in relation to the functional genomics of psychosis which might help to inform on the construct validity of mutant models of schizophrenia and highlight methodological challenges in phenotypic evaluation that relate to such models.

  2. Molecular genetic analyses of human NKG2C (KLRC2) gene deletion

    NARCIS (Netherlands)

    Miyashita, R; Tsuchiya, N; Hikami, K; Kuroki, K; Fukazawa, T; Bijl, M; Kallenberg, CGM; Hashimoto, H; Yabe, T; Tokunaga, K

    Human NKG2A, NKG2C and NKG2E genes are located on 12p13 in the NK gene complex. We recently identified deletion of NKG2C in a Japanese population. This study was performed to identify the breakpoint, and to examine the association of NKG2C deletion with susceptibility to rheumatoid arthritis and

  3. The rates and patterns of deletions in the human factor IX gene

    Energy Technology Data Exchange (ETDEWEB)

    Ketterling, R.P.; Vielhaber, E.L.; Lind, T.J.; Thorland, E.C.; Sommer S.S. (Mayo Clinic/Foundation, Rochester, MN (United States))

    1994-02-01

    Deletions are commonly observed in genes with either segments of highly homologous sequences or excessive gene length. However, in the factor IX gene and in most genes, deletions (of [ge]21 bp) are uncommon. The authors have analyzed DNA from 290 families with hemophilia B (203 independent mutations) and have found 12 deletions >20 bp. Eleven of these are >2 kb (range >3-163 kb), and one is 1.1 kb. The junctions of the four deletions that are completely contained within the factor IX gene have been determined. A novel mutation occurred in patient HB128: the data suggest that a 26.8-kb deletion occurred between two segments of alternating purines and pyrimidines and that a 2.3-kb sense strand segment derived from the deleted region was inserted. For a sample of 203 independent mutations, the authors estimate the [open quotes]baseline[close quotes] rates of deletional mutation per base pair per generation as a function of size. The rate for large (>2 kb)I deletions is exceedingly low. For every mutational event in which a given base is at the junction of a large deletion, there are an estimated 58 microdeletions (<20 bp) and 985 single-base substitutions at that base. Analysis of the nine reported deletion junctions in the factor IX gene literature reveals that (i) five are associated with inversion, orphan sequences, or sense strand insertions; (ii) four are simple deletions that display an excess of short direct repeats at their junctions; (iii) there is no dramatic clustering of junctions within the gene; and (iv) with the exception of alternating purines and pyrimidines, deletion junctions are not preferentially associated with repetitive DNA. 58 refs., 5 figs., 5 tabs.

  4. Systematic gene deletions evidences that laccases are involved in several stages of wood degradation in the filamentous fungus Podospora anserina.

    Science.gov (United States)

    Xie, Ning; Chapeland-Leclerc, Florence; Silar, Philippe; Ruprich-Robert, Gwenaël

    2014-01-01

    Transformation of plant biomass into biofuels may supply environmentally friendly alternative biological sources of energy. Laccases are supposed to be involved in the lysis of lignin, a prerequisite step for efficient breakdown of cellulose into fermentable sugars. The role in development and plant biomass degradation of the nine canonical laccases belonging to three different subfamilies and one related multicopper oxidase of the Ascomycota fungus Podospora anserina was investigated by targeted gene deletion. The 10 genes were inactivated singly, and multiple mutants were constructed by genetic crosses. lac6(Δ), lac8(Δ) and mco(Δ) mutants were significantly reduced in their ability to grow on lignin-containing materials, but also on cellulose and plastic. Furthermore, lac8(Δ), lac7(Δ), mco(Δ) and lac6(Δ) mutants were defective towards resistance to phenolic substrates and H2 O2 , which may also impact lignocellulose breakdown. Double and multiple mutants were generally more affected than single mutants, evidencing redundancy of function among laccases. Our study provides the first genetic evidences that laccases are major actors of wood utilization in a fungus and that they have multiple roles during this process apart from participation in lignin lysis.

  5. Evaluation of novel Brucella melitensis unmarked deletion mutants for safety and efficacy in the goat model of brucellosis.

    Science.gov (United States)

    Kahl-McDonagh, Melissa M; Elzer, Philip H; Hagius, Sue D; Walker, Joel V; Perry, Quinesha L; Seabury, Christopher M; den Hartigh, Andreas B; Tsolis, Renee M; Adams, L Garry; Davis, Donald S; Ficht, Thomas A

    2006-06-12

    Pregnant goats were employed to assess unmarked deletion mutant vaccine candidates BMDeltaasp24, BMDeltacydBA, and BMDeltavirB2, as the target host species naturally infected with Brucella melitensis. Goats were assessed for the degree of pathology associated with the vaccine strains as well as the protective immunity afforded by each strain against abortion and infection after challenge with wild-type Brucella melitensis 16M. Both BMDeltaasp24 and BMDeltavirB2 were considered safe vaccine candidates in the pregnant goat model because they did not cause abortion or colonize fetal tissues. BMDeltaasp24 was isolated from the maternal tissues only, indicating a slower rate of clearance of the vaccine strain than for BMDeltavirB2, which was not isolated from any maternal or fetal tissues. Both strains were protective against abortion and against infection in the majority of pregnant goats, although BMDeltaasp24 was more efficacious than BMDeltavirB2 against challenge infection.

  6. Transcriptome analysis of acetic-acid-treated yeast cells identifies a large set of genes whose overexpression or deletion enhances acetic acid tolerance.

    Science.gov (United States)

    Lee, Yeji; Nasution, Olviyani; Choi, Eunyong; Choi, In-Geol; Kim, Wankee; Choi, Wonja

    2015-08-01

    Acetic acid inhibits the metabolic activities of Saccharomyces cerevisiae. Therefore, a better understanding of how S. cerevisiae cells acquire the tolerance to acetic acid is of importance to develop robust yeast strains to be used in industry. To do this, we examined the transcriptional changes that occur at 12 h post-exposure to acetic acid, revealing that 56 and 58 genes were upregulated and downregulated, respectively. Functional categorization of them revealed that 22 protein synthesis genes and 14 stress response genes constituted the largest portion of the upregulated and downregulated genes, respectively. To evaluate the association of the regulated genes with acetic acid tolerance, 3 upregulated genes (DBP2, ASC1, and GND1) were selected among 34 non-protein synthesis genes, and 54 viable mutants individually deleted for the downregulated genes were retrieved from the non-essential haploid deletion library. Strains overexpressing ASC1 and GND1 displayed enhanced tolerance to acetic acid, whereas a strain overexpressing DBP2 was sensitive. Fifty of 54 deletion mutants displayed enhanced acetic acid tolerance. Three chosen deletion mutants (hsps82Δ, ato2Δ, and ssa3Δ) were also tolerant to benzoic acid but not propionic and sorbic acids. Moreover, all those five (two overexpressing and three deleted) strains were more efficient in proton efflux and lower in membrane permeability and internal hydrogen peroxide content than controls. Individually or in combination, those physiological changes are likely to contribute at least in part to enhanced acetic acid tolerance. Overall, information of our transcriptional profile was very useful to identify molecular factors associated with acetic acid tolerance.

  7. Deletion of degQ gene enhances outer membrane vesicle production of Shewanella oneidensis cells.

    Science.gov (United States)

    Ojima, Yoshihiro; Mohanadas, Thivagaran; Kitamura, Kosei; Nunogami, Shota; Yajima, Reiki; Taya, Masahito

    2017-04-01

    Shewanella oneidensis is a Gram-negative facultative anaerobe that can use a wide variety of terminal electron acceptors for anaerobic respiration. In this study, S. oneidensis degQ gene, encoding a putative periplasmic serine protease, was cloned and expressed. The activity of purified DegQ was inhibited by diisopropyl fluorophosphate, a typical serine protease-specific inhibitor, indicating that DegQ is a serine protease. In-frame deletion and subsequent complementation of the degQ were carried out to examine the effect of envelope stress on the production of outer membrane vesicles (OMVs). Analysis of periplasmic proteins from the resulting S. oneidensis strain showed that deletion of degQ induced protein accumulation and resulted in a significant decrease in protease activity within the periplasmic space. OMVs from the wild-type and mutant strains were purified and observed by transmission electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the OMVs showed a prominent band at ~37 kDa. Nanoliquid chromatography-tandem mass spectrometry analysis identified three outer membrane porins (SO3896, SO1821, and SO3545) as dominant components of the band, suggesting that these proteins could be used as indices for comparing OMV production by S. oneidensis strains. Quantitative evaluation showed that degQ-deficient cells had a fivefold increase in OMV production compared with wild-type cells. Thus, the increased OMV production following the deletion of DegQ in S. oneidensis may be responsible for the increase in envelope stress.

  8. Identification by gene deletion analysis of a regulator, VmsR, that controls virginiamycin biosynthesis in Streptomyces virginiae.

    Science.gov (United States)

    Kawachi, R; Wangchaisoonthorn, U; Nihira, T; Yamada, Y

    2000-11-01

    Virginiae butanolide (VB)-BarA of Streptomyces virginiae is one of the newly discovered pairs of a butyrolactone autoregulator and a corresponding receptor protein of Streptomyces species and regulates the production of the antibiotic virginiamycin (VM) in S. virginiae. The gene vmsR was found to be situated 4.7 kbp upstream of the barA gene, which encodes the VB-specific receptor. The vmsR product was predicted to be a regulator of VM biosynthesis based on its high homology to some Streptomyces pathway-specific transcriptional regulators for the biosynthetic gene clusters of polyketide antibiotics, such as Streptomyces peucetius DnrI (47.5% identity, 84. 3% similarity), which controls daunorubicin biosynthesis. A vmsR deletion mutant was created by homologous recombination. Neither virginiamycin M(1) nor virginiamycin S was produced in the vmsR mutant, while amounts of VB and BarA similar to those produced in the wild-type strain were detected. Reverse transcription-PCR analyses confirmed that the vmsR deletion had no deleterious effects on the transcription of the vmsR-surrounding genes, indicating that VmsR is a positive regulator of VM biosynthesis in S. virginiae.

  9. Emergence of porcine reproductive and respiratory syndrome virus deletion mutants: Correlation with the porcine antibody response to a hypervariable site in the ORF 3 structural glycoprotein

    DEFF Research Database (Denmark)

    Oleksiewicz, M.B.; Bøtner, Anette; Toft, P.;

    2000-01-01

    By using porcine immune sera to select a library of phage-displayed random peptides. we identified an antigenic sequence (RKASLSTS) in the C-terminus of the ORF 3 structural glycoprotein of European-type porcine reproductive and respiratory syndrome virus (PRRSV). Through the use of overlapping....... These distinctions suggested that deletion mutants were a hitherto unrecognized subtype of European-type PRRSV. Currently, deletion mutants appear to be outcompeting nondeleted viruses in the field, highlighting the importance of the porcine antibody response against the minor structural glycoproteins of European...

  10. Bridging the Gene-Behavior Divide through Neuroimaging Deletion Syndromes: Velocardiofacial (22q11.2 Deletion) and Williams (7q11.23 Deletion) Syndromes

    Science.gov (United States)

    Eisenberg, Daniel Paul; Jabbi, Mbemba; Berman, Karen Faith

    2010-01-01

    Investigating the relationship between genes and the neural substrates of complex human behavior promises to provide essential insight into the pathophysiology of mental disorders. One approach to this inquiry is through neuroimaging of individuals with microdeletion syndromes that manifest in specific neuropsychiatric phenotypes. Both Velocardiofacial Syndrome (VCFS) and Williams Syndrome (WS) involve haploinsufficiency of a relatively small set of identified genes on the one hand and association with distinct, clinically-relevant behavioral and cognitive profiles on the other hand. In VCFS, there is a deletion in chromosomal region 22q11.2 and a resultant predilection toward psychosis, poor arithmetic proficiency, and low performance intelligence quotients. In WS, there is a deletion in chromosomal region 7q11.23 and a resultant predilection toward hypersociability, non-social anxiety, impaired visuospatial construction, and often intellectual impairment. Structural and functional neuroimaging studies have begun not only to map these well-defined genetic alterations to systems-level brain abnormalities, but also to identify relationships between neural phenotypes and particular genes within the critical deletion regions. Though neuroimaging of both VCFS and WS presents specific, formidable methodological challenges, including comparison subject selection and accounting for neuroanatomical and vascular anomalies in patients, and many questions remain, the literature to date on these syndromes, reviewed herein, constitutes a fruitful “bottom-up” approach to defining gene-brain relationships. PMID:20206275

  11. Genome-wide screening for genes whose deletions confer sensitivity to mutagenic purine base analogs in yeast

    Directory of Open Access Journals (Sweden)

    Kozmin Stanislav G

    2005-06-01

    Full Text Available Abstract Background N-hydroxylated base analogs, such as 6-hydroxylaminopurine (HAP and 2-amino-6-hydroxylaminopurine (AHA, are strong mutagens in various organisms due to their ambiguous base-pairing properties. The systems protecting cells from HAP and related noncanonical purines in Escherichia coli include specialized deoxyribonucleoside triphosphatase RdgB, DNA repair endonuclease V, and a molybdenum cofactor-dependent system. Fewer HAP-detoxification systems have been identified in yeast Saccharomyces cerevisiae and other eukaryotes. Cellular systems protecting from AHA are unknown. In the present study, we performed a genome-wide search for genes whose deletions confer sensitivity to HAP and AHA in yeast. Results We screened the library of yeast deletion mutants for sensitivity to the toxic and mutagenic action of HAP and AHA. We identified novel genes involved in the genetic control of base analogs sensitivity, including genes controlling purine metabolism, cytoskeleton organization, and amino acid metabolism. Conclusion We developed a method for screening the yeast deletion library for sensitivity to the mutagenic and toxic action of base analogs and identified 16 novel genes controlling pathways of protection from HAP. Three of them also protect from AHA.

  12. Deletion of Cg-emb in corynebacterianeae leads to a novel truncated cell wall arabinogalactan, whereas inactivation of Cg-ubiA results in an arabinan-deficient mutant with a cell wall galactan core.

    Science.gov (United States)

    Alderwick, Luke J; Radmacher, Eva; Seidel, Mathias; Gande, Roland; Hitchen, Paul G; Morris, Howard R; Dell, Anne; Sahm, Hermann; Eggeling, Lothar; Besra, Gurdyal S

    2005-09-16

    The cell wall of Mycobacterium tuberculosis has a complex ultrastructure that consists of mycolic acids connected to peptidoglycan via arabinogalactan (AG) and abbreviated as the mAGP complex. The mAGP complex is crucial for the survival and pathogenicity of M. tuberculosis and is the target of several anti-tubercular agents. Apart from sharing a similar mAGP and the availability of the complete genome sequence, Corynebacterium glutamicum has proven useful in the study of orthologous M. tuberculosis genes essential for viability. Here we examined the effects of particular genes involved in AG polymerization by gene deletion in C. glutamicum. The anti-tuberculosis drug ethambutol is thought to target a set of arabinofuranosyltransferases (Emb) that are involved in arabinan polymerization. Deletion of emb in C. glutamicum results in a slow growing mutant with profound morphological changes. Chemical analysis revealed a dramatic reduction of arabinose resulting in a novel truncated AG structure possessing only terminal arabinofuranoside (t-Araf) residues with a corresponding loss of cell wall bound mycolic acids. Treatment of wild-type C. glutamicum with ethambutol and subsequent cell wall analyses resulted in an identical phenotype comparable to the C. glutamicum emb deletion mutant. Additionally, disruption of ubiA in C. glutamicum, the first enzyme involved in the biosynthesis of the sugar donor decaprenol phosphoarabinose (DPA), resulted in a complete loss of cell wall arabinan. Herein, we establish for the first time, (i) that in contrast to M. tuberculosis embA and embB mutants, deletion of C. glutamicum emb leads to a highly truncated AG possessing t-Araf residues, (ii) the exact site of attachment of arabinan chains in AG, and (iii) DPA is the only Araf sugar donor in AG biosynthesis suggesting the presence of a novel enzyme responsible for "priming" the galactan domain for further elaboration by Emb, resulting in the final maturation of the native AG

  13. Analysis of dystrophin gene deletions by multiplex PCR in eastern India

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    Basak Jayasri

    2006-01-01

    Full Text Available The most common genetic neuromuscular disease of childhood, Duchenne and Becker muscular dystrophy (DMD/BMD is caused by deletion, duplication or point mutation of the dystrophin gene located at Xp 21.2. In the present study DNA from seventy unrelated patients clinically diagnosed as having DMD/BMD referred from different parts of West Bengal, a few other states and Bangladesh are analyzed using the multiplex polymerase chain reaction (m-PCR to screen for exon deletions and its distribution within the dystrophin gene. Out of seventy patients forty six (63% showed large intragenic deletion in the dystrophin gene. About 79% of these deletions are located in the hot spot region i.e., between exon 42 to 53. This is the first report of frequency and distribution of deletion in dystrophin gene in eastern Indian DMD/BMD population.

  14. The capacity of Listeria monocytogenes mutants with in-frame deletions in putative ATP-binding cassette transporters to form biofilms and comparison with the wild type

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    Marina Ceruso

    2014-02-01

    Full Text Available Listeria monocytogenes (Lm is a food-borne pathogen responsible for human listeriosis, an invasive infection with high mortality rates. Lm has developed efficient strategies for survival under stress conditions such as starvation and wide variations in temperature, pH, and osmolarity. Therefore, Lm can survive in food under multiple stress conditions. Detailed studies to determine the mode of action of this pathogen for survival under stress conditions are important to control Lm in food. It has been shown that genes encoding for ATP-binding cassette (ABC transporters are induced in Lm in food, in particular under stress conditions. Previous studies showed that these genes are involved in sensitivity to nisin, acids, and salt. The aim of this study was to determine the involvement of some ABC transporters in biofilm formation. Therefore, deletion mutants of ABC transporter genes (LMOf2365_1875 and LMOf2365_1877 were created in Lm F2365, and then were compared to the wild type for their capacity to form biofilms. Lm strain F2365 was chosen as reference since the genome is fully sequenced and furthermore this strain is particularly involved in food-borne outbreaks of listeriosis. Our results showed that DLMOf2365_1875 had an increased capacity to form biofilms compared to the wild type, indicating that LMOf2365_1875 negatively regulates biofilm formation. A deeper knowledge on the ability to form biofilms in these mutants may help in the development of intervention strategies to control Lm in food and in the environment.

  15. The Capacity of Listeria Monocytogenes Mutants with In-Frame Deletions in Putative ATP-Binding Cassette Transporters to form Biofilms and Comparison with the Wild Type

    Science.gov (United States)

    Ceruso, Marina; Fratamico, Pina; Chirollo, Claudia; Taglialatela, Rosanna; Cortesi, Maria Luisa

    2014-01-01

    Listeria monocytogenes (Lm) is a food-borne pathogen responsible for human listeriosis, an invasive infection with high mortality rates. Lm has developed efficient strategies for survival under stress conditions such as starvation and wide variations in temperature, pH, and osmolarity. Therefore, Lm can survive in food under multiple stress conditions. Detailed studies to determine the mode of action of this pathogen for survival under stress conditions are important to control Lm in food. It has been shown that genes encoding for ATP-binding cassette (ABC) transporters are induced in Lm in food, in particular under stress conditions. Previous studies showed that these genes are involved in sensitivity to nisin, acids, and salt. The aim of this study was to determine the involvement of some ABC transporters in biofilm formation. Therefore, deletion mutants of ABC transporter genes (LMOf2365_1875 and LMOf2365_1877) were created in Lm F2365, and then were compared to the wild type for their capacity to form biofilms. Lm strain F2365 was chosen as reference since the genome is fully sequenced and furthermore this strain is particularly involved in food-borne outbreaks of listeriosis. Our results showed that ΔLMOf2365_1875 had an increased capacity to form biofilms compared to the wild type, indicating that LMOf2365_1875 negatively regulates biofilm formation. A deeper knowledge on the ability to form biofilms in these mutants may help in the development of intervention strategies to control Lm in food and in the environment. PMID:27800311

  16. DELETION AND 5'CPG ISLAND METHYLATION OF p15 GENE IN BRAIN GLIOMA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To investigate the abnormality of p15 gene in brain glioma and the correlation of it with occurrence or malignant progression of brain glioma. Methods: Deletion and 5'CPG island methylation of p15 gene were detected by the methods of PCR and PCR-based methylation in 56 cases of brain glioma. Results: Out of 43 cases of high grade glioma, 14 cases were found to have homozygous deletion of p15E1, while none of the 13 cases of low grade glioma was found to have deletion of p15E1 (P<0.05). Methylation of 5'CPG Island of p15 gene was found only in four cases of glioma. Conclusion: Abnormality of p15 gene may involved in the occurrence and malignant progression of brain glioma. Homozygous deletion of gene is the major mechanism of inactivation for p15 gene in brain glioma.

  17. Marfan syndrome with a complex chromosomal rearrangement including deletion of the FBN1 gene

    Directory of Open Access Journals (Sweden)

    Colovati Mileny ES

    2012-01-01

    Full Text Available Abstract Background The majority of Marfan syndrome (MFS cases is caused by mutations in the fibrillin-1 gene (FBN1, mapped to chromosome 15q21.1. Only few reports on deletions including the whole FBN1 gene, detected by molecular cytogenetic techniques, were found in literature. Results We report here on a female patient with clinical symptoms of the MFS spectrum plus craniostenosis, hypothyroidism and intellectual deficiency who presents a 1.9 Mb deletion, including the FBN1 gene and a complex rearrangement with eight breakpoints involving chromosomes 6, 12 and 15. Discussion This is the first report of MFS with a complex chromosome rearrangement involving a deletion of FBN1 and contiguous genes. In addition to the typical clinical findings of the Marfan syndrome due to FBN1 gene haploinsufficiency, the patient presents features which may be due to the other gene deletions and possibly to the complex chromosome rearrangement.

  18. Detection of Homozygous Deletions and Mutations in the CDKN2A Gene in Hydatidiform Moles

    Institute of Scientific and Technical Information of China (English)

    Jing Wang; Shuying Wu; Ying Gu; Yan Zhu; Xiaowei Zhang

    2008-01-01

    OBJECTIVE To investigate homozygous deletions and mutations in the CDKN2A gene (p16INK4a and p14ARF gene) in hydatidiform moles.METHODS A total of 38 hydatidiform mole samples and 30 villi samples were examined for homozygous deletions in the CDKN2A gene by PCR and for mutations by DHPLC.RESULTS I) Among 38 hydatidiform mole samples,homozygous deletions in the p16INK4a exon 1 were identified in 5 cases (13.2%), while no homozygous deletions were found in the p16INK4a exon 1 of 30 early-pregnancy samples. The rates of those deletions in hydatidiform compared to early-pregnancy villi samples was statistically significant (P = 0.036). Ii) No homozygous deletions in the p14ARF exon 1 or p16INK4a exon 2 were found in any of the hydatidiform moles or early-preganancy samples, iii)In all hydatidiform moles and early-pregnancy villi samples, no mutations were detected by DHPLC.CONCLUSION We suggest there may be a close correlation between homozygous deletions in the CDKN2A gene and occurrence of hydatidiform moles variation in the CDKN2A gene is mainly caused by homozygous deletions, while mutations may be not a major cause.

  19. FREQUENT DELETION OF MTS1/p16 GENE AND CORRELATION WITH CLINICOPATHOLOGICAL PARAMETERS IN ENDOMETRIAL CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    Zhou Chunxiao; Sun Jianheng; Lu Shixin; Jin Shunqian; Liu Hailing; Sheng Xiugui

    1998-01-01

    Objective:To investigate the possible relationship between deletion of MTS/p16 gene and progression of endometrial carcinoma. Methods: Forty-six primary endometrial carcinoma, 7 tumor-adjacent endometrial tissue, 10 normal endometrial tissue specimen and 5 xenografts from patients with endometrial carcinoma were examined for homozygous deletion of MTS/p16 gene by polymerase chain reaction-based analysis. Results: Of 46 endometrial cancer specimens, 9 showed homozygous deletion, no deletion was detected in the tumor-adjacent and normal endometial tissues. Nor was it detected in well-differentiated endometrial carcinoma and all xenografts. Conclusions: Deletion of MTS1/p16 gene might contribute to the progression of endometrial carcinoma and could be served as indicator for predicting prognosis.

  20. Relatively low proportion of dystrophin gene deletions in Israeili Duchenne and Becker muscular dystrophy patients

    Energy Technology Data Exchange (ETDEWEB)

    Shomrat, R.; Gluck, E.; Legum, C.; Shiloh, Y. [Tel Aviv Univ. (Israel)

    1994-02-15

    Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations in the X-linked dystrophin gene. The most common mutations in western populations are deletions that are spread non-randomly throughout the gene. Molecular analysis of the dystrophin gene structure by hybridization of the full length cDNA to Southern blots and by PCR in 62 unrelated Israeli male DMD/BMD patients showed deletions in 23 (37%). This proportion is significantly lower than that found in European and North American populations (55-65%). Seventy-eight percent of the deletions were confined to exons 44-52, half of these exons 44-45, and the remaining 22% to exons 1 and 19. There was no correlation between the size of the deletion and the severity of the disease. All the deletions causing frameshift resulted in the DMD phenotypes. 43 refs., 1 fig., 1 tab.

  1. The effect of waaL genes deletion from Yersinia enterocolitica O:3 genome on bacteria LPS’ phenotype

    Directory of Open Access Journals (Sweden)

    Shevchenko J. I.

    2014-11-01

    Full Text Available Aim. To estimate WaaL ligase contribution in the lipopolysaccharide (LPS phenotype profile formation of Y. enterocolitica O:3 (YeO3 bacteria. Methods. The waaL-knock-out mutants were created by an allelic exchange strategy. The LPS phenotypes of created mutants were visualized by silver-stained DOC-PAGE and immunoblotting with specific outer core (core oligosaccharide, hexasaccharide, OC and O-polysaccharide (OPS or O-Ag monoclonal antibodies. Results. Deletion of waaLOS gene from YeO3 genome has a marked effect on OC ligation in either single or double mutants. The waaLPS deletion has an opposite effect on the OPS ligation – barely detected increasing of OPS bands. Conclusions. The LPS ligases of YeO3 exhibit relaxed donor substrate specificity. Under given conditions the effect of WaaLOS ligase is more significant for OC and OPS ligation onto lipid A than that of WaaLPS.

  2. A glycoprotein E deletion mutant of bovine herpesvirus 1 infects the same limited number of tissues in calves as wild-type virus, but for a shorter period

    NARCIS (Netherlands)

    Engelenburg, van F.A.C.; Kaashoek, M.J.; Oirschot, van J.T.; Rijsewijk, F.A.M.

    1995-01-01

    To gain insight into the role of glycoprotein E of bovine herpesvirus 1 (BHV-1), we compared the distribution of wild-type (wt) BHV-1 with that of a gE deletion mutant (gE-) in calves after intranasal inoculation. The wt-infected calves had severe clinical signs, but the gE--infected calves were vir

  3. Transcriptomic profile of aguR deletion mutant of Lactococcus lactis subsp. cremoris CECT 8666

    NARCIS (Netherlands)

    Del Rio, Beatriz; Linares, Daniel M; Redruello, Begoña; Martin, Maria Cruz; Fernandez, Maria; de Jong, Anne; Kuipers, Oscar P; Ladero, Victor; Alvarez, Miguel A

    2015-01-01

    Lactococcus lactis subsp. cremoris CECT 8666 (formerly GE2-14) is a dairy strain that catabolizes agmatine (a decarboxylated derivative of arginine) into the biogenic amine putrescine by the agmatine deiminase (AGDI) pathway [1]. The AGDI cluster of L. lactis is composed by five genes aguR, aguB, ag

  4. Unmarked gene deletion and host-vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Deng, Ling; Zhu, Haojun; Chen, Zhengjun

    2009-01-01

    , and unmarked lacS mutants were obtained by each method. A new alternative recombination mechanism, i.e., marker circularization and integration, was shown to operate in the latter method, which did not yield the designed deletion mutation. Subsequently, Sulfolobus-E. coli plasmid shuttle vectors were...

  5. Site-specific deletion and rearrangement of integron insert genes catalyzed by the integron DNA integrase.

    Science.gov (United States)

    Collis, C M; Hall, R M

    1992-01-01

    Deletion of individual antibiotic resistance genes found within the variable region of integrons is demonstrated. Evidence for gene duplications and rearrangements resulting from the insertion of gene units at new locations is also presented. Deletion, duplication, and rearrangement occur only in the presence of the integron-encoded DNA integrase. These events are precise and involve loss or gain of one or more complete insert units or gene cassettes. This confirms the recent definition of gene cassettes as consisting of the gene coding sequences, all except the last 7 bases of the 59-base element found at the 3' end of the gene, and the core site located 5' to the gene (Hall et al., Mol. Microbiol. 5:1941-1959, 1991) and demonstrates that individual gene cassettes are functional units which can be independently mobilized. Both deletions and duplications can be generated by integrase-mediated cointegrate formation followed by integrase-mediated resolution involving a different pair of sites. However, deletion occurs 10 times more frequently than duplication, and we propose that the majority of deletion events are likely to involve integrase-dependent excision of the gene unit to generate a circular gene cassette. The implications of these findings in understanding the evolution of integrons and the spread of antibiotic resistance genes in bacterial populations is discussed. Images PMID:1311297

  6. Genes and Alcohol Consumption: Studies with Mutant Mice

    Science.gov (United States)

    Mayfield, Jody; Arends, Michael A.; Harris, R. Adron; Blednov, Yuri A.

    2017-01-01

    In this chapter, we review the effects of global null mutant and overexpressing transgenic mouse lines on voluntary self-administration of alcohol. We examine approximately 200 publications pertaining to the effects of 155 mouse genes on alcohol consumption in different drinking models. The targeted genes vary in function and include neurotransmitter, ion channel, neuroimmune, and neuropeptide signaling systems. The alcohol self-administration models include operant conditioning, two- and four-bottle choice continuous and intermittent access, drinking in the dark limited access, chronic intermittent ethanol, and scheduled high alcohol consumption tests. Comparisons of different drinking models using the same mutant mice are potentially the most informative, and we will highlight those examples. More mutants have been tested for continuous two-bottle choice consumption than any other test; of the 137 mouse genes examined using this model, 97 (72%) altered drinking in at least one sex. Overall, the effects of genetic manipulations on alcohol drinking often depend on the sex of the mice, alcohol concentration and time of access, genetic background, as well as the drinking test. PMID:27055617

  7. Genes and Alcohol Consumption: Studies with Mutant Mice.

    Science.gov (United States)

    Mayfield, J; Arends, M A; Harris, R A; Blednov, Y A

    2016-01-01

    In this chapter, we review the effects of global null mutant and overexpressing transgenic mouse lines on voluntary self-administration of alcohol. We examine approximately 200 publications pertaining to the effects of 155 mouse genes on alcohol consumption in different drinking models. The targeted genes vary in function and include neurotransmitter, ion channel, neuroimmune, and neuropeptide signaling systems. The alcohol self-administration models include operant conditioning, two- and four-bottle choice continuous and intermittent access, drinking in the dark limited access, chronic intermittent ethanol, and scheduled high alcohol consumption tests. Comparisons of different drinking models using the same mutant mice are potentially the most informative, and we will highlight those examples. More mutants have been tested for continuous two-bottle choice consumption than any other test; of the 137 mouse genes examined using this model, 97 (72%) altered drinking in at least one sex. Overall, the effects of genetic manipulations on alcohol drinking often depend on the sex of the mice, alcohol concentration and time of access, genetic background, as well as the drinking test.

  8. Germinal mosaicism for a deletion of the FMR1 gene leading to fragile X syndrome.

    Science.gov (United States)

    Jiraanont, P; Hagerman, R J; Neri, G; Zollino, M; Murdolo, M; Tassone, F

    2016-09-01

    Aberrant CGG trinucleotide amplification within the FMR1 gene, which spans approximately 38 Kb of genomic DNA is almost always what leads to fragile X syndrome (FXS). However, deletions of part or the entire FMR1 gene can also cause FXS. Both CGG amplification-induced silencing and deletions result in the absence of the FMR1 gene product, FMRP. Here, we report a rare case of germinal mosaicism of a deletion encompassing approximately 300 Kb of DNA, which by removing the entire FMR1 gene led to FXS. The male proband, carrying the deletion, presented in clinic with the typical features of FXS. His mother was analyzed by FISH on metaphase chromosomes with cosmid probe c22.3 spanning the FMR1 locus, and she was found not to carry the deletion on 30 analyzed cells from peripheral blood lymphocytes. Prenatal examination of the mother's third pregnancy showed that the male fetus also had the same deletion as the proband. Following this prenatal diagnosis, FISH analysis in the mother was expanded to 400 metaphases from peripheral lymphocytes, and a heterozygous FMR1 deletion was found in three. Although this result could be considered questionable from a diagnostic point of view, it indicates that the deletion is in the ovary's germinal cells.

  9. Contiguous gene syndromes due to deletions in the distal short arm of the human X chromosome

    Energy Technology Data Exchange (ETDEWEB)

    Ballabio, A.; Andria, G. (Univ. of Reggio Calabria, Catanzaro (Italy)); Bardoni, B.; Fraccaro, M.; Maraschio, P.; Zuffardi, O.; Guioli, S.; Camerino, G. (Univ. of Pavia (Italy)); Carrozzo, R. (Univ. of Naples (Italy)); Bick, D.; Campbell, L. (Univ. of Texas, San Antonio (USA)); Hamel, B. (Univ. of Nijmegen (Netherlands)); Ferguson-Smith, M.A. (Univ. of Cambridge (England)); Gimelli, G. (G. Gaslini Institute, Genoa (Italy))

    1989-12-01

    Mendelian inherited disorders to deletions of adjacent genes on a chromosome have been described as contiguous gene syndromes. Short stature, chondrodysplasia punctata, mental retardation, steroid sulfatase deficiency, and Kallmann syndrome have been found as isolated entities or associated in various combination in 27 patients with interstitial and terminal deletions involving the distal short are of the X chromosome. The use of cDNA and genomic probes from the Xp22-pter region allowed us to identify 12 different deletion intervals and to confirm, and further refine, the chromosomal assignment of X-linked recessive chondrodysplasia punctata and Kallmann syndrome genes. A putative pseudoautosomal gene affecting height and an X-linked nonspecific mental retardation gene have been tentatively assigned to specific intervals. The deletion panel described is a useful tool for mapping new sequences and orienting chromosome walks in the region.

  10. Deletion of ribosomal protein genes is a common vulnerability in human cancer, especially in concert with TP53 mutations.

    Science.gov (United States)

    Ajore, Ram; Raiser, David; McConkey, Marie; Jöud, Magnus; Boidol, Bernd; Mar, Brenton; Saksena, Gordon; Weinstock, David M; Armstrong, Scott; Ellis, Steven R; Ebert, Benjamin L; Nilsson, Björn

    2017-04-01

    Heterozygous inactivating mutations in ribosomal protein genes (RPGs) are associated with hematopoietic and developmental abnormalities, activation of p53, and altered risk of cancer in humans and model organisms. Here we performed a large-scale analysis of cancer genome data to examine the frequency and selective pressure of RPG lesions across human cancers. We found that hemizygous RPG deletions are common, occurring in about 43% of 10,744 cancer specimens and cell lines. Consistent with p53-dependent negative selection, such lesions are underrepresented in TP53-intact tumors (P ≪ 10(-10)), and shRNA-mediated knockdown of RPGs activated p53 in TP53-wild-type cells. In contrast, we did not see negative selection of RPG deletions in TP53-mutant tumors. RPGs are conserved with respect to homozygous deletions, and shRNA screening data from 174 cell lines demonstrate that further suppression of hemizygously deleted RPGs inhibits cell growth. Our results establish RPG haploinsufficiency as a strikingly common vulnerability of human cancers that associates with TP53 mutations and could be targetable therapeutically. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  11. Developmental genes during placentation: insights from mouse mutants

    Institute of Scientific and Technical Information of China (English)

    Jinhu a LU; Qiang WANG; Bingyan WANG; Fengchao WANG; Haibin WANG

    2011-01-01

    Placenta,a temporary organ first formed during the development of a new life is essential for the survival and growth of the fetus in eutherian mammals.It serves as an interface for the exchange of nutrients,gases and wastes between the maternal and fetal compartments.During the past decades,studies employing gene-engineered mouse mutants have revealed a wide range of signaling molecules governing the trophoblast development and function during placentation under various pathophysiological conditions.Here,we summarize the recent progress with particular respect to the involvement of developmental genes during placentation.

  12. Wing defects in Drosophila xenicid mutant clones are caused by C-terminal deletion of additional sex combs (Asx.

    Directory of Open Access Journals (Sweden)

    Kara Bischoff

    Full Text Available BACKGROUND: The coordinated action of genes that control patterning, cell fate determination, cell size, and cell adhesion is required for proper wing formation in Drosophila. Defects in any of these basic processes can lead to wing aberrations, including blisters. The xenicid mutation was originally identified in a screen designed to uncover regulators of adhesion between wing surfaces [1]. PRINCIPAL FINDINGS: Here, we demonstrate that expression of the betaPS integrin or the patterning protein Engrailed are not affected in developing wing imaginal discs in xenicid mutants. Instead, expression of the homeotic protein Ultrabithorax (Ubx is strongly increased in xenicid mutant cells. CONCLUSION: Our results suggest that upregulation of Ubx transforms cells from a wing blade fate to a haltere fate, and that the presence of haltere cells within the wing blade is the primary defect leading to the adult wing phenotypes observed.

  13. Overexpression of SOS genes in ciprofloxacin resistant Escherichia coli mutants.

    Science.gov (United States)

    Pourahmad Jaktaji, Razieh; Pasand, Shirin

    2016-01-15

    Fluoroquinolones are important antibiotics for the treatment of urinary tract infections caused by Escherichia coli. Mutational studies have shown that ciprofloxacin, a member of fluoroquinolones induces SOS response and mutagenesis in pathogenic bacteria which in turn develop antibiotic resistance. However, inhibition of SOS response can increase recombination activity which in turn leads to genetic variation. The aim of this study was to measure 5 SOS genes expressions in nine E. coli mutants with different MICs for ciprofloxacin following exposure to ciprofloxacin. Gene expression was assessed by quantitative real time PCR. Gene alteration assessment was conducted by PCR amplification and DNA sequencing. Results showed that the expression of recA was increased in 5 mutants. This overexpression is not related to gene alteration, and enhances the expression of polB and umuCD genes encoding nonmutagenic and mutagenic polymerases, respectively. The direct relationship between the level of SOS expression and the level of resistance to ciprofloxacin was also indicated. It was concluded that novel therapeutic strategy that inhibits RecA activity would enhance the efficiency of common antibiotics against pathogenic bacteria. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Cardiac characterization of 16 patients with large NF1 gene deletions.

    Science.gov (United States)

    Nguyen, R; Mir, T S; Kluwe, L; Jett, K; Kentsch, M; Mueller, G; Kehrer-Sawatzki, H; Friedman, J M; Mautner, V-F

    2013-10-01

    The aim of this study was to characterize cardiac features of patients with neurofibromatosis 1 (NF1) and large deletions of the NF1 gene region. The study participants were 16 patients with large NF1 deletions and 16 age- and sex-matched NF1 patients without such deletions. All the patients were comprehensively characterized clinically and by echocardiography. Six of 16 NF1 deletion patients but none of 16 non-deletion NF1 patients have major cardiac abnormalities (p = 0.041). Congenital heart defects (CHDs) include mitral insufficiency in two patients and ventricular septal defect, aortic stenosis, and aortic insufficiency in one patient each. Three deletion patients have hypertrophic cardiomyopathy. Two patients have intracardiac tumors. NF1 patients without large deletions have increased left ventricular (LV) diastolic posterior wall thickness (p NF1, suggestive of eccentric LV hypertrophy. CHDs and other cardiovascular anomalies are more frequent among patients with large NF1 deletion and may cause serious clinical complications. Eccentric LV hypertrophy may occur in NF1 patients without whole gene deletions, but the clinical significance of this finding is uncertain. All patients with clinical suspicion for NF1 should be referred to a cardiologist for evaluation and surveillance.

  15. Dysfunctional p53 deletion mutants in cell lines derived from Hodgkin's lymphoma

    DEFF Research Database (Denmark)

    Feuerborn, Alexander; Moritz, Constanze; von Bonin, Frederike;

    2006-01-01

    Classical Hodgkin's lymphoma (cHL) is a distinct malignancy of the immune system. Despite the progress made in the understanding of the pathology of cHL, the transforming events remain to be elucidated. It has been proposed that mutations in the TP53 gene in biopsy material as well as cell lines ...... loss of exons 10 - 11 (L1236) or exons 8 - 11 (HDLM-2), respectively. These changes were found in otherwise rarely mutated regions of TP53. Cell lines L1236 and HDLM-2 harbour fusions with alu-repeats in their TP53 mRNA 3'-ends, resulting in the carboxyterminal truncation and loss...

  16. Analysis of Dystrophin Gene Deletions by Multiplex PCR in Moroccan Patients

    Directory of Open Access Journals (Sweden)

    Aziza Sbiti

    2002-01-01

    Full Text Available Duchenne and Becker muscular dystrophy (DMD and BMD are X-linked diseases resulting from a defect in the dystrophin gene located on Xp21. DMD is the most frequent neuromuscular disease in humans (1/3500 male newborn. Deletions in the dystrophin gene represent 65% of mutations in DMD/BMD patients. We have analyzed DNA from 72 Moroccan patients with DMD/BMD using the multiplex polymerase chain reaction (PCR to screen for exon deletions within the dystrophin gene, and to estimate the frequency of these abnormalities. We found dystrophin gene deletions in 37 cases. Therefore the frequency in Moroccan DMD/BMD patients is about 51.3%. All deletions were clustered in the two known hot-spots regions, and in 81% of cases deletions were detected in the region from exon 43 to exon 52. These findings are comparable to those reported in other studies. It is important to note that in our population, we can first search for deletions of DMD gene in the most frequently deleted exons determined by this study. This may facilitate the molecular diagnosis of DMD and BMD in our country.

  17. Insight into the role of anthocyanin biosynthesis-related genes in Medicago truncatula mutants impaired in pigmentation in leaves.

    Science.gov (United States)

    Carletti, Giorgia; Lucini, Luigi; Busconi, Matteo; Marocco, Adriano; Bernardi, Jamila

    2013-09-01

    Flavonoids are the most common antioxidant compounds produced in plants. In this study, two wild types and two independent mutants of Medicago truncatula with altered anthocyanin content in leaves were characterized at the phenotype, metabolite profile, gene structure and transcript levels. Flavonoid profiles showed conserved levels of dihydroflavonols, leucoanthocyanidins and flavonols, while anthocyanidin, anthocyanin and isoflavone levels were lower in the mutants (up to 90% less) compared with the wild types. Genes encoding key enzymes of the anthocyanin pathway and transcriptional factors were analyzed by RT-PCR. Genes involved in the later steps of the anthocyanin pathway (dihydrokaempferol reductase 2, UDP-glucose:anthocyanin 3-O-glucosyltransferase and glutathione S-transferase) were found under-expressed in both mutants. Dihydrokaempferol reductase 1 was downregulated two-fold in the anthocyanin-less mutant while the UDP-glucose:anthocyanin 5-O-glucosyltransferase was strongly repressed only in the mutant with low pigmentation, suggesting a different regulation in the two genotypes. The common feature was that the first enzymes of the flavonoid biosynthesis pathway were not altered in rate of expression. A very high reduction in transcript accumulation was also found for two homologous R2R3 MYB genes, namely MtMYBA and AN2, suggesting that these genes have a role in anthocyanin accumulation in leaves. More evidence was found on analyzing their nucleotide sequence: several SNPs, insertions and deletions in the coding and non-coding regions of both MYB genes were found between mutants and wild types that could influence anthocyanin biosynthesis. Moreover, a subfamily of eight MYB genes with a high homology to MtMYBA was discovered in tandem on chromosome 5 of M. truncatula.

  18. Partial Rescue of pos5 Mutants by YEF1 and UTR1 Genes in Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    Yong-Fu LI; Feng SHI

    2006-01-01

    Three NAD kinase homologs, encoded by UTR1, POS5 and YEF1 genes, are found in the yeast Saccharomyces cerevisiae and proven to be important sources of NADPH for the cell. Pos5p, existing in the mitochondrial matrix, is critical for higher temperature endurance and mitochondrial functions, such as glycerol usability and arginine biosynthesis. Through constructing the high-copy expression plasmids of YEF1 and UTR1, which contained the green fluorescent protein reporter tag at their 3' terminus, and introducing them into POS5 gene deletion mutants (i.e. pos5, utr1pos5, yef1pos5 and utr1yef1pos5), the high-copy YEF1 and UTR1 plasmids carrying transformants for pos5 mutants were obtained. Their temperature sensitivity and growth phenotype on media with glycerol as the sole carbon source, or on media without arginine, were checked. Results showed the partial rescue of mitochondrial dysfunctions and temperature sensitivity of pos5 mutants by the high-copy YEF1 gene, and of glycerol growth defect and temperature sensitivity by the high-copy UTR1 gene, which confirmed the potential supplying ability of Yef1p and Utr1p for mitochondrial NADP(H) and implied the weak transport of NADP from cytosol to mitochondria. However, even through the green fluorescent protein reporter label, the subcellular localization of Yef1p and Utr1p in yeast cells could not be observed, which indicated the low expression level of these two NAD kinase homologs.

  19. Alu insertion/deletion of ACE gene polymorphism might not affect ...

    African Journals Online (AJOL)

    Widodo

    2016-09-03

    Sep 3, 2016 ... Received 2 May 2016; accepted 1 August 2016. Available ... The Egyptian Journal of Medical Human Genetics (2017) 18, 187–191. HOSTED BY ..... insertion–deletion polymorphism of ACE gene and Alzheimer's · disease in ...

  20. Sleeping Beauty transposon mutagenesis identifies genes that cooperate with mutant Smad4 in gastric cancer development.

    Science.gov (United States)

    Takeda, Haruna; Rust, Alistair G; Ward, Jerrold M; Yew, Christopher Chin Kuan; Jenkins, Nancy A; Copeland, Neal G

    2016-04-05

    Mutations in SMAD4 predispose to the development of gastrointestinal cancer, which is the third leading cause of cancer-related deaths. To identify genes driving gastric cancer (GC) development, we performed a Sleeping Beauty (SB) transposon mutagenesis screen in the stomach of Smad4(+/-) mutant mice. This screen identified 59 candidate GC trunk drivers and a much larger number of candidate GC progression genes. Strikingly, 22 SB-identified trunk drivers are known or candidate cancer genes, whereas four SB-identified trunk drivers, including PTEN, SMAD4, RNF43, and NF1, are known human GC trunk drivers. Similar to human GC, pathway analyses identified WNT, TGF-β, and PI3K-PTEN signaling, ubiquitin-mediated proteolysis, adherens junctions, and RNA degradation in addition to genes involved in chromatin modification and organization as highly deregulated pathways in GC. Comparative oncogenomic filtering of the complete list of SB-identified genes showed that they are highly enriched for genes mutated in human GC and identified many candidate human GC genes. Finally, by comparing our complete list of SB-identified genes against the list of mutated genes identified in five large-scale human GC sequencing studies, we identified LDL receptor-related protein 1B (LRP1B) as a previously unidentified human candidate GC tumor suppressor gene. In LRP1B, 129 mutations were found in 462 human GC samples sequenced, and LRP1B is one of the top 10 most deleted genes identified in a panel of 3,312 human cancers. SB mutagenesis has, thus, helped to catalog the cooperative molecular mechanisms driving SMAD4-induced GC growth and discover genes with potential clinical importance in human GC.

  1. Deletion of a single-copy DAAM1 gene in congenital heart defect: a case report

    Directory of Open Access Journals (Sweden)

    Bao Bihui

    2012-08-01

    Full Text Available Abstract Background With an increasing incidence of congenital heart defects (CHDs in recent years, genotype-phenotype correlation and array-based methods have contributed to the genome-wide analysis and understanding of genetic variations in the CHD population. Here, we report a copy number deletion of chromosomal 14q23.1 in a female fetus with complex congenital heart defects. This is the first description of DAAM1 gene deletion associated with congenital heart anomalies. Case Presentation Compared with the control population, one CHD fetus showed a unique copy number deletion of 14q23.1, a region that harbored DAAM1 and KIAA0666 genes. Conclusions Results suggest that the copy number deletion on chromosome 14q23.1 may be critical for cardiogenesis. However, the exact relationship and mechanism of how DAAM1 and KIAA0666 deletion contributes to the onset of CHD is yet to be determined.

  2. 5q14.3 deletion neurocutaneous syndrome: Contiguous gene syndrome caused by simultaneous deletion of RASA1 and MEF2C: A progressive disease.

    Science.gov (United States)

    Ilari, Rita; Agosta, Guillermo; Bacino, Carlos

    2016-03-01

    We report the case of a young girl who was presented with complex clinical symptoms caused by the deletion of contiguous genes: RASA1 and MEF2C, located on chromosome 5q14.3. Specifically, the diagnosis of her skin disorder and vascular malformations involving central nervous system is consistent with a RASopathy. The child's neurological manifestations are observed in most patients suffering from 5q14.3 by deletion or mutation of the MEF2C gene. A review of the literature allowed us to conclude that the contiguous deletion of genes RASA1 and MEF2C fulfills the criteria for the diagnosis of a Neurocutaneous syndrome as proposed by Carr et al. [2011]. We also assessed the penetrance of RASA1 and clinical manifestations of MEF2C according to the type of deletion. This child described presents the complete symptomatology of both deleted genes. We would also like to highlight the progression of the disorder.

  3. Deletion of genes involved in glutamate metabolism to improve poly-gamma-glutamic acid production in B. amyloliquefaciens LL3.

    Science.gov (United States)

    Zhang, Wei; He, Yulian; Gao, Weixia; Feng, Jun; Cao, Mingfeng; Yang, Chao; Song, Cunjiang; Wang, Shufang

    2015-02-01

    Here, we attempted to elevate poly-gamma-glutamic acid (γ-PGA) production by modifying genes involved in glutamate metabolism in Bacillus amyloliquefaciens LL3. Products of rocR, rocG and gudB facilitate the conversion from glutamate to 2-oxoglutarate in Bacillus subtillis. The gene odhA is responsible for the synthesis of a component of the 2-oxoglutarate dehydrogenase complex that catalyzes the oxidative decarboxylation of 2-oxoglutarate to succinyl coenzyme A. In-frame deletions of these four genes were performed. In shake flask experiments the gudB/rocG double mutant presented enhanced production of γ-PGA, a 38 % increase compared with wild type. When fermented in a 5-L fermenter with pH control, the γ-PGA yield of the rocR mutant was increased to 5.83 g/L from 4.55 g/L for shake flask experiments. The gudB/rocG double mutant produced 5.68 g/L γ-PGA compared with that of 4.03 g/L for the wild type, a 40 % increase. Those results indicated the possibility of improving γ-PGA production by modifying glutamate metabolism, and identified potential genetic targets to improve γ-PGA production.

  4. Truncating PREX2 mutations activate its GEF activity and alter gene expression regulation in NRAS-mutant melanoma

    KAUST Repository

    Lissanu Deribe, Yonathan

    2016-03-01

    PREX2 (phosphatidylinositol-3,4,5-triphosphate-dependent Rac-exchange factor 2) is a PTEN (phosphatase and tensin homolog deleted on chromosome 10) binding protein that is significantly mutated in cutaneous melanoma and pancreatic ductal adenocarcinoma. Here, genetic and biochemical analyses were conducted to elucidate the nature and mechanistic basis of PREX2 mutation in melanoma development. By generating an inducible transgenic mouse model we showed an oncogenic role for a truncating PREX2 mutation (PREX2E824*) in vivo in the context of mutant NRAS. Using integrative cross-species gene expression analysis, we identified deregulated cell cycle and cytoskeleton organization as significantly perturbed biological pathways in PREX2 mutant tumors. Mechanistically, truncation of PREX2 activated its Rac1 guanine nucleotide exchange factor activity, abolished binding to PTEN and activated the PI3K (phosphatidyl inositol 3 kinase)/Akt signaling pathway. We further showed that PREX2 truncating mutations or PTEN deletion induces down-regulation of the tumor suppressor and cell cycle regulator CDKN1C (also known as p57KIP2). This down-regulation occurs, at least partially, through DNA hypomethylation of a differentially methylated region in chromosome 11 that is a known regulatory region for expression of the CDKN1C gene. Together, these findings identify PREX2 as a mediator of NRAS-mutant melanoma development that acts through the PI3K/PTEN/Akt pathway to regulate gene expression of a cell cycle regulator.

  5. Clinical and molecuar characterization of Brazilian patients with growth hormone gene deletions

    Directory of Open Access Journals (Sweden)

    I.J.P. Arnhold

    1998-04-01

    Full Text Available Genomic DNA from 23 patients with isolated growth hormone (GH deficiency (12 males and 11 females: heights -4.9 ± 1.4 SDS was screened for GH gene deletions by restriction endonuclease analysis of polymerase chain reaction amplification products. Three unrelated patients had typical features of severe GH deficiency and deletions (6.7 kb in two and 7.6 kb in one of the GH gene. The two patients with 6.7-kb deletions developed growth-attenuating anti-GH antibodies whereas the patient with the 7.6-kb deletion continued to grow with GH replacement therapy. Our finding that 3/23 (~13% Brazilian subjects had GH gene deletions agrees with previous studies of severe isolated GH deficiency subjects in other populations. Two of three subjects (67% with deletions developed blocking antibodies despite administration of exogenous GH at low doses. Interestingly, only 1/10 of cases with affected relatives or parental consanguinity had GH-1 gene deletions

  6. Combinational deletion of three membrane protein-encoding genes highly attenuates yersinia pestis while retaining immunogenicity in a mouse model of pneumonic plague.

    Science.gov (United States)

    Tiner, Bethany L; Sha, Jian; Kirtley, Michelle L; Erova, Tatiana E; Popov, Vsevolod L; Baze, Wallace B; van Lier, Christina J; Ponnusamy, Duraisamy; Andersson, Jourdan A; Motin, Vladimir L; Chauhan, Sadhana; Chopra, Ashok K

    2015-04-01

    Previously, we showed that deletion of genes encoding Braun lipoprotein (Lpp) and MsbB attenuated Yersinia pestis CO92 in mouse and rat models of bubonic and pneumonic plague. While Lpp activates Toll-like receptor 2, the MsbB acyltransferase modifies lipopolysaccharide. Here, we deleted the ail gene (encoding the attachment-invasion locus) from wild-type (WT) strain CO92 or its lpp single and Δlpp ΔmsbB double mutants. While the Δail single mutant was minimally attenuated compared to the WT bacterium in a mouse model of pneumonic plague, the Δlpp Δail double mutant and the Δlpp ΔmsbB Δail triple mutant were increasingly attenuated, with the latter being unable to kill mice at a 50% lethal dose (LD50) equivalent to 6,800 LD50s of WT CO92. The mutant-infected animals developed balanced TH1- and TH2-based immune responses based on antibody isotyping. The triple mutant was cleared from mouse organs rapidly, with concurrent decreases in the production of various cytokines and histopathological lesions. When surviving animals infected with increasing doses of the triple mutant were subsequently challenged on day 24 with the bioluminescent WT CO92 strain (20 to 28 LD50s), 40 to 70% of the mice survived, with efficient clearing of the invading pathogen, as visualized in real time by in vivo imaging. The rapid clearance of the triple mutant, compared to that of WT CO92, from animals was related to the decreased adherence and invasion of human-derived HeLa and A549 alveolar epithelial cells and to its inability to survive intracellularly in these cells as well as in MH-S murine alveolar and primary human macrophages. An early burst of cytokine production in macrophages elicited by the triple mutant compared to WT CO92 and the mutant's sensitivity to the bactericidal effect of human serum would further augment bacterial clearance. Together, deletion of the ail gene from the Δlpp ΔmsbB double mutant severely attenuated Y. pestis CO92 to evoke pneumonic plague in a

  7. Inheritable and precise large genomic deletions of non-coding RNA genes in zebrafish using TALENs.

    Directory of Open Access Journals (Sweden)

    Yun Liu

    Full Text Available Transcription activator-like effector nucleases (TALENs have so far been applied to disrupt protein-coding genes which constitute only 2-3% of the genome in animals. The majority (70-90% of the animal genome is actually transcribed as non-coding RNAs (ncRNAs, yet the lack of efficient tools to knockout ncRNA genes hinders studies on their in vivo functions. Here we have developed novel strategies using TALENs to achieve precise and inheritable large genomic deletions and knockout of ncRNA genes in zebrafish. We have demonstrated that individual miRNA genes could be disrupted using one pair of TALENs, whereas large microRNA (miRNA gene clusters and long non-coding RNA (lncRNA genes could be precisely deleted using two pairs of TALENs. We have generated large genomic deletions of two miRNA clusters (the 1.2 kb miR-17-92 cluster and the 79.8 kb miR-430 cluster and one long non-coding RNA (lncRNA gene (the 9.0 kb malat1, and the deletions are transmitted through the germline. Taken together, our results establish TALENs as a robust tool to engineer large genomic deletions and knockout of ncRNA genes, thus opening up new avenues in the application of TALENs to study the genome in vivo.

  8. Tetrahydrodipicolinate N-succinyltransferase and dihydrodipicolinate synthase from Pseudomonas aeruginosa: structure analysis and gene deletion.

    Directory of Open Access Journals (Sweden)

    Robert Schnell

    Full Text Available The diaminopimelic acid pathway of lysine biosynthesis has been suggested to provide attractive targets for the development of novel antibacterial drugs. Here we report the characterization of two enzymes from this pathway in the human pathogen Pseudomonas aeruginosa, utilizing structural biology, biochemistry and genetics. We show that tetrahydrodipicolinate N-succinyltransferase (DapD from P. aeruginosa is specific for the L-stereoisomer of the amino substrate L-2-aminopimelate, and its D-enantiomer acts as a weak inhibitor. The crystal structures of this enzyme with L-2-aminopimelate and D-2-aminopimelate, respectively, reveal that both compounds bind at the same site of the enzyme. Comparison of the binding interactions of these ligands in the enzyme active site suggests misalignment of the amino group of D-2-aminopimelate for nucleophilic attack on the succinate moiety of the co-substrate succinyl-CoA as the structural basis of specificity and inhibition. P. aeruginosa mutants where the dapA gene had been deleted were viable and able to grow in a mouse lung infection model, suggesting that DapA is not an optimal target for drug development against this organism. Structure-based sequence alignments, based on the DapA crystal structure determined to 1.6 Å resolution revealed the presence of two homologues, PA0223 and PA4188, in P. aeruginosa that could substitute for DapA in the P. aeruginosa PAO1ΔdapA mutant. In vitro experiments using recombinant PA0223 protein could however not detect any DapA activity.

  9. Yeast expression and DNA immunization of hepatitis B virus S gene with second-loop deletion of α determinant region

    Institute of Scientific and Technical Information of China (English)

    Hui Hu; Xiao-Mou Peng; Yang-Su Huang; Lin Gu; Qi-Feng Xie; Zhi-Liang Gao

    2004-01-01

    AIM: Immune escape mutations of HBV often occur in the dominant epitope, the second-loop of the a determinant of hepatitis B surface antigen (HBsAg). To let the hosts respond to the subdominant epitopes in HBsAg may be an effective way to decrease the prevalence of immune escape mutants. For this reason, a man-made clone of HBV S gene with the second-loop deletion was constructed. Its antigenicity was evaluated by yeast expression analysis and DNA immunization in mice.METHODS: HBV S gene with deleted second-loop, amino acids from 139 to 145, was generated using splicing by overlap extension. HBV deleted S gene was then cloned into the yeast expression vector pPIC9 and the mammalian expression vector pcDNA3 to generate pHB-SDY and pHB-SD,respectively. The complete S gene was cloned into the same vectors as controls. The deleted recombinant HBsAg expressed in yeasts was detected using Abbott IMx HBsAg test kits, enzyme-linked immunoadsorbent assay (ELISA)and immune dot blotting to evaluate its antigenicity in vitro.The anti-HBs responses to DNA immunization in BALB/c mice were detected using Abbott IMx AUSAB test kits to evaluate the antigenicity of that recombinant protein in vivo.RESULTS: Both deleted and complete HBsAg were successfully expressed in yeasts. They were intracellular expressions. The deleted HBsAg could not be detected by ELISA, in which the monoclonal anti-HBs against the α determinant was used, but could be detected by Abbott IMx and immune dot blotting, in which multiple monoclonal antiHBs and polyclonal anti-HBs were used, respectively. The activity of the deleted HBsAg detected by Abbott IMx was much lower than that of complete HBsAg (the ratio of sample value/cut off value, 106±26.7 vs1 814.4±776.3, P<0.01,t = 5.02). The anti-HBs response of pHB-SD to DNA immunization was lower than that of complete HBV S gene vector pHB (the positive rate 2/10 vs6/10, 4.56±3.52 mIU/mL vs27.60±17.3 mIU/mL, P= 0.02, t= 2.7).CONCLUSIONS: HBsAg with deleted

  10. Neurophysiological defects and neuronal gene deregulation in Drosophila mir-124 mutants.

    Directory of Open Access Journals (Sweden)

    Kailiang Sun

    2012-02-01

    Full Text Available miR-124 is conserved in sequence and neuronal expression across the animal kingdom and is predicted to have hundreds of mRNA targets. Diverse defects in neural development and function were reported from miR-124 antisense studies in vertebrates, but a nematode knockout of mir-124 surprisingly lacked detectable phenotypes. To provide genetic insight from Drosophila, we deleted its single mir-124 locus and found that it is dispensable for gross aspects of neural specification and differentiation. On the other hand, we detected a variety of mutant phenotypes that were rescuable by a mir-124 genomic transgene, including short lifespan, increased dendrite variation, impaired larval locomotion, and aberrant synaptic release at the NMJ. These phenotypes reflect extensive requirements of miR-124 even under optimal culture conditions. Comparison of the transcriptomes of cells from wild-type and mir-124 mutant animals, purified on the basis of mir-124 promoter activity, revealed broad upregulation of direct miR-124 targets. However, in contrast to the proposed mutual exclusion model for miR-124 function, its functional targets were relatively highly expressed in miR-124-expressing cells and were not enriched in genes annotated with epidermal expression. A notable aspect of the direct miR-124 network was coordinate targeting of five positive components in the retrograde BMP signaling pathway, whose activation in neurons increases synaptic release at the NMJ, similar to mir-124 mutants. Derepression of the direct miR-124 target network also had many secondary effects, including over-activity of other post-transcriptional repressors and a net incomplete transition from a neuroblast to a neuronal gene expression signature. Altogether, these studies demonstrate complex consequences of miR-124 loss on neural gene expression and neurophysiology.

  11. Construction and characterization of invasion protein B gene deleted mutant of Salmonella typhimurium SL1344 strain%鼠伤寒沙门菌SL1344株侵袭性蛋白B缺失株的构建及生物学特性

    Institute of Scientific and Technical Information of China (English)

    陈松彪; 陈桂华; 赵战勤; 李静; 郁川; 何雷; 张春杰; 程相朝; 李银聚; 颜云飞; 金修哲

    2015-01-01

    Objective:In order to develop a safer vaccine strain exploit Salmonella typhimurium vaccine strain .A ΔsipB mutant of Salmonella typhimurium SL 1344 strain was constructed.Methods: Firstly, the recombinant suicide plasmid containing the missing 585 bp sipB ( PREΔsipB ) was built by homologous recombination , and screenned by two-step method.Results: PCR and sequencing results showed that SL 1344ΔsipB was successfully constructed.It was no significant changes compared with SL 1344.But compared with the parent strains SL 1344 , the mutant strain had obvious change in its virulence , oral challenge of bacteria in mice revealed that LD50 of the mutant strain was 1.70 ×108 CFU,the toxicity reduced about 1.4%.The protection rate induced by the sipB mutant was 50%,and the serum antibody peaked 14 d post-immunization.Conclusion:The SL1344ΔsipB mutant was constructed suc-cessfully,and genetic stability ,significantly reduced virulence.The study provides a new approach for further study of the relationship between the gene and pathogenicity of Salmonella typhimurium.It is likely that the ΔsipB mutant could be adapted to develope attenuated Salmonella vaccine.%目的:为了研制更加安全的鼠伤寒沙门菌弱毒株,本研究构建了鼠伤寒沙门菌SL1344ΔsipB基因缺失突变株。方法:首先构建含缺失585 bp sipB基因的重组自杀性质粒PREΔsipB,利用重组自杀性质粒介导的等位基因交换技术,两步法筛选出SL1344的sipB缺失株。结果:PCR及测序结果表明SL1344ΔsipB构建成功。进一步生物学特性研究表明,缺失株SL1344ΔsipB保留了亲本菌株SL1344的血清型1,4,5,12:i:1,2,且能够稳定遗传缺失585 bp的sipB基因,生长速度没有发生明显改变;但是,与亲本株SL1344相比,其毒力发生明显改变,缺失株SL1344ΔsipB口服感染6周龄BALB/c小鼠的LD50为1.70×108 CFU,毒力较亲本株SL1344降低至1.4%,免疫

  12. Construction of ply gene-deletion mutant of Streptococcus pneumoniae and research of its virulence change%肺炎链球菌ply基因缺陷菌株的构建及毒力变化的初步研究

    Institute of Scientific and Technical Information of China (English)

    李忱炜; 何於娟; 袁军; 王虹; 贺潇; 董杰; 崔瑾; 姜慧; 张雪梅; 胥文春

    2010-01-01

    目的 构建肺炎链球菌(Streptococcus pneumoniae,Sp)溶血素基因(pneumolysin,ply)缺陷菌株,并对其毒力作初步研究,为进一步探索宿主对溶血素的防御应答奠定基础.方法 采用长臂同源多聚酶链反应(LFH-PCR)技术将ply基因替换为红霉素耐药基因(erm)后同源重组于肺炎链球菌,在含红霉素的血平板上筛选出ply缺陷菌株.用PCR鉴定缺陷菌株,观察体外缺陷菌株生长情况,并在小鼠体内感染模型研究其毒力侵袭变化.结果 PCR结果显示ply基因完全被erm基因所替代,构建ply缺陷菌成功;单个菌落培养基生长情况表明ply基因缺陷并未对细菌的体外生长造成影响;但在小鼠鼻腔感染模型中,缺陷菌株入血时间(6 h)明显晚于野生菌株(2 h),且各时间点的菌量均显著低于野生菌株,两者比较差异有统计学意义(P<0.01);小鼠腹膜感染模型显示野生菌株半数致死时间为3 d,而缺陷菌株半数致死时间为18d,两者比较差异有统计学意义(P<0.01).结论 采用LFH-PCR技术作基因突变完全替代ply基因,方法简便快捷;ply的缺陷不影响细菌在体外的生长,但可显著降低细菌在宿主体内的毒力和侵袭.%Objective To lay the foundation for further exploration on parasitifer's defence reaction to pneumolysin through constructing ply gene-deletion strain of Streptococcus pneumoniae and researching on its virulence change. Methods A linker fragment with erm gene in middle and homologous upstream and downstream fragment of ply gene at both sides was prepared by long flanking homology-polymerase chain reaction(LFH-PCR). The linker fragment was transformed into Streptococcus pneumoniae. ply-deficient strain was then screened out from blood plate which contains erythromycin and identified by PCR. ply-deficient strain growth in vitro was observed and virulence change was observed through infecting mouse model. Results PCR results showed that ply gene was replaced

  13. Bacterial Genome Editing with CRISPR-Cas9: Deletion, Integration, Single Nucleotide Modification, and Desirable "Clean" Mutant Selection in Clostridium beijerinckii as an Example.

    Science.gov (United States)

    Wang, Yi; Zhang, Zhong-Tian; Seo, Seung-Oh; Lynn, Patrick; Lu, Ting; Jin, Yong-Su; Blaschek, Hans P

    2016-07-15

    CRISPR-Cas9 has been demonstrated as a transformative genome engineering tool for many eukaryotic organisms; however, its utilization in bacteria remains limited and ineffective. Here we explored Streptococcus pyogenes CRISPR-Cas9 for genome editing in Clostridium beijerinckii (industrially significant but notorious for being difficult to metabolically engineer) as a representative attempt to explore CRISPR-Cas9 for genome editing in microorganisms that previously lacked sufficient genetic tools. By combining inducible expression of Cas9 and plasmid-borne editing templates, we successfully achieved gene deletion and integration with high efficiency in single steps. We further achieved single nucleotide modification by applying innovative two-step approaches, which do not rely on availability of Protospacer Adjacent Motif sequences. Severe vector integration events were observed during the genome engineering process, which is likely difficult to avoid but has never been reported by other researchers for the bacterial genome engineering based on homologous recombination with plasmid-borne editing templates. We then further successfully employed CRISPR-Cas9 as an efficient tool for selecting desirable "clean" mutants in this study. The approaches we developed are broadly applicable and will open the way for precise genome editing in diverse microorganisms.

  14. Expression of a Mutant kcnj2 Gene Transcript in Zebrafish

    OpenAIRE

    2013-01-01

    Long QT 7 syndrome (LQT7, also known as Andersen-Tawil syndrome) is a rare autosomal-dominant disorder that causes cardiac arrhythmias, periodic paralysis, and dysmorphic features. Mutations in the human KCNJ2 gene, which encodes for the subunit of the potassium inwardly-rectifying channel (IK1), have been associated with the disorder. The majority of mutations are considered to be dominant-negative as mutant proteins interact to limit the function of wild type KCNJ2 proteins. Several LQT7 sy...

  15. Deletion analysis of SMN1 and NAIP genes in southern Chinese children with spinal muscular atrophy

    Institute of Scientific and Technical Information of China (English)

    Yu-hua LIANG; Xiao-ling CHEN; Zhong-sheng YU; Chun-yue CHEN; Sheng BI; Lian-gen MAO; Bo-lin ZHOU; Xian-ning ZHANG

    2009-01-01

    Spinal muscular atrophy (SMA) is a disorder characterized by degeneration of lower motor neurons and occasionally bulbar motor neurons leading to progressive limb and trunk paralysis as well as muscular atrophy. Three types of SMA are rec-ognized depending on the age of onset, the maximum muscular activity achieved, and survivorship: SMA1, SMA2, and SMA3. The survival of motor neuron (SMN) gene has been identified as an SMA determining gene, whereas the neuronal apoptosis inhibitory protein (NAIP) gene is considered to be a modifying factor of the severity of SMA. The main objective of this study was to analyze the deletion of SMN1 and NAIP genes in southern Chinese children with SMA. Here, polymerase chain reaction (PCR) combined with restriction fragment length polymorphism (RFLP) was performed to detect the deletion of both exon 7 and exon 8 of SMNI and exon 5 of NAIP in 62 southern Chinese children with strongly suspected clinical symptoms of SMA. All the 32 SMAI patients and 76% (13/17) of SMA2 patients showed homozygous deletions for exon 7 and exon 8, and all the 13 SMA3 patients showed single deletion of SMN1 exon 7 along with 24% (4/17) of SMA2 patients. Eleven out of 32 (34%) SMA1 patients showed NAIP deletion, and none of SMA2 and SMA3 patients was found to have NAIP deletion. The findings of homozygous deletions of exon 7 and/or exon 8 of SMN1 gene confirmed the diagnosis of SMA, and suggested that the deletion of SMN1 exon 7 is a major cause of SMA in southern Chinese children, and that the NA1P gene may be a modifying factor for disease severity of SMA 1. The molecular diagnosis system based on PCR-RFLP analysis can conveniently be applied in the clinical testing, genetic counseling, prenatal diagnosis and preimplantation genetic diagnosis of SMA.

  16. Prodynorphin gene deletion increased anxiety-like behaviours, impaired the anxiolytic effect of bromazepam and altered GABAA receptor subunits gene expression in the amygdala.

    Science.gov (United States)

    Femenía, Teresa; Pérez-Rial, Sandra; Urigüen, Leyre; Manzanares, Jorge

    2011-01-01

    This study evaluated the role of prodynorphin gene in the regulation of anxiety and associated molecular mechanisms. Emotional responses were assessed using the light-dark test, elevated plus maze and social interaction tests in prodynorphin knockout and wild-type mice. Corticotrophin releasing factor and proopiomelanocortin gene expressions in the hypothalamus were evaluated after restraint stress using in situ hybridization. The anxiolytic efficacy of bromazepam and GABA(A) receptor subunits gene expression in the amygdala were also assessed in both genotypes. The deletion of prodynorphin increased anxiety-like behaviours and proopiomelanocortin gene expression in the arcuate nucleus (two-fold). Moreover, the anxiolytic action of bromazepam was significantly attenuated in the mutant mice. Decreased GABA(A)γ(2) and increased GABA(A)β(2) gene expression receptor subunits were found in the amygdala of prodynorphin knockout mice. These results indicate that deletion of prodynorphin gene is associated with increased anxiety-like behaviours, enhanced sensibility response to stress stimuli, reduced anxiolytic efficacy of bromazepam and altered expression of the GABA(A) receptor subunits.

  17. Deletion Analysis Of The Duchenne/Becker Muscular Dystrophy Gene Using Multiplex Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Dastur R

    2003-01-01

    Full Text Available The diagnosis of Duchenne Muscular Dystrophy (DMD and Becker Muscular Dystrophy (BMD is mainly based on clinical profile, serum CPK values, muscle biopsy and immunostaining for dystrophin. Most recent and accurate method for diagnosing DMD/BMD is by detection of mutations in the DMD gene. This was done in 100 unrelated patients using 19 exons including the promoter region in two sets of multiplex polymerase chain reaction (PCR. These primers amplify most of the exons in the deletion prone ′hotspot′ regions allowing determination of deletion end point. Intragenic deletions were detected in 74 patients indicating that the use of PCR-based assays will allow deletion detection help in prenatal diagnosis for most of the DMD/BMD patients. The frequency of deletions observed in the present study was 74%.

  18. Frequent intragenic deletion of the P gene in Tanzanian patients with Type II oculocutaneous albinism (OCA2)

    Energy Technology Data Exchange (ETDEWEB)

    Spritz, R.; Fukai, K.; Holmes, S.A. [Univ. of Wisconsin, Madison, WI (United States)] [and others

    1995-06-01

    Type II oculocutaneous albinism (OCA2) is an autosomal recessive disorder in which the biosynthesis of melanin pigment is reduced in the skin, hair, and eyes. OCA2, which results from mutations of the P gene, is the most frequent type of albinism in African and African-American patients. OCA2 is especially frequent in Tanzania, where it occurs with an incidence of {approximately}1/1,400. We have identified abnormalities of the P gene in each of 13 unrelated patients with OCA2 from Tanzania. One of these, a deletion of exon 7, is strongly predominant, accounting for {approximately}77% of mutant alleles in this group of patients. 20 refs., 2 figs.

  19. Blue ghosts: a new method for isolating amber mutants defective in essential genes of Escherichia coli

    DEFF Research Database (Denmark)

    Brown, S; Brickman, E R; Beckwith, J

    1981-01-01

    We describe a technique which permits an easy screening for amber mutants defective in essential genes of Escherichia coli. Using this approach, we have isolated three amber mutants defective in the rho gene. An extension of the technique allows the detection of ochre mutants and transposon inser...

  20. PCR detection of retinoblastoma gene deletions in radiation-induced mouse lung adenocarcinomas

    Energy Technology Data Exchange (ETDEWEB)

    Churchill, M.E.; Gemmell, M.A.; Woloschak, G.E.

    1994-05-01

    From 1971--1986, Argonne National Laboratory conducted a series of large-scale studies of tumor incidence in 40,000 BCF{sub 1} mice irradiated with {sup 60}Co {gamma}-rays or JANUS fission-spectrum neutrons. Polymerase chain reaction (PCR) technique was used to detect deletions in the mouse retinoblastoma (mRb) gene. Six mRb gene exon fragments were amplified in a 40-cycle, 3-temperature PCR protocol. Absence of any of these fragments on a Southern blot indicated a deletion of that portion of the mRb gene. Tumors chosen for analysis were lung adenocarcinomas that were judged to be the cause of death in post-mortem analyses. Spontaneous tumors as well as those from irradiated mice were analyzed for mRb deletions. In all normal mouse tissues studies all six mRb exon fragments were present on Southern blots. Tumors in six neutron-irradiated mice also had no mRb deletions. However, 1 of 6 tumors from {gamma}-irradiated mice and 6 of 18 spontaneous tumors from unirradiated mice showed a deletion in one or both mRb alleles. All deletions detected were in the 5{prime} region of the mRb gene.

  1. Identification and molecular characterization of four new large deletions in the beta-globin gene cluster.

    Science.gov (United States)

    Joly, Philippe; Lacan, Philippe; Garcia, Caroline; Couprie, Nicole; Francina, Alain

    2009-01-01

    Despite the fact that mutations in the human beta-globin gene cluster are essentially point mutations, a significant number of large deletions have also been described. We present here four new large deletions in the beta-globin gene cluster that have been identified on patients displaying an atypical hemoglobin phenotype (high HbF) at routine analysis. The first deletion, which spreads over 2.0 kb, removes the entire beta-globin gene, including its promoter, and is associated with a typical beta-thal minor phenotype. The three other deletions are larger (19.7 to 23.9 kb) and remove both the delta and beta-globin genes. Phenotypically, they look like an HPFH-deletion as they are associated with normal hematological parameters. The precise localization of their 5' and 3' breakpoints gives new insights about the differences between HPFH and (deltabeta)(0)-thalassemia at the molecular level. The importance of detection of these deletions in prenatal diagnosis and newborn screening of hemoglobinopathies is also discussed.

  2. An atypical case of fragile X syndrome caused by a deletion that includes FMRI gene

    Energy Technology Data Exchange (ETDEWEB)

    Quan, F.; Zonana, J.; Gunter, K.; Peterson, K.L.; Magenis, R.E., Popovich, B.W. [Shriners Hospital for Crippled Children, Portland, OR (United States)

    1995-05-01

    Fragile X syndrome is the most common form of inherited mental retardation and results from the transcriptional inactivation of the FMR1 gene. In the vast majority of cases, this is caused by the expansion of an unstable CGG repeat in the first exon of the FMR1 gene. We describe here a phenotypically atypical case of fragile X syndrome, caused by a deletion that includes the entire FMR1 gene and {ge}9.0 Mb of flanking DNA. The proband, RK, was a 6-year-old mentally retarded male with obesity and anal atresia. A diagnosis of fragile X syndrome was established by the failure of RK`s DNA to hybridize to a 558-bp PstI-XhoI fragment (pfxa3) specific for the 5{prime}-end of the FMR1 gene. The analysis of flanking markers in the interval from Xq26.3-q28 indicated a deletion extending from between 160-500 kb distal and 9.0 Mb proximal to the FMR1 gene. High-resolution chromosome banding confirmed a deletion with breakpoints in Xq26.3 and Xq27.3. This deletion was maternally transmitted and arose as a new mutation on the grandpaternal X chromosome. The maternal transmission of the deletion was confirmed by FISH using a 34-kb cosmid (c31.4) containing most of the FMR1 gene. These results indicated that RK carried a deletion of the FMR1 region with the most proximal breakpoint described to date. This patient`s unusual clinical presentation may indicate the presence of genes located in the deleted interval proximal to the FMR1 locus that are able to modify the fragile X syndrome phenotype. 36 refs., 7 figs.

  3. Deletion of exon 8 from the EXT1 gene causes multiple osteochondromas (MO) in a family with three affected members.

    Science.gov (United States)

    Zhuang, Lei; Gerber, Simon D; Kuchen, Stefan; Villiger, Peter M; Trueb, Beat

    2016-01-01

    Multiple osteochondromas (also called hereditary multiple exostoses) is an autosomal dominant disorder characterized by multiple cartilaginous tumors, which are caused by mutations in the genes for exostosin-1 (EXT1) and exostosin-2 (EXT2). The goal of this study was to elucidate the genetic alterations in a family with three affected members. Isolation of RNA from the patients' blood followed by reverse transcription and PCR amplification of selected fragments showed that the three patients lack a specific region of 90 bp from their EXT1 mRNA. This region corresponds to the sequence of exon 8 from the EXT1 gene. No splice site mutation was found around exon 8. However, long-range PCR amplification of the region from intron 7 to intron 8 indicated that the three patients contain a deletion of 4318 bp, which includes exon 8 and part of the flanking introns. There is evidence that the deletion was caused by non-homologous end joining because the breakpoints are not located within a repetitive element, but contain multiple copies of the deletion hotspot sequence TGRRKM. Exon 8 encodes part of the active site of the EXT1 enzyme, including the DXD signature of all UDP-sugar glycosyltransferases. It is conceivable that the mutant protein exerts a dominant negative effect on the activity of the EXT glycosyltransferase since it might interact with normal copies of the enzyme to form an inactive hetero-oligomeric complex. We suggest that sequencing of RNA might be superior to exome sequencing to detect short deletions of a single exon.

  4. Cloning and Expression of Gene Responsible for High-Tillering Dwarf Phenotype in Indica Rice Mutant gsor23

    Directory of Open Access Journals (Sweden)

    Shou-jiang YUAN

    2013-09-01

    Full Text Available High-tillering dwarf mutant gsor23 was generated from an indica rice variety Indica9 radiatied by γ-ray. Genetic analysis showed that this phenotype was controlled by one single recessive gene, which was mapped within a physical distance of 386 kb between two insertion-deletion (InDel markers C1-WT2 and C1-WT4 on the long arm of chromosome 1. There is a known gene D10 within this region, the mutation of which causes high-tillering in rice. Sequence analysis of the D10 allele in gsor23 revealed that the base cytosine (C at the 404th position in the coding region was deleted, which would cause frameshift mutation after the 134th amino acids. The mutation site and indica background of gsor23 were different from the previously reported japonica mutants d10-1 and d10-2. Therefore, gsor23 is a novel allelic mutant of D10 which encodes the carotenoid-cleaving dioxygenase 8 (CCD8, a key enzyme involved in the biosynthesis of the new plant hormone strigolactones (SLs. After treatment with GR24, a synthetic analogue of SLs, the high-tillering phenotype of gsor23 was restored to normal. Real-time RT-PCR analysis showed that D10 expression was high in roots, but low in leaves. Compared with the wild type Indica9, the expression of the SL biosynthesis gene D10 was upregulated, while genes likely involved in the SL signal transduction pathway such as D3 and D14 were down-regulated in the gsor23 mutant.

  5. Establishing a Markerless Genetic Exchange System for Methanosarcina mazei Strain Gö1 for Constructing Chromosomal Mutants of Small RNA Genes

    Directory of Open Access Journals (Sweden)

    Claudia Ehlers

    2011-01-01

    Full Text Available A markerless genetic exchange system was successfully established in Methanosarcina mazei strain Gö1 using the hpt gene coding for hypoxanthine phosphoribosyltransferase. First, a chromosomal deletion mutant of the hpt gene was generated conferring resistance to the purine analog 8-aza-2,6-diaminopurine (8-ADP. The nonreplicating allelic exchange vector (pRS345 carrying the pac-resistance cassette for direct selection of chromosomal integration, and the hpt gene for counterselection was introduced into this strain. By a pop-in and ultimately pop-out event of the plasmid from the chromosome, allelic exchange is enabled. Using this system, we successfully generated a M. mazei deletion mutant of the gene encoding the regulatory non-coding RNA sRNA154. Characterizing M. mazei Δ154 under nitrogen limiting conditions demonstrated differential expression of at least three cytoplasmic proteins and reduced growth strongly arguing for a prominent role of sRNA154 in regulation of nitrogen fixation by posttranscriptional regulation.

  6. A HOMOLOGOUS GENE-REPORTER SYSTEM FOR THE BASIDIOMYCETE SCHIZOPHYLLUM-COMMUNE BASED ON INTERNALLY DELETED HOMOLOGOUS GENES

    NARCIS (Netherlands)

    SCHUREN, FHJ; HARMSEN, MC; WESSELS, JGH

    Problems encountered in our attempts to achieve expression of heterologous genes, driven by ascomycetous regulatory sequences, in homobasidiomycetes led us to develop a gene-reporter system based on the expression of homologous genes in Schizophyllum commune. Internal deletions were made in the

  7. Alu-mediated large deletion of the CDSN gene as a cause of peeling skin disease.

    Science.gov (United States)

    Wada, T; Matsuda, Y; Muraoka, M; Toma, T; Takehara, K; Fujimoto, M; Yachie, A

    2014-10-01

    Peeling skin disease (PSD) is an autosomal recessive skin disorder caused by mutations in CDSN and is characterized by superficial peeling of the upper epidermis. Corneodesmosin (CDSN) is a major component of corneodesmosomes that plays an important role in maintaining epidermis integrity. Herein, we report a patient with PSD caused by a novel homozygous large deletion in the 6p21.3 region encompassing the CDSN gene, which abrogates CDSN expression. Several genes including C6orf15, PSORS1C1, PSORS1C2, CCHCR1, and TCF19 were also deleted, however, the patient showed only clinical features typical of PSD. The deletion size was 59.1 kb. Analysis of the sequence surrounding the breakpoint showed that both telomeric and centromeric breakpoints existed within Alu-S sequences that were oriented in opposite directions. These results suggest an Alu-mediated recombination event as the mechanism underlying the deletion in our patient.

  8. Gene deletion of cytosolic ATP: citrate lyase leads to altered organic acid production in Aspergillus niger

    DEFF Research Database (Denmark)

    Meijer, Susan Lisette; Nielsen, Michael Lynge; Olsson, Lisbeth

    2009-01-01

    With the availability of the genome sequence of the filamentous fungus Aspergillus niger, the use of targeted genetic modifications has become feasible. This, together with the fact that A. niger is well established industrially, makes this fungus an attractive micro-organism for creating a cell...... factory platform for production of chemicals. Using molecular biology techniques, this study focused on metabolic engineering of A. niger to manipulate its organic acid production in the direction of succinic acid. The gene target for complete gene deletion was cytosolic ATP: citrate lyase (acl), which...... the acl gene. Additionally, the total amount of organic acids produced in the deletion strain was significantly increased. Genome-scale stoichiometric metabolic model predictions can be used for identifying gene targets. Deletion of the acl led to increased succinic acid production by A. niger....

  9. [Comparative proteomics analysis of extracellular proteins from Listeria monocytogenes and its isogenic prfA deletion mutant].

    Science.gov (United States)

    Yin, Yuelan; Bai, Chunguang; Wang, Guoliang; Jia, Yanyan; Qu, Jin; Fu, Hong; Gao, Yunfei; Jiao, Xin'an

    2013-04-04

    Positive regulatory factor A (PrfA) protein plays a key role in the pathogenicity of Listeria monocytogenes by regulating the expression of virulence genes. We studied the regulation functions of PrfA and its role in Listeria monocytogenes (Lm) virulence. Extracellular proteins were obtained from the supernatants of parental strain LM4 and mutant strain LM4deltaprfA cultured in minimal medium. We used two-dimensional gel electrophoresis and matrix associated laser dissociation/ionization time of flight mass spectrometry (MALDI- TOF-MS) to analyze the differences of secreted proteins between LM4 and LM4deltaprfA. The electrophoresis results show that 31 different spots, 19 spots corresponding 12 proteins were identified by MALDI- TOF-MS. Some virulence related proteins were verified, such as InlC, ActA and LLO. Some new proteins that are regulated by PrfA include D-alanyl-D-alanine carboxypeptidase, dipeptide Glycine and Trytophan (GW) repeat-containing surface protein, transcriptional regulator and some hypothetical proteins with unknown functions. Real-time quantitative PCR was conducted to verify the proteomics results. The mRNA expression level of hly, actA and inlC gene was significantly reduced, and that of D-alanyl-D-alanine carboxypeptidase and GW repeat-containing surface protein's synthesis also had a reduction in LM4deltaprfA strain. PrfA plays key roles on the regulation of genes in LIPI- I and LIPI- II.

  10. Mapping of polyketide biosynthesis pathways in Aspergillus nidulans using a genome wide PKS gene deletion library

    DEFF Research Database (Denmark)

    Larsen, Thomas Ostenfeld; Rank, Christian; Klejnstrup, Marie Louise;

    In order to map new links between PKS genes and their products in Aspergillus nidulans we have systematically deleted all thirty-two individual genes predicted to encode polyketide synthases in this model organism. This number greatly exceeds the number of currently known PKs calling for new...

  11. 不同基因缺失型酵母对全氟辛酸的灵敏性%Sensitivity of different gene deletion yeast to perfluorooctanoic acid

    Institute of Scientific and Technical Information of China (English)

    金士威; 程巧红; 戴和平

    2012-01-01

    针对全氟化合物这类新型的有机污染物,在环境中具有持久性,生物累积性和毒性的问题,以全氟辛酸为模型化合物,以真核生物酵母以及4基因缺失型酵母突变体和5基因缺失型酵母突变体为模式生物,开发便捷、灵敏的毒理学评价方法,探究全氟辛酸对不同基因缺失型酵母的毒性效应,以期建立以酵母为平台的环境污染物快速筛选系统.研究结果表明,5基因缺失型酵母突变体对全氟辛酸较为敏感,可以作为建立环境污染物快速筛选系统的细胞模型.%Perfluorinated compounds ( PFCs) are a class of emerging organic contaminants with persistent stability, strong bioaccumulation and high toxicity. To develop a convenient, sensitive toxicological evaluation method, perfluorooctanoic acid ( PFOA) was used as a model compound and eukaryotic yeast and its 4 genes deletion yeast mutant and 5 genes deletion yeast mutant were used as model organisms. The toxic effects of PFOA on different gene deletion yeast were studied. The results show that 5 genes deletion yeast mutant is more sensitive to PFOA, which can be used as a cell model to establish rapid screening system of environmental pollutants.

  12. [Biological characteristics of an Hog1 MAPK homologous gene FoHog1 knock-out mutant of Fusarium oxysporum f. sp. cubense].

    Science.gov (United States)

    Mao, Chao; Chen, Pingya; Dai, Qingdong; Yang, Laying; Huang, Junsheng

    2014-11-04

    This study was aimed to obtain a mitogen-activated protein kinase (MAPK) gene namely FoHog1 from Fusarium oxysporum f. sp. cubense and to verify its function. We amplified FoHog1 gene by PCR and RT-PCR methods and analyzed it through bioinformatics method. PEG-mediated protoplast transformation was used to create the deletion mutants of FoHog1 gene. We analyzed different biological characteristics between knock-out strain and wild-type strain. FoHog1 gene encoding a putative protein of 357 amino acids and its genetic relationship with different Fusarium' s protein. Compared with the wild-type strain, FoHog1 deletion mutants have loose hyphae colony, less spores production, lower dry weight of hyphae and more sensitive to temperature, pH and osmotic stress. FoHog1 deletion mutants also have reduced colonization ability compared with the wild-type strain. FoHog1 gene participated in mycelial growth, sporulation, catabolism of sodium acetate and ammonium chloride, osmotic stress response and pathogenic process with Fusarium oxysporum f. sp. cubense Race 4.

  13. Effect of the deletion of qmoABC and the promoter distal gene encoding a hypothetical protein on sulfate-reduction in Desulfovibrio vulgaris Hildenborough

    Energy Technology Data Exchange (ETDEWEB)

    Zane, Grant M.; Yen, Huei-chi Bill; Wall, Judy D.

    2010-03-18

    The pathway of electrons required for the reduction of sulfate in sulfate-reducing bacteria (SRB) is not yet fully characterized. In order to determine the role of a transmembrane protein complex suggested to be involved in this process, a deletion of Desulfovibrio vulgaris Hildenborough was created by marker exchange mutagenesis that eliminated four genes putatively encoding the QmoABC complex and a hypothetical protein (DVU0851). The Qmo complex (quinone-interacting membrane-bound oxidoreductase) is proposed to be responsible for transporting electrons to the dissimilatory adenosine-5?phosphosulfate (APS) reductase in SRB. In support of the predicted role of this complex, the deletion mutant was unable to grow using sulfate as its sole electron acceptor with a range of electron donors. To explore a possible role for the hypothetical protein in sulfate reduction, a second mutant was constructed that had lost only the gene that codes for DVU0851. The second constructed mutant grew with sulfate as the sole electron acceptor; however, there was a lag that was not present with the wild-type or complemented strain. Neither deletion strain was significantly impaired for growth with sulfite or thiosulfate as terminal electron acceptor. Complementation of the D(qmoABC-DVU0851) mutant with all four genes or only the qmoABC genes restored its ability to grow by sulfate respiration. These results confirmed the prediction that the Qmo complex is in the electron pathway for sulfate-reduction and revealed that no other transmembrane complex could compensate when Qmo was lacking.

  14. Deletion of Braun lipoprotein and plasminogen-activating protease-encoding genes attenuates Yersinia pestis in mouse models of bubonic and pneumonic plague.

    Science.gov (United States)

    van Lier, Christina J; Sha, Jian; Kirtley, Michelle L; Cao, Anthony; Tiner, Bethany L; Erova, Tatiana E; Cong, Yingzi; Kozlova, Elena V; Popov, Vsevolod L; Baze, Wallace B; Chopra, Ashok K

    2014-06-01

    Currently, there is no FDA-approved vaccine against Yersinia pestis, the causative agent of bubonic and pneumonic plague. Since both humoral immunity and cell-mediated immunity are essential in providing the host with protection against plague, we developed a live-attenuated vaccine strain by deleting the Braun lipoprotein (lpp) and plasminogen-activating protease (pla) genes from Y. pestis CO92. The Δlpp Δpla double isogenic mutant was highly attenuated in evoking both bubonic and pneumonic plague in a mouse model. Further, animals immunized with the mutant by either the intranasal or the subcutaneous route were significantly protected from developing subsequent pneumonic plague. In mice, the mutant poorly disseminated to peripheral organs and the production of proinflammatory cytokines concurrently decreased. Histopathologically, reduced damage to the lungs and livers of mice infected with the Δlpp Δpla double mutant compared to the level of damage in wild-type (WT) CO92-challenged animals was observed. The Δlpp Δpla mutant-immunized mice elicited a humoral immune response to the WT bacterium, as well as to CO92-specific antigens. Moreover, T cells from mutant-immunized animals exhibited significantly higher proliferative responses, when stimulated ex vivo with heat-killed WT CO92 antigens, than mice immunized with the same sublethal dose of WT CO92. Likewise, T cells from the mutant-immunized mice produced more gamma interferon (IFN-γ) and interleukin-4. These animals had an increasing number of tumor necrosis factor alpha (TNF-α)-producing CD4(+) and CD8(+) T cells than WT CO92-infected mice. These data emphasize the role of TNF-α and IFN-γ in protecting mice against pneumonic plague. Overall, our studies provide evidence that deletion of the lpp and pla genes acts synergistically in protecting animals against pneumonic plague, and we have demonstrated an immunological basis for this protection.

  15. Stable disruption of ethanol production by deletion of the genes encoding alcohol dehydrogenase isozymes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Ida, Yoshihiro; Furusawa, Chikara; Hirasawa, Takashi; Shimizu, Hiroshi

    2012-02-01

    We analyzed the effects of the deletions of genes encoding alcohol dehydrogenase (ADH) isozymes of Saccharomyces cerevisiae. The decrease in ethanol production by ADH1 deletion alone could be partially compensated by the upregulation of other isozyme genes, while the deletion of all known ADH isozyme genes stably disrupted ethanol production. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  16. EXPRESSION AND DELETION ANALYSIS OF EcoRII ENDONUCLEASE AND METHYLASE GENE

    Institute of Scientific and Technical Information of China (English)

    刘金毅; 赵晓娟; 孟雁; 沈洁; 薛越强; 史顺娣; 蔡有余

    2001-01-01

    Objective. To clone complete EcoRII restriction endonuclease gene (ecoRllR) and methyltransferase gene(ecoRllM) in one ector and to analyze the coordinating expression of this whole R-M system.Methods. Unidirectional deletion subclones were constructed with ExolII. ecoRllR/M genes were preliminari-ly located in the cloned fragment according to the enzyme activities of subclones. Exact deletion sites were deter-mined by sequencing, and transcriptional start sites were determined by S1 mapping.Results. The DNA fragment which was cloned into pBluescript SK + contained intact ecoRIlR gene andecoRllM gene, anc two transcriptional start sites of ecoRllR gene were determined. 132bp to 458bp from 3' endof ecoRllR gene ar.e indispensable to enzyme activities and deletion of 202bp from 3' end of ecoRllM gene madeenzyme lose the capability in DNA protection to resist specific cut with EcoRII endonuclease (EcoRII. R). Dele-tion of the coding ar d flanking sequences of one gene did not affect the expression of the other gene, and the recombi-nants only containing ecoRllR gene appeared to be lethal to dcm+ host.Conclusion. scoRllM gene linking closely to ecoRIIR gene is very important for the existence of the R-M sys-tem in process of evolution, but the key to control EcoRlI R-M order may not exist in transcriptional level .``Liu Jmy,Corresponding author.

  17. Escherichia coli deletion mutants illuminate trade-offs between growth rate and flux through a foreign anabolic pathway.

    Science.gov (United States)

    Falls, Kelly C; Williams, Aimee L; Bryksin, Anton V; Matsumura, Ichiro

    2014-01-01

    Metabolic engineers strive to improve the production yields of microbial fermentations, sometimes by mutating the genomes of production strains. Some mutations are detrimental to the health of the organism, so a quantitative and mechanistic understanding of the trade-offs could inform better designs. We employed the bacterial luciferase operon (luxABCDE), which uses ubiquitous energetic cofactors (NADPH, ATP, FMNH2, acetyl-CoA) from the host cell, as a proxy for a novel anabolic pathway. The strains in the Escherichia coli Keio collection, each of which contains a single deletion of a non-essential gene, represent mutational choices that an engineer might make to optimize fermentation yields. The Keio strains and the parental BW25113 strain were transformed with a luxABCDE expression vector. Each transformant was propagated in defined M9 medium at 37 °C for 48 hours; the cell density (optical density at 600 nanometers, OD600) and luminescence were measured every 30 minutes. The trade-offs were visualized by plotting the maximum growth rate and luminescence/OD600 of each transformant across a "production possibility frontier". Our results show that some loss-of-function mutations enhance growth in vitro or light production, but that improvement in one trait generally comes at the expense of the other.

  18. Scarless deletion of up to seven methyl-accepting chemotaxis genes with an optimized method highlights key function of CheM in Salmonella Typhimurium.

    Science.gov (United States)

    Hoffmann, Stefanie; Schmidt, Christiane; Walter, Steffi; Bender, Jennifer K; Gerlach, Roman G

    2017-01-01

    Site-directed scarless mutagenesis is an essential tool of modern pathogenesis research. We describe an optimized two-step protocol for genome editing in Salmonella enterica serovar Typhimurium to enable multiple sequential mutagenesis steps in a single strain. The system is based on the λ Red recombinase-catalyzed integration of a selectable antibiotics resistance marker followed by replacement of this cassette. Markerless mutants are selected by expressing the meganuclease I-SceI which induces double-strand breaks in bacteria still harboring the resistance locus. Our new dual-functional plasmid pWRG730 allows for heat-inducible expression of the λ Red recombinase and tet-inducible production of I-SceI. Methyl-accepting chemotaxis proteins (MCP) are transmembrane chemoreceptors for a vast set of environmental signals including amino acids, sugars, ions and oxygen. Based on the sensory input of MCPs, chemotaxis is a key component for Salmonella virulence. To determine the contribution of individual MCPs we sequentially deleted seven MCP genes. The individual mutations were validated by PCR and genetic integrity of the final seven MCP mutant WRG279 was confirmed by whole genome sequencing. The successive MCP mutants were functionally tested in a HeLa cell infection model which revealed increased invasion rates for non-chemotactic mutants and strains lacking the MCP CheM (Tar). The phenotype of WRG279 was reversed with plasmid-based expression of CheM. The complemented WRG279 mutant showed also partially restored chemotaxis in swarming assays on semi-solid agar. Our optimized scarless deletion protocol enables efficient and precise manipulation of the Salmonella genome. As demonstrated with whole genome sequencing, multiple subsequent mutagenesis steps can be realized without the introduction of unwanted mutations. The sequential deletion of seven MCP genes revealed a significant role of CheM for the interaction of S. Typhimurium with host cells which might give

  19. Becker muscular dystrophy in Indian patients: Analysis of dystrophin gene deletion patterns

    Directory of Open Access Journals (Sweden)

    Dastur Rashna

    2008-01-01

    Full Text Available Background: Becker muscular dystrophy (BMD is caused by mutations in the dystrophin gene with variable phenotypes. Becker muscular dystrophy patients have low levels of nearly full-length dystrophin and carry in-frame mutations, which allow partial functioning of the protein. Aim: To study the deletion patterns of BMD and to correlate the same with reading frame rule and different phenotypes. Setting: A tertiary care teaching hospital. Design: This is a prospective hospital-based study. Materials and Methods: Thirty-two exons spanning different "hot spot" regions using Multiplex PCR techniques were studied in 347 patients. Two hundred and twenty-two showed deletions in one or more of the 32 exons. Out of these, 46 diagnosed as BMD patients were analyzed. Results: Forty-six BMD patients showed deletions in both regions of the dystrophin gene. Out of these 89.1% (41/46 were in-frame deletions. Deletions starting with Exon 45 were found in 76.1% (35/46 of the cases. Mutations in the majority of cases i.e. 39/46 (84.8% were seen in 3′ downstream region (Exon 45-55, distal rod domain. Few, i.e. 5/46 (10.8% showed deletions in 5′ upstream region (Exons 3-20, N-terminus and proximal rod domain of the gene, while in 2/46 (4.4% large mutations (>40 bp spanning both regions (Exons 3-55 were detected. Conclusion: This significant gene deletion analysis has been carried out for BMD patients particularly from Western India using 32 exons.

  20. [Mutant gene expression in murine aggregation chimeras. 5. The ocular retardation and fidget genes].

    Science.gov (United States)

    Kindiakov, B N; Koniukhov, B V

    1986-01-01

    Analysis of ocular retardation (or) and fidget (fi) genes expression in 18 day old embryos, 10 and 20 day old or/or C/C----+/+ c/c and fi/fi or/or C/C----+/+ +/+ c/c mice has shown that genes or and fi are active in developing retina and suppress cell proliferation. Structural defects of retina and decrease in the eye size in the chimaeras, compared to the normal embryos, were observed already in the presence of 13-16% of mutant cells. As the fraction of mutant cells increased, the degree of eye disturbances increased as well. In the fi/fi or/or----+/+ +/+ chimaeras structural defects of retina and decrease in the eye size are more pronounced than in the or/or----+/+ chimaeras, due to the synergetical effect of both mutant genes in the fi/fi or/or cell clones. In the ontogenesis of the or/or----+/+ chimaeras the development of the retinal photoreceptor layer is normalized due to the substitution of mutant cells for actively proliferating normal cells. No metabolic cooperation between the mutant and normal cells was observed in the developing retina of chimaeras.

  1. Construction and Verification of LuxS-negative Mutants of Streptococcus Mutans and the Effect of the Absence of LuxS Gene on the Acid Tolerance

    Institute of Scientific and Technical Information of China (English)

    YU Dan-ni; CHEN Jie; ZHANG Yao-chao; HAN Yu-zhi

    2009-01-01

    Objective: To knock out the entire Luxs gene of Streptococcus mutans(S.mutans) UA159 strain via homologous recombination and construct a Luxs-deleted mutant strain of S. Mutans. To study the difference between the acid resistance of S. Mutans Ingbritt C international standard strain and the acid resistance of LuxS mutant strain. Methods: Two DNA fragments locating in the upper and downstream of Luxs gene were amplified and a erythromycin resistance gene of PJT10 between them were engineered into PUC19 plasmid for constructing the recombination plasmid pUCluxKO. Electrotransformation of S.mutans cells with pUCluxKO-mutant resulted in isolation of erythromycin resistant S. Mutans transformants, which was identified by polymerase chain reaction, V.harveyi BB170 luminescence bioassay and sequencing analysis. Solutions of S. Mutans standard strain and LuxS mutant strain with same density were made and cultured at pH 3.5 to 7.0 BHI liquid for the same period.Terminal growth situation was compared.Firstly acidized in pH 5.5 BHI liquid,the two strains were cultured at pH 3.0 BHI liquid. The acid tolerance responses of the two strains were compared.Results:Restriction endonuclease analyses showed that pUCluxKO-mutant vector had been successfully recombined. The Luxs-deleted status of S.mutans mutants was confirmed by PCR with primers which were specific for the genes of Luxs and Erythromycin resistance. S.mutans mutant can not induce bioluminescence, indiating the mutant had been successfully recombined. After twenty generations of culture, the constructed Chinese S.mutans mutants were confirmed to be stable. Significant difference of aciduricity was observed between S.mutans standard strain and LuxS mutant strain.The acid resistance of standard strain was stronger than that of LuxS mutant strain.The two strains both displayed the capability of acid tolerance responses. Conclusion:The S.mutans gene allelic exchange plasmid is constructed correctively and a Luxs

  2. A Catalog of Genes Homozygously Deleted in Human Lung Cancer and the Candidacy of PTPRD as a Tumor Suppressor Gene

    Science.gov (United States)

    Kohno, Takashi; Otsuka, Ayaka; Girard, Luc; Sato, Masanori; Iwakawa, Reika; Ogiwara, Hideaki; Sanchez-Cespedes, Montse; Minna, John D.; Yokota, Jun

    2010-01-01

    A total of 176 genes homozygously deleted in human lung cancer were identified by DNA array-based whole genome scanning of 52 lung cancer cell lines and subsequent genomic PCR in 74 cell lines, including the 52 cell lines scanned. One or more exons of these genes were homozygously deleted in one (1%) to 20 (27%) cell lines. These genes included known tumor suppressor genes, e.g., CDKN2A/p16, RB1, and SMAD4, and candidate tumor suppressor genes whose hemizygous or homozygous deletions were reported in several types of human cancers, such as FHIT, KEAP1, and LRP1B/LRP-DIP. CDKN2A/p16 and p14ARF located in 9p21 were most frequently deleted (20/74, 27%). The PTPRD gene was most frequently deleted (8/74, 11%) among genes mapping to regions other than 9p21. Somatic mutations, including a nonsense mutation, of the PTPRD gene were detected in 8/74 (11%) of cell lines and 4/95 (4%) of surgical specimens of lung cancer. Reduced PTPRD expression was observed in the majority (>80%) of cell lines and surgical specimens of lung cancer. Therefore, PTPRD is a candidate tumor suppressor gene in lung cancer. Microarray-based expression profiling of 19 lung cancer cell lines also indicated that some of the 176 genes, such as KANK and ADAMTS1, are preferentially inactivated by epigenetic alterations. Genetic/epigenetic as well as functional studies of these 176 genes will increase our understanding of molecular mechanisms behind lung carcinogenesis. PMID:20073072

  3. Copy Number Deletion Has Little Impact on Gene Expression Levels in Racehorses

    Directory of Open Access Journals (Sweden)

    Kyung-Do Park

    2014-09-01

    Full Text Available Copy number variations (CNVs, important genetic factors for study of human diseases, may have as large of an effect on phenotype as do single nucleotide polymorphisms. Indeed, it is widely accepted that CNVs are associated with differential disease susceptibility. However, the relationships between CNVs and gene expression have not been characterized in the horse. In this study, we investigated the effects of copy number deletion in the blood and muscle transcriptomes of Thoroughbred racing horses. We identified a total of 1,246 CNVs of deletion polymorphisms using DNA re-sequencing data from 18 Thoroughbred racing horses. To discover the tendencies between CNV status and gene expression levels, we extracted CNVs of four Thoroughbred racing horses of which RNA sequencing was available. We found that 252 pairs of CNVs and genes were associated in the four horse samples. We did not observe a clear and consistent relationship between the deletion status of CNVs and gene expression levels before and after exercise in blood and muscle. However, we found some pairs of CNVs and associated genes that indicated relationships with gene expression levels: a positive relationship with genes responsible for membrane structure or cytoskeleton and a negative relationship with genes involved in disease. This study will lead to conceptual advances in understanding the relationship between CNVs and global gene expression in the horse.

  4. Deletion of Salmonella enterica serovar typhimurium sipC gene

    Institute of Scientific and Technical Information of China (English)

    Maryam; Safarpour; Dehkordi; Abbas; Doosti; Asghar; Arshi

    2015-01-01

    Objective:To construct a novel plasmid as Salmonella enterica serovar typhimurium(S.typhimurium)sip C gene knockouts candidate.Methods:In this research,50upstream and 30downstream regions of S.typhimurium sip C gene and kanamycin gene were PCR amplified.Each of these DNA fragment was cloned into p GEM T-easy vector.The construct was confirmed by PCR and restriction digest.Results:PCR amplified 320,206 and 835 bp DNA fragments were subcloned into p ET-32 vector resulting with a plasmid called p ET-32-sip C up-kan-sip C down.Conclusions:The new plasmid(p ET-32-sip C up-kan-sip C down)is useful for genetic engineering and for future manipulation of S.typhimurium sip C gene.

  5. Deletion of Salmonella enterica serovar typhimurium sipC gene

    Institute of Scientific and Technical Information of China (English)

    Maryam Safarpour Dehkordi; Abbas Doosti; Asghar Arshi

    2015-01-01

    Objective: To construct a novel plasmid as Salmonella enterica serovar typhimurium (S. typhimurium) sipC gene knockouts candidate. Methods: In this research, 50 upstream and 30 downstream regions of S. typhimurium sipC gene and kanamycin gene were PCR amplified. Each of these DNA fragment was cloned into pGEM T-easy vector. The construct was confirmed by PCR and restriction digest. Results: PCR amplified 320, 206 and 835 bp DNA fragments were subcloned into pET-32 vector resulting with a plasmid called pET-32-sipC up-kan-sip C down. Conclusions: The new plasmid (pET-32-sipC up-kan-sip C down) is useful for genetic engineering and for future manipulation of S. typhimurium sipC gene.

  6. Rb and p53 gene deletions in lung adenocarcinomas from irradiated and control mice

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Y.; Woloschak, G.E. [Argonne National Lab., IL (United States). Center for Mechanistic Biology and Biotechnology

    1997-08-01

    This study was conducted on mouse lung adenocarcinoma tissues that were formalin-treated and paraffin-embedded 25 years ago to investigate the large gene deletions of mRb and p53 in B6CF{sub 1} male mice. A total of 80 lung tissue samples from irradiated mice and 40 lung samples from nonirradiated controls were randomly selected and examined in the mRb portion of this study. The results showed a significant (P < 0.05) higher percentage of mRb deletions in lung adenocarcinomas from mice exposed to 60 once-weekly {gamma}-ray doses than those from mice receiving 24 once-weekly {gamma}-ray doses at low doses and low dose rates; however, the percentage was not significantly different (P > 0.05) from that for spontaneous lung adenocarcinomas or lung adenocarcinomas from mice exposed to single-dose {gamma} irradiation at a similar total dose. mRb fragments 3 (71%) and 5 (67%), the parts of the gene that encoded the pocket binding region of Rb protein to adenovirus E1A and SV40 T-antigen, were the most frequently deleted fragments. p53 gene deletion analysis was carried out on normal lungs and lung adenocarcinomas that were initially found to bear mRb deletions. Exons 1,4,5,6, and 9 were chosen to be analyzed.

  7. Screening of Dystrophin Gene Deletions in Egyptian Patients with DMD/BMD Muscular Dystrophies

    Directory of Open Access Journals (Sweden)

    Laila K. Effat

    2000-01-01

    Full Text Available Duchenne muscular dystrophy (DMD and Becker muscular dystrophy (BMD are allelic disorders caused by mutations within the dystrophin gene. Our study has identified 100 Egyptian families collected from the Human Genetics Clinic, National Research Center, Cairo. All cases were subjected to complete clinical evaluation pedigree analysis, electromyography studies, estimation of serum creatine phosphokinase enzyme (CPK levels and DNA analysis. Multiplex PCR using 18 pairs of specific primers were used for screening of deletion mutations within the dystrophin gene. A frequency of 55% among the families. Sixty per cent of detected deletions involved multiple exons spanning the major or the minor hot spot of the dystrophin gene. The remainder 40% which mainly involved exon 45. Comparing these findings with frequencies of other countries it was found that our figures fall within the reported range of 40%– for deletions. The distribution of deletions in our study and other different studies was variable and specific ethnic differences do not apparently account for specific deletions. In addition this study concluded that employment of the 18 exon analysis is a cost effective and a highly accurate (97% to launch a nationwide program.

  8. Optimal in silico target gene deletion through nonlinear programming for genetic engineering.

    Science.gov (United States)

    Hong, Chung-Chien; Song, Mingzhou

    2010-02-24

    Optimal selection of multiple regulatory genes, known as targets, for deletion to enhance or suppress the activities of downstream genes or metabolites is an important problem in genetic engineering. Such problems become more feasible to address in silico due to the availability of more realistic dynamical system models of gene regulatory and metabolic networks. The goal of the computational problem is to search for a subset of genes to knock out so that the activity of a downstream gene or a metabolite is optimized. Based on discrete dynamical system modeling of gene regulatory networks, an integer programming problem is formulated for the optimal in silico target gene deletion problem. In the first result, the integer programming problem is proved to be NP-hard and equivalent to a nonlinear programming problem. In the second result, a heuristic algorithm, called GKONP, is designed to approximate the optimal solution, involving an approach to prune insignificant terms in the objective function, and the parallel differential evolution algorithm. In the third result, the effectiveness of the GKONP algorithm is demonstrated by applying it to a discrete dynamical system model of the yeast pheromone pathways. The empirical accuracy and time efficiency are assessed in comparison to an optimal, but exhaustive search strategy. Although the in silico target gene deletion problem has enormous potential applications in genetic engineering, one must overcome the computational challenge due to its NP-hardness. The presented solution, which has been demonstrated to approximate the optimal solution in a practical amount of time, is among the few that address the computational challenge. In the experiment on the yeast pheromone pathways, the identified best subset of genes for deletion showed advantage over genes that were selected empirically. Once validated in vivo, the optimal target genes are expected to achieve higher genetic engineering effectiveness than a trial

  9. Optimal in silico target gene deletion through nonlinear programming for genetic engineering.

    Directory of Open Access Journals (Sweden)

    Chung-Chien Hong

    Full Text Available BACKGROUND: Optimal selection of multiple regulatory genes, known as targets, for deletion to enhance or suppress the activities of downstream genes or metabolites is an important problem in genetic engineering. Such problems become more feasible to address in silico due to the availability of more realistic dynamical system models of gene regulatory and metabolic networks. The goal of the computational problem is to search for a subset of genes to knock out so that the activity of a downstream gene or a metabolite is optimized. METHODOLOGY/PRINCIPAL FINDINGS: Based on discrete dynamical system modeling of gene regulatory networks, an integer programming problem is formulated for the optimal in silico target gene deletion problem. In the first result, the integer programming problem is proved to be NP-hard and equivalent to a nonlinear programming problem. In the second result, a heuristic algorithm, called GKONP, is designed to approximate the optimal solution, involving an approach to prune insignificant terms in the objective function, and the parallel differential evolution algorithm. In the third result, the effectiveness of the GKONP algorithm is demonstrated by applying it to a discrete dynamical system model of the yeast pheromone pathways. The empirical accuracy and time efficiency are assessed in comparison to an optimal, but exhaustive search strategy. SIGNIFICANCE: Although the in silico target gene deletion problem has enormous potential applications in genetic engineering, one must overcome the computational challenge due to its NP-hardness. The presented solution, which has been demonstrated to approximate the optimal solution in a practical amount of time, is among the few that address the computational challenge. In the experiment on the yeast pheromone pathways, the identified best subset of genes for deletion showed advantage over genes that were selected empirically. Once validated in vivo, the optimal target genes are

  10. Type I oculocutaneous albinism (OCA1) associated with a large deletion of the tyrosinase (TYR) gene

    Energy Technology Data Exchange (ETDEWEB)

    Spritz, R.A.; Wick, P.A.; Holmes, S.A.; Schnur, R.E. [Univ. of Wisconsin, Madison, WI (United States)]|[Children`s Hospital of Philadelphia, PA (United States)

    1994-09-01

    OCA1 is an autosomal recessive disorder in which the biosynthesis of melanin is reduced or absent in skin, hair, and eyes, due to deficient enzymatic activity of tyrosinase. TYR consists of 5 exons spanning over 65 kb at 11q14-q21. Analyses of TYR in >400 unrelated patients with OCA1 have identified more than 50 different point mutations; however, no large deletions have been detected. Here we report a large deletion of TYR in a Caucasian boy with OCA1B. Simultaneous SSCP/heteroduplex screening and DNA sequence analysis indicated that the patient was apparently homozygous for a previously described TYR mutation, adjacent to the 3` splice site of IVS2 (-7, t{r_arrow}a). To distinguish between possible gene deletion vs. maternal uniparental isodisomy, we characterized several chromosome 11 polymorphisms. Maternal uniparental isodisomy was excluded by the patient`s heterozygosity for alleles at D11S35 (11q21-122) and HBG2 (11p15.5). In addition, the patient failed to inherit paternal alleles at an MboI RFLP in exon 1 of TYR and at a TaqI RFLP in the promoter region of the gene. To detect a possible submicroscopic deletion, we performed quantitative Southern blot hybridization using a full length TYR cDNA. Compared with controls, both the patient and his father appeared deleted for two or three TYR-derived PstI fragments; the two TYRL-derived fragments appeared normal. These data indicate that the patient and his father have a partial TYR deletion, including at least exons 1, 2, and IVS2. Based on the organization of the gene, this deletion is at least 50 kb in size. The patient is thus hemizygous for the maternally-inherited mutation in IVS2, accounting for his OCA1B phenotype.

  11. The single N-glycan deletion mutant of soluble ErbB3 protein attenuates heregulin β1-induced tumor progression by blocking of the HIF-1 and Nrf2 pathway

    Energy Technology Data Exchange (ETDEWEB)

    Takamiya, Rina, E-mail: rinataka0429@gmail.com; Takahashi, Motoko; Uehara, Yasuaki; Ariki, Shigeru; Hashimoto, Jiro; Hasegawa, Yoshihiro; Kuroki, Yoshio

    2014-11-21

    Highlights: • The sErbB3 N418Q mutant blocks heregulin β1 induced nuclear accumulation of HIF-1α. • The sErbB3 N418Q mutant attenuates cancer cell migration induced by heregulin β1. • The sErbB3 N418Q mutant blocks heregulin β1 induced nuclear accumulation of Nrf2. • The sErbB3 N418Q mutant may be a potential therapeutic application for tumor. - Abstract: It has been well documented that activation of the ErbB3–PI3K–Akt pathway is implicated in tumor survival and progression. We previously demonstrated that the single N-glycan deletion mutant of soluble ErbB3 protein (sErbB3 N418Q) attenuates heregulin β1-induced ErbB3 signaling. The active PI3K–Akt pathway augments the nuclear accumulation of hypoxia inducible factor (HIF)-1α, which activates the transcription of many target genes and drives cancer progression. In this study, we focused on the effects of sErbB3 N418Q mutant on nuclear accumulation of HIF-1α. Pretreatment with the sErbB3 N418Q mutant suppressed heregulin β1-induced HIF-1α activation in MCF7 cells. Similar results were also obtained in other breast cancer cell lines, T47D and BT474. Interestingly, these suppressive effects were not observed with the sErbB3 wild type. In addition, pretreatment with the sErbB3 N418Q mutant suppressed the cell migration of MCF7 cells induced by heregulin β1. Furthermore, incubation with heregulin β1 also induced the nuclear accumulation of Nrf2, and this effect was also reduced by the sErbB3 N418Q mutant, but not the sErbB3 wild type. These findings indicated that the sErbB3 N418Q mutant suppressed malignant formation of cancer cells by blocking of the HIF-1α and Nrf2 pathways.

  12. Deletion of glucose-inhibited division (gidA) gene alters the morphological and replication characteristics of Salmonella enterica Serovar typhimurium.

    Science.gov (United States)

    Shippy, Daniel C; Heintz, Joseph A; Albrecht, Ralph M; Eakley, Nicholas M; Chopra, Ashok K; Fadl, Amin A

    2012-06-01

    Salmonella is an important food-borne pathogen that continues to plague the United States food industry. Characterization of bacterial factors involved in food-borne illnesses could help develop new ways to control salmonellosis. We have previously shown that deletion of glucose-inhibited division gene (gidA) significantly altered the virulence potential of Salmonella in both in vitro and in vivo models of infection. Most importantly, the gidA mutant cells displayed a filamentous morphology compared to the wild-type Salmonella cells. In our current study, we investigated the role of GidA in Salmonella cell division using fluorescence and electron microscopy, transcriptional, and proteomic assays. Scanning electron microscopy data indicated a filamentous morphology with few constrictions in the gidA mutant cells. The filamentation of the gidA mutant cells is most likely due to the defect in chromosome segregation, with little to no sign of septa formation observed using fluorescence and transmission electron microscopy. Furthermore, deletion of gidA altered the expression of many genes and proteins responsible for cell division and chromosome segregation as indicated by global transcriptional profiling and semi-quantitative western blot analysis. Taken together, our data indicate GidA as a potential regulator of Salmonella cell division genes.

  13. Recombinant Escherichia coli K5 strain with the deletion of waaR gene decreases the molecular weight of the heparosan capsular polysaccharide.

    Science.gov (United States)

    Huang, Haichan; Liu, Xiaobo; Lv, Shencong; Zhong, Weihong; Zhang, Fuming; Linhardt, Robert J

    2016-09-01

    Heparosan, the capsular polysaccharide of Escherichia coli K5 having a carbohydrate backbone similar to that of heparin, has become a potential precursor for bioengineering heparin. In the heparosan biosynthesis pathway, the gene waaR encoding α-1-, 2- glycosyltransferase catalyze s the third glucosyl residues linking to the oligosaccharide chain. In the present study, a waaR deletion mutant of E. coli K5 was constructed. The mutant showed improvement of capsule polysaccharide yield. It is interesting that the heparosan molecular weight of the mutant is reduced and may become more suitable as a precursor for the production of low molecular weight heparin derived from the wild-type K5 capsular polysaccharide.

  14. Deletion of the msdS/AfmsdC gene induces abnormal polarity and septation in Aspergillus fumigatus.

    Science.gov (United States)

    Li, Yanjie; Zhang, Lei; Wang, Depeng; Zhou, Hui; Ouyang, Haomiao; Ming, Jia; Jin, Cheng

    2008-07-01

    alpha-Mannosidases play an important role in the processing of mannose-containing glycans in eukaryotes. A deficiency in alpha-mannosidase is lethal in humans and cattle. In contrast to mammals, Saccharomyces cerevisiae does not require the endoplasmic reticulum alpha-mannosidase gene for growth. However, little is known of the consequence of loss of function of class I alpha-mannosidases in filamentous fungi. In this study, the msdS/AfmsdC gene was identified to encode 1,2-alpha-mannosidase MsdS in Aspergillus fumigatus. Soluble MsdS expressed in Escherichia coli was characterized as a typical class I alpha-mannosidase. The msdS gene was deleted by replacement of the msdS gene with a pyrG gene. Although the mutant showed a defect in N-glycan processing, as well as a reduction of cell wall components and a reduced ability of conidiation, it appeared that the rate of hyphal growth was not affected. Morphology analysis revealed abnormal polarity and septation at the stages of germination, hyphal growth and conidiation. Although the mechanism by which the N-glycan processing affects polarity and septation is unclear, our results show that msdS is involved in polarity and septation in A. fumigatus.

  15. Deletion of Cmu genes in mouse B lymphocytes upon stimulation with LPS.

    Science.gov (United States)

    Radbruch, A; Sablitzky, F

    1983-01-01

    Mouse B lymphocytes can be activated polyclonally by bacterial lipopolysaccharide (LPS) to differentiate into plasmablasts. Within several days many cells perform immunoglobulin (Ig) class switching in vitro. We have purified LPS blasts expressing IgM or only IgG3 on the cell surface and analysed the DNA of these cells by Southern hybridisation blotting to detect rearrangement or deletion of CH genes. Quantitative evaluation of the Southern blots suggests that populations of surface IgG3+ (sIgG3+) cells from 6-day and sIgM+ cells from 8-day-old cultures contain only about half as many Cmu genes as spleen cells. Cmu deletion is nearly complete in populations of sIgG3+ cells from 9-day-old cultures. Therefore, upon stimulation with LPS, within a few days Cmu is deleted in most sIgG3+ cells from both chromosomes.

  16. Histone Deacetylase 3 Inhibition Overcomes BIM Deletion Polymorphism-Mediated Osimertinib Resistance in EGFR-Mutant Lung Cancer.

    Science.gov (United States)

    Tanimoto, Azusa; Takeuchi, Shinji; Arai, Sachiko; Fukuda, Koji; Yamada, Tadaaki; Roca, Xavier; Ong, S Tiong; Yano, Seiji

    2016-12-16

    Purpose: The BIM deletion polymorphism is associated with apoptosis resistance to EGFR tyrosine kinase inhibitors (EGFR-TKI), such as gefitinib and erlotinib, in non-small cell lung cancer (NSCLC) harboring EGFR mutations. Here, we investigated whether the BIM deletion polymorphism contributes to resistance against osimertinib, a third-generation EGFR-TKI. In addition, we determined the efficacy of a histone deacetylase (HDAC) inhibitor, vorinostat, against this form of resistance and elucidated the underlying mechanism.Experimental Design: We used EGFR-mutated NSCLC cell lines, which were either heterozygous or homozygous for the BIM deletion polymorphism, to evaluate the effect of osimertinib in vitro and in vivo Protein expression was examined by Western blotting. Alternative splicing of BIM mRNA was analyzed by RT-PCR.Results:EGFR-mutated NSCLC cell lines with the BIM deletion polymorphism exhibited apoptosis resistance to osimertinib in a polymorphism dosage-dependent manner, and this resistance was overcome by combined use with vorinostat. Experiments with homozygous BIM deletion-positive cells revealed that vorinostat affected the alternative splicing of BIM mRNA in the deletion allele, increased the expression of active BIM protein, and thereby induced apoptosis in osimertinib-treated cells. These effects were mediated predominantly by HDAC3 inhibition. In xenograft models, combined use of vorinostat with osimertinib could regress tumors in EGFR-mutated NSCLC cells homozygous for the BIM deletion polymorphism. Moreover, this combination could induce apoptosis even when tumor cells acquired EGFR-T790M mutations.Conclusions: These findings indicate the importance of developing HDAC3-selective inhibitors, and their combined use with osimertinib, for treating EGFR-mutated lung cancers carrying the BIM deletion polymorphism. Clin Cancer Res; 1-11. ©2016 AACR.

  17. A large deletion/insertion-induced frameshift mutation of the androgen receptor gene in a family with a familial complete androgen insensitivity syndrome.

    Science.gov (United States)

    Cong, Peikuan; Ye, Yinghui; Wang, Yue; Lu, Lingping; Yong, Jing; Yu, Ping; Joseph, Kimani Kagunda; Jin, Fan; Qi, Ming

    2012-06-01

    Androgen insensitivity syndrome (AIS) is an X-linked recessive genetic disorder with a normal 46, XY karyotype caused by abnormality of the androgen receptor (AR) gene. One Chinese family consisting of the proband and 5 other members with complete androgen insensitivity syndrome (CAIS) was investigated. Mutation analysis by DNA sequencing on all 8 exons and flanking intron regions of the AR gene revealed a unique large deletion/insertion mutation in the family. A 287 bp deletion and 77 bp insertion (c.933_1219delins77) mutation at codon 312 resulted in a frameshift which caused a premature stop (p.Phe312Aspfs*7) of polypeptide formation. The proband's mother and grandmother were heterozygous for the mutant allele. The proband's father, uncle and grandfather have the normal allele. From the pedigree constructed from mutational analysis of the family, it is revealed that the probably pathogenic mutation comes from the maternal side.

  18. Structure of recombinant capsids formed by the beta-annulus deletion mutant -- rCP (Delta48-59) of Sesbania mosaic virus.

    Science.gov (United States)

    Pappachan, Anju; Subashchandrabose, Chinnathambi; Satheshkumar, P S; Savithri, H S; Murthy, M R N

    2008-05-25

    A unique feature of several T=3 icosahedral viruses is the presence of a structure called the beta-annulus formed by extensive hydrogen bonding between protein subunits related by icosahedral three-fold axis of symmetry. This unique structure has been suggested as a molecular switch that determines the T=3 capsid assembly. In order to examine the importance of the beta-annulus, a deletion mutant of Sesbania mosaic virus coat protein in which residues 48-59 involved in the formation of the beta-annulus were deleted retaining the rest of the residues in the amino terminal segment (rCP (Delta48-59)) was constructed. When expressed in Escherichia coli, the mutant protein assembled into virus like particles of sizes close to that of the wild type virus particles. The purified capsids were crystallized and their three dimensional structure was determined at 3.6 A resolution by X-ray crystallography. The mutant capsid structure closely resembled that of the native virus particles. However, surprisingly, the structure revealed that the assembly of the particles has proceeded without the formation of the beta-annulus. Therefore, the beta-annulus is not essential for T=3 capsid assembly as speculated earlier and may be formed as a consequence of the particle assembly. This is the first structural demonstration that the virus particle morphology with and without the beta-annulus could be closely similar.

  19. PCR detection of retinoblastoma gene deletions in radiation-induced mouse lung adenocarcinomas

    Energy Technology Data Exchange (ETDEWEB)

    Churchill, M.E.; Gemmell, M.A.; Woloschak, G.E.

    1993-04-01

    From 1971 to 1986, Argonne National Laboratory conducted a series of large-scale studies of tumor incidence in 40,000 BCF{sub 1} mice irradiated with {sup 60}Co {gamma} rays or JANUS fission-spectrum neutrons; normal and tumor tissues from mice in these studies were preserved in paraffin blocks. A polymerase chain reaction (PCR) technique has been developed to detect deletions in the mouse retinoblastoma (mRb) gene in the paraffin-embedded tissues. Microtomed sections were used as the DNA source in PCR reaction mixtures. Six mRb gene exon fragments were amplified in a 40-cycle, 3-temperature PCR protocol. The absence of any of these fragments (relative to control PCR products) on a Southern blot indicated a deletion of that portion of the mRb gene. The tumors chosen for analysis were lung adenocarcinomas that were judged to be the cause of death in post-mortem analyses. Spontaneous tumors as well as those from irradiated mice (569 cGy of {sup 60}Co {gamma} rays or 60 cGy of JANUS neutrons, doses that have been found to have approximately equal biological effectiveness in the BCF, mouse) were analyzed for mRb deletions. In all normal mouse tissues studies, all six mRb exon fragments were present on Southem blots. Tumors in six neutron-irradiated mice also had no mRb deletions. However, I of 6 tumors from {gamma}-irradiated mice and 6 of 18 spontaneous tumors from unirradiated mice had a deletion in one or both mRb alleles. All deletions detected were in the 5{prime} region of the mRb gene.

  20. PCR detection of retinoblastoma gene deletions in radiation-induced mouse lung adenocarcinomas

    Energy Technology Data Exchange (ETDEWEB)

    Churchill, M.E.; Gemmell, M.A.; Woloschak, G.E.

    1993-01-01

    From 1971 to 1986, Argonne National Laboratory conducted a series of large-scale studies of tumor incidence in 40,000 BCF[sub 1] mice irradiated with [sup 60]Co [gamma] rays or JANUS fission-spectrum neutrons; normal and tumor tissues from mice in these studies were preserved in paraffin blocks. A polymerase chain reaction (PCR) technique has been developed to detect deletions in the mouse retinoblastoma (mRb) gene in the paraffin-embedded tissues. Microtomed sections were used as the DNA source in PCR reaction mixtures. Six mRb gene exon fragments were amplified in a 40-cycle, 3-temperature PCR protocol. The absence of any of these fragments (relative to control PCR products) on a Southern blot indicated a deletion of that portion of the mRb gene. The tumors chosen for analysis were lung adenocarcinomas that were judged to be the cause of death in post-mortem analyses. Spontaneous tumors as well as those from irradiated mice (569 cGy of [sup 60]Co [gamma] rays or 60 cGy of JANUS neutrons, doses that have been found to have approximately equal biological effectiveness in the BCF, mouse) were analyzed for mRb deletions. In all normal mouse tissues studies, all six mRb exon fragments were present on Southem blots. Tumors in six neutron-irradiated mice also had no mRb deletions. However, I of 6 tumors from [gamma]-irradiated mice and 6 of 18 spontaneous tumors from unirradiated mice had a deletion in one or both mRb alleles. All deletions detected were in the 5[prime] region of the mRb gene.

  1. Deletions in the Y-derived amelogenin gene fragment in the Indian population

    Directory of Open Access Journals (Sweden)

    Sahoo Sanghamitra

    2006-04-01

    Full Text Available Abstract Background Rare failures in amelogenin-based gender typing of individuals have been observed globally. In this study, we report the deletion of a large fragment of the amelogenin gene in 10 individuals out of 4,257 male samples analyzed from 104 different endogamous populations of India. Methods Samples were analyzed using commercial genetic profiling kits. Those that exhibited failures in amelogenin-based gender identification were further analyzed with published as well as newly designed primers to ascertain the nature and extent of mutation. Results The failure rate among Indian males was 0.23 %. Though the exact size and nature of the deletion (single point mutations at a number of positions or a single large deletion could not be determined in the present study, it is inferred that the deletion spans a region downstream of the reverse primer-binding site of commercially available amelogenin primer sets. Deletions were conspicuously absent among the Mongoloid tribes of Northeast India, while both caste and tribal groups harbored these mutations, which was predominantly among the Y-chromosomes belonging to J2 lineage. Conclusion Our study indicates that the different amelogenin primer sets currently included in genetic profiling multiplex kits may result in erroneous interpretations due to mutations undetectable during routine testing. Further there are indications that these mutations could possibly be lineage-specific, inherited deletions.

  2. Characteristics of invasion-reduced hilA gene mutant of Salmonella Enteritidis in vitro and in vivo.

    Science.gov (United States)

    Lv, Shuang; Si, Wei; Yu, Shenye; Li, Zhaoli; Wang, Xiumei; Chen, Liping; Zhang, Wanjiang; Liu, Siguo

    2015-08-01

    Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) is a facultative intracellular pathogen that causes huge losses in poultry industry and also food poisoning in humans due to its being a food-borne pathogen. Functions of Invasion-related genes need to be explored, as invasion is a key step for Salmonella infection. In this study, a transposon mutant library of Salmonella Enteritidis isolate SM6 was constructed and screened for the invasion-related genes via incubation with Caco-2 cells. Three stably attenuated mutants were identified for significantly reduced invasion with insertions all in hilA (hyperinvasive locus A) gene. We constructed and evaluated the hilA deletion mutant in vivo and in vitro. SM6△hilA showed significantly reduced ability to invade Caco-2 cells and decreased pathogenicity in chicks. However, the bacterial load and pathological damage in the cecum were significantly higher than those in the SM6 in vivo. Present results provide new evidences for pathogenicity research on Salmonella Enteritidis.

  3. A Self-deleting Cre-lox-ermAM Cassette, CHESHIRE, for marker-less gene deletion in Streptococcus pneumoniae

    Science.gov (United States)

    Weng, Liming; Biswas, Indranil; Morrison, Donald A.

    2009-01-01

    Although targeted mutagenesis of Streptococcus pneumoniae is readily accomplished with the aid of natural genetic transformation and chimeric donor DNA constructs assembled in vitro, the drug resistance markers often employed for selection of recombinant products can themselves be undesirable by-products of the genetic manipulation. A new cassette carrying the erythromycin-resistance marker ermAM is described that can be used as a temporary marker for selection of desired recombinants. The cassette may subsequently be removed at will by virtue of an embedded fucose-regulated Cre recombinase gene and terminal lox66 and lox71 Cre recognition sites, with retention of 34 bp from the cassette as an inert residual double-mutant lox72 site. PMID:19850089

  4. Intragenic deletion in the LARGE gene causes Walker-Warburg syndrome.

    NARCIS (Netherlands)

    Reeuwijk, J. van; Grewal, P.K.; Salih, M.A.; Beltran Valero de Bernabe, D.; McLaughlan, J.M.; Michielse, C.B.; Herrmann, R.; Hewitt, J.E.; Steinbrecher, A.; Seidahmed, M.Z.; Shaheed, M.M.; Abomelha, A.; Brunner, H.G.; Bokhoven, J.H.L.M. van; Voit, T.

    2007-01-01

    Intragenic homozygous deletions in the Large gene are associated with a severe neuromuscular phenotype in the myodystrophy (myd) mouse. These mutations result in a virtual lack of glycosylation of alpha-dystroglycan. Compound heterozygous LARGE mutations have been reported in a single human patient,

  5. Establishment of a Cre recombinase based mutagenesis protocol for markerless gene deletion in Streptococcus suis.

    Science.gov (United States)

    Koczula, A; Willenborg, J; Bertram, R; Takamatsu, D; Valentin-Weigand, P; Goethe, R

    2014-12-01

    The lack of knowledge about pathogenicity mechanisms of Streptococcus (S.) suis is, at least partially, attributed to limited methods for its genetic manipulation. Here, we established a Cre-lox based recombination system for markerless gene deletions in S. suis serotype 2 with high selective pressure and without undesired side effects.

  6. Insertion/deletion polymorphism of the ACE gene and adherence to ACE inhibitors

    NARCIS (Netherlands)

    H. Schelleman (Hedi); O.H. Klungel (Olaf); C.M. van Duijn (Cock); J.C.M. Witteman (Jacqueline); A. Hofman (Albert); A.C. de Boer (Anthonius); B.H.Ch. Stricker (Bruno)

    2005-01-01

    textabstractAims: We investigated whether the insertion/deletion (I/D) polymorphism of the ACE gene modified the adherence to ACE inhibitors as measured by the discontinuation of an ACE inhibitor, or addition of another antihypertensive drug. Methods: This was a cohort study among 239 subjects who

  7. Insertion/deletion polymorphism of the ACE gene and adherence to ACE inhibitors

    NARCIS (Netherlands)

    Schelleman, H; Klungel, O H; van Duijn, C M; Witteman, J C M; Hofman, A; de Boer, A; Stricker, B H Ch

    AIMS: We investigated whether the insertion/deletion (I/D) polymorphism of the ACE gene modified the adherence to ACE inhibitors as measured by the discontinuation of an ACE inhibitor, or addition of another antihypertensive drug. METHODS: This was a cohort study among 239 subjects who started ACE

  8. IDH mutant and 1p/19q co-deleted oligodendrogliomas: tumor grade stratification using diffusion-, susceptibility-, and perfusion-weighted MRI

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Yu; Xing, Zhen; She, Dejun; Yang, Xiefeng; Zheng, Yingyan; Xiao, Zebin; Cao, Dairong [First Affiliated Hospital of Fujian Medical University, Department of Radiology, Fuzhou, Fujian (China); Wang, Xingfu [First Affiliated Hospital of Fujian Medical University, Department of Pathology, Fuzhou (China)

    2017-06-15

    Currently, isocitrate dehydrogenase (IDH) mutation and 1p/19q co-deletion are proven diagnostic biomarkers for both grade II and III oligodendrogliomas (ODs). Non-invasive diffusion-weighted imaging (DWI), susceptibility-weighted imaging (SWI), and dynamic susceptibility contrast perfusion-weighted imaging (DSC-PWI) are widely used to provide physiological information (cellularity, hemorrhage, calcifications, and angiogenesis) of neoplastic histology and tumor grade. However, it is unclear whether DWI, SWI, and DSC-PWI are able to stratify grades of IDH-mutant and 1p/19q co-deleted ODs. We retrospectively reviewed the conventional MRI (cMRI), DWI, SWI, and DSC-PWI obtained on 33 patients with IDH-mutated and 1p/19q co-deleted ODs. Features of cMRI, normalized ADC (nADC), intratumoral susceptibility signals (ITSSs), normalized maxim CBV (nCBV), and normalized maximum CBF (nCBF) were compared between low-grade ODs (LGOs) and high-grade ODs (HGOs). Receiver operating characteristic curve and logistic regression were applied to determine diagnostic performances. HGOs tended to present with prominent edema and enhancement. nADC, ITSSs, nCBV, and nCBF were significantly different between groups (all P < 0.05). The combination of SWI and DSC-PWI for grading resulted in sensitivity and specificity of 100.00 and 93.33%, respectively. IDH-mutant and 1p/19q co-deleted ODs can be stratified by grades using cMRI and advanced magnetic resonance imaging techniques including DWI, SWI, and DSC-PWI. Combined ITSSs with nCBV appear to be a promising option for grading molecularly defined ODs in clinical practice. (orig.)

  9. The entire β-globin gene cluster is deleted in a form of τδβ-thalassemia.

    NARCIS (Netherlands)

    E.R. Fearon; H.H.Jr. Kazazian; P.G. Waber (Pamela); J.I. Lee (Joseph); S.E. Antonarakis; S.H. Orkin (Stuart); E.F. Vanin; P.S. Henthorn; F.G. Grosveld (Frank); A.F. Scott; G.R. Buchanan

    1983-01-01

    textabstractWe have used restriction endonuclease mapping to study a deletion involving the beta-globin gene cluster in a Mexican-American family with gamma delta beta-thalassemia. Analysis of DNA polymorphisms demonstrated deletion of the beta-globin gene from the affected chromosome. Using a DNA

  10. The entire β-globin gene cluster is deleted in a form of τδβ-thalassemia.

    NARCIS (Netherlands)

    E.R. Fearon; H.H.Jr. Kazazian; P.G. Waber (Pamela); J.I. Lee (Joseph); S.E. Antonarakis; S.H. Orkin (Stuart); E.F. Vanin; P.S. Henthorn; F.G. Grosveld (Frank); A.F. Scott; G.R. Buchanan

    1983-01-01

    textabstractWe have used restriction endonuclease mapping to study a deletion involving the beta-globin gene cluster in a Mexican-American family with gamma delta beta-thalassemia. Analysis of DNA polymorphisms demonstrated deletion of the beta-globin gene from the affected chromosome. Using a DNA f

  11. Contiguous gene deletion of ELOVL7, ERCC8 and NDUFAF2 in a patient with a fatal multisystem disorder

    DEFF Research Database (Denmark)

    Janssen, Rolf J R J; Distelmaier, Felix; Smeets, Roel

    2009-01-01

    gene deletion on chromosome 5. The deletion encompassed the NDUFAF2, ERCC8 and ELOVL7 genes, encoding complex I assembly factor 2 (also known as human B17.2L), a protein of the transcription-coupled nucleotide excision repair (TC-NER) machinery, and a putative elongase of very long-chain fatty acid...

  12. Adipose-Specific Deletion of Autophagy-Related Gene 7 (atg7) in Mice Reveals a Role in Adipogenesis

    National Research Council Canada - National Science Library

    Yong Zhang; Scott Goldman; Rebecca Baerga; Yun Zhao; Masaaki Komatsu; Shengkan Jin

    2009-01-01

    .... Autophagy is a membrane trafficking process leading to lysosomal degradation. Here, we investigated the effect of the deletion of an essential autophagy gene, autophagy-related gene 7 (atg7), on adipogenesis...

  13. Systematic hybrid LOH: a new method to reduce false positives and negatives during screening of yeast gene deletion libraries

    DEFF Research Database (Denmark)

    Alvaro, D.; Sunjevaric, I.; Reid, R. J.

    2006-01-01

    We have developed a new method, systematic hybrid loss of heterozygosity, to facilitate genomic screens utilizing the yeast gene deletion library. Screening is performed using hybrid diploid strains produced through mating the library haploids with strains from a different genetic background...... mating a library gene deletion haploid to such a conditional centromere strain, which corresponds to the chromosome carrying the gene deletion, loss of heterozygosity (LOH) at the gene deletion locus can be generated in these otherwise hybrid diploids. The use of hybrid diploid strains permits...... complementation of any spurious recessive mutations in the library strain, facilitating attribution of the observed phenotype to the documented gene deletion and dramatically reducing false positive results commonly obtained in library screens. The systematic hybrid LOH method can be applied to virtually any...

  14. Tackling the issue of environmental survival of live Salmonella Typhimurium vaccines: deletion of the lon gene.

    Science.gov (United States)

    Leyman, Bregje; Boyen, Filip; Van Parys, Alexander; Verbrugghe, Elin; Haesebrouck, Freddy; Pasmans, Frank

    2012-12-01

    Vaccination is an important measure to control Salmonella contamination in the meat production chain. A previous study showed that both the ΔrfaJ and ΔrfaL strains are suitable markers and allow serological differentiation of infected and vaccinated animals. The aim of this study was to verify whether deletion of the lon gene in a Salmonella Typhimurium ΔrfaJ marker strain resulted in decreased environmental survival. Our results indicate that deletion of the lon gene in the ΔrfaJ strain did not affect invasiveness in IPEC-J2 cells and resulted in an increased susceptibility to UV, disinfectants (such as hydrogen peroxide and tosylchloramide sodium) and citric acid. Immunization of pigs with inactivated ΔrfaJ or ΔlonΔrfaJ vaccines allowed differentiation of infected and vaccinated pigs. Furthermore, deletion of the lon gene did not reduce the protection conferred by live wild type or ΔrfaJ vaccines against subsequent challenge with a virulent Salmonella Typhimurium strain in BALB/c mice. Based on our results in mice, we conclude that deletion of lon in ΔrfaJ contributes to environmental safety of the ΔrfaJ DIVA strain.

  15. An analysis of substitution, deletion and insertion mutations in cancer genes.

    Science.gov (United States)

    Iengar, Prathima

    2012-08-01

    Cancer-associated mutations in cancer genes constitute a diverse set of mutations associated with the disease. To gain insight into features of the set, substitution, deletion and insertion mutations were analysed at the nucleotide level, from the COSMIC database. The most frequent substitutions were c → t, g → a, g → t, and the most frequent codon changes were to termination codons. Deletions more than insertions, FS (frameshift) indels more than I-F (in-frame) ones, and single-nucleotide indels, were frequent. FS indels cause loss of significant fractions of proteins. The 5'-cut in FS deletions, and 5'-ligation in FS insertions, often occur between pairs of identical bases. Interestingly, the cut-site and 3'-ligation in insertions, and 3'-cut and join-pair in deletions, were each found to be the same significantly often (p Proto-oncogenes undergo fewer, less-disruptive mutations, in selected protein regions, to activate a single allele. Finally, catalogues, in ranked order, of genes mutated in each cancer, and cancers in which each gene is mutated, were created. The study highlights the nucleotide level preferences and disruptive nature of cancer mutations.

  16. Gene dosage analysis identifies large deletions of the FECH gene in 10% of families with erythropoietic protoporphyria.

    Science.gov (United States)

    Whatley, Sharon D; Mason, Nicola G; Holme, S Alexander; Anstey, Alex V; Elder, George H; Badminton, Michael N

    2007-12-01

    Erythropoietic protoporphyria (EPP) is an inherited cutaneous porphyria characterized by partial deficiency of ferrochelatase (FECH), accumulation of protoporphyrin IX in erythrocytes, skin, and liver, and acute photosensitivity. Genetic counseling in EPP requires identification of FECH mutations, but current sequencing-based procedures fail to detect mutations in about one in six families. We have used gene dosage analysis by quantitative PCR to identify large deletions of the FECH gene in 19 (58%) of 33 unrelated UK patients with EPP in whom mutations could not be detected by sequencing. Seven deletions were identified, six of which were previously unreported. Breakpoints were identified for six deletions (c.1-7887-IVS1+2425insTTCA; c.1-9629-IVS1+2437; IVS2-1987-IVS4+352del; c.768-IVS7+244del; IVS7+2784-IVS9+108del; IVS6+2350-TGA+95del). Five breakpoints were in intronic repeat sequences (AluSc, AluSq, AluSx, L1MC4). The remaining deletion (Del Ex3-4) is likely to be a large insertion-deletion. Combining quantitative PCR with routine sequencing increased the sensitivity of mutation detection in 189 unrelated UK patients with EPP from 83% (95% CI: 76-87%) to 93% (CI: 88-96%) (P=0.003). Our findings show that large deletions of the FECH gene are an important cause of EPP. Gene dosage analysis should be incorporated into routine procedures for mutation detection in EPP.

  17. Phosphoinositide-specific Phospholipase C β1 gene deletion in bipolar disorder affected patient.

    Science.gov (United States)

    Lo Vasco, Vincenza Rita; Longo, Lucia; Polonia, Patrizia

    2013-03-01

    The involvement of phosphoinositides (PI) signal transduction pathway and related molecules, such as the Phosphoinositide-specific Phospholipase C (PI-PLC) enzymes, in the pathophysiology of mood disorders is corroborated by a number of recent evidences. Our previous works identified the deletion of PLCB1 gene, which codifies for the PI-PLC β1 enzyme, in 4 out 15 patients affected with schizophrenia, and no deletion both in major depression affected patients and in normal controls. By using interphase fluorescent in situ hybridization methodology, we analyzed PLCB1 in paraffin embedded samples of orbito-frontal cortex of 15 patients affected with bipolar disorder. Deletion of PLCB1 was identified in one female patient.

  18. Partial Gene Deletions of PMP22 Causing Hereditary Neuropathy with Liability to Pressure Palsies

    Directory of Open Access Journals (Sweden)

    Sun-Mi Cho

    2014-01-01

    Full Text Available Hereditary neuropathy with liability to pressure palsies (HNPP is an autosomal neuropathy that is commonly caused by a reciprocal 1.5 Mb deletion on chromosome 17p11.2, at the site of the peripheral myelin protein 22 (PMP22 gene. Other patients with similar phenotypes have been shown to harbor point mutations or small deletions, although there is some clinical variation across these patients. In this report, we describe a case of HNPP with copy number changes in exon or promoter regions of PMP22. Multiplex ligation-dependent probe analysis revealed an exon 1b deletion in the patient, who had been diagnosed with HNPP in the first decade of life using molecular analysis.

  19. Total alpha-globin gene cluster deletion has high frequency in Filipinos

    Energy Technology Data Exchange (ETDEWEB)

    Hunt, J.A.; Haruyama, A.Z.; Chu, B.M. [Kapiolani Medical Center, Honolulu, HI (United States)] [and others

    1994-09-01

    Most {alpha}-thalassemias [Thal] are due to large deletions. In Southeast Asians, the (--{sup SEA}) double {alpha}-globin gene deletion is common, 3 (--{sup Tot}) total {alpha}-globin cluster deletions are known: Filipino (--{sup Fil}), Thai (--{sup Thai}), and Chinese (--{sup Chin}). In a Hawaii Thal project, provisional diagnosis of {alpha}-Thal-1 heterozygotes was based on microcytosis, normal isoelectric focusing, and no iron deficiency. One in 10 unselected Filipinos was an {alpha}-Thal-1 heterozygote, 2/3 of these had a (--{sup Tot}) deletion: a {var_sigma}-cDNA probe consistently showed fainter intensity of the constant 5.5 kb {var_sigma}{sub 2} BamHI band, with no heterzygosity for {var_sigma}-globin region polymorphisms; {alpha}-cDNA or {var_sigma}-cDNA probes showed no BamHI or BglII bands diagnostic of the (--{sup SEA}) deletion; bands for the (-{alpha}) {alpha}-Thal-2 single {alpha}-globin deletions were only seen in Hb H cases. A reliable monoclonal anti-{var_sigma}-peptide antibody test for the (--{sup SEA}) deletion was always negative in (--{sup Tot}) samples. Southern digests with the Lo probe, a gift from D. Higgs of Oxford Univ., confirmed that 49 of 50 (--{sup Tot}) chromosomes in Filipinos were (--{sup Fil}). Of 20 {alpha}-Thal-1 hydrops born to Filipinos, 11 were (--{sup Fil}/--{sup SEA}) compound heterozygotes; 9 were (--{sup SEA}/--{sup SEA}) homozygotes, but none was a (--{sup Fil}/--{sup Fil}).

  20. Attenuated Actinobacillus pleuropneumoniae double-deletion mutant S-8∆clpP/apxIIC confers protection against homologous or heterologous strain challenge.

    Science.gov (United States)

    Xie, Fang; Li, Gang; Zhou, Long; Zhang, Yanhe; Cui, Ning; Liu, Siguo; Wang, Chunlai

    2017-01-06

    Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, which leads to large economic losses to the swine industry worldwide. In this study, S-8△clpP△apxIIC, a double-deletion mutant of A. pleuropneumoniae was constructed, and its safety and protective efficacy were evaluated in pigs. The S-8△clpP△apxIIC mutant exhibited attenuated virulence in a murine (BALB/c) model, and caused no detrimental effects on pigs even at a dose of up to 1.0 × 10(9) CFU. Furthermore, the S-8△clpP△apxIIC mutant was able to induce a strong immune response in pigs, which included high levels of IgG1 and IgG2, stimulated gamma interferon (IFN-γ), interleukin 12 (IL-12), and interleukin 4 (IL-4) production, and conferred effective protection against the lethal challenge with A. pleuropneumoniae serovars 7 or 5a. The pigs in the S-8△clpP△apxIIC immunized groups have no lesions and reduced bacterial loads in the lung tissue after challenge. The data obtained in this study suggest that the S-8△clpP△apxIIC mutant can serve as a highly immunogenic and potential live attenuated vaccine candidate against A. pleuropneumoniae infection.

  1. Deletion in the FMR1 gene in a fragile-X male

    Energy Technology Data Exchange (ETDEWEB)

    Mannermaa, A.; Pulkkinen, L.; Kajanoja, E. [Kuopio Univ., Hospital (Finland)] [and others

    1996-08-09

    The pathogenesis of fragile-X syndrome is a consequence of absence of the FMR1 gene product associated with expansion of the CGG repeat and abnormal methylation of this and a CpG island 250 hp proximal to the CGG repeat located at exon 1 in the FMR1 gene. While this is usually the case, some suspected fragile-X syndrome patients have been described with a mutation other than CGG expansion. We describe here an affected fragile-X male, who was found to be mosaic of a full mutation of the CGG expansion and a deletion in the FMR1 gene. The patient`s phenotype is probably mainly due to the effect of the full mutation of the repeat sequence. Thus, the influence of the deletion is difficult to evaluate. 20 refs., 2 figs.

  2. First contiguous gene deletion causing biotinidase deficiency: The enzyme deficiency in three Sri Lankan children

    Directory of Open Access Journals (Sweden)

    Danika Nadeen Senanayake

    2015-03-01

    Full Text Available We report three symptomatic children with profound biotinidase deficiency from Sri Lanka. All three children presented with typical clinical features of the disorder. The first is homozygous for a missense mutation in the BTD gene (c.98_104 del7insTCC; p.Cys33PhefsX36 that is commonly seen in the western countries, the second is homozygous for a novel missense mutation (p.Ala439Asp, and the third is the first reported instance of a contiguous gene deletion causing the enzyme deficiency. In addition, this latter finding exemplifies the importance of considering a deletion within the BTD gene for reconciling enzymatic activity with genotype, which can occur in asymptomatic children who are identified by newborn screening.

  3. Molecular basis and consequences of a deletion in the amelogenin gene, analyzed by capture PCR

    Energy Technology Data Exchange (ETDEWEB)

    Lagerstroem-Fermer, M.; Pettersson, U.; Landegren, U. (Univ. of Uppsala (Sweden))

    1993-07-01

    A mutation that disrupts the gene for one of the major proteins in tooth enamel has been investigated. The mutation is located in the amelogenin gene and causes X-linked amelogenesis imperfecta, characterized by defective mineralization of tooth enamel. The authors have isolated the breakpoints of a 5-kb deletion in the amelogenin gene on the basis of nucleotide sequence information located upstream of the lesion, using a technique termed capture PCR. The deletion removes five of the seven exons, spanning from the second intron to the last exon. Only the first two codons for the mature protein remain, consistent with the relatively severe phenotype of affected individuals in the present family. The mutation appears to have arisen as an illegitimate recombination event since of 11 nucleotide positions immediately surrounding the two breakpoints, 9 are identical. 17 refs., 3 figs., 1 tab.

  4. A common deletion at D6S265 in the hemochromatosis gene region

    Energy Technology Data Exchange (ETDEWEB)

    Pyper, W.R.; Burt, M.J.; Powell, L.W. [Queensland Institute of Medical Research and Department of Medicine, Brisbane (Australia)] [and others

    1994-09-01

    Positional cloning of the hemochromatosis (HC) gene on chromosome 6p has utilized a number of highly polymorphic microsatellite markers. While the putative HC gene has been localized within 1 cM of HLA-A, definition of the genetic limits of the HC locus has been controversial. Isolation and characterization of additional markers within this region will enable construction of a physical map upon which the HC gene can located. D6S265 is one such microsatellite, physically mapped within 120 kb centromeric of HLA-A. Recombinant and linkage analysis of this dinucleotide repeat in 24 Australian families segregating for HC positioned D6S265 within 1 cM of the HC gene, while allele association analysis showed allele 1 to be significantly increased in HC patients ({chi}{sup 2}=41.4, p<0.001, RR=5.75). In 6 of the 24 HC families, a D6265 locus deletion was found to segregate with HLA-A25 and HLA-A26 alleles. The D6S265 locus deletion was not associated with expression of HC. This study enables us to exclude candidate HC genes from the deleted region involving D6S265, and gives further support for an area of instability in the HLA class I region.

  5. 无标记的变异链球菌的clpP基因缺陷株的构建%Construction of the markless clpP-deletion mutant of Streptococcus mutans

    Institute of Scientific and Technical Information of China (English)

    彭诚; 于丹妮; 张文娟; 韩育植; 任志明

    2010-01-01

    目的 构建无标记的clpP基因缺陷的变异链球菌(简称变链菌)突变株.方法 设计引物PCR扩增大观霉素(Sp)抗性基因,使loxP位点位于Sp抗性基因的两侧,构建出大观霉素抗性基因盒(loxP-Sp-loxP).将clpP基因克隆到pGEM-T-Easy TA载体后,双酶切以去除clpP基因的部分序列,并连入loxP-Sp-loxP,得到clpP基因缺陷的同源重组载体pIB△clpP-Sp.将该载体线性化并电转变链菌标准株,大观霉素筛选得到clpP基因缺陷株.再以温敏质粒pCrePA电转缺陷株,Cre重组酶表达并删除选择标记基因,继而在限制性温度下培养以消除pCrePA,获得无标记的clpP基因缺陷株,并进行PCR及DNA测序鉴定.结果 PCR及DNA测序结果表明clpP基因内部分序列已被删除,且无Sp抗性基因,该部位只留有一个34 bp的loxP位点.结论 在变链菌中成功构建出无标记的clpP基因缺陷株,为进一步研究clpP基因的功能及其在变链菌致龋过程中的作用奠定了基础.%Objective To construct markless gene deletion mutant at the clpP loci on the chromosome of Streptococcus mutans(S.mutans).Methods ASp resistance gene was amplified by PCR,to construct the Sp resistance cassette where the Sp resistance gene was flanked with two loxP site.After the clpP gene was cloned into the pGEM-T-Easy TA cloning vector,it was digested and linked with the Sp resistance cassette,yielding homologous recombination vector pIB △ clpP-Sp.The vector was linearized and used for the transformation of S.mutans UA159,with transformants selected on TPY plates containing Sp.The selected strain was transformed with the thermosensitive plasmid pCrePA to excise the Sp resistance gene.The pCre-PA was then easily eliminated at nonpermissive temperature,resulting in a markless mutant strain carrying a deletion at the clpP loci,which was verified by PCR and DNA sequencing.Results The result of the PCR analysis and DNA sequencing indicated that a part of the clpP gene was deleted

  6. Transfection of the cloned human excision repair gene ERCC-1 to UV-sensitive CHO mutants only corrects the repair defect in complementation group 2 mutants.

    NARCIS (Netherlands)

    M. van Duin (Mark); J.H. Janssen; J. de Wit (Jan); J.H.J. Hoeijmakers (Jan); L.H. Thompson; D. Bootsma (Dirk); A. Westerveld (Andries)

    1988-01-01

    textabstractThe human DNA-excision repair gene ERCC-1 is cloned by its ability to correct the excision-repair defect of the ultraviolet light- and mitomycin-C-sensitive CHO mutant cell line 43-3B. This mutant is assigned to complementation group 2 of the excision-repair-deficient CHO mutants. In ord

  7. Genetic analysis of the Rhizobium meliloti bacA gene: functional interchangeability with the Escherichia coli sbmA gene and phenotypes of mutants.

    Science.gov (United States)

    Ichige, A; Walker, G C

    1997-01-01

    The Rhizobium meliloti bacA gene encodes a function that is essential for bacterial differentiation into bacteroids within plant cells in the symbiosis between R. meliloti and alfalfa. An Escherichia coli homolog of BacA, SbmA, is implicated in the uptake of microcin B17, microcin J25 (formerly microcin 25), and bleomycin. When expressed in E. coli with the lacZ promoter, the R. meliloti bacA gene was found to suppress all the known defects of E. coli sbmA mutants, namely, increased resistance to microcin B17, microcin J25, and bleomycin, demonstrating the functional similarity between the two proteins. The R. meliloti bacA386::Tn(pho)A mutant, as well as a newly constructed bacA deletion mutant, was found to show increased resistance to bleomycin. However, it also showed increased resistance to certain aminoglycosides and increased sensitivity to ethanol and detergents, suggesting that the loss of bacA function causes some defect in membrane integrity. The E. coli sbmA gene suppressed all these bacA mutant phenotypes as well as the Fix- phenotype when placed under control of the bacA promoter. Taken together, these results strongly suggest that the BacA and SbmA proteins are functionally similar and thus provide support for our previous hypothesis that BacA may be required for uptake of some compound that plays an important role in bacteroid development. However, the additional phenotypes of bacA mutants identified in this study suggest the alternative possibility that BacA may be needed for membrane integrity, which is likely to be critically important during the early stages of bacterial differentiation within plant cells.

  8. Deletion of the TNFAIP3/A20 gene detected by FICTION analysis in classical Hodgkin lymphoma

    Directory of Open Access Journals (Sweden)

    Nomoto Junko

    2012-10-01

    Full Text Available Abstract Background The TNFAIP3 gene, which encodes a ubiquitin-modifying enzyme (A20 involved in the negative regulation of NF-κB signaling, is frequently inactivated by gene deletions/mutations in a variety of B-cell malignancies. However, the detection of this in primary Hodgkin lymphoma (HL specimens is hampered by the scarcity of Hodgkin Reed-Sternberg (HR-S cells even after enrichment by micro-dissection. Methods We used anti-CD30 immunofluorescence with fluorescence in-situ hybridization (FISH to evaluate the relative number of TNFAIP3/CEP6 double-positive signals in CD30-positive cells. Results From a total of 47 primary classical Hodgkin lymphoma (cHL specimens, 44 were evaluable. We found that the relative numbers of TNFAIP3/CD30 cells were distributed among three groups, corresponding to those having homozygous (11%, heterozygous (32%, and no (57% deletions in TNFAIP3. This shows that TNFAIP3 deletions could be sensitively detected using our chosen methods. Conclusions Comparing the results with mutation analysis, TNFAIP3 inactivation was shown to have escaped detection in many samples with homozygous deletions. This suggests that TNFAIP3 inactivation in primary cHL specimens might be more frequent than previously reported.

  9. Deletion of the TNFAIP3/A20 gene detected by FICTION analysis in classical Hodgkin lymphoma

    Science.gov (United States)

    2012-01-01

    Background The TNFAIP3 gene, which encodes a ubiquitin-modifying enzyme (A20) involved in the negative regulation of NF-κB signaling, is frequently inactivated by gene deletions/mutations in a variety of B-cell malignancies. However, the detection of this in primary Hodgkin lymphoma (HL) specimens is hampered by the scarcity of Hodgkin Reed-Sternberg (HR-S) cells even after enrichment by micro-dissection. Methods We used anti-CD30 immunofluorescence with fluorescence in-situ hybridization (FISH) to evaluate the relative number of TNFAIP3/CEP6 double-positive signals in CD30-positive cells. Results From a total of 47 primary classical Hodgkin lymphoma (cHL) specimens, 44 were evaluable. We found that the relative numbers of TNFAIP3/CD30 cells were distributed among three groups, corresponding to those having homozygous (11%), heterozygous (32%), and no (57%) deletions in TNFAIP3. This shows that TNFAIP3 deletions could be sensitively detected using our chosen methods. Conclusions Comparing the results with mutation analysis, TNFAIP3 inactivation was shown to have escaped detection in many samples with homozygous deletions. This suggests that TNFAIP3 inactivation in primary cHL specimens might be more frequent than previously reported. PMID:23039325

  10. Increased biomass production and glycogen accumulation in apcE gene deleted Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Joseph, Ancy; Aikawa, Shimpei; Sasaki, Kengo; Matsuda, Fumio; Hasunuma, Tomohisa; Kondo, Akihiko

    2014-01-01

    The effect of phycobilisome antenna-truncation in the cyanobacterium Synechocystis sp. PCC 6803 on biomass production and glycogen accumulation have not yet been fully clarified. To investigate these effects here, the apcE gene, which encodes the anchor protein linking the phycobilisome to the thylakoid membrane, was deleted in a glucose tolerant strain of Synechocystis sp. PCC 6803. Biomass production of the apcE-deleted strain under photoautotrophic and atmospheric air conditions was 1.6 times higher than that of strain PCC 6803 (1.32 ± 0.01 versus 0.84 ± 0.07 g cell-dry weight L(-1), respectively) after 15 days of cultivation. In addition, the glycogen content of the apcE-deleted strain (24.2 ± 0.7%) was also higher than that of strain PCC 6803 (11.1 ± 0.3%). Together, these results demonstrate that antenna truncation by deleting the apcE gene was effective for increasing biomass production and glycogen accumulation under photoautotrophic and atmospheric air conditions in Synechocystis sp. PCC 6803.

  11. Intragenic deletion in the Desmoglein 4 gene underlies the skin phenotype in the Iffa Credo "hairless" rat.

    Science.gov (United States)

    Bazzi, Hisham; Kljuic, Ana; Christiano, Angela M; Christiano, Angela M; Panteleyev, Andrey A

    2004-10-01

    The Iffa Credo (IC) "hairless" rat is an autosomal recessive hypotrichotic animal model actively used in pharmacological and dermatological studies. Although the molecular basis of the IC rat phenotype was never defined, the designation "hr/hr" (hairless) has been used for this rat mutation. Despite the observation that IC rats share many phenotypic similarities with Charles River (CR) 'hairless rats', crossbreeding between CR and IC rats indicated that these mutations are not allelic, and moreover, genetic analysis of both CR and IC hairless mutant rats showed no mutations in the hr gene. Here, we present a detailed analysis of the skin phenotype in the IC rat. While the initial stages of hair follicle (HF) morphogenesis reveal no significant abnormalities, the subsequent processes of inner root sheath and hair shaft formation are severely disturbed due to impaired proliferation in the hair matrix and abnormal differentiation in the precortex zone. This results in significant reduction of hair bulb volume, and the formation of dysmorphic "blebbed" hair shafts lacking medullar structure and resembling "lanceolate" hairs. Based on the presence of lance-head hairs typical of rodent lanceolate mutants, we performed molecular analysis of the desmoglein 4 gene and found a large intragenic deletion encompassing nine exons of the gene. This finding, together with specific morphological features of skin and hairs, confirms that the IC rat is allelic with the lanceolate hair (lah) mutations in mice and rats. Our results elucidate the genetic and morphological basis of the IC rat mutation, thus providing a new model to study molecular mechanisms of hair growth control.

  12. Germinal mosaicism in a sample of families with Duchenne/Becker muscular dystrophy with partial deletions in the DMD gene.

    Science.gov (United States)

    Bermúdez-López, Cesárea; García-de Teresa, Benilde; González-del Angel, Ariadna; Alcántara-Ortigoza, Miguel Angel

    2014-02-01

    Germinal mosaicism should be considered when estimating the recurrence risk in families with Duchenne/Becker muscular dystrophy (D/BMD). Germinal mosaicism, however, has not been assessed in Mexican families with deletions in the DMD gene. To determine the distribution of deletions in the two hot spots and the proportion of de novo and transmitted deletions, we analyzed 153 individuals with D/BMD and a DMD partial deletion and 322 of their maternal female relatives. Predilection for the distal hot spot was observed in 112 families (73%), while gene dosage analysis of female relatives of D/BMD patients identified germinal mosaicism deletions in at least 11.6% of the patients' families, thought to result from de novo mutations. Recurrence risk due to germinal mosaicism justifies carrier detection in maternal female relatives and prenatal diagnosis in mothers of individuals with apparently de novo DMD deletions.

  13. Treacher Collins syndrome with a de Novo 5-bp deletion in the TCOF1 gene.

    Science.gov (United States)

    Su, Pen-Hua; Chen, Jia-Yu; Chen, Suh-Jen; Yu, Ju-Shan

    2006-06-01

    Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial development with features including malar hypoplasia, micrognathia, microtia, downward slanting palpebral fissures, lower eyelid coloboma, conductive hearing loss, and cleft palate. TCS is caused by mutations in the TCOF1 gene, which encodes the nuclear phosphoprotein treacle. Here, we describe a 1-day-old male infant with classical TCS presentation. A 5-bp deletion in exon 22 of the TCOF1 gene (3469del ACTCT) was found to cause a premature stop codon. This is the first report of TCOF1 gene mutation in the Taiwanese population.

  14. Effect of 5'-flanking sequence deletions on expression of the human insulin gene in transgenic mice

    DEFF Research Database (Denmark)

    Fromont-Racine, M; Bucchini, D; Madsen, O

    1990-01-01

    Expression of the human insulin gene was examined in transgenic mouse lines carrying the gene with various lengths of DNA sequences 5' to the transcription start site (+1). Expression of the transgene was demonstrated by 1) the presence of human C-peptide in urine, 2) the presence of specific......, and -168 allowed correct initiation of the transcripts and cell specificity of expression, while quantitative expression gradually decreased. Deletion to -58 completely abolished the expression of the gene. The amount of human product that in mice harboring the longest fragment contributes up to 50...... of the transgene was observed in cell types other than beta-islet cells....

  15. Differentiation of the shutoff of protein synthesis by virion host shutoff and mutant gamma (1)34.5 genes of herpes simplex virus 1.

    Science.gov (United States)

    Poon, A P; Roizman, B

    1997-03-01

    vhs protein is the product of the UL41 open reading frame of herpes simplex virus 1. The protein, made late in infection, is packaged into virions and, in newly infected cells, shuts off host protein synthesis by degrading mRNA. gamma (1)34.5 gene encodes a protein which precludes total shutoff of protein synthesis after the onset of viral DNA synthesis in infected cells of human derivation. The experiments reported here were designed to test the hypothesis that in cells infected with gamma (1)34.5- mutant the total shutoff of protein synthesis reflects the failure to alter the function of vhs made late in infection. Hence, double mutants, vhs- and gamma (1)34.5 should not cause total shutoff of protein synthesis. The mutants constructed to test the hypothesis were (i) viruses lacking 1 kbp from the coding domain of gamma (1)34.5 and carrying lacZ inserted into the coding domain of UI41, (ii) viruses with deletions in gamma (1)34.5 genes, (iii) viruses with lacZ inserted into UL41, and (iv) viruses in which the sequences of the deleted or interrupted genes were restored. We report that viruses with wild-type UL41 gene shut off the synthesis of actin, whereas viruses with interrupted genes made amounts of actin comparable to those of mock-infected cells. However, late in infection, protein synthesis in human neuroblastoma cells infected with the gamma (1)34.5- mutants was shut off irrespective of the status of the UL41 gene. Conversely, the phenotype of UI41 viruses with wild-type gamma (1)34.5 gene could not be differentiated from those of wild-type virus in the same assays. These studies indicate that the functions of the UL41 and gamma (1)34.5 genes and their products are independent of each other.

  16. Early-onset obesity and paternal 2pter deletion encompassing the ACP1, TMEM18, and MYT1L genes

    Science.gov (United States)

    Doco-Fenzy, Martine; Leroy, Camille; Schneider, Anouck; Petit, Florence; Delrue, Marie-Ange; Andrieux, Joris; Perrin-Sabourin, Laurence; Landais, Emilie; Aboura, Azzedine; Puechberty, Jacques; Girard, Manon; Tournaire, Magali; Sanchez, Elodie; Rooryck, Caroline; Ameil, Agnès; Goossens, Michel; Jonveaux, Philippe; Lefort, Geneviève; Taine, Laurence; Cailley, Dorothée; Gaillard, Dominique; Leheup, Bruno; Sarda, Pierre; Geneviève, David

    2014-01-01

    Obesity is a common but highly, clinically, and genetically heterogeneous disease. Deletion of the terminal region of the short arm of chromosome 2 is rare and has been reported in about 13 patients in the literature often associated with a Prader–Willi-like phenotype. We report on five unrelated patients with 2p25 deletion of paternal origin presenting with early-onset obesity, hyperphagia, intellectual deficiency, and behavioural difficulties. Among these patients, three had de novo pure 2pter deletions, one presented with a paternal derivative der(2)t(2;15)(p25.3;q26) with deletion in the 2pter region and the last patient presented with an interstitial 2p25 deletion. The size of the deletions was characterized by SNP array or array-CGH and was confirmed by fluorescence in situ hybridization (FISH) studies. Four patients shared a 2p25.3 deletion with a minimal critical region estimated at 1.97 Mb and encompassing seven genes, namely SH3HYL1, ACP1, TMEMI8, SNTG2, TPO, PXDN, and MYT1L genes. The fifth patient had a smaller interstitial deletion encompassing the TPO, PXDN, and MYT1L genes. Paternal origin of the deletion was determined by genotyping using microsatellite markers. Analysis of the genes encompassed in the deleted region led us to speculate that the ACP1, TMEM18, and/or MYT1L genes might be involved in early-onset obesity. In addition, intellectual deficiency and behavioural troubles can be explained by the heterozygous loss of the SNTG2 and MYT1L genes. Finally, we discuss the parent-of-origin of the deletion. PMID:24129437

  17. Early-onset obesity and paternal 2pter deletion encompassing the ACP1, TMEM18, and MYT1L genes.

    Science.gov (United States)

    Doco-Fenzy, Martine; Leroy, Camille; Schneider, Anouck; Petit, Florence; Delrue, Marie-Ange; Andrieux, Joris; Perrin-Sabourin, Laurence; Landais, Emilie; Aboura, Azzedine; Puechberty, Jacques; Girard, Manon; Tournaire, Magali; Sanchez, Elodie; Rooryck, Caroline; Ameil, Agnès; Goossens, Michel; Jonveaux, Philippe; Lefort, Geneviève; Taine, Laurence; Cailley, Dorothée; Gaillard, Dominique; Leheup, Bruno; Sarda, Pierre; Geneviève, David

    2014-04-01

    Obesity is a common but highly, clinically, and genetically heterogeneous disease. Deletion of the terminal region of the short arm of chromosome 2 is rare and has been reported in about 13 patients in the literature often associated with a Prader-Willi-like phenotype. We report on five unrelated patients with 2p25 deletion of paternal origin presenting with early-onset obesity, hyperphagia, intellectual deficiency, and behavioural difficulties. Among these patients, three had de novo pure 2pter deletions, one presented with a paternal derivative der(2)t(2;15)(p25.3;q26) with deletion in the 2pter region and the last patient presented with an interstitial 2p25 deletion. The size of the deletions was characterized by SNP array or array-CGH and was confirmed by fluorescence in situ hybridization (FISH) studies. Four patients shared a 2p25.3 deletion with a minimal critical region estimated at 1.97 Mb and encompassing seven genes, namely SH3HYL1, ACP1, TMEMI8, SNTG2, TPO, PXDN, and MYT1L genes. The fifth patient had a smaller interstitial deletion encompassing the TPO, PXDN, and MYT1L genes. Paternal origin of the deletion was determined by genotyping using microsatellite markers. Analysis of the genes encompassed in the deleted region led us to speculate that the ACP1, TMEM18, and/or MYT1L genes might be involved in early-onset obesity. In addition, intellectual deficiency and behavioural troubles can be explained by the heterozygous loss of the SNTG2 and MYT1L genes. Finally, we discuss the parent-of-origin of the deletion.

  18. Analysis of the aspartic acid metabolic pathway using mutant genes.

    Science.gov (United States)

    Azevedo, R A

    2002-01-01

    Amino acid metabolism is a fundamental process for plant growth and development. Although a considerable amount of information is available, little is known about the genetic control of enzymatic steps or regulation of several pathways. Much of the information about biochemical pathways has arisen from the use of mutants lacking key enzymes. Although mutants were largely used already in the 60's, by bacterial and fungal geneticists, it took plant research a long time to catch up. The advance in this area was rapid in the 80's, which was followed in the 90's by the development of techniques of plant transformation. In this review we present an overview of the aspartic acid metabolic pathway, the key regulatory enzymes and the mutants and transgenic plants produced for lysine and threonine metabolism. We also discuss and propose a new study of high-lysine mutants.

  19. LuxS基因缺失的变形链球菌突变株的构建及鉴定%Construction and identification of a LuxS-deleted mutant strain of S.mutans

    Institute of Scientific and Technical Information of China (English)

    于丹妮; 韩福胜; 韩玉植; 陈杰

    2008-01-01

    目的 通过同源重组法构建LuxS基因缺失的变形链球菌(Streptococcus mutans)突变株.方法 运用基因同源重组方法将红霉素抗性基因(Eymr)连接到PCR扩增LuxS基因两端区域产生的2个基因片段之间,并共同插入到pUCl9载体的多克隆位点中,构建出带红霉素抗性标志的缺失突变载体pUCluxKO.将突变载体转化到含完整LuxS基因的突变受体变形链球菌标准株中,红霉素筛选出LuxS基因缺失的变形链球菌突变株,并经PCR、生物荧光检测及DNA测序鉴定.结果 构建的突变载体经限制性内切核酸酶酶切分析显示,产生的条带与设计结果完全一致.PCR方法扩增突变株LuxS和Eymr基因显示,LuxS基因已被完整敲除掉,经生物荧光检测,突变株不能诱导哈氏弧菌(Vibrio harveyi)BBl70的生物发光,说明不能产生信号分子AI-2(autoinducer-2).DNA测序证实筛选得到了LuxS基因缺失的变形链球菌突变株.连续传代培养后证实,变形链球菌LuxS基因突变株具有良好的稳定性.结论 成功构建出LuxS基因缺失的变形链球菌突变株,为研究LuxS基因对变形链球菌致龋毒力的影响奠定了基础.%Objective To knock out the entire LuxS gene of Streptococcus mutans UA159 strain via homologous recombination and construct a LuxS-deleted mutant strain of S.mutans.Methods The erythromycin resistance gene(Eymr)was inserted between the two DNA fragments located in the upper and downstream of LuxS gene that had been amplified by PCR.Then the two DNA fragments along with the inserted Eymr were engineered into pUCl9 plasmid to construct the recombination plasmid pUCluxKO.Electrotransformation of S. mutans cells with pUCluxKO-mutant resulted in the isolation of erythromycin resistant S.mutans,transformants,which was then subjected to polymerase chain reaction,Vibrio harveyi BBl70 luminescence bioassay and sequencing analysis.Results Restriction endonuclease analysis showed that pUCluxKOmutant vector

  20. Insights into genes involved in electricity generation in Geobacter sulfurreducens via whole genome microarray analysis of the OmcF-deficient mutant.

    Science.gov (United States)

    Kim, Byoung-Chan; Postier, Bradley L; Didonato, Raymond J; Chaudhuri, Swades K; Nevin, Kelly P; Lovley, Derek R

    2008-06-01

    Geobacter sulfurreducens effectively produces electricity in microbial fuel cells by oxidizing acetate with an electrode serving as the sole electron acceptor. Deletion of the gene encoding OmcF, a monoheme outer membrane c-type cytochrome, substantially decreased current production. Previous studies demonstrated that inhibition of Fe(III) reduction in the OmcF-deficient mutant could be attributed to poor transcription of the gene for OmcB, an outer membrane c-type cytochrome that is required for Fe(III) reduction. However, a mutant in which omcB was deleted produced electricity as well as wild type. Microarray analysis of the OmcF-deficient mutant versus the wild type revealed that many of the genes with the greatest decreases in transcript levels were genes whose expression was previously reported to be upregulated in cells grown with an electrode as the sole electron acceptor. These included genes with putative functions related to metal efflux and/or type I secretion and two hypothetical proteins. The outer membrane cytochromes, OmcS and OmcE, which previous studies have demonstrated are required for optimal current generation, were not detected on the outer surface of the OmcF-deficient mutant even though the omcS and omcE genes were still transcribed, suggesting that the putative secretion system could be involved in the export of outer membrane proteins necessary for electron transfer to the fuel cell anode. These results suggest that the requirement for OmcF for optimal current production is not because OmcF is directly involved in extracellular electron transfer but because OmcF is required for the appropriate transcription of other genes either directly or indirectly involved in electricity production.

  1. Deletion of the zupT gene for a zinc importer influences zinc pools in Cupriavidus metallidurans CH34.

    Science.gov (United States)

    Herzberg, M; Bauer, L; Nies, D H

    2014-03-01

    Cupriavidus metallidurans strain CH34 accomplishes a high level of transition metal resistance by a combination of rather unspecific transition metal import and controlled efflux of surplus metals. Using the plasmid-free mutant strain AE104 that possesses only a limited number of metal efflux systems, cellular metal pools were identified as counterparts of these transport reactions. At low zinc concentrations strain AE104 took up Zn(II) until the zinc content reached an optimum level of 70,000 Zn(II) per cell in the exponential phase of growth, whereas a ΔzupT mutant lacking the zinc importer ZupT contained only 20,000 Zn(II)/cell, possibly the minimum zinc content. Mutant and parent cells accumulated up to 125,000 Zn(II) per cell at high (100 μM) external zinc concentrations (optimum zinc content). When the mutant strain Δe4, which has all the known genes for zinc efflux systems deleted, was cultivated in the presence of zinc concentrations close to its upper tolerance level (10 μM), these cells contained 250,000 Zn(II) per cell, probably the maximum zinc content. Instead of zinc, 120,000 cobalt or cadmium ions could also fill-up parts of this zinc pool, showing that it is in fact an undefined pool of divalent transition metal cations bound with low substrate specificity. Even when the cells contained sufficient numbers of total zinc, the zinc importer ZupT was required for important cellular processes, indicating the presence of a pool of tightly bound zinc ions, which depends on ZupT for efficient replenishment. The absence of ZupT led to the formation of inclusion bodies, perturbed oxidative stress resistance and decreased efficiency in the synthesis of the zinc-dependent subunit RpoC of the RNA polymerase, leading to RpoC accumulation. Moreover, when a czc allele for a zinc-exporting transenvelope efflux system CzcCBA was constitutively expressed in a ΔzupT mutant, this led to the disappearance of the CzcA protein and the central subunit of the protein

  2. Reverse genetic screen for loss-of-function mutations uncovers a frameshifting deletion in the melanophilin gene accountable for a distinctive coat color in Belgian Blue cattle.

    Science.gov (United States)

    Li, Wanbo; Sartelet, Arnaud; Tamma, Nico; Coppieters, Wouter; Georges, Michel; Charlier, Carole

    2016-02-01

    In the course of a reverse genetic screen in the Belgian Blue cattle breed, we uncovered a 10-bp deletion (c.87_96del) in the first coding exon of the melanophilin gene (MLPH), which introduces a premature stop codon (p.Glu32Aspfs*1) in the same exon, truncating 94% of the protein. Recessive damaging mutations in the MLPH gene are well known to cause skin, hair, coat or plumage color dilution phenotypes in numerous species, including human, mice, dog, cat, mink, rabbit, chicken and quail. Large-scale array genotyping undertaken to identify p.Glu32Aspfs*1 homozygous mutant animals revealed a mutation frequency of 5% in the breed and allowed for the identification of 10 homozygous mutants. As expression of a colored coat requires at least one wild-type allele at the co-dominant Roan locus encoded by the KIT ligand gene (KITLG), homozygous mutants for p.Ala227Asp corresponding with the missense mutation were excluded. The six remaining colored calves displayed a distinctive dilution phenotype as anticipated. This new coat color was named 'cool gray'. It is the first damaging mutation in the MLPH gene described in cattle and extends the already long list of species with diluted color due to recessive mutations in MLPH and broadens the color palette of gray in this breed.

  3. X-ray survival characteristics and genetic analysis for nineSaccharomyces deletion mutants that affect radiation sensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Game, John C.; Williamson, Marsha S.; Baccari, Clelia

    2006-07-21

    We examine ionizing radiation (IR) sensitivity and epistasisrelationships of several Saccharomyces mutants affectingpost-translational modifications of histones H2B and H3. Mutantsbre1delta, lge1delta, and rtf1delta, defective in histone H2B lysine 123ubiquitination, show IR sensitivity equivalent to that of the dot1deltamutant that we reported on earlier, consistent with published findingsthat Dot1p requires H2B K123 ubiquitination to fully methylate histone H3K79. This implicates progressive K79 methylation rather thanmono-methylation in IR resistance. The set2delta mutant, defective in H3K36 methylation, shows mild IR sensitivity whereas mutants that abolishH3 K4 methylation resemble wild type. The dot1delta, bre1delta, andlge1delta mutants show epistasis for IR sensitivity. The paf1deltamutant, also reportedly defective in H2B K123 ubiquitination, confers nosensitivity. The rad6delta, rad51null, rad50delta, and rad9deltamutations are epistatic to bre1? and dot1delta, but rad18delta andrad5delta show additivity with bre1delta, dot1delta, and each other. Thebre1delta rad18delta double mutant resembles rad6delta in sensitivity;thus the role of Rad6p in ubiquitinating H2B accounts for its extrasensitivity compared to rad18delta. We conclude that IR resistanceconferred by BRE1 and DOT1 is mediated through homologous recombinationalrepair, not postreplication repair, and confirm findings of a G1checkpoint role for the RAD6/BRE1/DOT1 pathway.

  4. Germline deletions in the tumour suppressor gene FOCAD are associated with polyposis and colorectal cancer development.

    Science.gov (United States)

    Weren, Robbert D A; Venkatachalam, Ramprasath; Cazier, Jean-Baptiste; Farin, Henner F; Kets, C Marleen; de Voer, Richarda M; Vreede, Lilian; Verwiel, Eugène T P; van Asseldonk, Monique; Kamping, Eveline J; Kiemeney, Lambertus A; Neveling, Kornelia; Aben, Katja K H; Carvajal-Carmona, Luis; Nagtegaal, Iris D; Schackert, Hans K; Clevers, Hans; van de Wetering, Marc; Tomlinson, Ian P; Ligtenberg, Marjolijn J L; Hoogerbrugge, Nicoline; Geurts van Kessel, Ad; Kuiper, Roland P

    2015-06-01

    Heritable genetic variants can significantly affect the lifetime risk of developing cancer, including polyposis and colorectal cancer (CRC). Variants in genes currently known to be associated with a high risk for polyposis or CRC, however, explain only a limited number of hereditary cases. The identification of additional genetic causes is, therefore, crucial to improve CRC prevention, detection and treatment. We have performed genome-wide and targeted DNA copy number profiling and resequencing in early-onset and familial polyposis/CRC patients, and show that deletions affecting the open reading frame of the tumour suppressor gene FOCAD are recurrent and significantly enriched in CRC patients compared with unaffected controls. All patients carrying FOCAD deletions exhibited a personal or family history of polyposis. RNA in situ hybridization revealed FOCAD expression in epithelial cells in the colonic crypt, the site of tumour initiation, as well as in colonic tumours and organoids. Our data suggest that monoallelic germline deletions in the tumour suppressor gene FOCAD underlie moderate genetic predisposition to the development of polyposis and CRC.

  5. CO-DELETION OF BOTH p15/p16 GENES CORRELATES WITH POOR PROGNOSIS NON-SMALL CELL LUNG CNACER

    Institute of Scientific and Technical Information of China (English)

    胡颖; 廖美琳; 丁嘉安; 周瑾; 许凯黎

    2002-01-01

    Objective: To investigate the relationship between co-deletion of p15/p16 genes and the prognostic significance in patients with non-small cell lung cancer (NSCLC). Methods: By using polymerase chain reaction (PCR), the loss of p15/pl6 genes was examined in DNA samples from 140 NSCLC patients. Results: The rate of co-deletion in adenocarcinoma was significantly higher than that in squamous cell carcinoma (P0.05). By a five years' follow-up survey, the survival rate of NSCLC patients with co-deletion of pl5/pl6 genes was obviously lower than that of patients without co-deletion (P<0.01). In the multivariate analysis, co-deletion of p15/p16 genes and TNM stages were identified as independent predictors for overall survival (P<0.01). Conclusion: Since the co-deletion of pl5/pl6 genes is significantly related to the prognosis of NSCLC patients, detecting co-deletion of both genes might be used as a potential marker for NSCLC prognosis.

  6. Mutant INS-Gene Induced Diabetes of Youth: Proinsulin Cysteine Residues Impose Dominant-Negative Inhibition on Wild-Type Proinsulin Transport

    OpenAIRE

    Ming Liu; Leena Haataja; Jordan Wright; Wickramasinghe, Nalinda P.; Qing-Xin Hua; Phillips, Nelson F.; Fabrizio Barbetti; Weiss, Michael A; Peter Arvan

    2010-01-01

    Recently, a syndrome of M utant I NS-gene-induced D iabetes of Y outh (MIDY , derived from one of 26 distinct mutations) has been identified as a cause of insulin-deficient diabetes, resulting from expression of a misfolded mutant proinsulin protein in the endoplasmic reticulum (ER) of insulin-producing pancreatic beta cells. Genetic deletion of one, two, or even three alleles encoding insulin in mice does not necessarily lead to diabetes. Yet MIDY patients are INS-gene heterozygotes; inherit...

  7. Osteopathia striata congenita with cranial sclerosis and intellectual disability due to contiguous gene deletions involving the WTX locus

    DEFF Research Database (Denmark)

    Holman, Sk; Morgan, T; Baujat, G;

    2013-01-01

    sclerosis, with a high prevalence of cleft palate and hearing loss. Intellectual disability or neurodevelopmental delay is not observed in females with point mutations in WTX leading to OSCS. One female has been described with a deletion spanning multiple neighbouring genes suggesting that deletion of some...

  8. Differential gene expression in salt-tolerant rice mutant and its parental variety

    Institute of Scientific and Technical Information of China (English)

    张劲松; 周骏马; 张驰; 陈受宜

    1996-01-01

    The differential expressions of three genes rbcL, salT and rab!6 in response to ABA, NaCl, PEG and heat shock were investigated in seedlings of a salt-tolerant rice mutant 20 (mutant 20) and its parental variety Oryza sativa var. japonica 77-170(170). By Northern blot analysis it was found that ABA induced the expression of all three genes of rbcL, salT and rab16 in shoots and roots of both 170 and mutant 20 with the exceptions of rab16 in shoots of mutant 20 and rbcL in roots of 170. Lower concentrations of NaCl induced rbcL expression in shoots of mutant 20 but not 170. Higher concentrations of NaCl decreased rbcL expression but induced expressions of salT and rab16 in shoots of both 170 and mutant 20. PEG(15%) and 37℃ heat shock showed almost no effects on the expression of the three genes in mutant 20. However, they caused a decrease in rbcL expression and slight induction of the rab16 gene in 170, with salT expression unaffected. These results indicated that mutant 20 was relatively less responsiv

  9. Molecular mapping of three nuclear male sterility mutant genes in cultivated sunflower (Helianthus annuus L.)

    Science.gov (United States)

    The nuclear male sterility (NMS) trait is a useful tool for sunflower (Helianthus annuus L.) breeding and genetic programs. Previously, we induced NMS mutants in cultivated line HA 89. The mutants possessed single recessive genes, ms6, ms7, and ms8, respectively, in NMS HA 89-872, NMS HA 89-552, and...

  10. Opposing phenotypes in mice with Smith-Magenis deletion and Potocki-Lupski duplication syndromes suggest gene dosage effects on fluid consumption behavior.

    Science.gov (United States)

    Heck, Detlef H; Gu, Wenli; Cao, Ying; Qi, Shuhua; Lacaria, Melanie; Lupski, James R

    2012-11-01

    A quantitative long-term fluid consumption and fluid-licking assay was performed in two mouse models with either an ∼2 Mb genomic deletion, Df(11)17, or the reciprocal duplication copy number variation (CNV), Dp(11)17, analogous to the human genomic rearrangements causing either Smith-Magenis syndrome [SMS; OMIM #182290] or Potocki-Lupski syndrome [PTLS; OMIM #610883], respectively. Both mouse strains display distinct quantitative alterations in fluid consumption compared to their wild-type littermates; several of these changes are diametrically opposing between the two chromosome engineered mouse models. Mice with duplication versus deletion showed longer versus shorter intervals between visits to the waterspout, generated more versus less licks per visit and had higher versus lower variability in the number of licks per lick-burst as compared to their respective wild-type littermates. These findings suggest that copy number variation can affect long-term fluid consumption behavior in mice. Other behavioral differences were unique for either the duplication or deletion mutants; the deletion CNV resulted in increased variability of the licking rhythm, and the duplication CNV resulted in a significant slowing of the licking rhythm. Our findings document a readily quantitated complex behavioral response that can be directly and reciprocally influenced by a gene dosage effect.

  11. Possible deletion of a developmentally regulated heavy-chain variable region gene in autoimmune diseases

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Pei-Ming; Olee, Tsaiwei; Kozin, F.; Carson, D.A.; Chen, P.P. (Research Institute of Scripps Clinic, La Jolla, CA (USA)); Olsen, N.J. (Vanderbilt Univ., Nashville, TN (USA)); Siminovitch, K.A. (Univ. of Toronto (Canada))

    1990-10-01

    Several autoantibody-associated variable region (V) genes are preferentially expressed during early ontogenic development, suggesting strongly that they are of developmental and physiological importance. As such, it is possible that polymorphisms in one or more of these genes may alter susceptibility to autoimmune disease. The authors have searched extensively for a probe related to a developmentally regulated V gene that has the power to differentiate among highly homologous V genes in human populations. Using such a probe (i.e., Humhv3005/P1) related to both anti-DNA and anti-IgG autoantibodies, they studied restriction fragment length polymorphisms in patients with rheumatoid arthritis and systemic lupus erythematosus and found an apparent heavy-chain V (V{sub H}) gene deletion that was nearly restricted to the autoimmune patients. These data suggest that deletions of physiologically important V{sub H} genes may increase the risk of autoimmunity through indirect effects on the development and homeostasis of the B-cell repertoire.

  12. A novel contiguous gene deletion of AVPR2 and ARHGAP4 genes in male dizygotic twins with nephrogenic diabetes insipidus and intellectual disability.

    Science.gov (United States)

    Huang, Lingli; Poke, Gemma; Gecz, Jozef; Gibson, Kate

    2012-10-01

    The clinical features of loss of ARHGAP4 function remain unclear despite several reports of different patterns of deletions inactivating different functional regions of the protein. The protein encoded by ARHGAP4 is thought to function as a Rho GTPase activating protein. Characterization of the genetic defect causing X-linked nephrogenic diabetes insipidus (NDI) and intellectual disability in two dizygotic twin brothers revealed a novel contiguous deletion of 17,905 bp encompassing the entire AVPR2 gene and extending into intron 7 of the ARHGAP4 gene. Examination of their mother showed that she was a carrier of this deletion. An attempt was made to distinguish the putative clinical signs of an ARHGAP4 deletion from the well-defined phenotype of X-linked NDI caused by an AVPR2 gene deletion. By reviewing all characterized deletions encompassing ARHGAP4, we reconsider the potential role of ARHGAP4 in cognition.

  13. Effect of 5'-flanking sequence deletions on expression of the human insulin gene in transgenic mice

    DEFF Research Database (Denmark)

    Fromont-Racine, M; Bucchini, D; Madsen, O;

    1990-01-01

    Expression of the human insulin gene was examined in transgenic mouse lines carrying the gene with various lengths of DNA sequences 5' to the transcription start site (+1). Expression of the transgene was demonstrated by 1) the presence of human C-peptide in urine, 2) the presence of specific tra...... of the transgene was observed in cell types other than beta-islet cells.......Expression of the human insulin gene was examined in transgenic mouse lines carrying the gene with various lengths of DNA sequences 5' to the transcription start site (+1). Expression of the transgene was demonstrated by 1) the presence of human C-peptide in urine, 2) the presence of specific......, and -168 allowed correct initiation of the transcripts and cell specificity of expression, while quantitative expression gradually decreased. Deletion to -58 completely abolished the expression of the gene. The amount of human product that in mice harboring the longest fragment contributes up to 50...

  14. Esophageal atresia with tracheoesophageal fistula in a patient with 7q35-36.3 deletion including SHH gene.

    Science.gov (United States)

    Busa, Tiffany; Panait, Nicoleta; Chaumoitre, Kathia; Philip, Nicole; Missirian, Chantal

    2016-10-01

    Terminal 7q deletion is rarely reported in the literature. Holoprosencephaly and sacral dysgenesis are found in association with this deletion, due to haploinsufficiency of SHH and HLBX9 genes respectively. We report on a 2-year-old boy with 7q35-36.3 deletion encompassing SHH identified by oligonucleotide array comparative genomic hybridization. In addition to other frequent features, the patient presented with esophageal atresia and tracheoeosophageal fistula diagnosed at birth. This case, together with two others previously described, one presenting with esophageal atresia, the other with congenital esophageal stenosis, confirms the possible association between congenital esophageal malformations and 7q terminal deletion including SHH.

  15. Candida albicans Gene Deletion with a Transient CRISPR-Cas9 System.

    Science.gov (United States)

    Min, Kyunghun; Ichikawa, Yuichi; Woolford, Carol A; Mitchell, Aaron P

    2016-01-01

    Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-Cas9) systems are used for a wide array of genome-editing applications in organisms ranging from fungi to plants and animals. Recently, a CRISPR-Cas9 system has been developed for the diploid fungal pathogen Candida albicans; the system accelerates genetic manipulation dramatically [V. K. Vyas, M. I. Barrasa, and G. R. Fink, Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248]. We show here that the CRISPR-Cas9 genetic elements can function transiently, without stable integration into the genome, to enable the introduction of a gene deletion construct. We describe a transient CRISPR-Cas9 system for efficient gene deletion in C. albicans. Our observations suggest that there are two mechanisms that lead to homozygous deletions: (i) independent recombination of transforming DNA into each allele and (ii) recombination of transforming DNA into one allele, followed by gene conversion of the second allele. Our approach will streamline gene function analysis in C. albicans, and our results indicate that DNA can function transiently after transformation of this organism. IMPORTANCE The fungus Candida albicans is a major pathogen. Genetic analysis of this organism has revealed determinants of pathogenicity, drug resistance, and other unique biological features, as well as the identities of prospective drug targets. The creation of targeted mutations has been greatly accelerated recently through the implementation of CRISPR genome-editing technology by Vyas et al. [Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248]. In this study, we find that CRISPR elements can be expressed from genes that are present only transiently, and we develop a transient CRISPR system that further accelerates C. albicans genetic manipulation.

  16. Gene expression and single nucleotide polymorphism array analyses of spindle cell lipomas and conventional lipomas with 13q14 deletion.

    Science.gov (United States)

    Bartuma, Hammurabi; Nord, Karolin H; Macchia, Gemma; Isaksson, Margareth; Nilsson, Jenny; Domanski, Henryk A; Mandahl, Nils; Mertens, Fredrik

    2011-08-01

    Spindle cell lipomas (SCL) are circumscribed, usually s.c. tumors that typically occur on the posterior neck, shoulder, and back of middle aged men. Cytogenetically, almost all SCL are characterized by deletions of chromosome arm 13q, often in combination with loss of 16q. Deletions of 13q are seen also in approximately 15% of conventional lipomas. Through single nucleotide polymorphism (SNP) array analyses, we identified two minimal deleted regions (MDR) in 13q14 in SCL. In MDR1, four genes were located, including the tumor suppressor gene RB1. MDR1 in SCL overlapped with the MDR detected in conventional lipomas with 13q14 deletion. In MDR2 in SCL there were 34 genes and the two microRNA (miRNA) genes miR-15a and miR-16-1. Global gene expression analysis was used to study the impact of the deletions on genes mapping to the two SCL-associated MDR. Five genes (C13orf1, DHRS12, ATP7B, ALG11, and VPS36) in SCL and one gene (C13orf1) in conventional lipomas with 13q-deletions were found to be significantly underexpressed compared with control tissues. Quantitative real-time PCR showed that miR-16-1 was expressed at lower levels in SCL than in the control samples. No mutations were found at sequencing of RB1, miR-15a, and miR-16-1. Our findings further delineate the target region for the 13q deletion in SCL and conventional lipomas and show that the deletions are associated with down-regulated expression of several genes, notably C13orf1, which was the only gene to be significantly down-regulated in both tumor types. Copyright © 2011 Wiley-Liss, Inc.

  17. Truncated photosystem chlorophyll antenna size in the green microalga Chlamydomonas reinhardtii upon deletion of the TLA3-CpSRP43 gene.

    Science.gov (United States)

    Kirst, Henning; Garcia-Cerdan, Jose Gines; Zurbriggen, Andreas; Ruehle, Thilo; Melis, Anastasios

    2012-12-01

    The truncated light-harvesting antenna size3 (tla3) DNA insertional transformant of Chlamydomonas reinhardtii is a chlorophyll-deficient mutant with a lighter green phenotype, a lower chlorophyll (Chl) per cell content, and higher Chl a/b ratio than corresponding wild-type strains. Functional analyses revealed a higher intensity for the saturation of photosynthesis and greater light-saturated photosynthetic activity in the tla3 mutant than in the wild type and a Chl antenna size of the photosystems that was only about 40% of that in the wild type. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western-blot analyses showed that the tla3 strain was deficient in the Chl a/b light-harvesting complex. Molecular and genetic analyses revealed a single plasmid insertion in chromosome 4 of the tla3 nuclear genome, causing deletion of predicted gene g5047 and plasmid insertion within the fourth intron of downstream-predicted gene g5046. Complementation studies defined that gene g5047 alone was necessary and sufficient to rescue the tla3 mutation. Gene g5047 encodes a C. reinhardtii homolog of the chloroplast-localized SRP43 signal recognition particle, whose occurrence and function in green microalgae has not hitherto been investigated. Biochemical analysis showed that the nucleus-encoded and chloroplast-localized CrCpSRP43 protein specifically operates in the assembly of the peripheral components of the Chl a/b light-harvesting antenna. This work demonstrates that cpsrp43 deletion in green microalgae can be employed to generate tla mutants with a substantially diminished Chl antenna size. The latter exhibit improved solar energy conversion efficiency and photosynthetic productivity under mass culture and bright sunlight conditions.

  18. Deletion of Helicobacter pylorivacuolating cytotoxin gene by introduction of directed mutagenesis

    Institute of Scientific and Technical Information of China (English)

    Jian-Ping Yuan; Tao Li; Xiao-Dong Shi; Bao-Yu Hu; Gui-Zhen Yang; Shan-Qing Tong; Xiao-Kui Guo

    2003-01-01

    AIM: To construct a vacA-knockout Helicobacter pylori mutant strain, whose only difference from the wild strain is its disrupted vacA gene.METHODS AND RESULTS: A clone containing kanamycin resistance gene used for homologous recombination was constructed in a directional cloning procedure into pBluescript ⅡSK, and then transformed into vacA+ H pylori by electroporation.Colonies growing on the selective media containing kanamycin were harvested for chromosomal DNA extraction,and the allelic exchange was determined by polymerase chain reactions and sequencing. Loss of vacuolating activity of the vaci-knockout strain was confirmed by examining the gastric cells co-cultured with cell-free supernatants from H pylori wild strain or the mutant.CONCLUSION: We constructed a vacA-knockout strain of H pylori through direct mutagenesis, which creates an important precondition for the future research on virulence comparison with gene expression analysis.

  19. Differentially expressed genes in white egg 2 mutant of silkworm ...

    African Journals Online (AJOL)

    use

    2011-12-21

    Dec 21, 2011 ... Our results hopefully shed light on the further study of molecular mechanism of white ... 2002). The mutant white egg 3 (w-3) has white eyes and eggs with ... As of other insects, the color of the eggs of silkworm mainly depends ...

  20. Programmed cell death in Saccharomyces cerevisiae is hampered by the deletion of GUP1 gene

    Directory of Open Access Journals (Sweden)

    Tulha Joana

    2012-05-01

    Full Text Available Abstract Background During the past years, yeast has been successfully established as a model to study mechanisms of programmed cell death regulation. Saccharomyces cerevisiae commits to cell death showing typical hallmarks of metazoan apoptosis, in response to different stimuli. Gup1p, an O-acyltransferase, is required for several cellular processes that are related to apoptosis development, such as rafts integrity and stability, lipid metabolism including GPI anchor correct remodeling, proper mitochondrial and vacuole function, bud site selection and actin dynamics. Therefore, we hypothesize that apoptotic process would be affected by GUP1 deletion. Results In the present work we used two known apoptosis inducing conditions, chronological aging and acetic acid, to assess several apoptotic markers in gup1∆ mutant strain. We found that this mutant presents a significantly reduced chronological lifespan as compared to Wt and it is also highly sensitive to acetic acid treatment. In addition, it presents extremely high levels of ROS. There were notorious differences on apoptotic markers between Wt and gup1∆ mutant strains, namely on the maintenance of plasma membrane integrity, on the phosphatidylserine externalization, on the depolarization of mitochondrial membrane and on the chromatin condensation. Those suggested that the mutant, under either condition, probably dies of necrosis and not from apoptosis. Conclusions To Gup1p has been assigned an important function on lipid rafts assembly/integrity, lipid metabolism and GPI anchor remodeling. Our results provide, for the first time, the connection of the integrity of yeast lipid rafts and apoptosis induction and/or signaling, giving new insights into the molecular mechanisms underlying this process in yeast.

  1. Studies on the Mutant Systems of the Bombyx mori Gene Bank

    Institute of Scientific and Technical Information of China (English)

    LU Cheng; DAI Fang-yin; XIANG Zhong-huai

    2002-01-01

    Through over ten years of study, more than 1 000 genetic materials including mutant genes,chromosomal variation strains and special genetic materials of Bombyx mori, Linnaeus, collected, introduced or created since 1940s especially late 1980s, have been sorted out and put in order. After identifications and genetic analyses of their morphological, physiological and biochemical characters, the silkworm gene bank was constructed and the preservation system was perfected, and more than 600 silkworm strains were kept in this gene bank. The preserved silkworm mutant genes have covered more than 90% of existent ones across the world, in which, more than 100 are rare and precious mutant genes, and over 60 mutant genes were found and studied for the first time. Through hybrid analyses, linkage tests and three-point gene location tests, a perfect linkage retrieval labeling system of silkworm was established, which included 230 marker genes covering all the 28 linkage groups of Bombyx mori. The gene location system (composite system of recessive genes) of different linkage groups was set up. The intergenic complementation of mutant egg color and third type of maternal heredity egg color have been found, and indicated that the epistatic effect of mutant gene of white egg is universal. Twenty eight independent near isogenic lines murked with morphological mutation gene have been created and a series of novel breeding materials possessing great potential for application such as high feeding efficiency, special sex markers, natural colored silk, resistance to disease, wider feeding range and adjustable parthenogenesis, etc., have been developed. The sustainable maintenance and management technique system of silkworm gene resources were well established.

  2. Unusual presentation of Kallmannn syndrome with contiguous gene deletion in three siblings of a family.

    Science.gov (United States)

    Madhu, Sri Venkat; Kant, Saket; Holla, Vikram Venkappayya; Arora, Rakesh; Rathi, Sahaj

    2012-12-01

    We report the case of 3 brothers aged 34, 24, and 22 years, unmarried, who presented to our endocrinology clinic with absence of secondary sexual characters. There was no such history in other siblings, but their maternal uncle had similar complaints. On examination, all 3 had pre-pubertal appearance, voice, and genitalia along with anosmia and bimanual synkinesia. Cryptorchidism was noticed in 2 while third person had small hypoplastic testes. It was also noted that all 3 patients had icthyosis mainly involving trunk, back, and limbs. The hormonal assays were consistent with isolated hypogonadotrophic hypogonadism. IQ testing revealed mental retardation in the 2 patients. Ultrasound showed ectopic right kidney in one patient, atrophic right kidney in the second patient while the third patient had normal kidneys. MRI brain of all the patients showed poorly visualized olfactory tract and bulb. Kallmann syndrome (KS) was diagnosed based on hormonal evaluation and MRI results. Of the four types of KS: Synkinesia, renal anomaly, and X-linked pedigree pattern in our patients pointed towards X-linked type 1 KS as the possible cause. But, icthyosis and mental retardation are not usual presentation of type 1 KS. They are usually seen as a result of contiguous gene deletion of KAL1, steroid sulfatase (STS), and mental retardation (MRX) gene on X chromosome. Hence, the possible gene defect in our cases is inherited defect in contiguous gene deletion. The contiguous gene deletion as the cause of KS in 3 patients of same family is very rare and worth reporting. Also, the significance of phenotype-genotypic association in Kallmann syndrome is discussed.

  3. Identification of a novel functional deletion variant in the 5'-UTR of the DJ-1 gene

    Directory of Open Access Journals (Sweden)

    Warnich Louise

    2009-10-01

    Full Text Available Abstract Background DJ-1 forms part of the neuronal cellular defence mechanism against oxidative insults, due to its ability to undergo self-oxidation. Oxidative stress has been implicated in the pathogenesis of central nervous system damage in different neurodegenerative disorders including Alzheimer's disease and Parkinson's disease (PD. Various mutations in the DJ-1 (PARK7 gene have been shown to cause the autosomal recessive form of PD. In the present study South African PD patients were screened for mutations in DJ-1 and we aimed to investigate the functional significance of a novel 16 bp deletion variant identified in one patient. Methods The possible effect of the deletion on promoter activity was investigated using a Dual-Luciferase Reporter assay. The DJ-1 5'-UTR region containing the sequence flanking the 16 bp deletion was cloned into a pGL4.10-Basic luciferase-reporter vector and transfected into HEK293 and BE(2-M17 neuroblastoma cells. Promoter activity under hydrogen peroxide-induced oxidative stress conditions was also investigated. Computational (in silico cis-regulatory analysis of DJ-1 promoter sequence was performed using the transcription factor-binding site database, TRANSFAC via the PATCH™ and rVISTA platforms. Results A novel 16 bp deletion variant (g.-6_+10del was identified in DJ-1 which spans the transcription start site and is situated 93 bp 3' from a Sp1 site. The deletion caused a reduction in luciferase activity of approximately 47% in HEK293 cells and 60% in BE(2-M17 cells compared to the wild-type (P Conclusion This is the first report of a functional DJ-1 promoter variant, which has the potential to influence transcript stability or translation efficiency. Further work is necessary to determine the extent to which the g.-6_+10del variant affects the normal function of the DJ-1 promoter and whether this variant confers a risk for PD.

  4. Identifying relationships among genomic disease regions: predicting genes at pathogenic SNP associations and rare deletions.

    Directory of Open Access Journals (Sweden)

    Soumya Raychaudhuri

    2009-06-01

    Full Text Available Translating a set of disease regions into insight about pathogenic mechanisms requires not only the ability to identify the key disease genes within them, but also the biological relationships among those key genes. Here we describe a statistical method, Gene Relationships Among Implicated Loci (GRAIL, that takes a list of disease regions and automatically assesses the degree of relatedness of implicated genes using 250,000 PubMed abstracts. We first evaluated GRAIL by assessing its ability to identify subsets of highly related genes in common pathways from validated lipid and height SNP associations from recent genome-wide studies. We then tested GRAIL, by assessing its ability to separate true disease regions from many false positive disease regions in two separate practical applications in human genetics. First, we took 74 nominally associated Crohn's disease SNPs and applied GRAIL to identify a subset of 13 SNPs with highly related genes. Of these, ten convincingly validated in follow-up genotyping; genotyping results for the remaining three were inconclusive. Next, we applied GRAIL to 165 rare deletion events seen in schizophrenia cases (less than one-third of which are contributing to disease risk. We demonstrate that GRAIL is able to identify a subset of 16 deletions containing highly related genes; many of these genes are expressed in the central nervous system and play a role in neuronal synapses. GRAIL offers a statistically robust approach to identifying functionally related genes from across multiple disease regions--that likely represent key disease pathways. An online version of this method is available for public use (http://www.broad.mit.edu/mpg/grail/.

  5. Active expression of Gγ globin gene on chromosome 11 with Yunnanese (Ayγδβ)~0-thalasseinia deletion in MEL cells

    Institute of Scientific and Technical Information of China (English)

    张俊武; 乔军; 宋文风; 邱志明

    1996-01-01

    A permanent lymphocyte cell line of a heterozygote with Yunnanese (Aγδβ)0-thalassemia deletion, associated with an increased production of Cry globin in adult, was founded using Epstein-Barr virus transformation. The hybrids of the lymphocyte cell and mouse erythroleukemia cell (MEL) were achieved and the hybrids containing human chromosome 11 were selected with the monoclonal antibody 53/6. The subclones containing only either the normal or the abnormal human chromosome 11 were separated and the expression of the human globin genes was studied. Expression of the β-globin gene, but not the Cγ and Aγ, was observed in the hybrids containing only the normal human chromosome 11, while active expression of the Cγ globin gene was observed in the hybrids containing only the abnormal human chromosome 11. These results have confirmed that the DNA deletion in the β-globin gene cluster is the cause of persistent active expression of the Cγ globin gene in the Yunnanese mutant.

  6. Ku70 and ku80 null mutants improve the gene targeting frequency in Monascus ruber M7.

    Science.gov (United States)

    He, Yi; Liu, Qingpei; Shao, Yanchun; Chen, Fusheng

    2013-06-01

    Normally, gene targeting by homologous recombination occurs rarely during a transformation process since non-homologous recombination is predominant in filamentous fungi. In our previous researches, the average gene replacement frequency (GRF) in Monascus ruber M7 was as low as 15 %. To develop a highly efficient gene targeting system for M. ruber M7, two M. ruber M7 null mutants of ku70 (MrΔku70) and ku80 (MrΔku80) were constructed which had no apparent defects in the development including vegetative growth, colony phenotype, microscopic morphology and spore yield compared with M. ruber M7. In addition, the production of some significant secondary metabolites such as pigments and citrinin had no differences between the two disruptants and the wild-type strain. Further results revealed that the GRFs of triA (encoding a putative acetyltransferase) were 42.2 % and 61.5 % in the MrΔku70 and MrΔku80 strains, respectively, while it was only about 20 % in M. ruber M7. Furthermore, GRFs of these two disruptants at other loci (the pigE, fmdS genes in MrΔku70 and the ku70 gene in MrΔku80) were investigated, and the results indicated that GRFs in the MrΔku70 strain and the MrΔku80 strain were doubled and tripled compared with that in M. ruber M7, respectively. Therefore, the ku70 and ku80 null mutants of M. ruber M7, especially the ku80-deleted strain, will be excellent hosts for efficient gene targeting.

  7. Cloning and functional analysis of the sequences flanking mini-Tn5 in the magnetosomes deleted mutant NM4 of Magnetospirillum gryphiswaldense MSR-1

    Institute of Scientific and Technical Information of China (English)

    LI; Feng; LI; Ying; JIANG; Wei; WANG; Zhenfang; LI; Jilun

    2005-01-01

    A magnetosome deleted mutant NM4 of Magnetospirillum gryphiswaldense MSR-1 was generated by mini-Tn5 transposon mutagenesis, and a 5045-bp fragment flanking mini-Tn5 in NM4 was cloned by Anchored PCR. Sequencing analysis showed that this fragment involved six putative open reading frames (ORFs); the mini-Tn5 was inserted into ORF4. Functional complementary test indicated that the 5045-bp fragment was required for biosynthesis of magnetosomes in M. gryphiswaldense MSR-1. The protein encoded by ORF4 had 25% of identity with the chemotaxis protein CheYIII of Caulobacter crescentus CB15, and the protein encoded by ORF4 contained a conserved signal receiver domain that can receive the signal from the sensor partner of the bacterial two-component systems. It was suggested that the protein encoded by ORF4 may take part in the signal transduction relating to biosynthesis of magnetosomes.

  8. 布鲁氏菌M5-90△WboA基因缺失株的构建及免疫效果的初步评价%Construction and preliminary evaluation of immune effects of deletion mutant of WboA gene of Brucella melitensis vaccine M5-90

    Institute of Scientific and Technical Information of China (English)

    张艳; 陈创夫; 张辉; 张沾; 李志强; 张俊波; 孟仁; 王震

    2011-01-01

    Vaccination is a major measure for prevention of brucellosis, but it is unable to be distinguished from the vaccinated to natural infected animals. In this study, the WboA gene was knocked out of the genomic DNA of Brucelh melitensis vaccine M5-90 strain to construct the recombinant M5-90 A WboA by homologous recombination. The test results showed that M5-90 A WboA was less virulent than that of the parent M5-90 strain (p<0.01). Humoral immunity and cellular immunity tests showed that there was no significant difference between M5-90A WboA and M5-90 parent strain (p<0.05). BALB/c mice were immunized with M5-90 A WboA or M5-90 and challenged with virulent strain 16M and the survival rate were 10% and 20%, respectively, indicating that the M5-90 A WboA provided the similar protection of M5-90 strain. Agglutination test and western blot showed that the serum response of M5-90 △ WboA in vaccinated mice were negative. These results indicated that M5-90 A WboA strain might be a promising vaccine against brucellosis, and could be distinguished from the vaccine immunization to natural infection in animals by serum test.%为获得毒力较弱并能区分自然感染和疫苗免疫的布鲁氏菌候选疫苗株,本研究用PCR方法扩增WboA基因的上下游同源臂序列,构建重组质粒pGEM-7zf-△WboA-Sac,电转化布鲁氏菌M5-90感受态细胞,筛选布鲁氏菌疫苗株M5-90的WboA基因缺失株,并对获得的M5-90△WboA遗传稳定性、毒力、免疫保护性、抗体水平等指标进行检测.实验结果表明M5-90△WboA株的毒力比M5-90株明显减弱,差异极显著(p<0.01),体液免疫和细胞免疫结果表明M5-90△WboA株与亲本M5-90株相比差异不显著(p<0.05),M5-90△WboA株和亲本株的保护率分别为10%和20%,表明M5-90△WboA株与M5-90株具有相似的保护性.凝集试验和western blot试验显示M5-90△WboA株免疫小鼠的血清反应结果为阴性.本研究构建的布鲁氏菌基因缺失株M5-90△WboA

  9. Novel large deletion in the ACTA1 gene in a child with autosomal recessive nemaline myopathy.

    Science.gov (United States)

    Friedman, Bethany; Simpson, Kara; Tesi-Rocha, Carolina; Zhou, Delu; Palmer, Cheryl A; Suchy, Sharon F

    2014-04-01

    Nemaline myopathy (NM) is a genetically and clinically heterogeneous disorder resulting from a disruption of the thin filament proteins of the striated muscle sarcomere. The disorder is typically characterized by muscle weakness including the face, neck, respiratory, and limb muscles and is clinically classified based on the age of onset and severity. Mutations in the ACTA1 gene contribute to a significant proportion of NM cases. The majority of ACTA1 gene mutations are missense mutations causing autosomal dominant NM by producing an abnormal protein. However, approximately 10% of ACTA1 gene mutations are associated with autosomal recessive NM; these mutations are associated with loss of protein function. We report the first case of a large deletion in the ACTA1 gene contributing to autosomal recessive NM. This case illustrates the importance of understanding disease mechanisms at the molecular level to accurately infer the inheritance pattern and potentially aid with clinical management. Copyright © 2014. Published by Elsevier B.V.

  10. High risk genetic factor in Chinese patients with idiopathic male infertility:deletion of DAZ gene copy on Y chromosome

    Institute of Scientific and Technical Information of China (English)

    杨元; 肖翠英; 张思仲; 张思孝; 黄明孔; 林立

    2004-01-01

    @@ Idiopathic azoospermia or oligozoospermia affects approximately 2%-4% of all married males. Recently studies have confirmed that the deletion of DAZ in AZFc region of Y chromosome may be one of the important genetic aetiologies of Caucasian male infertility. To determine the relationship between DAZ gene deletion and idiopathic male infertility in Chinese population, we analysed the DAZ gene copy number of AZFc region in patients with idiopathic azoospermia or oligozoospermia, as well as fertile Chinese men.

  11. Virulence Associated Genes-Deleted Salmonella Montevideo Is Attenuated, Highly Immunogenic and Confers Protection against Virulent Challenge in Chickens

    Science.gov (United States)

    Lalsiamthara, Jonathan; Lee, John H.

    2016-01-01

    To construct a novel live vaccine against Salmonella enterica serovar Montevideo (SM) infection in chickens, two important bacterial regulatory genes, lon and cpxR, which are associated with invasion and virulence, were deleted from the wild type SM genome. Attenuated strains, JOL1625 (Δlon), JOL1597 (ΔcpxR), and JOL1599 (ΔlonΔcpxR) were thereby generated. Observations with scanning electron microscopy suggested that JOL1625 and JOL1599 cells showed increased ruffled surface which may be related to abundant extracellular polysaccharide (EPS) production. JOL1597 depicted milder ruffled surface but showed increased surface corrugation. ConA affinity-based fluorometric quantification and fluorescence microscopy revealed significant increases in EPS production in JOL1625 and JOL1599. Four weeks old chickens were used for safety and immunological studies. The mutants were not observed in feces beyond day 3 nor in spleen and cecum beyond day 7, whereas wild type SM was detected for at least 2 weeks in spleen and cecum. JOL1599 was further evaluated as a vaccine candidate. Chickens immunized with JOL1599 showed strong humoral responses, as indicated by systemic IgG and secretory IgA levels, as well as strong cell-mediated immune response, as indicated by increased lymphocyte proliferation. JOL1599-immunized groups also showed significant degree of protection against wild type challenge. Our results indicate that Δlon- and/or ΔcpxR-deleted SM exhibited EPS-enhanced immunogenicity and attenuation via reduced bacterial cell intracellular replication, conferred increased protection, and possess safety qualities favorable for effective vaccine development against virulent SM infections. PMID:27785128

  12. The evolution of small insertions and deletions in the coding genes of Drosophila melanogaster.

    Science.gov (United States)

    Chong, Zechen; Zhai, Weiwei; Li, Chunyan; Gao, Min; Gong, Qiang; Ruan, Jue; Li, Juan; Jiang, Lan; Lv, Xuemei; Hungate, Eric; Wu, Chung-I

    2013-12-01

    Studies of protein evolution have focused on amino acid substitutions with much less systematic analysis on insertion and deletions (indels) in protein coding genes. We hence surveyed 7,500 genes between Drosophila melanogaster and D. simulans, using D. yakuba as an outgroup for this purpose. The evolutionary rate of coding indels is indeed low, at only 3% of that of nonsynonymous substitutions. As coding indels follow a geometric distribution in size and tend to fall in low-complexity regions of proteins, it is unclear whether selection or mutation underlies this low rate. To resolve the issue, we collected genomic sequences from an isogenic African line of D. melanogaster (ZS30) at a high coverage of 70× and analyzed indel polymorphism between ZS30 and the reference genome. In comparing polymorphism and divergence, we found that the divergence to polymorphism ratio (i.e., fixation index) for smaller indels (size ≤ 10 bp) is very similar to that for synonymous changes, suggesting that most of the within-species polymorphism and between-species divergence for indels are selectively neutral. Interestingly, deletions of larger sizes (size ≥ 11 bp and ≤ 30 bp) have a much higher fixation index than synonymous mutations and 44.4% of fixed middle-sized deletions are estimated to be adaptive. To our surprise, this pattern is not found for insertions. Protein indel evolution appear to be in a dynamic flux of neutrally driven expansion (insertions) together with adaptive-driven contraction (deletions), and these observations provide important insights for understanding the fitness of new mutations as well as the evolutionary driving forces for genomic evolution in Drosophila species.

  13. Deletion of the Synechocystis sp. PCC 6803 kaiAB1C1 gene cluster causes impaired cell growth under light-dark conditions.

    Science.gov (United States)

    Dörrich, Anja K; Mitschke, Jan; Siadat, Olga; Wilde, Annegret

    2014-11-01

    In contrast to Synechococcus elongatus PCC 7942, few data exist on the timing mechanism of the widely used cyanobacterium Synechocystis sp. PCC 6803. The standard kaiAB1C1 operon present in this organism was shown to encode a functional KaiC protein that interacted with KaiA, similar to the S. elongatus PCC 7942 clock. Inactivation of this operon in Synechocystis sp. PCC 6803 resulted in a mutant with a strong growth defect when grown under light-dark cycles, which was even more pronounced when glucose was added to the growth medium. In addition, mutants showed a bleaching phenotype. No effects were detected in mutant cells grown under constant light. Microarray experiments performed with cells grown for 1 day under a light-dark cycle revealed many differentially regulated genes with known functions in the ΔkaiABC mutant in comparison with the WT. We identified the genes encoding the cyanobacterial phytochrome Cph1 and the light-repressed protein LrtA as well as several hypothetical ORFs with a complete inverse behaviour in the light cycle. These transcripts showed a stronger accumulation in the light but a weaker accumulation in the dark in ΔkaiABC cells in comparison with the WT. In general, we found a considerable overlap with microarray data obtained for hik31 and sigE mutants. These genes are known to be important regulators of cell metabolism in the dark. Strikingly, deletion of the ΔkaiABC operon led to a much stronger phenotype under light-dark cycles in Synechocystis sp. PCC 6803 than in Synechococcus sp. PCC 7942. © 2014 The Authors.

  14. Severe fetal and neonatal hemolytic anemia due to a 198 kb deletion removing the complete β-globin gene cluster.

    Science.gov (United States)

    Verhovsek, Madeleine; Shah, Nirmish R; Wilcox, Ibifiri; Koenig, Sara C; Barros, Tiago; Thornburg, Courtney D; Steinberg, Martin H; Luo, Hong-yuan; Chui, David H K

    2012-11-01

    Fetal and neonatal hemolytic anemia can be caused by (γδβ)(0)-thalassemia deletions of the β-globin gene cluster. Many of these deletions have not been well characterized, and diagnostic tests are not readily available, thus hampering carrier detection, family counseling, and antenatal diagnosis. We report and define a 198 kb deletion removing the entire β-globin gene cluster, which was found in members of a multigeneration family of Irish/Scottish descent. The proband had life-threatening fetal and neonatal hemolytic anemia which subsided by 1 year of age. Copyright © 2012 Wiley Periodicals, Inc.

  15. Morphological Structure and Genetic Mapping of New Leaf-Color Mutant Gene in Rice (Oryza sativa)

    Institute of Scientific and Technical Information of China (English)

    LI Yu-hong; WANG Bao-he; DAI Zheng-yuan; LI Ai-hong; LIU Guang-qing; ZUO Shi-min; ZHANG Hong-xi; PAN Xue-biao

    2012-01-01

    Leaf-color mutations are a widely-observed class of mutations,playing an important role in the study of chlorophyll biosynthesis and plant chloroplast structure,function,genetics and development.A naturally-occurring leaf-color rice mutant,Baihuaidao 7,was analyzed.Mutant plants typically exhibited a green-white-green leaf-color progression,but this phenotype was only expressed in the presence of a stress signal induced by mechanical scarification such as transplantation.Prior to the appearance of white leaves,mutant plant growth,leaf color,chlorophyll content,and chloroplast ultrastructure appeared to be identical to those of the wild type.After the changeover to white leaf color,an examination of the mutated leaves revealed a decrease in total chlorophyll,chlorophyll a,chlorophyll b,and carotenoid content,a reduction in the number of chloroplast grana lamella and grana,and a gradual degradation of the thylakoid lamellas.At maturity,the mutant plant was etiolated and dwarfed compared with wild-type plants.Genetic analysis indicated that the leaf mutant character is controlled by a recessive nuclear gene.Genetic mapping of the mutant gene was performed using an F2 population derived from a Baihuaidao 7 ×Jiangxi 1587 cross.The mutant gene was mapped to rice chromosome 11,positioned between InDel markers L59.2-7 and L64.8-11,which are separated by approximately 740.5 kb.The mutant gene is believed to be a new leaf-color mutant gene in rice,and is tentatively designated as gwgl.

  16. Autosomal Recessive Nonsyndromic Hearing Impairment due to a Novel Deletion in the RDX Gene

    Directory of Open Access Journals (Sweden)

    Kwanghyuk Lee

    2011-01-01

    Full Text Available The RDX gene anchors cytoskeletal actin of stereocilia to hair cell transmembrane and is responsible for autosomal recessive nonsyndromic hearing impairment (ARNSHI due to DFNB24. A genome scan was performed using DNA samples from a consanguineous Pakistani family with ARNSHI. A significant maximum two-point LOD score of 4.5 (θ=0 and multipoint LOD score of 5.8 were achieved at marker D11S1998 (chr11 : 117.20 Mb. The region of homozygosity is bounded by markers D11S2000 (105.06 Mb and D11S4464 (123.13 Mb and contains the NSHI genes TECTA and RDX. Although no potentially causal variants were identified in the TECTA gene, within the RDX gene a novel deletion c.1076_1079delTTAA (p.Ile359Lysfs*6 was identified. The RDX deletion segregates with ARNSHI within the family and was not observed in 500 control chromosomes. It is predicted to cause premature truncation of radixin at the α-helical domain and to result in nonfunctional transcripts within the cochlea. RDX isoforms which encode the coiled-coil region of the α-helical domain are deemed necessary for proper function of hair cell stereocilia.

  17. Infectious bronchitis viruses with naturally occurring genomic rearrangement and gene deletion.

    Science.gov (United States)

    Hewson, Kylie A; Ignjatovic, Jagoda; Browning, Glenn F; Devlin, Joanne M; Noormohammadi, Amir H

    2011-02-01

    Infectious bronchitis viruses (IBVs) are group III coronaviruses that infect poultry worldwide. Genetic variations, including whole-gene deletions, are key to IBV evolution. Australian subgroup 2 IBVs contain sequence insertions and multiple gene deletions that have resulted in a substantial genomic divergence from international IBVs. The genomic variations present in Australian IBVs were investigated and compared to those of another group III coronavirus, turkey coronavirus (TCoV). Open reading frames (ORFs) found throughout the genome of Australian IBVs were analogous in sequence and position to TCoV ORFs, except for ORF 4b, which appeared to be translocated to a different position in the subgroup 2 strains. Subgroup 2 strains were previously reported to lack genes 3a, 3b and 5a, with some also lacking 5b. Of these, however, genes 3b and 5b were found to be present but contained various mutations that may affect transcription. In this study, it was found that subgroup 2 IBVs have undergone a more substantial genomic rearrangements than previously thought.

  18. Identification of a PKP2 gene deletion in a family with arrhythmogenic right ventricular cardiomyopathy.

    Science.gov (United States)

    Li Mura, Ilena Egle Astrid; Bauce, Barbara; Nava, Andrea; Fanciulli, Manuela; Vazza, Giovanni; Mazzotti, Elisa; Rigato, Ilaria; De Bortoli, Marzia; Beffagna, Giorgia; Lorenzon, Alessandra; Calore, Martina; Dazzo, Emanuela; Nobile, Carlo; Mostacciuolo, Maria Luisa; Corrado, Domenico; Basso, Cristina; Daliento, Luciano; Thiene, Gaetano; Rampazzo, Alessandra

    2013-11-01

    Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a primary heart muscle disease characterized by progressive myocardial loss, with fibro-fatty replacement, and high frequency of ventricular arrhythmias that can lead to sudden cardiac death. ARVC is a genetically determined disorder, usually caused by point mutations in components of the cardiac desmosome. Conventional mutation screening of ARVC genes fails to detect causative mutations in about 50% of index cases, suggesting a further genetic heterogeneity. We performed a genome-wide linkage study and a copy number variations (CNVs) analysis, using high-density SNP arrays, in an ARVC family showing no mutations in any of the desmosomal genes. The CNVs analysis identified a heterozygous deletion of about 122 kb on chromosome 12p11.21, including the entire plakophilin-2 gene and shared by all affected family members. It was not listed on any of available public CNVs databases and was confirmed by quantitative real-time PCR. This is the first SNP array-based genome-wide study leading to the identification of a CNV segregating with the disease phenotype in an ARVC family. This result underscores the importance of performing additional analysis for possible genomic deletions/duplications in ARVC patients without point mutations in known disease genes.

  19. [Phenotype analysis and mutant gene location of ventral yellow mouse (VY(Slac))].

    Science.gov (United States)

    Shi, Mei-Lian; Xu, Ping; Yin, Xiao-Shu; Yang, Wei-Wei; Gu, Mei-Er; Yu, Li-Ping; Liu, Gui-Jie; Wu, Bao-Jin

    2012-06-01

    The ventri-yellow pigmentation mouse (temporarily named VY(Slac)) arose spontaneously in the C57BL/6J inbred mouse strain, found and bred by Shanghai SLAC Laboratory Animal Co., Ltd. VY(Slac) presented a special phenotype marked by yellow coat on the ventral surface of neck and trunk that was without melanin deposition but maintained a normal structure. The number of melanocytes in epidermis and melanin in hair follicle of the abdominal skin of the mutant mouse were less than that of their background strain, while there was no significant difference between the dorsal skins of the two strains. This mutant phenotype was inherited as single-gene dominant inheritance, confirmed by genetic experiment, and there was no significant difference between VY(Slac) and B(6) for other biological parameters such as weight, anatomic and histological structures of major organs and blood physiology. When the linkage relationship between the genomic DNA samples of F(2) 48 mice (VY(Slac)D(2)F(1)×D(2)) and mutant phenotype were evaluated, the mutant gene was confirmed on chromosome 2 near D2Mit229. New microsatellite and SNP markers were selected to amplify genomic DNA samples of 196 F(2) mice and the mutant gene was narrowed down to 5.3 Mb region between rs13476833 and rs27310903 on chromosome 2. The preliminary results of our phenotype analysis and gene location provides a solid basis for further identification of this mutant gene.

  20. Genetic Analysis and Molecular Mapping of a Novel Chlorophyll-Deficit Mutant Gene in Rice

    Institute of Scientific and Technical Information of China (English)

    HUANG Xiao-qun; WANG Ping-rong; ZHAO Hai-xin; DENG Xiao-jian

    2008-01-01

    A rice etiolation mutant 824ys featured with chlorophyll deficiency was identified from a normal green rice variety 824B.It showed whole green-yellow plant from the seedling stage,reduced number of tillers and longer growth duration.The contents of chlorophyll,chlorophyll a,chlorophyll b and net photosynthetic rate in leaves of the mutant obviously decreased,as well as the number of spikelets per panicle,seed setting rate and 1000-grain weight compared with its wild-type parent.Genetic analyses on F1 and F2 generetions of 824ys crossed with three normal green varieties showed that the chlorophyll-deficit mutant character was controlled by a pair of recessive nuclear gene.Genetic mapping of the mutant gene was conducted by using microsatellite markers and F2 mapping population of 495R/824ys,and the mutant gene of 824ys was mapped on the shon arm of rice chromosome 3.The genetic distances from the target gene to the markers RM218,RM282 and RM6959 were 25.6 cM,5.2 cM and 21.8 cM,respectively.It was considered to be a now chlorophyll-deficit mutant gene and tentatively named as chl11(t).

  1. Reporter Gene Silencing in Targeted Mouse Mutants Is Associated with Promoter CpG Island Methylation.

    Science.gov (United States)

    Kirov, Julia V; Adkisson, Michael; Nava, A J; Cipollone, Andreana; Willis, Brandon; Engelhard, Eric K; Lloyd, K C Kent; de Jong, Pieter; West, David B

    2015-01-01

    Targeted mutations in mouse disrupt local chromatin structure and may lead to unanticipated local effects. We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Messenger RNA levels of the reporter gene and targeted gene were assessed by qRT-PCR, and methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. Mutants were stratified by LacZ staining into presumed Silenced and Expressed reporter genes. Silenced mutants had reduced relative quantities LacZ mRNA and greater CpG Island methylation compared with the Expressed mutant group. Within the silenced group, LacZ coding sequence methylation was significantly and positively correlated with CpG Island methylation, while promoter CpG methylation was only weakly correlated with LacZ gene mRNA. The results support the conclusion that there is promoter silencing in a subset of mutants carrying the tm1a allele. The features of targeted genes which promote local silencing when targeted remain unknown.

  2. Compensatory evolution for a gene deletion is not limited to its immediate functional network

    Directory of Open Access Journals (Sweden)

    Bull JJ

    2009-05-01

    Full Text Available Abstract Background Genetic disruption of an important phenotype should favor compensatory mutations that restore the phenotype. If the genetic basis of the phenotype is modular, with a network of interacting genes whose functions are specific to that phenotype, compensatory mutations are expected among the genes of the affected network. This perspective was tested in the bacteriophage T3 using a genome deleted of its DNA ligase gene, disrupting DNA metabolism. Results In two replicate, long-term adaptations, phage compensatory evolution accommodated the low ligase level provided by the host without reinventing its own ligase. In both lines, fitness increased substantially but remained well below that of the intact genome. Each line accumulated over a dozen compensating mutations during long-term adaptation, and as expected, many of the compensatory changes were within the DNA metabolism network. However, several compensatory changes were outside the network and defy any role in DNA metabolism or biochemical connection to the disruption. In one line, these extra-network changes were essential to the recovery. The genes experiencing compensatory changes were moderately conserved between T3 and its relative T7 (25% diverged, but the involvement of extra-network changes was greater in T3. Conclusion Compensatory evolution was only partly limited to the known functionally interacting partners of the deleted gene. Thus gene interactions contributing to fitness were more extensive than suggested by the functional properties currently ascribed to the genes. Compensatory evolution offers an easy method of discovering genome interactions among specific elements that does not rest on an a priori knowledge of those elements or their interactions.

  3. Effect of deletion of the lpxM gene on virulence and vaccine potential of Yersinia pestis in mice.

    Science.gov (United States)

    Anisimov, Andrey P; Shaikhutdinova, Rima Z; Pan'kina, Lyudmila N; Feodorova, Valentina A; Savostina, Elena P; Bystrova, Ol'ga V; Lindner, Buko; Mokrievich, Aleksandr N; Bakhteeva, Irina V; Titareva, Galina M; Dentovskaya, Svetlana V; Kocharova, Nina A; Senchenkova, Sof'ya N; Holst, Otto; Devdariani, Zurab L; Popov, Yuriy A; Pier, Gerald B; Knirel, Yuriy A

    2007-04-01

    Yersinia pestis undergoes an obligate flea-rodent-flea enzootic life cycle. The rapidly fatal properties of Y. pestis are responsible for the organism's sustained survival in natural plague foci. Lipopolysaccharide (LPS) plays several roles in Y. pestis pathogenesis, prominent among them being resistance to host immune effectors and induction of a septic-shock state during the terminal phases of infection. LPS is acylated with 4-6 fatty acids, the number varying with growth temperature and affecting the molecule's toxic properties. Y. pestis mutants were constructed with a deletion insertion in the lpxM gene in both virulent and attenuated strains, preventing the organisms from synthesizing the most toxic hexa-acylated lipid A molecule when grown at 25 degrees C. The virulence and/or protective potency of pathogenic and attenuated Y. pestis DeltalpxM mutants were then examined in a mouse model. The DeltalpxM mutation in a virulent strain led to no change in the LD(50) value compared to that of the parental strain, while the DeltalpxM mutation in attenuated strains led to a modest 2.5-16-fold reduction in virulence. LPS preparations containing fully hexa-acylated lipid A were ten times more toxic in actinomycin D-treated mice then preparations lacking this lipid A isoform, although this was not significant (P>0.05). The DeltalpxM mutation in vaccine strain EV caused a significant increase in its protective potency. These studies suggest there is little impact from lipid A modifications on the virulence of Y. pestis strains but there are potential improvements in the protective properties in attenuated vaccine strains.

  4. ET-46ONCOLYTIC VIRAL THERAPY FOR MALIGNANT GLIOMAS USING MYXOMA VIRUS DELETED FOR ANTI-APOPTOTIC M11L GENE

    Science.gov (United States)

    Pisklakova, Alexandra; McKenzie, Brienne; Kenchappa, Rajappa; McFadden, Grant; Forsyth, Peter

    2014-01-01

    Brain Tumour Initiating Cells (BTICs) are stem-like cells hypothesized to mediate recurrence in high-grade gliomas. Myxoma virus (MyxV) is a promising oncolytic virus, which is highly effective in conventional long term resistant glioma cell lines and less effective in BTICs. We hypothesized that one possible factor limiting efficacy in BTICs is that cell death following infection with MyxV is inhibited by virally encoded anti-apoptotic proteins, such as the Bcl-2 structural homologue, M011L. To test this we evaluated and compared the efficacy of wtMYXV versus the viral construct MyxV-M011L-KO (in which the anti-apoptotic protein M11L has been deleted) in BTICs. We found that WT-MyxV does not induce significant level of apoptosis in infected BTICs, but that MyxV-M011L-KO induces dramatically more apoptosisas shown by caspase activation, PARP cleavage, and Cytochrome C release from the mitochondria M11L from the WT-MyxV localized to the mitochondrial membrane and prevented the association of Bax with the mitochondrial membrane. Finally, silencing of Bax using specific siRNAs significantly blocked the induction of apoptosis and cell death that occurs after infection with mutant MyxV-M011L-KO virus. Therefore MyxV-M011L-KO, which is has the anti-apoptotic virally derived gene M11L, dramatically improves the oncolytic efficacy in BTICs and this is dependent on the presence of the pro-apoptotic host protein, Bax. This is the first demonstration, that the MyxV mutant, genetically modified to promote apoptosis in tumor initiating cells, is significantly more efficacious than the wildtype virus. Strategies, such as this one, that promotes apoptosis in tumor initiating cells might be particularly effective.

  5. Inflammatory peeling skin syndrome caused by homozygous genomic deletion in the PSORS1 region encompassing the CDSN gene.

    Science.gov (United States)

    Ishida-Yamamoto, Akemi; Furio, Laetitia; Igawa, Satomi; Honma, Masaru; Tron, Elodie; Malan, Valerie; Murakami, Masamoto; Hovnanian, Alain

    2014-01-01

    Peeling skin syndrome (PSS) type B is a rare recessive genodermatosis characterized by lifelong widespread, reddish peeling of the skin with pruritus. The disease is caused by small-scale mutations in the Corneodesmosin gene (CDSN) leading to premature termination codons. We report for the first time a Japanese case resulting from complete deletion of CDSN. Corneodesmosin was undetectable in the epidermis, and CDSN was unamplifiable by PCR. QMPSF analysis demonstrated deletion of CDSN exons inherited from each parent. Deletion mapping using microsatellite haplotyping, CGH array and PCR analysis established that the genomic deletion spanned 49-72 kb between HCG22 and TCF19, removing CDSN as well as five other genes within the psoriasis susceptibility region 1 (PSORS1) on 6p21.33. This observation widens the spectrum of molecular defects underlying PSS type B and shows that loss of these five genes from the PSORS1 region does not result in an additional cutaneous phenotype.

  6. Identification of two point mutations and a one base deletion in exon 19 of the dystrophin gene by heteroduplex formation.

    Science.gov (United States)

    Prior, T W; Papp, A C; Snyder, P J; Burghes, A H; Sedra, M S; Western, L M; Bartello, C; Mendell, J R

    1993-03-01

    Two thirds of the Duchenne muscular dystrophy population have either gene deletions or duplications. The nondeletion/duplication cases are most likely the result of point mutations or small deletions and duplications that cannot be easily identified by current strategies. The major obstacle in identifying small mutations is due to the large size of the dystrophin gene. We selectively screened 5 DMD exons containing CpG dinucleotides in 110 DMD patients without detectable deletions or duplications. Nonsenses mutations are frequently due to a C- to -T transition within a CG dinucleotide pair. To screen for the nonsense mutations, we used the heteroduplex method. Utilizing this approach, we identified 2 different nonsense mutations and a single base deletion all occurring in exon 19. This is the first report of a clustering of small mutations in the dystrophin gene.

  7. An Arabidopsis embryonic lethal mutant with reduced expression of alanyl—t RNA synthetase gene

    Institute of Scientific and Technical Information of China (English)

    SUNJIANGE; XIAOLIYAO; 等

    1998-01-01

    In present paper,one of the T-DNA insertional embryonic lethal mutant of Arabidopsis is identified and designated as acd mutant.The embryo developmant of this mutant is arrested in globular stage,The cell division pattern is abnormal during early embryogenesis and results in distubed cellular differentiation.Most of mutant embryos are finally degenerated and aborted in globular stage,However,a few of them still can germinate in agar palte and produce seedlings with shoter hypoctyl and distorted shoot meristem.To understand the molecular basis of the phenotype of this mutant,the joint fragment of T-DNA/plant DNA is isolated by plasmid rescue and Dig-labeled as probe for cDNA library screening.According to the sequence analysis and similarity searching,a 936 bp cDNA sequence(EMBL accession #:Y12555)from selectoed positive clone shows a 99.8%(923/925bp) sequence homolgy with Alanyl-tRNA Synthetase(AlaRS) gene of Arabidopsis thaliana.Furthermore,the data of in situ hybridization experiment indicate that the expression of Ala RS gene is weak in early embryogenesis and declines along with globular embryodevelopment in this mutant Accordingly,the reduced expression of Ala RS gene may be closely related to the morphological changes in early embryogenesis of this lethal mutant.

  8. Establishment of markerless gene deletion tools in thermophilic Bacillus smithii and construction of multiple mutant strains

    NARCIS (Netherlands)

    Bosma, E.F.; Weijer, van de A.H.P.; Vlist, L.; Vos, de W.M.; Oost, van der J.; Kranenburg, van R.

    2015-01-01

    BACKGROUND: Microbial conversion of biomass to fuels or chemicals is an attractive alternative for fossil-based fuels and chemicals. Thermophilic microorganisms have several operational advantages as a production host over mesophilic organisms, such as low cooling costs, reduced contamination risks

  9. The impact of gene duplication, insertion, deletion, lateral gene transfer and sequencing error on orthology inference: a simulation study.

    Science.gov (United States)

    Dalquen, Daniel A; Altenhoff, Adrian M; Gonnet, Gaston H; Dessimoz, Christophe

    2013-01-01

    The identification of orthologous genes, a prerequisite for numerous analyses in comparative and functional genomics, is commonly performed computationally from protein sequences. Several previous studies have compared the accuracy of orthology inference methods, but simulated data has not typically been considered in cross-method assessment studies. Yet, while dependent on model assumptions, simulation-based benchmarking offers unique advantages: contrary to empirical data, all aspects of simulated data are known with certainty. Furthermore, the flexibility of simulation makes it possible to investigate performance factors in isolation of one another.Here, we use simulated data to dissect the performance of six methods for orthology inference available as standalone software packages (Inparanoid, OMA, OrthoInspector, OrthoMCL, QuartetS, SPIMAP) as well as two generic approaches (bidirectional best hit and reciprocal smallest distance). We investigate the impact of various evolutionary forces (gene duplication, insertion, deletion, and lateral gene transfer) and technological artefacts (ambiguous sequences) on orthology inference. We show that while gene duplication/loss and insertion/deletion are well handled by most methods (albeit for different trade-offs of precision and recall), lateral gene transfer disrupts all methods. As for ambiguous sequences, which might result from poor sequencing, assembly, or genome annotation, we show that they affect alignment score-based orthology methods more strongly than their distance-based counterparts.

  10. The impact of gene duplication, insertion, deletion, lateral gene transfer and sequencing error on orthology inference: a simulation study.

    Directory of Open Access Journals (Sweden)

    Daniel A Dalquen

    Full Text Available The identification of orthologous genes, a prerequisite for numerous analyses in comparative and functional genomics, is commonly performed computationally from protein sequences. Several previous studies have compared the accuracy of orthology inference methods, but simulated data has not typically been considered in cross-method assessment studies. Yet, while dependent on model assumptions, simulation-based benchmarking offers unique advantages: contrary to empirical data, all aspects of simulated data are known with certainty. Furthermore, the flexibility of simulation makes it possible to investigate performance factors in isolation of one another.Here, we use simulated data to dissect the performance of six methods for orthology inference available as standalone software packages (Inparanoid, OMA, OrthoInspector, OrthoMCL, QuartetS, SPIMAP as well as two generic approaches (bidirectional best hit and reciprocal smallest distance. We investigate the impact of various evolutionary forces (gene duplication, insertion, deletion, and lateral gene transfer and technological artefacts (ambiguous sequences on orthology inference. We show that while gene duplication/loss and insertion/deletion are well handled by most methods (albeit for different trade-offs of precision and recall, lateral gene transfer disrupts all methods. As for ambiguous sequences, which might result from poor sequencing, assembly, or genome annotation, we show that they affect alignment score-based orthology methods more strongly than their distance-based counterparts.

  11. Exploration of geosmin synthase from Streptomyces peucetius ATCC 27952 by deletion of doxorubicin biosynthetic gene cluster.

    Science.gov (United States)

    Singh, Bijay; Oh, Tae-Jin; Sohng, Jae Kyung

    2009-10-01

    Thorough investigation of Streptomyces peucetius ATCC 27952 genome revealed a sesquiterpene synthase, named spterp13, which encodes a putative protein of 732 amino acids with significant similarity to S. avermitilis MA-4680 (SAV2163, GeoA) and S. coelicolor A3(2) (SCO6073). The proteins encoded by SAV2163 and SCO6073 produce geosmin in the respective strains. However, the spterp13 gene seemed to be silent in S. peucetius. Deletion of the doxorubicin gene cluster from S. peucetius resulted in increased cell growth rate along with detectable production of geosmin. When we over expressed the spterp13 gene in S. peucetius DM07 under the control of an ermE* promoter, 2.4 +/- 0.4-fold enhanced production of geosmin was observed.

  12. R3-R4 deletion in the PRNP gene is associated with Creutzfeldt-Jakob disease (CJD)

    Energy Technology Data Exchange (ETDEWEB)

    Cervenakova, L.; Brown, P.; Nagle, J. [and others

    1994-09-01

    There are conflicting reports on the association of deletions in the PRNP gene on chromosome 20 with CJD, a rapidly progressive fatal spongiform encephalopathy. We accumulated data suggesting that a deletion of R3-R4 type (parts of the third and fourth repeats are deleted from the area of four repeating 24 bp sequences in the 5{prime} region of the gene) is causing CJD. Screening of 129 unaffected control individuals demonstrated presence of a deletion of R2 type in four (1.55% of the studied chromosomes), but none of them had the R3-R4 type. Of 181 screened patients with spongiform encephalopathies, two had a deletion of R3-R4 type with no other mutations in the coding sequence. Both patients had a classical rapidly progressive dementing disease and diffuse spongiform degeneration, and both cases were apparently sporadic. The same R3-R4 type of deletion was detected in three additional neuropathologically confirmed spongiform encephalopathy patients, of which two had other known pathogenic mutations in the PRNP gene: at codon 178 on the methionine allele exhibiting the phenotype of fatal familial insomnia, and codon 200 causing CJD with severe dementia; the third was a patient with iatrogenic CJD who developed the disease after treatment with growth hormone extracted from cadaveric human pituitary glands. In all cases the deletion coincided with a variant sequence at position 129 coding for methionine.

  13. A 57-bp deletion in the ovine KAP6-1 gene affects wool fibre diameter.

    Science.gov (United States)

    Zhou, H; Gong, H; Li, S; Luo, Y; Hickford, J G H

    2015-08-01

    High glycine-tyrosine keratin-associated proteins (HGT-KAPs) are predominantly present in the orthocortex of wool fibres. They vary in abundance in different wools and have been implicated in regulating wool fibre properties, but little is known about the functional roles of these proteins in the fibre matrix. In this study, we used polymerase chain reaction--single-strand conformational polymorphism (PCR-SSCP) analysis to screen for variation in a gene encoding the ovine HGT-KAP6-1 protein. We identified three gene variants (A, B and C). Variants A and B were similar to each other, with only three nucleotide differences occurring downstream of the coding sequence. However, variant C had a 57-bp deletion that would notionally result in a loss of 19 amino acids in the protein. The presence of C was found to be associated with an increase in mean fibre diameter (MFD), fibre diameter standard deviation (FDSD), coefficient of variation of fibre diameter (CVFD) and prickle factor (percentage of fibres over 30 microns; PF). Sheep of genotype BC produced wool of greater MFD, FDSD and PF than sheep of genotypes AA, AB and BB. The CVFD was greater in the BC sheep than the AB sheep. The results suggest that variation in ovine KRTAP6-1 affects wool fibre diameter-associated traits and that the 57-bp deletion in this gene would lead to coarser wool with greater FDSD, CVFD and PF.

  14. Clinical and pathological implications of GSTM1 and GSTT1 gene deletions in sporadic breast cancer

    Directory of Open Access Journals (Sweden)

    Maria Salete Costa Gurgel

    2011-12-01

    Full Text Available There is a lack of consensus about the influence of GST M1/T1 gene deletions (DEL on sporadic breast cancer (SBC. To evaluate the occurrence of DEL in 177 SBC cases and in 169 controls, and compare clinical and biological characteristics. A lower frequency of GSTM1 DEL was observed in mulatto women, OR=0.48 (0.24–0.98. The risk of nuclear grade 3 tumors (GN3 was lower in patients with GSTT1 DEL, OR=0.37 (0.15–0.90. DEL of at least one gene (ALOG was associated with women who had not breastfed, OR=0.41 (0.19–0.88, and with negative hormone receptor, HR–, ORadj=2.25 (1.03–4.90. Both genes deleted (BGD was associated with non-classic invasive ductal carcinoma (NCDC, ORadj=12.09 (1.03–142.03. Mulatto women with SBC had a lower frequency of GSTM1 DEL, while tumors differentiated were related to GSTT1 DEL. HRtumors were related with DEL ALOG, and the BGD was associated with a greater risk of NCDC.

  15. Mutation mismatch repair gene deletions in diffuse large B-cell lymphoma.

    Science.gov (United States)

    Couronné, Lucile; Ruminy, Philippe; Waultier-Rascalou, Agathe; Rainville, Vinciane; Cornic, Marie; Picquenot, Jean-Michel; Figeac, Martin; Bastard, Christian; Tilly, Hervé; Jardin, Fabrice

    2013-05-01

    To further unravel the molecular pathogenesis of diffuse large B-cell lymphoma (DLBCL), we performed high-resolution comparative genomic hybridization on lymph node biopsies from 70 patients. With this strategy, we identified microdeletions of genes involved in the mutation mismatch repair (MMR) pathway in two samples. The first patient presented with a homozygous deletion of MSH2-MSH6 due to duplication of an unbalanced pericentric inversion of chromosome 2. The other case showed a PMS2 heterozygous deletion. PMS2 and MSH2-MSH6 abnormalities, respectively, resulted in a decrease and complete loss of gene expression. However, unlike tumors associated with the hereditary non-polyposis colorectal cancer syndrome or immunodeficiency-related lymphomas, no microsatellite instability was detected. Mutational profiles revealed especially in one patient an aberrant hypermutation without a clear activation-induced cytidine deaminase signature, indicating a breakdown of the high-fidelity repair in favor of the error-prone repair pathway. Our findings suggest that in a rare subset of patients, inactivation of the genes of the MMR pathway is likely an important step in the molecular pathogenesis of DLBCL and does not involve the same molecular mechanisms as other common neoplasms with MMR deficiency.

  16. Deletion of fucose residues in plant N-glycans by repression of the GDP-mannose 4,6-dehydratase gene using virus-induced gene silencing and RNA interference.

    Science.gov (United States)

    Matsuo, Kouki; Matsumura, Takeshi

    2011-02-01

    Production of pharmaceutical glycoproteins in plants has many advantages in terms of safety and reduced costs. However, plant-produced glycoproteins have N-glycans with plant-specific sugar residues (core β-1,2-xylose and α-1,3-fucose) and a Lewis a (Le(a) ) epitope, i.e., Galβ(1-3)[Fucα(1-4)]GlcNAc. Because these sugar residues and glycan structures seemed to be immunogenic, several attempts have been made to delete them by repressing their respective glycosyltransferase genes. However, until date, such deletions have not been successful in completely eliminating the fucose residues. In this study, we simultaneously reduced the plant-specific core α-1,3-fucose and α-1,4-fucose residues in the Le(a) epitopes by repressing the Guanosine 5'-diphosphate (GDP)-D-mannose 4,6-dehydratase (GMD) gene, which is associated with GDP-L-fucose biosynthesis, in Nicotiana benthamiana plants. Repression of GMD was achieved using virus-induced gene silencing (VIGS) and RNA interference (RNAi). The proportion of fucose-free N-glycans found in total soluble protein from GMD gene-repressed plants increased by 80% and 95% following VIGS and RNAi, respectively, compared to wild-type plants. A small amount of putative galactose substitution in N-glycans from the NbGMD gene-repressed plants was observed, similar to what has been previously reported GMD-knockout Arabidopsis mutant. On the other hand, the recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) with fucose-deleted N-glycans was successfully produced in NbGMD-RNAi transgenic N. benthamiana plants. Thus, repression of the GMD gene is thus very useful for deleting immunogenic total fucose residues and facilitating the production of pharmaceutical glycoproteins in plants.

  17. pnp4a Is the Causal Gene of the Medaka Iridophore Mutant guanineless

    Directory of Open Access Journals (Sweden)

    Tetsuaki Kimura

    2017-04-01

    Full Text Available See-through medaka lines are suitable for observing internal organs throughout life. They were bred by crossing multiple color mutants. However, some of the causal genes for these mutants have not been identified. The medaka has four pigment cell types: black melanophores, yellow xanthophores, white leucophores, and silvery iridophores. The causal genes of melanophore, xanthophore, and leucophore mutants have been elucidated, but the causal gene for the iridophore mutant remains unknown. Here, we describe the iridophore mutant, guanineless (gu, which exhibits a strong reduction in visible iridophores throughout its larval to adult stages. The gu locus was previously mapped to chromosome 5, but was located near the telomeric region, making it difficult to integrate into the chromosome. We sought the causal gene of gu using synteny analysis with the zebrafish genome and found a strong candidate, purine nucleoside phosphorylase 4a (pnp4a. Gene targeting and complementation testing showed that pnp4a is the causal gene of gu. This result will allow the establishment of inbred medaka strains or other useful strains with see-through phenotypes without major disruption in the genetic background of each strain.

  18. DIA1R is an X-linked gene related to Deleted In Autism-1.

    Directory of Open Access Journals (Sweden)

    Azhari Aziz

    Full Text Available BACKGROUND: Autism spectrum disorders (ASDS are frequently occurring disorders diagnosed by deficits in three core functional areas: social skills, communication, and behaviours and/or interests. Mental retardation frequently accompanies the most severe forms of ASDs, while overall ASDs are more commonly diagnosed in males. Most ASDs have a genetic origin and one gene recently implicated in the etiology of autism is the Deleted-In-Autism-1 (DIA1 gene. METHODOLOGY/PRINCIPAL FINDINGS: Using a bioinformatics-based approach, we have identified a human gene closely related to DIA1, we term DIA1R (DIA1-Related. While DIA1 is autosomal (chromosome 3, position 3q24, DIA1R localizes to the X chromosome at position Xp11.3 and is known to escape X-inactivation. The gene products are of similar size, with DIA1 encoding 430, and DIA1R 433, residues. At the amino acid level, DIA1 and DIA1R are 62% similar overall (28% identical, and both encode signal peptides for targeting to the secretory pathway. Both genes are ubiquitously expressed, including in fetal and adult brain tissue. CONCLUSIONS/SIGNIFICANCE: Examination of published literature revealed point mutations in DIA1R are associated with X-linked mental retardation (XLMR and DIA1R deletion is associated with syndromes with ASD-like traits and/or XLMR. Together, these results support a model where the DIA1 and DIA1R gene products regulate molecular traffic through the cellular secretory pathway or affect the function of secreted factors, and functional deficits cause disorders with ASD-like symptoms and/or mental retardation.

  19. A Next-generation Genetically Attenuated Plasmodium falciparum Parasite Created by Triple Gene Deletion

    OpenAIRE

    Mikolajczak, Sebastian A.; Lakshmanan, Viswanathan; Fishbaugher, Matthew; Camargo, Nelly; Harupa, Anke; Kaushansky, Alexis; Douglass, Alyse N.; Baldwin, Michael; Healer, Julie; O'Neill, Matthew; Phuong, Thuan; Cowman, Alan; Kappe, Stefan H. I.

    2014-01-01

    Immunization with live-attenuated Plasmodium sporozoites completely protects against malaria infection. Genetic engineering offers a versatile platform to create live-attenuated sporozoite vaccine candidates. We previously generated a genetically attenuated parasite (GAP) by deleting the P52 and P36 genes in the NF54 wild-type (WT) strain of Plasmodium falciparum (Pf p52−/p36− GAP). Preclinical assessment of p52−/p36− GAP in a humanized mouse model indicated an early and severe liver stage gr...

  20. Total beta-globin gene deletion has high frequency in Filipinos

    Energy Technology Data Exchange (ETDEWEB)

    Patrick, N.; Miyakawa, F.; Hunt, J.A. [Univ. of Hawaii, Honolulu, HI (United States)] [and others

    1994-09-01

    The distribution of {beta}-thalassemia [{beta}{sup Th}] mutations is unique to each ethnic group. Most mutations affect one or a few bases; large deletions have been rare. Among families screened in Hawaii, [{beta}{sup Th}] heterozygotes were diagnosed by microcytosis, absence of abnormal hemoglobins on isoelectric focusing, and raised Hb A{sub 2} by chromatography. Gene frequency for {beta}{sup Th} was 0.02 in Filipinos. In Filipinos, polymerase chain reaction [PCR] with denaturing gradient gel electrophoresis for {beta}{sup Th} mutations detected a mutation in only 6 of 42 {beta}{sup Th} heterozygotes; an IVS2-666 C/T polymorphism showed non-heterozygosity in 37 and heterozygosity in only 5 of these {beta}{sup Th} heterozygotes. One {beta}{sup Th}/{beta}{sup Th} major patient and his mother had no mutation detected by allele-specific oligomer hybridization; PCR failed to amplify any DNA from his {beta}-globin gene. After a total {beta}-globin gene deletion [{beta}{sup Del}] was found in a Filipino family in Ontario, specific PCR amplification for {beta}{sup Del} detected this in 43 of 53 {beta}{sup Th} Filipino samples tested; the above {beta}{sup Th}/{beta}{sup Th} patient was a ({beta}{sup Del}/{beta}{sup Del}) homozygote. The {beta}{sup Del} may account for over 60% of all {beta}{sup Th} alleles in Filipinos; this is the highest proportion of a deletion {beta}{sup Th} mutation reported from any population. Most but not all {beta}{sup Del} heterozygotes had high Hb F [5.13 {plus_minus} 3.94 mean {plus_minus} 1 s.d.] compared to the codon 41/42 four base deletion common in Chinese [2.30 {plus_minus} 0.86], or to {beta}{sup Th} heterozygotes with normal {alpha}-globin genes [2.23 {plus_minus} 0.80].

  1. Familial spinal neurofibromatosis due to a multiexonic NF1 gene deletion.

    Science.gov (United States)

    Pizzuti, Antonio; Bottillo, Irene; Inzana, Francesca; Lanari, Valentina; Buttarelli, Francesca; Torrente, Isabella; Giallonardo, Anna Teresa; De Luca, Alessandro; Dallapiccola, Bruno

    2011-08-01

    We report the detailed clinical presentation and molecular features of a spinal neurofibromatosis familial case where a 40-year-old woman, presenting with multiple bilateral spinal neurofibromas and no other clinical feature of neurofibromatosis type 1 (NF1), inherited a paternal large multiexonic deletion (c.5944-?_7126+?del) which resulted in NF1 gene haploinsufficiency at the RNA level. In the clinically unaffected 73-year-old father, spinal cord MRI disclosed bilateral and symmetrical hypertrophy of spinal lumbosacral roots. Our study widens the phenotypic and mutational spectrum of NF1 and illustrates the difficulties of counseling patients with border-line or atypical presentation of this disorder.

  2. An intronic deletion in the PROM1 gene leads to autosomal recessive cone-rod dystrophy

    Science.gov (United States)

    Eidinger, Osnat; Leibu, Rina; Newman, Hadas; Rizel, Leah; Perlman, Ido

    2015-01-01

    Purpose To investigate the genetic basis for autosomal recessive cone-rod dystrophy (CRD) in a consanguineous Israeli Jewish family. Methods Patients underwent a detailed ophthalmic evaluation, including eye examination, visual field testing, optical coherence tomography (OCT), and electrophysiological tests, electroretinography (ERG) and visual evoked potential (VEP). Genome-wide homozygosity mapping using a single nucleotide polymorphism (SNP) array was performed to identify homozygous regions shared among two of the affected individuals. Mutation screening of the underlying gene was performed with direct sequencing. In silico and in vitro analyses were used to predict the effect of the identified mutation on splicing. Results The affected family members are three siblings who have various degrees of progressive visual deterioration, glare, color vision abnormalities, and night vision difficulties. Visual field tests revealed central scotomas of different extension. Cone and rod ERG responses were reduced, with cones more severely affected. Homozygosity mapping revealed several homozygous intervals shared among two of the affected individuals. One included the PROM1 gene. Sequence analysis of the 26 coding exons of PROM1 in one affected individual revealed no mutations in the coding sequence or in intronic splice sites. However, in intron 21, proximate to the intron–exon junction, we observed a homozygous 10 bp deletion between positions −26 and −17 (c.2281–26_-17del). The deletion was linked to a known SNP, c.2281–6C>G. The deletion cosegregated with the disease in the family, and was not detected in public databases or in 101 ethnically-matched control individuals. In silico analysis predicted that this deletion would lead to altered intron 21 splicing. Bioinformatic analysis predicted that a recognition site for the SRSF2 splicing factor is located within the deleted sequence. The in vitro splicing assay demonstrated that c.2281–26_-17del leads to

  3. Genomic structure and characterization of the Drosophila S3 ribosomal/DNA repair gene and mutant alleles.

    Science.gov (United States)

    Kelley, M R; Xu, Y; Wilson, D M; Deutsch, W A

    2000-03-01

    The Drosophila S3 protein is known to be associated with ribosomes, where it is thought to play a role in the initiation of protein translation. The S3 protein also contains a DNA repair activity, efficiently processing 8-oxoguanine residues in DNA via an N-glycosylase/apurinic-apyrimidinic (AP) lyase activity. The gene that encodes S3 has previously been localized to one of the Minute loci on chromosome 3 in Drosophila. This study focused on the genomic organization of S3 at M(3)95A, initial promoter characterization, and analysis of three mutant alleles at this locus. The S3 gene was found to be a single-copy gene 2 to 3 kb in length and containing a single intron. The upstream 1.6-kb region was analyzed for promoter activity, identifying a presumptive regulatory domain containing potential enhancer and suppressor elements. This finding is of interest, as the S3 gene is constitutively expressed throughout development and mRNA is most likely maternally inherited. Lastly, three Minute alleles from the same locus were sequenced and two alleles found to contain a 22-bp deletion in exon 2, resulting in a truncated S3 protein, although wildtype levels of S3 mRNA and protein were detected in the viable heterozygous Minute alleles, possibly reflecting dosage compensation.

  4. Virulence characteristics of Salmonella following deletion of genes encoding the tRNA modification enzymes GidA and MnmE.

    Science.gov (United States)

    Shippy, Daniel C; Eakley, Nicholas M; Lauhon, Charles T; Bochsler, Philip N; Fadl, Amin A

    2013-04-01

    Salmonella is an important foodborne pathogen causing major public health problems throughout the world due to the consumption of contaminated food. Our previous studies have shown that deletion of glucose-inhibited division (gidA) gene significantly altered Salmonella virulence in both in vitro and in vivo models of infection. In Escherichia coli, GidA and MnmE have been shown to modify several bacterial factors by a post-transcriptional mechanism to modify tRNA. Therefore, we hypothesize that GidA and MnmE complex together to modulate virulence genes in Salmonella using a similar mechanism. To test our hypothesis, and to examine the relative contribution of GidA and MnmE in modulation of Salmonella virulence, we constructed gidA and mnmE single mutants as well as a gidA mnmE double mutant strain of Salmonella. Results from the in vitro data displayed a reduction in growth, motility, intracellular replication, and invasion of T84 intestinal epithelial cells in the mutant strains compared to the wild-type Salmonella strain. The in vivo data showed a significant attenuation of the mutant strains as indicated by the induction of inflammatory cytokines and chemokines, as well as in the severity of histopathological lesions in the liver and spleen, compared to mice infected with the wild-type strain. Also, a significant increase in the LD50 was observed in mice infected with the mutant strains, and mice immunized with the mutants were protected against a lethal dose of wild-type Salmonella. A pull-down assay indicated that Salmonella GidA and MnmE bind together, and HPLC analysis revealed that deletion of gidA and/or mnmE altered Salmonella tRNA modification. Overall, the data suggest MnmE and GidA bind together and use a post-transcriptional mechanism to modify tRNA to regulate Salmonella pathogenesis.

  5. Assessment of the Immune Responses Induced in Cattle after Inoculation of a Mycobacterium bovis Strain Deleted in Two mce2 Genes

    Directory of Open Access Journals (Sweden)

    Federico Carlos Blanco

    2012-01-01

    Full Text Available The generation of efficient candidate vaccines against bovine tuberculosis will contribute to the control of this zoonotic disease. Rationally attenuated Mycobacterium bovis strains generated by knockout of virulence genes are promising candidate vaccines. However, to be effective, these candidate vaccines should at least maintain the immunological properties of their virulent parental M. bovis strains. Therefore, the aim of this study was to obtain an M. bovis strain deleted in the mce2 genes and evaluate the effect of the mutation on the immunological profile elicited by the bacteria in cattle. We showed that the activation of CD4+ T cells in cattle inoculated with the mutant strain was equivalent to that in animals inoculated with the parental strain. Moreover, after in vitro stimulation, peripheral blood mononuclear cells from animals inoculated with the mutant produced higher levels of mRNA Th-1 cytokines than the parental strain. Therefore, these results indicate that the mce2 mutant is a promising candidate vaccine against bovine tuberculosis.

  6. Large deletions encompassing the TCOF1 and CAMK2A genes are responsible for Treacher Collins syndrome with intellectual disability.

    Science.gov (United States)

    Vincent, Marie; Collet, Corinne; Verloes, Alain; Lambert, Laetitia; Herlin, Christian; Blanchet, Catherine; Sanchez, Elodie; Drunat, Séverine; Vigneron, Jacqueline; Laplanche, Jean-Louis; Puechberty, Jacques; Sarda, Pierre; Geneviève, David

    2014-01-01

    Mandibulofacial dysostosis is part of a clinically and genetically heterogeneous group of disorders of craniofacial development, which lead to malar and mandibular hypoplasia. Treacher Collins syndrome is the major cause of mandibulofacial dysostosis and is due to mutations in the TCOF1 gene. Usually patients with Treacher Collins syndrome do not present with intellectual disability. Recently, the EFTUD2 gene was identified in patients with mandibulofacial dysostosis associated with microcephaly, intellectual disability and esophageal atresia. We report on two patients presenting with mandibulofacial dysostosis characteristic of Treacher Collins syndrome, but associated with unexpected intellectual disability, due to a large deletion encompassing several genes including the TCOF1 gene. We discuss the involvement of the other deleted genes such as CAMK2A or SLC6A7 in the cognitive development delay of the patients reported, and we propose the systematic investigation for 5q32 deletion when intellectual disability is associated with Treacher Collins syndrome.

  7. Pyrin gene and mutants thereof, which cause familial Mediterranean fever

    Energy Technology Data Exchange (ETDEWEB)

    Kastner, Daniel L [Bethesda, MD; Aksentijevichh, Ivona [Bethesda, MD; Centola, Michael [Tacoma Park, MD; Deng, Zuoming [Gaithersburg, MD; Sood, Ramen [Rockville, MD; Collins, Francis S [Rockville, MD; Blake, Trevor [Laytonsville, MD; Liu, P Paul [Ellicott City, MD; Fischel-Ghodsian, Nathan [Los Angeles, CA; Gumucio, Deborah L [Ann Arbor, MI; Richards, Robert I [North Adelaide, AU; Ricke, Darrell O [San Diego, CA; Doggett, Norman A [Santa Cruz, NM; Pras, Mordechai [Tel-Hashomer, IL

    2003-09-30

    The invention provides the nucleic acid sequence encoding the protein associated with familial Mediterranean fever (FMF). The cDNA sequence is designated as MEFV. The invention is also directed towards fragments of the DNA sequence, as well as the corresponding sequence for the RNA transcript and fragments thereof. Another aspect of the invention provides the amino acid sequence for a protein (pyrin) associated with FMF. The invention is directed towards both the full length amino acid sequence, fusion proteins containing the amino acid sequence and fragments thereof. The invention is also directed towards mutants of the nucleic acid and amino acid sequences associated with FMF. In particular, the invention discloses three missense mutations, clustered in within about 40 to 50 amino acids, in the highly conserved rfp (B30.2) domain at the C-terminal of the protein. These mutants include M6801, M694V, K695R, and V726A. Additionally, the invention includes methods for diagnosing a patient at risk for having FMF and kits therefor.

  8. Naturally Occurring Deletion Mutants of the Pig-Specific, Intestinal Crypt Epithelial Cell Protein CLCA4b without Apparent Phenotype.

    Directory of Open Access Journals (Sweden)

    Stephanie Plog

    Full Text Available The human CLCA4 (chloride channel regulator, calcium-activated modulates the intestinal phenotype of cystic fibrosis (CF patients via an as yet unknown pathway. With the generation of new porcine CF models, species-specific differences between human modifiers of CF and their porcine orthologs are considered critical for the translation of experimental data. Specifically, the porcine ortholog to the human CF modulator gene CLCA4 has recently been shown to be duplicated into two separate genes, CLCA4a and CLCA4b. Here, we characterize the duplication product, CLCA4b, in terms of its genomic structure, tissue and cellular expression patterns as well as its in vitro electrophysiological properties. The CLCA4b gene is a pig-specific duplication product of the CLCA4 ancestor and its protein is exclusively expressed in small and large intestinal crypt epithelial cells, a niche specifically occupied by no other porcine CLCA family member. Surprisingly, a unique deleterious mutation of the CLCA4b gene is spread among modern and ancient breeds in the pig population, but this mutation did not result in an apparent phenotype in homozygously affected animals. Electrophysiologically, neither the products of the wild type nor of the mutated CLCA4b genes were able to evoke a calcium-activated anion conductance, a consensus feature of other CLCA proteins. The apparently pig-specific duplication of the CLCA4 gene with unique expression of the CLCA4b protein variant in intestinal crypt epithelial cells where the porcine CFTR is also present raises the question of whether it may modulate the porcine CF phenotype. Moreover, the naturally occurring null variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype.

  9. Large deletion of the GJB6 gene in deaf patients heterozygous for the GJB2 gene mutation: genotypic and phenotypic analysis.

    Science.gov (United States)

    Feldmann, Delphine; Denoyelle, Françoise; Chauvin, Pierre; Garabédian, Eréa-Noël; Couderc, Rémy; Odent, Sylvie; Joannard, Alain; Schmerber, Sébastien; Delobel, Bruno; Leman, Jacques; Journel, Hubert; Catros, Hélène; Le Maréchal, Cédric; Dollfus, Hélène; Eliot, Marie-Madeleine; Delaunoy, Jean-Pierre; David, Albert; Calais, Catherine; Drouin-Garraud, Valérie; Obstoy, Marie-Françoise; Bouccara, Didier; Sterkers, Olivier; Huy, Patrice Tran Ba; Goizet, Cyril; Duriez, Françoise; Fellmann, Florence; Hélias, Jocelyne; Vigneron, Jacqueline; Montaut, Bétina; Lewin, Patricia; Petit, Christine; Marlin, Sandrine

    2004-06-15

    Recent investigations identified a large deletion of the GJB6 gene in trans to a mutation of GJB2 in deaf patients. We looked for GJB2 mutations and GJB6 deletions in 255 French patients presenting with a phenotype compatible with DFNB1. 32% of the patients had biallelic GJB2 mutations and 6% were a heterozygous for a GJB2 mutation and a GJB6 deletion. Biallelic GJB2 mutations and combined GJB2/GJB6 anomalies were more frequent in profoundly deaf children. Based on these results, we are now assessing GJB6 deletion status in cases of prelingual hearing loss. Copyright 2004 Wiley-Liss, Inc.

  10. Clinical implications of cytosine deletion of exon 5 of P53 gene in non small cell lung cancer patients

    Directory of Open Access Journals (Sweden)

    Rashid Mir

    2016-01-01

    Full Text Available Aim: Lung cancer is considered to be the most common cancer in the world. In humans, about 50% or more cancers have a mutated tumor suppressor p53 gene thereby resulting in accumulation of p53 protein and losing its function to activate the target genes that regulate the cell cycle and apoptosis. Extensive research conducted in murine cancer models with activated p53, loss of p53, or p53 missense mutations have facilitated researchers to understand the role of this key protein. Our study was aimed to evaluate the frequency of cytosine deletion in nonsmall cell lung cancer (NSCLC patients. Methods: One hundred NSCLC patients were genotyped for P53 (exon5, codon168 cytosine deletion leading to loss of its function and activate the target genes by allele-specific polymerase chain reaction. The P53 cytosine deletion was correlated with all the clinicopathological parameters of the patients. Results and Analysis: 59% cases were carrying P53 cytosine deletion. Similarly, the significantly higher incidence of cytosine deletion was reported in current smokers (75% in comparison to exsmoker and nonsmoker. Significantly higher frequency of cytosine deletion was reported in adenocarcinoma (68.08% than squamous cell carcinoma (52.83%. Also, a significant difference was reported between p53 cytosine deletion and metastasis (64.28%. Further, the majority of the cases assessed for response carrying P53 cytosine deletion were found to show faster disease progression. Conclusion: The data suggests that there is a significant association of the P53 exon 5 deletion of cytosine in codon 168 with metastasis and staging of the disease.

  11. Dopaminergic Neuron-Specific Deletion of p53 Gene Attenuates Methamphetamine Neurotoxicity.

    Science.gov (United States)

    Lu, Tao; Kim, Paul P; Greig, Nigel H; Luo, Yu

    2017-08-01

    p53 plays an essential role in the regulation of cell death in dopaminergic (DA) neurons and its activation has been implicated in the neurotoxic effects of methamphetamine (MA). However, how p53 mediates MA neurotoxicity remains largely unknown. In this study, we examined the effect of DA-specific p53 gene deletion in DAT-p53KO mice. Whereas in vivo MA binge exposure reduced locomotor activity in wild-type (WT) mice, this was significantly attenuated in DAT-p53KO mice and associated with significant differences in the levels of the p53 target genes BAX and p21 between WT and DAT-p53KO. Notably, DA-specific deletion of p53 provided protection of substantia nigra pars reticulata (SNpr) tyrosine hydroxylase (TH) positive fibers following binge MA, with DAT-p53KO mice having less decline of TH protein levels in striatum versus WT mice. Whereas DAT-p53KO mice demonstrated a consistently higher density of TH fibers in striatum compared to WT mice at 10 days after MA exposure, DA neuron counts within the substantia nigra pars compacta (SNpc) were similar. Finally, supportive of these results, administration of a p53-specific inhibitor (PFT-α) provided a similarly protective effect on MA binge-induced behavioral deficits. Neither DA specific p53 deletion nor p53 pharmacological inhibition affected hyperthermia induced by MA binge. These findings demonstrate a specific contribution of p53 activation in behavioral deficits and DA neuronal terminal loss by MA binge exposure.

  12. A Japanese boy with myalgia and cramps has a novel in-frame deletion of the dystrophin gene.

    Science.gov (United States)

    Ishigaki, C; Patria, S Y; Nishio, H; Yabe, M; Matsuo, M

    1996-05-01

    We report a Japanese Becker muscular dystrophy (BMD) patient with occasional myalgia and cramps during normal activity that developed at the age of 28 months. His family history was negative for neuromuscular diseases. Muscle biopsy analyses, including dystrophin immunostaining, disclosed no clinically relevant findings. The diagnosis of BMD was initially made at the age of 10 years, when indications of persistent high serum levels of CK prompted us to screen deletions in the dystrophin gene by amplification of 19 deletion-prone exons from the genomic DNA by the polymerase chain reaction (PCR). Among the exons examined, exons 13 and 17 were deleted. To clarify the size of the deletion, the dystrophin transcript was analyzed by reverse transcription PCR. The determined nucleotide sequence of the amplified product encompassing exons 10 to 20 disclosed that the entire segment corresponding to exons 13 to 18 (810 bp) was absent, a deletion that would be expected to cause the production of a dystrophin protein lacking 270 amino acids from the rod domain. This result indicates that occasional myalgia and cramps could be early clinical manifestations of mild BMD, especially in patients who have a deletion in the rod domain, and that deletion screening of the dystrophin gene might be the only reliable method to diagnose such cases.

  13. Complementation analyses of Sinorhizobium meliloti nifA mutant with different originated nifA genes

    Institute of Scientific and Technical Information of China (English)

    YAO Zhenhua; R(U)VERG Silvia; WANG Yiping; ZOU Huasong; TIAN Zhexian; DAI Xiaomi; BECKER Anke; LI Jian; YAN Haiqin; XIAO Yan; ZHU Jiabi; YU Guanqiao

    2006-01-01

    A previous work inferred that the nifA gene of Enterobacter cloacae did not restore the symbiotic phenotype of Sinorhizobium meliloti nifA mutant. In the present study, two nifA genes of Bradyrhizobium japonicum and Mesorhizobium huakuii also did not restore the symbiotic phenotype of S.meliloti nifA mutant. In whole genomic microarray experiments, 238 genes were found to be differentially expressed after S. meliloti nifA had been constitutively expressed in its nifA mutant. In contrast,only 20, 7 and 9 genes changed their transcriptional levels when expressing B. japonium, M. huakuii and Enterobacter cloacae nifA genes in Sm nifA mutant,separately. These genes were classified into several functional groups including house keeping, energy and central intermediary metabolism, transport systems and symbiosis. Interestingly, the genes that of nifH operons showed high expression levels in the presence of either B. japonium or M. huakuii NifA,which was confirmed by subsequent lacZ fusion experiments.

  14. Similarity of DMD gene deletion and duplication in the Chinese patients compared to global populations

    Directory of Open Access Journals (Sweden)

    Yan Ming

    2008-04-01

    Full Text Available Abstract Background DNA deletion and duplication were determined as the major mutation underlying Duchenne muscular dystrophy (DMD and Becker muscular dystrophy (BMD. Method Applying multiplex ligation-dependent probe amplification (MLPA, we have analyzed 179 unrelated DMD/BMD subjects from northern China. Results Seventy-three percent of the subjects were found having a deletion (66.25% or duplication (6.25%. Exons 51–52 were detected as the most common fragment deleted in single-exon deletion, and the region of exons 45–50 was the most common exons deleted in multi-exon deletions. About 90% of DMD/BMD cases carry a small size deletion that involves 10 exons or less, 26.67% of which carry a single-exon deletion. Most of the smaller deletions resulted in an out-of-frame mutation. The most common exons deleted were determined to be between exon 48 and exon 52, with exon 50 was the model allele. Verifying single-exon deletion, one sample with a deletion of exon 53 that was initially observed from MLPA showed that there was a single base deletion that abolished the ligation site in MLPA. Confirmation of single-exon deletion is recommended to exclude single base deletion or mutation at the MLPA ligation site. Conclusion The frequency of deletion and duplication in northern China is similar to global ethnic populations.

  15. Impact of alg3 gene deletion on growth, development, pigment production, protein secretion, and functions of recombinant Trichoderma reesei cellobiohydrolases in Aspergillus niger

    Energy Technology Data Exchange (ETDEWEB)

    Dai, Ziyu; Aryal, Uma K.; Shukla, Anil; Qian, Wei-Jun; Smith, Richard D.; Magnuson, Jon K.; Adney, William S.; Beckham, Gregg T.; Brunecky, Roman; Himmel, Michael E.; Decker, Stephen R.; Ju, Xiaohui; Zhang, Xiao; Baker, Scott E.

    2013-12-01

    ALG3 is a Family 58 glycosyltransferase enzyme involved in early N-linked glycan synthesis. Here, we investigated the effect of the alg3 gene disruption on growth, development, metabolism, and protein secretion in Aspergillus niger. The alg3 gene deletion resulted in a significant reduction of growth on complete (CM) and potato dextrose agar (PDA) media and a substantial reduction of spore production on CM. It also delayed spore germination in the liquid cultures of both CM and PDA media, but led to a significant accumulation of red pigment on both CM and liquid modified minimal medium (MM) supplemented with yeast extract. The relative abundance of 55 proteins of the total 190 proteins identified in the secretome was significantly different as a result of alg3 gene deletion. Comparison of a Trichoderma reesei cellobiohydrolase (Cel7A) heterologously expressed in A. niger parental and Δalg3 strains showed that the recombinant Cel7A expressed in the mutant background was smaller in size than that from the parental strains. This study suggests that ALG3 is critical for growth and development, pigment production, and protein secretion in A. niger. Functional analysis of recombinant Cel7A with aberrant glycosylation demonstrates the feasibility of this alternative approach to evaluate the role of N-linked glycosylation in glycoprotein secretion and function.

  16. Targeted deletion of multiple CTCF-binding elements in the human C-MYC gene reveals a requirement for CTCF in C-MYC expression.

    Directory of Open Access Journals (Sweden)

    Wendy M Gombert

    Full Text Available BACKGROUND: Insulators and domain boundaries both shield genes from adjacent enhancers and inhibit intrusion of heterochromatin into transgenes. Previous studies examined the functional mechanism of the MYC insulator element MINE and its CTCF binding sites in the context of transgenes that were randomly inserted into the genome by transfection. However, the contribution of CTCF binding sites to both gene regulation and maintenance of chromatin has not been tested at the endogenous MYC gene. METHODOLOGY/PRINCIPAL FINDINGS: To determine the impact of CTCF binding on MYC expression, a series of mutant human chromosomal alleles was prepared in homologous recombination-efficient DT40 cells and individually transferred by microcell fusion into murine cells. Functional tests reported here reveal that deletion of CTCF binding elements within the MINE does not impact the capacity of this locus to correctly organize an 'accessible' open chromatin domain, suggesting that these sites are not essential for the formation of a competent, transcriptionally active locus. Moreover, deletion of the CTCF site at the MYC P2 promoter reduces transcription but does not affect promoter acetylation or serum-inducible transcription. Importantly, removal of either CTCF site leads to DNA methylation of flanking sequences, thereby contributing to progressive loss of transcriptional activity. CONCLUSIONS: These findings collectively demonstrate that CTCF-binding at the human MYC locus does not repress transcriptional activity but is required for protection from DNA methylation.

  17. Selection of Arabidopsis mutants overexpressing genes driven by the promoter of an auxin-inducible glutathione S-transferase gene.

    Science.gov (United States)

    van der Kop, D A; Schuyer, M; Pinas, J E; van der Zaal, B J; Hooykaas, P J

    1999-03-01

    Transgenic arabidopsis plants were isolated that contained a T-DNA construct in which the promoter of an auxin-inducible glutathione S-transferase (GST) gene from tobacco was fused to the kanamycin resistance (nptII) as well as to the beta-glucuronidase (gusA) reporter gene. Subsequently, seeds were treated with EMS to obtain mutants in which both reporter gene fusions were up-regulated. Northern analysis showed that the mRNA level of a related, endogenous auxin-inducible GST gene of Arabidopsis was increased in some of these mutants as well. Two of the gup (GST up-regulated) mutants were characterized in more detail and roughly mapped. Both had epinastic cotyledons and leaves, a phenotype that turned out to be linked to the gup mutation.

  18. Detection of APC gene deletions in colorectal malignancies using quantitative PCR in a Chinese population.

    Science.gov (United States)

    Fang, Zhengyu; Xiong, Yi; Li, Jiana; Liu, Li; Li, Manhui; Zhang, Wei; Shi, Lei; Wan, Jun

    2011-09-01

    The adenomatous polyposis coli (APC) gene has been shown to be involved in genetic instability and to be downregluated in several human carcinomas. The chromosome locus of APC, 5q21-22, is frequently deleted in colorectal cancers (CRCs). The functional impact of such regions needs to be extensively investigated in large amount of clinical samples. Case-matched tissues of CRC and adjacent normal epithelium (n = 134) were included in this study. Quantitative PCR was carried out to examine the copy number as well as mRNA expression of APC gene in colorectal malignancies. Our results showed that copy number deletions of APC were present in a relatively high percentage of colorectal cancer samples (26.1%, 35 out of 134). There was a positive correlation between copy number decrease of APC and tumor progression in CRCs. Furthermore, copy number loss of APC was correlated with decreased mRNA expression. However, mRNA levels of APC were also impaired in CRC samples with unaltered copy numbers, indicating that sporadic CRCs exhibit different mechanisms of APC regulation.

  19. A nine-nucleotide deletion and splice variation in the coding region of the interferon induced ISG12 gene

    DEFF Research Database (Denmark)

    Smidt, Kamille; Hansen, Lise Lotte; Søgaard, T Max M;

    2003-01-01

    distributed between ISG12 and ISG12-S in breast carcinoma cells, in cancer cell lines and in cervical cytobrush material with neoplastic lesions. In addition, we have found a nine-nucleotide deletion situated in exon 4 of the ISG12 gene. This deletion leads to a three-amino-acid deletion (AMA) in the putative...... inducible splice variant of ISG12 lacking exon 2 leading to a putative truncated protein isoform of Mr 7400, ISG12-S. In cells from blood and cervical cytobrush material from healthy women, the level of ISG12-S expression was higher than ISG12 expression, whereas the expression pattern was more evenly...

  20. The amylose-free potato mutant as a model plant to study gene expression and gene silencing.

    NARCIS (Netherlands)

    Flipse, E.

    1995-01-01

    In this thesis, gene-expression and gene silencing were examined for Granule Bound Starch Synthase (GBSS) which catalyses the formation of amylose and Branching Enzyme (BE) which catalyses the formation of amylopectin. The (GBSS) deficient, with iodine, red staining amylose-free (amf) potato mutant

  1. Comparative genome analysis of deleted genes in Shigella flexneri 2a strain 301

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Comparative genome analysis is performed between Shigella flexneri 2a strain 301 and its close relatives, the nonpathogenic E. Coli K-12 strain MG1655. Result shows that there are 136 DNA segments whose size is larger than 1000 bp absent from Shigella flexneri 2a strain 301, which is up to 717253 bp in total length. These deleted segments altogether contain 670 open reading frames (ORFs). Prediction of these ORFs indicates that there are 40% genes of unknown function. The other genes of definite functions encode metabolic enzymes, structure proteins, transcription regulatory factors and some elements correlated with horizontal transfer. Here we compare the complete genomic sequences of the two closely related species, which differ in pathogenic phenotype. To our knowledge, this not only reveals the difference of genomic sequence between the two important enteric pathogens for the first time, but also provides valuable clues to further researches in its process of physiological activity, pathogenesis and the evolution of enteric bacteria.

  2. Deletion of pigR gene in Monascus ruber leads to loss of pigment production.

    Science.gov (United States)

    Xie, Nana; Liu, Qingpei; Chen, Fusheng

    2013-09-01

    Pigments produced by Monascus are traditional food colorants and are widely used as dietary supplements. Since genes involving in pigment biosynthesis have not been reported, we describe the identification of a putative pigment-regulatory gene (pigR) obtained by molecular analysis of an albino strain of Monascus ruber M7. In the pigR-deleted strain (ΔpigR), neither the pigments nor pigR expression were detected by HPLC or reverse-transcription PCR, respectively, whereas the introduction of the pigR, together with a constitutive trpC promoter into ΔpigR, caused it to produce 5.4 U of red pigments/g dry mycelia, about 12-fold higher than Monascus ruber M7 (0.46 U/g dry mycelia). Thus pigR up-regulates pigment production in Monascus ruber M7.

  3. Mutants of Streptomyces roseosporus that express enhanced recombination within partially homologous genes.

    Science.gov (United States)

    Hosted, T J; Baltz, R H

    1996-10-01

    Streptomyces roseosporus mutants that express enhanced recombination between partially homologous (homeologous) sequences were isolated by selection for recombination between the bacteriophage phi C31 derivative KC570 containing the Streptomyces coelicolor glucose kinase (glk) gene and the S. roseosporus chromosome. The frequencies of homeologous recombination in the ehr mutants were determined by measuring the chromosomal insertion frequencies of plasmids containing S. coelicolor glnA or whiG genes. S. roseosporus ehr mutants showed 10(2)- to 10(4)-fold increases in homeologous recombination relative to Ehr+ strains, but no increase in homologous recombination. Southern hybridization analysis revealed single unique sites for the insertion of each of the plasmids, and the crossovers occurred in frame and in proper translational register, yielding functional chimeric glnA and whiG genes.

  4. Analysis of blood stem cell activity and cystatin gene expression in a mouse model presenting a chromosomal deletion encompassing Csta and Stfa2l1.

    Science.gov (United States)

    Bilodeau, Mélanie; MacRae, Tara; Gaboury, Louis; Laverdure, Jean-Philippe; Hardy, Marie-Pierre; Mayotte, Nadine; Paradis, Véronique; Harton, Sébastien; Perreault, Claude; Sauvageau, Guy

    2009-10-19

    The cystatin protein superfamily is characterized by the presence of conserved sequences that display cysteine protease inhibitory activity (e.g., towards cathepsins). Type 1 and 2 cystatins are encoded by 25 genes of which 23 are grouped in 2 clusters localized on mouse chromosomes 16 and 2. The expression and essential roles of most of these genes in mouse development and hematopoiesis remain poorly characterized. In this study, we describe a set of quantitative real-time PCR assays and a global expression profile of cystatin genes in normal mouse tissues. Benefiting from our collection of DelES embryonic stem cell clones harboring large chromosomal deletions (to be reported elsewhere), we selected a clone in which a 95-kb region of chromosome 16 is missing (Del(16qB3Delta/+)). In this particular clone, 2 cystatin genes, namely Csta and Stfa2l1 are absent along with 2 other genes (Fam162a, Ccdc58) and associated intergenic regions. From this line, we established a new homozygous mutant mouse model (Del(16qB3Delta/16qB3Delta)) to assess the in vivo biological functions of the 2 deleted cystatins. Stfa2l1 gene expression is high in wild-type fetal liver, bone marrow, and spleen, while Csta is ubiquitously expressed. Homozygous Del(16qB3Delta/16qB3Delta) animals are phenotypically normal, fertile, and not overtly susceptible to spontaneous or irradiation-induced tumor formation. The hematopoietic stem and progenitor cell activity in these mutant mice are also normal. Interestingly, quantitative real-time PCR expression profiling reveals a marked increase in the expression levels of Stfa2l1/Csta phylogenetically-related genes (Stfa1, Stfa2, and Stfa3) in Del(16qB3Delta/16qB3Delta) hematopoietic tissues, suggesting that these candidate genes might be contributing to compensatory mechanisms. Overall, this study presents an optimized approach to globally monitor cystatin gene expression as well as a new mouse model deficient in Stfa2l1/Csta genes, expanding the

  5. A novel deletion partly removing the AVP gene causes autosomal recessive inheritance of early-onset neurohypophyseal diabetes insipidus.

    Science.gov (United States)

    Christensen, J H; Kvistgaard, H; Knudsen, J; Shaikh, G; Tolmie, J; Cooke, S; Pedersen, S; Corydon, T J; Gregersen, N; Rittig, S

    2013-01-01

    Familial neurohypophyseal diabetes insipidus (FNDI) typically presents with age-dependent penetrance and autosomal dominant inheritance caused by missense variations in one allele of the AVP gene encoding the arginine vasopressin (AVP) prohormone. We present the molecular genetic characteristics underlying an unusual form of FNDI occurring with very early onset and seemingly autosomal recessive inheritance. By DNA amplification and sequencing, we identified a novel variant allele of the AVP gene carrying a 10,396 base pair deletion involving the majority of the AVP gene as well as its regulatory sequences in the intergenic region between the AVP and the OXT gene, encoding the oxytocin prohormone. We found two chromosomes carrying the deletion in affected family members and one in unaffected family members suspected to transmit the deleted allele. Whole-genome array analysis confirmed the results and excluded the presence of any additional major pathogenic abnormalities. The deletion is predicted to abolish the transcription of the AVP gene, thus the fact that family members heterozygous for the deletion remain healthy argues, in general, against haploinsufficiency as the pathogenic mechanism FNDI. Accordingly, our data is strong support to the prevailing idea that dominant inheritance of FNDI is due to a dominant-negative effect exerted by variant AVP prohormone.

  6. Genetic Analysis and Gene Mapping of Light Brown Spotted Leaf Mutant in Rice

    Institute of Scientific and Technical Information of China (English)

    FENG Bao-hua; YANG Yang; SHI Yong-feng; LIN Lu; CHEN Jie; WEI Yan-lin; Hei LEUNG

    2013-01-01

    A light brown spotted-leaf mutant of rice was isolated from an ethane methyl sulfonate (EMS)induced IR64 mutant bank.The mutant,designated as Ibsl1 (light brown spotted-leaf 1),displayed light brown spot in the whole growth period from the first leaf to the flag leaf under natural summer field conditions.Agronomic traits including plant height,growth duration,number of filled grains per panicle,seed-setting rate and 1000-grain weight of the mutant were significantly affected.Genetic analysis showed that the mutation was controlled by a single recessive gene,tentatively named Ibsl1(t),which was mapped to the short arm of chromosome 6.By developing simple sequence repeat (SSR) markers,the gene was finally delimited to an interval of 130 kb between markers RM586 and RM588.The Ibsl1(t) gene is likely a novel rice spotted-leaf gene since no other similar genes have been identified near the chromosomal region.The genetic data and recombination populations provided will facilitate further fine-mapping and cloning of the gene.

  7. Dopamine D2 receptor gene -141C Insertion/Deletion polymorphism in Turkish schizophrenic patients.

    Science.gov (United States)

    Kurt, Hulyam; Dikmen, Miris; Basaran, Ayşe; Yenilmez, Cinar; Ozdemir, Figen; Degirmenci, Irfan; Gunes, Hasan Veysi; Kucuk, Meral Urhan; Mutlu, Fezan

    2011-02-01

    Schizophrenia is a chronic and neuropsychiatric disease that affects about 0.5-1% of the world's population. An increase in dopamine and dopamine D2 receptor (DRD2) gene products has been well described in schizophrenic patients. Several groups have studied the relationship between dopaminergic hyperactivity and cellular communications have obtained discordant results. Studies searching for the relationship between the schizophrenia and DRD2 gene have gained more interest. Our objective was to determine the relationships among schizophrenic symptoms in schizophrenia subtypes and severity of symptoms in terms of DRD2 gene -141C Insertion/Deletion [Ins/Del; I/D] polymorphism by PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) assay method. Genomic DNA was prepared from peripheral blood by using salt extraction method. After amplification of genomic DNA, PCR products were digested with BstNI restriction enzyme for the detection of DRD2 gene -141C Ins/Del polymorphism in 73 schizophrenic patients and 60 healthy control subjects. The allelic frequencies of the DRD2 gene -141C Ins/Del polymorphism in case and control groups were 79.5 and 77.5% for I allele; 20.5 and 22.5% for D allele respectively. There was no significant difference in frequencies of genotypes and alleles between the two groups. In schizophrenic and control subjects, there were no significant relationship in severity of the disease and schizophrenia types among the -141C Ins/Del genotypes and alleles.

  8. Novel Mutations and Deletions of the KIT (Steel Factor Receptor) Gene in Human Piebaldism

    Science.gov (United States)

    Ezoe, Kazuhiko; Holmes, Stuart A.; Ho, Lingling; Bennett, Christopher P.; Bolognia, Jean L.; Brueton, Louise; Burn, John; Falabella, Rafael; Gatto, Emilia M.; Ishii, Norihisa; Moss, Celia; Pittelkow, Mark R.; Thompson, Elizabeth; Ward, K. Anne; Spritz, Richard A.

    1995-01-01

    Piebaldism is an autosomal dominant genetic disorder of pigmentation characterized by white patches of skin and hair. Melanocytes are lacking in these hypopigmented regions, the result of mutations of the KIT gene, which encodes the cell surface receptor for steel factor (SLF). We describe the analysis of 26 unrelated patients with piebaldism-like hypopigmentation—17 typical patients, 5 with atypical clinical features or family histories, and 4 with other disorders that involve white spotting. We identified novel pathologic mutations or deletions of the KIT gene in 10 (59%) of the typical patients, and in 2 (40%) of the atypical patients. Overall, we have identified pathologic KIT gene mutations in 21 (75%) of 28 unrelated patients with typical piebaldism we have studied. Of the patients without apparent KIT mutations, none have apparent abnormalities of the gene encoding SLF itself (MGF), and genetic linkage analyses in two of these families are suggestive of linkage of the piebald phenotype to KIT. Thus, most patients with typical piebaldism appear to have abnormalities of the KIT gene. ImagesFigure 2 PMID:7529964

  9. Improving Adaptive and Memory Immune Responses of an HIV/AIDS Vaccine Candidate MVA-B by Deletion of Vaccinia Virus Genes (C6L and K7R) Blocking Interferon Signaling Pathways.

    Science.gov (United States)

    García-Arriaza, Juan; Arnáez, Pilar; Gómez, Carmen E; Sorzano, Carlos Óscar S; Esteban, Mariano

    2013-01-01

    Poxvirus vector Modified Vaccinia Virus Ankara (MVA) expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (termed MVA-B) is a promising HIV/AIDS vaccine candidate, as confirmed from results obtained in a prophylactic phase I clinical trial in humans. To improve the immunogenicity elicited by MVA-B, we have generated and characterized the innate immune sensing and the in vivo immunogenicity profile of a vector with a double deletion in two vaccinia virus (VACV) genes (C6L and K7R) coding for inhibitors of interferon (IFN) signaling pathways. The innate immune signals elicited by MVA-B deletion mutants (MVA-B ΔC6L and MVA-B ΔC6L/K7R) in human macrophages and monocyte-derived dendritic cells (moDCs) showed an up-regulation of the expression of IFN-β, IFN-α/β-inducible genes, TNF-α, and other cytokines and chemokines. A DNA prime/MVA boost immunization protocol in mice revealed that these MVA-B deletion mutants were able to improve the magnitude and quality of HIV-1-specific CD4(+) and CD8(+) T cell adaptive and memory immune responses, which were mostly mediated by CD8(+) T cells of an effector phenotype, with MVA-B ΔC6L/K7R being the most immunogenic virus recombinant. CD4(+) T cell responses were mainly directed against Env, while GPN-specific CD8(+) T cell responses were induced preferentially by the MVA-B deletion mutants. Furthermore, antibody levels to Env in the memory phase were slightly enhanced by the MVA-B deletion mutants compared to the parental MVA-B. These findings revealed that double deletion of VACV genes that act blocking intracellularly the IFN signaling pathway confers an immunological benefit, inducing innate immune responses and increases in the magnitude, quality and durability of the HIV-1-specific T cell immune responses. Our observations highlighted the immunomodulatory role of the VACV genes C6L and K7R, and that targeting common pathways, like IRF3/IFN-β signaling, could be a general strategy to improve the immunogenicity

  10. Improving Adaptive and Memory Immune Responses of an HIV/AIDS Vaccine Candidate MVA-B by Deletion of Vaccinia Virus Genes (C6L and K7R Blocking Interferon Signaling Pathways.

    Directory of Open Access Journals (Sweden)

    Juan García-Arriaza

    Full Text Available Poxvirus vector Modified Vaccinia Virus Ankara (MVA expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (termed MVA-B is a promising HIV/AIDS vaccine candidate, as confirmed from results obtained in a prophylactic phase I clinical trial in humans. To improve the immunogenicity elicited by MVA-B, we have generated and characterized the innate immune sensing and the in vivo immunogenicity profile of a vector with a double deletion in two vaccinia virus (VACV genes (C6L and K7R coding for inhibitors of interferon (IFN signaling pathways. The innate immune signals elicited by MVA-B deletion mutants (MVA-B ΔC6L and MVA-B ΔC6L/K7R in human macrophages and monocyte-derived dendritic cells (moDCs showed an up-regulation of the expression of IFN-β, IFN-α/β-inducible genes, TNF-α, and other cytokines and chemokines. A DNA prime/MVA boost immunization protocol in mice revealed that these MVA-B deletion mutants were able to improve the magnitude and quality of HIV-1-specific CD4(+ and CD8(+ T cell adaptive and memory immune responses, which were mostly mediated by CD8(+ T cells of an effector phenotype, with MVA-B ΔC6L/K7R being the most immunogenic virus recombinant. CD4(+ T cell responses were mainly directed against Env, while GPN-specific CD8(+ T cell responses were induced preferentially by the MVA-B deletion mutants. Furthermore, antibody levels to Env in the memory phase were slightly enhanced by the MVA-B deletion mutants compared to the parental MVA-B. These findings revealed that double deletion of VACV genes that act blocking intracellularly the IFN signaling pathway confers an immunological benefit, inducing innate immune responses and increases in the magnitude, quality and durability of the HIV-1-specific T cell immune responses. Our observations highlighted the immunomodulatory role of the VACV genes C6L and K7R, and that targeting common pathways, like IRF3/IFN-β signaling, could be a general strategy to improve the

  11. Altered intestinal epithelial homeostasis in mice with intestine-specific deletion of the Krüppel-like factor 4 gene.

    Science.gov (United States)

    Ghaleb, Amr M; McConnell, Beth B; Kaestner, Klaus H; Yang, Vincent W

    2011-01-15

    The zinc finger transcription factor, Krüppel-like factor 4 (KLF4), is expressed in the post-mitotic, differentiated epithelial cells lining the intestinal tract and exhibits a tumor suppressive effect on intestinal tumorigenesis. Here we report a role for KLF4 in maintaining homeostasis of intestinal epithelial cells. Mice with conditional ablation of the Klf4 gene from the intestinal epithelium were viable. However, both the rates of proliferation and migration of epithelial cells were increased in the small intestine of mutant mice. In addition, the brush-border alkaline phosphatase was reduced as was expression of ephrine-B1 in the small intestine, resulting in mispositioning of Paneth cells to the upper crypt region. In the colon of mutant mice, there was a reduction of the differentiation marker, carbonic anhydrase-1, and failure of differentiation of goblet cells. Mechanistically, deletion of Klf4 from the intestine resulted in activation of genes in the Wnt pathway and reduction in expression of genes encoding regulators of differentiation. Taken together, these data provide new insights into the function of KLF4 in regulating postnatal proliferation, migration, differentiation, and positioning of intestinal epithelial cells and demonstrate an essential role for KLF4 in maintaining normal intestinal epithelial homeostasis in vivo. Copyright © 2010 Elsevier Inc. All rights reserved.

  12. P450XXI (steroid 21-hydroxylase) gene deletions are not found in family studies of congenital adrenal hyperplasia

    Energy Technology Data Exchange (ETDEWEB)

    Matteson, K.J.; Phillips, J.A. III; Miller, W.L.; Chung, B.C.; Orlando, P.J.; Frisch, H.; Ferrandez, A.; Burr, I.M.

    1987-08-01

    Congenital adrenal hyperplasia (CAH) is a common genetic disorder due to defective 21-hydroxylation of steroid hormones. The human P450XXIA2 gene encodes cytochrome P450c21 (steroid 21-monooxygenase (steroid 21-hydroxylase)), which mediates 21-hydroxylation. The P450XXIA2 gene may be distinguished from the duplicated P450XXIA1 pseudogene by cleavage with the restriction endonuclease Taq I, with the XXIA2 gene characterized by a 3.7-kilobase (kb) fragment and the XXIA1 pseudogene characterized by a 3.2-kb fragment. Restriction endonuclease mapping by several laboratories has suggested that deletion of the P450XXIA2 gene occurs in about 25% of patients with CAH, as their genomic DNA lacks detectable 3.7-kb Taq I fragments. The authors have cloned human P450c21 cDNA and used it to study genomic DNA prepared from 51 persons in 10 families, each of which includes 2 or more persons with CAH. After Taq I digestion, apparent deletions are seen in 7 of the 20 alleles of the probands; using EcoRI, apparent deletions are seen in 9 of the 20 alleles. However, the apparently deleted alleles seen with Taq I do not coincide with those seen with EcoRI. Furthermore, studies with Bgl II, EcoRI, Kpn I, and Xba I yield normal patterns with at least two enzymes in all cases. Since all probands yielded normal patterns with at least two of the five enzymes used, they conclude that the P450XXIA2 gene deletions widely reported in CAH patients probably represent gene conversions, unequal crossovers,or polymorphisms rather than simple gene deletions.

  13. Recombination-dependent deletion formation in mammalian cells deficient in the nucleotide excision repair gene ERCC1.

    Science.gov (United States)

    Sargent, R G; Rolig, R L; Kilburn, A E; Adair, G M; Wilson, J H; Nairn, R S

    1997-11-25

    Nucleotide excision repair proteins have been implicated in genetic recombination by experiments in Saccharomyces cerevisiae and Drosophila melanogaster, but their role, if any, in mammalian cells is undefined. To investigate the role of the nucleotide excision repair gene ERCC1, the hamster homologue to the S. cerevisiae RADIO gene, we disabled the gene by targeted knockout. Partial tandem duplications of the adenine phosphoribosyltransferase (APRT) gene then were constructed at the endogenous APRT locus in ERCC1- and ERCC1+ cells. To detect the full spectrum of gene-altering events, we used a loss-of-function assay in which the parental APRT+ tandem duplication could give rise to APRT- cells by homologous recombination, gene rearrangement, or point mutation. Measurement of rates and analysis of individual APRT- products indicated that gene rearrangements (principally deletions) were increased at least 50-fold, whereas homologous recombination was affected little. The formation of deletions is not caused by a general effect of the ERCC1 deficiency on gene stability, because ERCC1- cell lines with a single wild-type copy of the APRT gene yielded no increase in deletions. Thus, deletion formation is dependent on the tandem duplication, and presumably the process of homologous recombination. Recombination-dependent deletion formation in ERCC1- cells is supported by a significant decrease in a particular class of crossover products that are thought to arise by repair of a heteroduplex intermediate in recombination. We suggest that the ERCC1 gene product in mammalian cells is involved in the processing of heteroduplex intermediates in recombination and that the misprocessed intermediates in ERCC1- cells are repaired by illegitimate recombination.

  14. A thiostrepton resistance gene and its mutants serve as selectable markers in Geobacillus kaustophilus HTA426.

    Science.gov (United States)

    Wada, Keisuke; Kobayashi, Jyumpei; Furukawa, Megumi; Doi, Katsumi; Ohshiro, Takashi; Suzuki, Hirokazu

    2016-01-01

    Effective utilization of microbes often requires complex genetic modification using multiple antibiotic resistance markers. Because a few markers have been used in Geobacillus spp., the present study was designed to identify a new marker for these thermophiles. We explored antibiotic resistance genes functional in Geobacillus kaustophilus HTA426 and identified a thiostrepton resistance gene (tsr) effective at 50 °C. The tsr gene was further used to generate the mutant tsr(H258Y) functional at 55 °C. Higher functional temperature of the mutant was attributable to the increase in thermostability of the gene product because recombinant protein produced from tsr(H258Y) was more thermostable than that from tsr. In fact, the tsr(H258Y) gene served as a selectable marker for plasmid transformation of G. kaustophilus. This new marker could facilitate complex genetic modification of G. kaustophilus and potentially other Geobacillus spp.

  15. Deletion of psychiatric risk gene Cacna1c impairs hippocampal neurogenesis in cell-autonomous fashion.

    Science.gov (United States)

    Völkening, Bianca; Schönig, Kai; Kronenberg, Golo; Bartsch, Dusan; Weber, Tillmann

    2017-05-01

    Ca(2+) is a universal signal transducer which fulfills essential functions in cell development and differentiation. CACNA1C, the gene encoding the alpha-1C subunit (i.e., Cav 1.2) of the voltage-dependent l-type calcium channel (LTCC), has been implicated as a risk gene in a variety of neuropsychiatric disorders. To parse the role of Cav 1.2 channels located on astrocyte-like stem cells and their descendants in the development of new granule neurons, we created Tg(GLAST-CreERT2) /Cacna1c(fl/fl) /RCE:loxP mice, a transgenic tool that allows cell-type-specific inducible deletion of Cacna1c. The EGFP reporter was used to trace the progeny of recombined type-1 cells. FACS-sorted Cacna1c-deficient neural precursor cells from the dentate gyrus showed reduced proliferative activity in neurosphere cultures. Moreover, under differentiation conditions, Cacna1c-deficient NPCs gave rise to fewer neurons and more astroglia. Similarly, under basal conditions in vivo, Cacna1c gene deletion in type-1 cells decreased type-1 cell proliferation and reduced the neuronal fate-choice decision of newly born cells, resulting in reduced net hippocampal neurogenesis. Unexpectedly, electroconvulsive seizures completely compensated for the proliferation deficit of Cacna1c deficient type-1 cells, indicating that there must be Cav 1.2-independent mechanisms of controlling proliferation related to excitation. In the aggregate, this is the first report demonstrating the presence of functional L-type 1.2 channels on type-1 cells. Cav 1.2 channels promote type-1 cell proliferation and push the glia-to-neuron ratio in the direction of a neuronal fate choice and subsequent neuronal differentiation. Cav 1.2 channels expressed on NPCs and their progeny possess the ability to shape neurogenesis in a cell-autonomous fashion. © 2017 Wiley Periodicals, Inc.

  16. Homozygous Deletions and Recurrent Amplifications Implicate New Genes Involved in Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Wennuan Liu

    2008-08-01

    Full Text Available Prostate cancer cell lines provide ideal in vitro systems for the identification and analysis of prostate tumor suppressors and oncogenes. A detailed characterization of the architecture of prostate cancer cell line genomes would facilitate the study of precise roles of various genes in prostate tumorigenesis in general. To contribute to such a characterization, we used the GeneChip 500K single nucleotide polymorphic (SNP array for analysis of genotypes and relative DNA copy number changes across the genome of 11 cell lines derived from both normal and cancerous prostate tissues. For comparison purposes, we also examined the alterations observed in the cell lines in tumor/normal pairs of clinical samples from 72 patients. Along with genome-wide maps of DNA copy number changes and loss of heterozygosity for these cell lines, we report previously unreported homozygous deletions and recurrent amplifications in prostate cancers in this study. The homozygous deletions affected a number of biologically important genes, including PPP2R2A and BNIP3L identified in this study and CDKN2A/CDKN2B reported previously. Although most amplified genomic regions tended to be large, amplifications at 8q24.21 were of particular interest because the affected regions are relatively small, are found in multiple cell lines, are located near MYC, an oncogene strongly implicated in prostate tumorigenesis, and are known to harbor SNPs that are associated with inherited susceptibility for prostate cancer. The genomic alterations revealed in this study provide an important catalog of positional information relevant to efforts aimed at deciphering the molecular genetic basis of prostate cancer.

  17. Familial glucocorticoid resistance caused by a splice site deletion in the human glucocorticoid receptor gene

    Energy Technology Data Exchange (ETDEWEB)

    Karl, M.; Lamberts, S.W.J.; Detera-Wadleigh, S.D.; Encio, I.J.; Stratakis, C.A.; Hurley, D.M.; Accili, D.; Chrousos, G.P. (National Institutes of Health, Bethesda, MD (United States) Erasmus Univ. of Rotterdam (Netherlands))

    1993-03-01

    The clinical syndrome of generalized, compensated glucocorticoid resistance is characterized by increased cortisol secretion without clinical evidence of hyper- or hypocortisolism, and manifestations of androgen and/or mineralocorticoid excess. This condition results from partial failure of the glucocorticoid receptor (GR) to modulate transcription of its target genes. The authors studied the molecular mechanisms of this syndrome in a Dutch kindred, whose affected members had hypercortisolism and approximately half of normal GRs, and whose proband was a young woman with manifestations of hyperandrogenism. Using the polymerase chain reaction to amplify and sequence each of the nine exons of the GR gene [alpha], along with their 5[prime]- and 3[prime]-flanking regions, the authors identified a 4-base deletion at the 3[prime]-boundary of exon 6 in one GR allele ([Delta][sub 4]), which removed a donor splice site in all three affected members studied. In contrast, the sequence of exon 6 in the two unaffected siblings was normal. A single nucleotide substitution causing an amino acid substitution in the amino terminal domain of the GR (asparagine to serine, codon 363) was also discovered in exon 2 of the other allele (G[sub 1220]) in the proband, in one of her affected brothers and in her unaffected sister. This deletion in the glucocorticoid receptor gene was associated with the expression of only one allele and a decrease of GR protein by 50% in affected members of this glucocorticoid resistant family. The mutation identified in exon 2 did not segregate with the disease and appears to be of no functional significance. The presence of the null allele was apparently compensated for by increased cortisol production at the expense of concurrent hyperandrogenism. 40 refs., 3 figs.

  18. Residual plastids of bleached mutants of Euglena gracilis and their effects on the expression of nucleus-encoded genes

    Institute of Scientific and Technical Information of China (English)

    WANG Jiangxin; SHI Zhixin; XU Xudong

    2004-01-01

    Bleached mutants of Euglena gracilis were obtained by treatment with ofloxacin (Ofl)and streptomycin (Sm) respectively. As shown by electron microscopy, the residual plastids contain prothylakoids in an Ofl mutant, and the highly developed and tightly stacked membranous structure found in cells of two Sm mutants. Nine genes of the plastid genome were examined with PCR, showing that ribosomal protein genes and most other plastid genes were lost in all but one Sm mutant. Using differential display and RT-PCR, it was shown that chloroplast degeneration could cause changes in transcription of certain nucleus-encoded genes during heterotrophic growth in darkness.

  19. Estrogens Suppress a Behavioral Phenotype in Zebrafish Mutants of the Autism Risk Gene, CNTNAP2.

    Science.gov (United States)

    Hoffman, Ellen J; Turner, Katherine J; Fernandez, Joseph M; Cifuentes, Daniel; Ghosh, Marcus; Ijaz, Sundas; Jain, Roshan A; Kubo, Fumi; Bill, Brent R; Baier, Herwig; Granato, Michael; Barresi, Michael J F; Wilson, Stephen W; Rihel, Jason; State, Matthew W; Giraldez, Antonio J

    2016-02-17

    Autism spectrum disorders (ASDs) are a group of devastating neurodevelopmental syndromes that affect up to 1 in 68 children. Despite advances in the identification of ASD risk genes, the mechanisms underlying ASDs remain unknown. Homozygous loss-of-function mutations in Contactin Associated Protein-like 2 (CNTNAP2) are strongly linked to ASDs. Here we investigate the function of Cntnap2 and undertake pharmacological screens to identify phenotypic suppressors. We find that zebrafish cntnap2 mutants display GABAergic deficits, particularly in the forebrain, and sensitivity to drug-induced seizures. High-throughput behavioral profiling identifies nighttime hyperactivity in cntnap2 mutants, while pharmacological testing reveals dysregulation of GABAergic and glutamatergic systems. Finally, we find that estrogen receptor agonists elicit a behavioral fingerprint anti-correlative to that of cntnap2 mutants and show that the phytoestrogen biochanin A specifically reverses the mutant behavioral phenotype. These results identify estrogenic compounds as phenotypic suppressors and illuminate novel pharmacological pathways with relevance to autism.

  20. Dosage effect of high-amylose modifier gene(s) on the starch structure of maize amylose-extender mutant.

    Science.gov (United States)

    Jiang, Hongxin; Campbell, Mark; Wu, Yusheng; Du, Shuangkui; Srichuwong, Sathaporn; Jane, Jay-Lin

    2015-01-21

    The objective of this study was to investigate how dosages of high-amylose modifier (HAM) gene(s) affected the structure of maize amylose extender (ae) mutant starch. GEMS-0067 (G), a homozygous mutant of ae and the HAM gene(s), and H99ae (H), an ae single mutant, were self-pollinated or inter-crossed to produce maize endosperms of G/G, G/H, H/G, and H/H with 3, 2, 1, and 0 doses of HAM gene(s), respectively. Endosperm starch was fractionated into amylopectin, amylose, and intermediate component (IC) of large and small molecular weights using 1-butanol precipitation of amylose followed by gel-permeation chromatography. Increases in the dosage of HAM gene(s) from 0 to 3 decreased the amylopectin content. The HAM-gene dosage significantly changed the branch chain-length of small-molecular-weight IC, but had little effect on the branch chain-length distributions of amylopectin and large-molecular-weight IC and the molecular structure of amylose.

  1. Safety and protective efficacy of a spiC and crp deletion mutant of Salmonella gallinarum as a live attenuated vaccine for fowl typhoid.

    Science.gov (United States)

    Cheng, Zhao; Yin, Junlei; Kang, Xilong; Geng, Shizhong; Hu, Maozhi; Pan, Zhiming; Jiao, Xinan

    2016-08-01

    With an aim to develop a safe, immunogenic fowl typhoid (FT) vaccine, the safety and efficacy of 1009ΔspiCΔcrp, a spiC and crp deletion mutant of Salmonella gallinarum, were evaluated in chickens. Three-day-old chickens were intramuscularly immunized with 1009ΔspiCΔcrp (1×10(7)CFU) and boosted 7days later (at 10-days old) with the same dose and via the same route (vaccinated group). The vaccinated group showed no clinical symptoms and no differences in body weight compared to the unvaccinated control group. 1009ΔspiCΔcrp bacteria colonized and persisted in the liver and spleen of vaccinated chickens for >14days, and significant specific humoral and cellular immune responses were induced. Vaccinated chickens were challenged with S. gallinarum strain SG9 at 21days post-immunization (24-day-old chickens), and efficient protection was observed based on the mortality and clinical symptoms, as compared to those in the control group. These results demonstrate that 1009ΔspiCΔcrp can be used as a live attenuated vaccine.

  2. Cloning and functional analysis of the sequences flanking mini-Tn5 in the magnetosome-deleted mutant NM21 of Magnetospirillum gryphiswaldense MSR-1

    Institute of Scientific and Technical Information of China (English)

    LI Feng; LI Ying; JIANG Wei; WANG ZhenFang; LI JiLun

    2009-01-01

    A magnetosome-deleted mutant NM21 of MagnetospMIlum gryphiswaldense MSR-1 was generated by mini-Tn5 lacZ2 transposon mutagenesis, and a 3073-bp fragment flanking mini-Tn5 lacZ2 in NM21 was cloned by Anchored PCR. Sequencing analysis showed that this fragment involved three putative ORFs; the mini-Tn5 lacZ2 was inserted into ORF1. Functional complementary test indicated that the 3073-bp fragment was required for biosynthesis of magnetosomes in M. gryphiswaldense MSR-1. The majority of proteins, which bad homology with the protein encoded by ORF1, were the cation transporter. Transmembrane domain analysis showed that the protein encoded by ORF1 contained four trans-membrane domains. It may be a transmembrane protein. The protein encoded by ORF1 contained two putative conserved domains: COG0053 and PRK09509. The MMT1 and FieF, containing conserved domains COG0053 and PRK09509 too, were Fe2+ transporter (cation diffusion facilitator superfamily). It was suggested that the protein encoded by ORF1 might take part in the magnetosomes biosynthesis as Fe2+ transporter.

  3. A novel break point of the BMPR2 gene exonic deletion in a patient with pulmonary arterial hypertension.

    Science.gov (United States)

    Aimi, Yuki; Hirayama, Tomomi; Kataoka, Masaharu; Momose, Yuichi; Nishimaki, Saiko; Matsushita, Kenichi; Yoshino, Hideaki; Satoh, Toru; Gamou, Shinobu

    2013-12-01

    The presence of genetic rearrangements of bone morphogenetic protein type 2 receptor (BMPR2) was identified in pulmonary arterial hypertension (PAH) patients as the deletion or duplication of one or more exons of the gene. We recently investigated the deletion break points in exonic deletions of BMPR2 in two Japanese familial cases with PAH, and found that these were Alu-mediated via either non-allelic homologous recombination or non-homologous recombination. We herein report the third case of exonic deletion, which was in a 25-year-old female PAH patient with a deletion of BMPR2 exon 3. The break point in this case was not located in an Alu sequence. The 5'- and 3'-break point maps between the inverted Alu sequences in intron 2 and in exon 3, respectively, resulted in a 759-bp deletion. This novel exonic deletion in this PAH case may be a unique and non-recurrent rearrangement, and appears to be of a different size from that in other patients.

  4. The maize auxotrophic mutant orange pericarp is defective in duplicate genes for tryptophan synthase beta.

    Science.gov (United States)

    Wright, A D; Moehlenkamp, C A; Perrot, G H; Neuffer, M G; Cone, K C

    1992-06-01

    orange pericarp (orp) is a seedling lethal mutant of maize caused by mutations in the duplicate unlinked recessive loci orp1 and orp2. Mutant seedlings accumulate two tryptophan precursors, anthranilate and indole, suggesting a block in tryptophan biosynthesis. Results from feeding studies and enzyme assays indicate that the orp mutant is defective in tryptophan synthase beta activity. Thus, orp is one of only a few amino acid auxotrophic mutants to be characterized in plants. Two genes encoding tryptophan synthase beta were isolated from maize and sequenced. Both genes encode polypeptides with high homology to tryptophan synthase beta enzymes from other organisms. The cloned genes were mapped by restriction fragment length polymorphism analysis to approximately the same chromosomal locations as the genetically mapped factors orp1 and orp2. RNA analysis indicates that both genes are expressed in all tissues examined from normal plants. Together, the biochemical, genetic, and molecular data verify the identity of orp1 and orp2 as duplicate structural genes for the beta subunit of tryptophan synthase.

  5. Oculo-facio-cardio-dental (OFCD) syndrome: the first Italian case of BCOR and co-occurring OTC gene deletion.

    Science.gov (United States)

    Di Stefano, C; Lombardo, B; Fabbricatore, C; Munno, C; Caliendo, I; Gallo, F; Pastore, L

    2015-04-01

    Oculo-facio-cardio-dental (OFCD) syndrome is a rare genetic disorder affecting ocular, facial, dental and cardiac systems. The syndrome is an X-linked dominant trait and it might be lethal in males. This syndrome is usually caused by mutations in the BCL6 interacting co-repressor gene (BCOR). We described a female child with mild phenotype of oculo-facio-cardio-dental syndrome. Array-comparative genomic hybridization (a-CGH) analysis revealed a de novo heterozygous deletion in the Xp11.4 region of approximately 2.3 Mb, involving BCOR and ornithine carbamoyl-transferase (OTC) genes. The deletion observed was subsequently confirmed by real time PCR. In this study we report a first case with co-occurrence of BCOR and OTC genes completely deleted in OFCD syndrome.

  6. Heme oxygenase-2 gene deletion attenuates oxidative stress in neurons exposed to extracellular hemin

    Directory of Open Access Journals (Sweden)

    Benvenisti-Zarom Luna

    2004-09-01

    Full Text Available Abstract Background Hemin, the oxidized form of heme, accumulates in intracranial hematomas and is a potent oxidant. Growing evidence suggests that it contributes to delayed injury to surrounding tissue, and that this process is affected by the heme oxygenase enzymes. In a prior study, heme oxygenase-2 gene deletion increased the vulnerability of cultured cortical astrocytes to hemin. The present study tested the effect of HO-2 gene deletion on protein oxidation, reactive oxygen species formation, and cell viability after mixed cortical neuron/astrocyte cultures were incubated with neurotoxic concentrations of hemin. Results Continuous exposure of wild-type cultures to 1–10 μM hemin for 14 h produced concentration-dependent neuronal death, as detected by both LDH release and fluorescence intensity after propidium iodide staining, with an EC50 of 1–2 μM; astrocytes were not injured by these low hemin concentrations. Cell death was consistently reduced by at least 60% in knockout cultures. Exposure to hemin for 4 hours, a time point that preceded cell lysis, increased protein oxidation in wild-type cultures, as detected by staining of immunoblots for protein carbonyl groups. At 10 μM hemin, carbonylation was increased 2.3-fold compared with control sister cultures subjected to medium exchanges only; this effect was reduced by about two-thirds in knockout cultures. Cellular reactive oxygen species, detected by fluorescence intensity after dihydrorhodamine 123 (DHR staining, was markedly increased by hemin in wild-type cultures and was localized to neuronal cell bodies and processes. In contrast, DHR fluorescence intensity in knockout cultures did not differ from that of sham-washed controls. Neuronal death in wild-type cultures was almost completely prevented by the lipid-soluble iron chelator phenanthroline; deferoxamine had a weaker but significant effect. Conclusions These results suggest that HO-2 gene deletion protects neurons in mixed

  7. Anatomical distributional defects in mutant genes associated with dominant intermediate Charcot-Marie-Tooth disease type C in an adenovirus-mediated mouse model

    Directory of Open Access Journals (Sweden)

    SeoJin Lee

    2017-01-01

    Full Text Available Dominant intermediate Charcot-Marie-Tooth disease type C (DI-CMTC is a dominantly inherited neuropathy that has been classified primarily based on motor conduction velocity tests but is now known to involve axonal and demyelination features. DI-CMTC is linked to tyrosyl-tRNA synthetase (YARS-associated neuropathies, which are caused by E196K and G41R missense mutations and a single de novo deletion (153-156delVKQV. It is well-established that these YARS mutations induce neuronal dysfunction, morphological symptoms involving axonal degeneration, and impaired motor performance. The present study is the first to describe a novel mouse model of YARS-mutation-induced neuropathy involving a neuron-specific promoter with a deleted mitochondrial targeting sequence that inhibits the expression of YARS protein in the mitochondria. An adenovirus vector system and in vivo techniques were utilized to express YARS fusion proteins with a Flag-tag in the spinal cord, peripheral axons, and dorsal root ganglia. Following transfection of YARS-expressing viruses, the distributions of wild-type (WT YARS and E196K mutant proteins were compared in all expressed regions; G41R was not expressed. The proportion of Flag/green fluorescent protein (GFP double-positive signaling in the E196K mutant-type mice did not significantly differ from that of WT mice in dorsal root ganglion neurons. All adenovirus genes, and even the empty vector without the YARS gene, exhibited GFP-positive signaling in the ventral horn of the spinal cord because GFP in an adenovirus vector is driven by a cytomegalovirus promoter. The present study demonstrated that anatomical differences in tissue can lead to dissimilar expressions of YARS genes. Thus, use of this novel animal model will provide data regarding distributional defects between mutant and WT genes in neurons, the DI-CMTC phenotype, and potential treatment approaches for this disease.

  8. Genetic Analysis and Mapping of TWH Gene in Rice Twisted Hull Mutant

    Institute of Scientific and Technical Information of China (English)

    LI Jin-bo; XIA Ming-yuan; WAN Bing-liang; DU Xue-shu; ZHA Zhong-ping; YU Da-zhao; QI Hua-xiong

    2009-01-01

    A mutant with twisted hulls was found in a breeding population of rice (Oryza sativa L.). The mutant shows less grain weight and inferior grain quality in addition to twisted hulls. Genetic analysis indicated that the phenotype of mutant was controlled by a single recessive gene (temporarily designated as TWH). To map the TWH gene, an F2 population was generated by crossing the twh mutant to R725, an indica rice variety with normal hulls. For bulked segregant analysis, the bulk of mutant plants was prepared by mixing equal amount of plant tissue from 10 twisted-hull plants and the bulk of normal plants was obtained by pooling equal amount tissue of 10 normal-hull plants. Two hundred and seven pairs of simple sequence repeat (SSR) primers, which are distributed on 12 rice chromosomes, were used for polymorphism analysis of the parents and the two bulks. The TWH locus was initially mapped close to the SSR marker RM526 on chromosome 2. Therefore, further mapping was performed using 50 pairs of SSR primers around the marker RM526. The TWH was delimited between the SSR markers RM14128 and RM208 on the long arm of chromosome 2 at the genetic distances of 1.4 cM and 2.7 cM, respectively. These results provide the foundation for further fine mapping, cloning and functional analysis of the TWH gene.

  9. Altered behavior in mice with deletion of the alpha2-antiplasmin gene.

    Science.gov (United States)

    Kawashita, Eri; Kanno, Yosuke; Ikeda, Kanako; Kuretake, Hiromi; Matsuo, Osamu; Matsuno, Hiroyuki

    2014-01-01

    The α2-antiplasmin (α2AP) protein is known to be a principal physiological inhibitor of plasmin, and is expressed in various part of the brain, including the hippocampus, cortex, hypothalamus and cerebellum, thus suggesting a potential role for α2AP in brain functions. However, the involvement of α2AP in brain functions is currently unclear. The goal of this study was to investigate the effects of the deletion of the α2AP gene on the behavior of mice. The motor function was examined by the wire hang test and rotarod test. To evaluate the cognitive function, a repeated rotarod test, Y-maze test, Morris water maze test, passive or shuttle avoidance test and fear conditioning test were performed. An open field test, dark/light transition test or tail suspension test was performed to determine the involvement of α2AP in anxiety or depression-like behavior. The α2AP knockout (α2AP-/-) mice exhibited impaired motor function compared with α2AP+/+ mice. The α2AP-/- mice also exhibited impairments in motor learning, working memory, spatial memory and fear conditioning memory. Furthermore, the deletion of α2AP induced anxiety-like behavior, and caused an anti-depression-like effect in tail suspension. Therefore, our findings suggest that α2AP is a crucial mediator of motor function, cognitive function, anxiety-like behavior and depression-like behavior, providing new insights into the role of α2AP in the brain functions.

  10. Deletion of an X-inactivation boundary disrupts adjacent gene silencing.

    Directory of Open Access Journals (Sweden)

    Lindsay M Horvath

    2013-11-01

    Full Text Available In mammalian females, genes on one X are largely silenced by X-chromosome inactivation (XCI, although some "escape" XCI and are expressed from both Xs. Escapees can closely juxtapose X-inactivated genes and provide a tractable model for assessing boundary function at epigenetically regulated loci. To delimit sequences at an XCI boundary, we examined female mouse embryonic stem cells carrying X-linked BAC transgenes derived from an endogenous escape locus. Previously we determined that large BACs carrying escapee Kdm5c and flanking X-inactivated transcripts are properly regulated. Here we identify two lines with truncated BACs that partially and completely delete the distal Kdm5c XCI boundary. This boundary is not required for escape, since despite integrating into regions that are normally X inactivated, transgenic Kdm5c escapes XCI, as determined by RNA FISH and by structurally adopting an active conformation that facilitates long-range preferential association with other escapees. Yet, XCI regulation is disrupted in the transgene fully lacking the distal boundary; integration site genes up to 350 kb downstream of the transgene now inappropriately escape XCI. Altogether, these results reveal two genetically separable XCI regulatory activities at Kdm5c. XCI escape is driven by a dominant element(s retained in the shortest transgene that therefore lies within or upstream of the Kdm5c locus. Additionally, the distal XCI boundary normally plays an essential role in preventing nearby genes from escaping XCI.

  11. Whole-Genome Microarray and Gene Deletion Studies Reveal Regulation of the Polyhydroxyalkanoate Production Cycle by the Stringent Response in Ralstonia eutropha H16

    Energy Technology Data Exchange (ETDEWEB)

    Brigham, CJ; Speth, DR; Rha, C; Sinskey, AJ

    2012-10-22

    Poly(3-hydroxybutyrate) (PHB) production and mobilization in Ralstonia eutropha are well studied, but in only a few instances has PHB production been explored in relation to other cellular processes. We examined the global gene expression of wild-type R. eutropha throughout the PHB cycle: growth on fructose, PHB production using fructose following ammonium depletion, and PHB utilization in the absence of exogenous carbon after ammonium was resupplied. Our results confirm or lend support to previously reported results regarding the expression of PHB-related genes and enzymes. Additionally, genes for many different cellular processes, such as DNA replication, cell division, and translation, are selectively repressed during PHB production. In contrast, the expression levels of genes under the control of the alternative sigma factor sigma(54) increase sharply during PHB production and are repressed again during PHB utilization. Global gene regulation during PHB production is strongly reminiscent of the gene expression pattern observed during the stringent response in other species. Furthermore, a ppGpp synthase deletion mutant did not show an accumulation of PHB, and the chemical induction of the stringent response with DL-norvaline caused an increased accumulation of PHB in the presence of ammonium. These results indicate that the stringent response is required for PHB accumulation in R. eutropha, helping to elucidate a thus-far-unknown physiological basis for this process.

  12. COOH-terminal deletion of HBx gene is a frequent event in HBV-associated hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Xiao-Hong Liu; Jing Lin; Shu-Hui Zhang; Shun-Min Zhang; Mark A Feitelson; Heng-Jun Gao; Ming-Hua Zhu

    2008-01-01

    AIM:To investigate the hepatitis B virus (HBV) x gene (HBx) state in the tissues of HBV-related hepatocellular carcinoma (HCC) in Chinese patients and whether there were particular HBx mutations.METHODS:HBx gene was amplified and direct sequencing was used in genomic DNA samples from 20HCC and corresponding non-cancerous liver tissues from HBsAg-positive patients.HBV DNA integration and HBx deleted mutation were validated in 45 HCC patients at different stages by Southern blot analysis and polymerase chain reaction methods.RESULTS:The frequencies of HBx point mutations were significantly lower in HCC than their corresponding non-cancerous liver tissues (11/19 vs 18/19,P = 0.019).In contrast,deletions in HBx gene were significantly higher in HCC than their non-cancerous liver tissues (16/19 vs 4/19,P<0.001).The deletion of HBx COOH-terminal was detected in 14 HCC tissues.A specific integration of HBx at 17p13 locus was also found in 8 of 16 HCC,and all of them also exhibited full-length HBx deletions.Integrated or integrated coexistence with replicated pattern was obtained in 45.5% (20145)-56.8% (25145)tumors and 40.9% (18/45)-52.3% (23/45) non-tumor tissues.CONCLUSION:HBx deletion,especially the COOH-terminal deletion of HBx is a frequent event in HBV-associated HCC tissues in China.HBV integration had also taken place in partial HCC tissues.This supporting the hypothesis that deletion and probably integrated forms of the HBx gene may be implicated in liver carcinogenesis.

  13. Analysis of a new homozygous deletion in the tumor suppressor region at 3p12.3 reveals two novel intronic noncoding RNA genes

    NARCIS (Netherlands)

    Angeloni, Debora; ter Elst, Arja; Wei, Ming Hui; van der Veen, Anneke Y.; Braga, Eleonora A.; Klimov, Eugene A.; Timmer, Tineke; Korobeinikova, Luba; Lerman, Michael I.; Buys, Charles H. C. M.

    2006-01-01

    Homozygous deletions or loss of heterozygosity (LOH) at human chromosome band 3p12 are consistent features of lung and other malignancies, suggesting the presence of a tumor suppressor gene(s) (TSG) at this location. Only one gene has been cloned thus far from the overlapping region deleted in lung

  14. Large deletions within the spinal muscular atrophy gene region in a patient with spinal muscular atrophy type 3

    Institute of Scientific and Technical Information of China (English)

    Wei Wei; Chunyue Chen; Wenting Liu; Zhenfang Du; Xiaoling Chen; Xianning Zhang

    2011-01-01

    Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder characterized by degeneration and loss of anterior horn cells in the spinal cord and brain stem nuclei, leading to progressive limb and trunk paralysis and muscular atrophy. Depending on the age of onset and maximum muscular function achieved, SMA is recognized as SMA1, SMA2, SMA3 or SMA4, and most patients have a deletion or truncation of the survival motor neuron 1 (SMN1) gene. In this report, we present a patient with a mild SMA phenotype, SMA3, and define his genetic abnormality. Tetra-primer amplification refractory mutation system PCR combined with restriction fragment length polymorphism analysis and array comparative genomic hybridization were used to determine the genetic variations in this patient. A 500 kb deletion in chromosome 5q13.2, including homozygous deletion of neuronal apoptosis inhibitory protein, and heterozygous deletion of occludin and B-double prime 1 was identified. This SMA region deletion did not involve SMN, indicating that SMN was likely to function normally. The phenotype was dependent of the large deletion and neuronal apoptosis inhibitory protein, occludin and B-double prime 1 may be candidate genes for SMA3.

  15. Overlapping submicroscopic deletions in Xq28 in two unrelated boys with developmental disorders: Identification of a gene near FRAXE

    Energy Technology Data Exchange (ETDEWEB)

    Gedeon, A.K.; Sutherland, G.R. [Women`s and Children`s Hospital, North Adelaide (Australia)]|[Univ. of Adelaide (Australia); Ades, L.C.; Gecz, J.; Baker, E.; Mulley, J.C. [Women`s and Children`s Hospital, North Adelaide (Australia); Keinaenen, M. [Clinical Laboratory Medix, Espoo (Finland); Kaeaeriaeinen, H. [Univ. of Helsinki (Finland)

    1995-04-01

    Two unrelated boys are described with delay in development and submicroscopic deletions in Xq28, near FRAXE. Molecular diagnosis to exclude the fragile X (FRAXA) syndrome used the direct probe pfxa3, together with a control probe pS8 (DXS296), against PstI restriction digests of DNA. Deletions were detected initially by the control probe pS8, which is an anonymous fragment subcloned from YAC 539, within 1 Mb distal to FRAXA. Further molecular analyses determined that the maximum size of the deletion is <100 kb in one boy (MK) and is wholly overlapped by the deletion of up to {approximately}200 kb in the other (CB). These deletions lie between the sequences detected by the probe VK21C (DXS296) and a dinucleotide repeat VK18AC (DXS295). The patient MK had only speech delay with otherwise normal development, while patient CB had global developmental delay that included speech delay. Detection of overlapping deletions in these two cases led to speculation that coding sequences of a gene(s) important in language development may be affected. Hybridization of the pS8 and VK21A probes to zooblots revealed cross-species homology. This conservation during evolution suggested that this region contains sequences with functional significance in normal development. The VK21A probe detected a 9.5-kb transcript in placenta and brain and a smaller, 2.5-kb, transcript in other tissues analyzed. 26 refs., 6 figs.

  16. Soluble epoxide hydrolase gene deletion improves blood flow and reduces infarct size after cerebral ischemia in reproductively senescent female mice

    Directory of Open Access Journals (Sweden)

    Kristen L Zuloaga

    2015-01-01

    Full Text Available Soluble epoxide hydrolase (sEH, a key enzyme in the metabolism of vasodilatory epoxyeicosatrienoic acids (EETs, is sexually dimorphic, suppressed by estrogen, and contributes to underlying sex differences in cerebral blood flow and injury after cerebral ischemia. We tested the hypothesis that sEH inhibition or gene deletion in reproductively senescent (RS female mice would increase cerebral perfusion and decrease infarct size following stroke. RS (15-18 month old and young (3-4 month old female sEH knockout (sEHKO mice and wild type (WT mice were subjected to 45 min middle cerebral artery occlusion (MCAO with laser Doppler perfusion monitoring. WT mice were treated with vehicle or a sEH inhibitor t-AUCB at the time of reperfusion and every 24hrs thereafter for 3 days. Differences in regional cerebral blood flow were measured in vivo using optical microangiography. Infarct size was measured 3 days after reperfusion. Infarct size and cerebral perfusion 24h after MCAO were not altered by age. Both sEH gene deletion and sEH inhibition increased cortical perfusion 24h after MCAO. Neither sEH gene deletion nor sEH inhibition reduced infarct size in young mice. However, sEH gene deletion, but not sEH inhibition of the hydrolase domain of the enzyme, decreased infarct size in RS mice. Results of these studies show that sEH gene deletion and sEH inhibition enhance cortical perfusion following MCAO and sEH gene deletion reduces damage after ischemia in RS female mice; however this neuroprotection in absent is young mice.

  17. The effect of the CCR5-delta32 deletion on global gene expression considering immune response and inflammation

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    Hütter Gero

    2011-10-01

    Full Text Available Abstract Background The natural function of the C-C chemokine receptor type 5 (CCR5 is poorly understood. A 32 base pair deletion in the CCR5 gene (CCR5-delta32 located on chromosome 3 results in a non-functional protein. It is supposed that this deletion causes an alteration in T-cell response to inflammation. For example, the presence of the CCR5-delta32 allele in recipients of allografts constitutes as an independent and protective factor associated with a decreased risk of graft-versus-host disease (GVHD and graft rejection. However, the mechanism of this beneficial effect of the deletion regarding GVHD is unknown. In this survey we searched for a CCR5-delta32 associated regulation of critical genes involved in the immune response and the development of GVHD. Methods We examined CD34+ hematopoietic progenitor cells derived from bone marrow samples from 19 healthy volunteers for the CCR5-delta32 deletion with a genomic PCR using primers flanking the site of the deletion. Results 12 individuals were found to be homozygous for CCR5 WT and 7 carried the CCR5-delta32 deletion heterozygously. Global gene expression analysis led to the identification of 11 differentially regulated genes. Six of them are connected with mechanisms of immune response and control: LRG1, CXCR2, CCRL2, CD6, CD7, WD repeat domain, and CD30L. Conclusions Our data indicate that the CCR5-delta32 mutation may be associated with differential gene expression. Some of these genes are critical for immune response, in the case of CD30L probably protective in terms of GVHD.

  18. Overexpressing enzymes of the Ehrlich pathway and deleting genes of the competing pathway in Saccharomyces cerevisiae for increasing 2-phenylethanol production from glucose.

    Science.gov (United States)

    Shen, Li; Nishimura, Yuya; Matsuda, Fumio; Ishii, Jun; Kondo, Akihiko

    2016-07-01

    2-Phenylethanol (2-PE) is a higher aromatic alcohol that is used in the cosmetics and food industries. The budding yeast Saccharomyces cerevisiae is considered to be a suitable host for the industrial production of higher alcohols, including 2-PE. To produce 2-PE from glucose in S. cerevisiae, we searched for suitable 2-keto acid decarboxylase (KDC) and alcohol dehydrogenase (ADH) enzymes of the Ehrlich pathway for overexpression in strain YPH499, and found that overexpression of the ARO10 and/or ADH1 genes increased 2-PE production from glucose. Further, we screened ten BY4741 single-deletion mutants of genes involved in the competing pathways for 2-PE production, and found that strains aro8Δ and aat2Δ displayed increased 2-PE production. Based on these results, we engineered a BY4741 strain that overexpressed ARO10 and contained an aro8Δ deletion, and demonstrated that the strain produced 96 mg/L 2-PE from glucose as the sole carbon source. As this engineered S. cerevisiae strain showed a significant increase in 2-PE production from glucose without the addition of an intermediate carbon substrate, it is a promising candidate for the large-scale production of 2-PE.

  19. Dopaminergic function in relation to genes associated with risk for schizophrenia: translational mutant mouse models.

    Science.gov (United States)

    Moran, Paula M; O'Tuathaigh, Colm M P; Papaleo, Francesco; Waddington, John L

    2014-01-01

    Mutant mice play an increasingly important role in understanding disease processes at multiple levels. In particular, they illuminate the impact of risk genes for disease on such processes. This article reviews recent advances in the application of mutant mice to study the intricacies of dopaminergic (DAergic) function in relation to the putative pathophysiology of psychotic illness, particularly schizophrenia, and antipsychotic drug action. It considers models for understanding the role(s) of risk genes, with a particular focus on DTNBP1 and NRG1, their interactions with environmental factors, and with each other (epistasis). In overview, it considers new schemas for understanding psychotic illness that integrate DAergic pathophysiology with developmental, social, and cognitive processes, and how mutant mouse models can reflect and inform on such schemas.

  20. Characterization and mapping of a white panicle mutant gene in rice

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A spontaneous white panicle mutant was found from the F6 progenies of an indica/japonica cross. The mutant exhibits white stripes on its basal leaves while the panicles, rachis and pedicel are milky white colored at flowering stage. Genetic analysis in an F2 population from the cross of Zhi7/white panicle mutant indicates that the white panicle phenotype is controlled by a single recessive nuclear gene, tentatively termed as wp(t). Using microsatellite markers, the wp(t) gene was anchored between the markers of SSR101 and SSR63.9 with a map distance of 2.3 and 0.8 cM, respectively, and co-segregated with the marker of SSR17 on rice chromosome 1.

  1. Construction and Identification of omp31-Deleted Mutant of Brucella Standard Strain 16M%布鲁氏菌16M△omp31基因缺失株的构建与鉴定

    Institute of Scientific and Technical Information of China (English)

    王慧; 张亚丽; 王远志; 陈创夫; 任晓丽

    2013-01-01

    To construct the omp31 deletion mutant of Brucella melitensis 16M,the upstream and downstream of the omp31 gene and SacB gene were amplified by PCR from Brucella melitensis 16M and Bacillus subtilis. After constructing omp31-SacB re-combinant fragment into plasmid 18-T simple vector, the suicide plasmid pGEM-7zf+-△omp31-SacB was further obtained and transformed into Brucella melitensis 16M by electroporation. The Aomp31 mutant strain was screened out by homologous recombination and its stability was detected by continuous bacteria culture. The results showed that Brucella melitensis 16M △omp31 mutant strain was successfully generated and reversion was not observed in 15 generations. This research lays a foundation of further study on the anti-apoptosis mechanism and construction of new types of vaccines of Brucella.%为了构建布鲁氏菌16M△omp31基因缺失株,采用PCR方法分别从亲本株16 M上扩增omp31基因的侧翼看序列及枯草芽孢杆菌SacB基因,并将所得片段与pMD18-T载体相连并测序,利用双酶切的方法分别将其连入自杀载体pGEM-7zf+,获得亚克隆pGEM-7zf+-△omp31-SacB.将所构建好的自杀载体通过电转化入布鲁氏菌16M感受态细胞中,经2次同源重组后筛选出16M△omp31基因缺失株,并对获得缺失株进行遗传稳定性检测.结果显示:成功获得布鲁氏菌16 M△omp31基因缺失株,该缺失株在15代内未发生回复性突变.本研究为今后研究布鲁氏菌抗凋亡机制奠定基础.

  2. Variants in linkage disequilibrium with the late cornified envelope gene cluster deletion are associated with susceptibility to psoriatic arthritis.

    LENUS (Irish Health Repository)

    Bowes, John

    2010-12-01

    A common deletion mapping to the psoriasis susceptibility locus 4 on chromosome 1q21, encompassing two genes of the late cornified envelope (LCE) gene cluster, has been associated with an increased risk of psoriasis vulgaris (PsV). One previous report found no association of the deletion with psoriatic arthritis (PsA), suggesting it may be a specific risk factor for PsV. Given the genetic overlap between PsA and PsV, a study was undertaken to investigate whether single nucleotide polymorphisms (SNPs) mapping to this locus are risk factors for PsA in a UK and Irish population.

  3. Pre-thymic somatic mutation leads to high mutant frequency at hypoxanthine-guanine phosphoribosyltransferase gene

    Energy Technology Data Exchange (ETDEWEB)

    Jett, J. [Lawrence Livermore National Lab., CA (United States)

    1994-12-01

    While characterizing the background mutation spectrum of the Hypoxathine-guanine phosphoribosyltransferase (HPRT) gene in a healthy population, an outlier with a high mutant frequency of thioguanine resistant lymphocytes was found. When studied at the age of 46, this individual had been smoking 60 cigarettes per day for 38 years. His mutant frequency was calculated at 3.6 and 4.2x10{sup {minus}4} for two sampling periods eight months apart. Sequencing analysis of the HPRT gene in his mutant thioguanine resistant T lymphocytes was done to find whether the cells had a high rate of mutation, or if the mutation was due to a single occurrence of mutation and, if so, when in the T lymphocyte development the mutation occurred. By T-cell receptor analysis it has been found that out of 35 thioguanine resistant clones there was no dominant gamma T cell receptor gene rearrangement. During my appointment in the Science & Engineering Research Semester, I found that 34 of those clones have the same base substitution of G{yields}T at cDNA position 197. Due to the consistent mutant frequency from both sampling periods and the varying T cell receptors, the high mutant frequency cannot be due to recent proliferation of a mature mutant T lymphocyte. From the TCR and DNA sequence analysis we conclude that the G{yields}T mutation must have occurred in a T lymphocyte precursor before thymic differentiation so that the thioguanine resistant clones share the same base substitution but not the same gamma T cell receptor gene.

  4. 11p13 deletions can be more frequent than the PAX6 gene point mutations in Polish patients with aniridia.

    Science.gov (United States)

    Wawrocka, Anna; Sikora, Agata; Kuszel, Lukasz; Krawczynski, Maciej R

    2013-08-01

    Aniridia is a rare, bilateral, congenital ocular disorder causing incomplete formation of the iris, usually characterized by iris aplasia/hypoplasia. It can also appear with other ocular anomalies, such as cataracts, glaucoma, corneal pannus, optic nerve hypoplasia, macular hypoplasia, or ectopia lentis. In the majority of cases, it is caused by mutation in the PAX6 gene, but it can also be caused by microdeletions that involve the 11p13 region. Twelve unrelated patients of Polish origin with a clinical diagnosis of aniridia were screened for the presence of microdeletions in the 11p13 region by means of multiplex ligation probe amplification (MLPA). Additionally, the coding regions of the PAX6 gene were sequenced in all probands. MLPA examination revealed different size deletions of the 11p13 region in five patients. In three cases, deletions encompassed the entire PAX6 gene and a few adjacent genes. In one case, a fragment of the PAX6 gene was deleted only. In the final case, the deletion did not include any PAX6 sequence. Our molecular findings provide further evidence of the existence of the distant 3' regulatory elements in the downstream region of the PAX6 gene, which is known from other studies to influence the level of protein expression. Sequence analysis of the PAX6 gene revealed the three different point mutations in the remaining four patients with aniridia. All the detected mutations were reported earlier. Based on accomplished results, the great diversity of the molecular basis of aniridia was found. It varies from point mutations to different size deletions in the 11p13 region which encompass partly or completely the PAX6 gene or cause a position effect.

  5. Mutant INS-gene induced diabetes of youth: proinsulin cysteine residues impose dominant-negative inhibition on wild-type proinsulin transport.

    Directory of Open Access Journals (Sweden)

    Ming Liu

    Full Text Available Recently, a syndrome of Mutant INS-gene-induced Diabetes of Youth (MIDY, derived from one of 26 distinct mutations has been identified as a cause of insulin-deficient diabetes, resulting from expression of a misfolded mutant proinsulin protein in the endoplasmic reticulum (ER of insulin-producing pancreatic beta cells. Genetic deletion of one, two, or even three alleles encoding insulin in mice does not necessarily lead to diabetes. Yet MIDY patients are INS-gene heterozygotes; inheritance of even one MIDY allele, causes diabetes. Although a favored explanation for the onset of diabetes is that insurmountable ER stress and ER stress response from the mutant proinsulin causes a net loss of beta cells, in this report we present three surprising and interlinked discoveries. First, in the presence of MIDY mutants, an increased fraction of wild-type proinsulin becomes recruited into nonnative disulfide-linked protein complexes. Second, regardless of whether MIDY mutations result in the loss, or creation, of an extra unpaired cysteine within proinsulin, Cys residues in the mutant protein are nevertheless essential in causing intracellular entrapment of co-expressed wild-type proinsulin, blocking insulin production. Third, while each of the MIDY mutants induces ER stress and ER stress response; ER stress and ER stress response alone appear insufficient to account for blockade of wild-type proinsulin. While there is general agreement that ultimately, as diabetes progresses, a significant loss of beta cell mass occurs, the early events described herein precede cell death and loss of beta cell mass. We conclude that the molecular pathogenesis of MIDY is initiated by perturbation of the disulfide-coupled folding pathway of wild-type proinsulin.

  6. Construction of DNA damage response gene pprI function-deficient and function-complementary mutants in Deinococcus radiodurans

    Institute of Scientific and Technical Information of China (English)

    GAO Guanjun; LU Huiming; HUANG Lifen; HUA Yuejin

    2005-01-01

    PprI, a DNA damage response factor from the extraordinary radioresistant bacterium Deinococcus radiodurans, plays a central regulatory role in multiple DNA damage repair. In this study, a fusion DNA fragment carrying kanamycin resistance gene with the D. Radiodurans groEL promoter was cloned by PCR amplification and reversely inserted into the pprI locus in the genome of the wild-type strain R1. The resulting pprI-deficient strain, designated YR1, was very sensitive to ionizing radiation. Meanwhile, the re- combinant DNA fragment was cloned into the shuttle vector pRADZ3, and resulted in plasmid pRADK with kanamycin resistance in D. Radiodurans. The fragments containing complete pprI gene and 3'-terminal deletion pprI△ were cloned into plasmid pRADK. The resulted plasmids designated pRADKpprI and pRADKpprI△ were then transformed to YR1. Results show that YR1 carrying pRADKpprI was able to fully restore the extreme radioresistance to the same level as the wild-type D. Raiodurans R1, whereas YR1 pRADKpprI△ failed to do so. Construction of DNA repair switch PprI function-deficient and function-complementary mutants in D. Radiodurans is not only useful to elucidating the relationship between domains and functions of PprI protein, but also opens the door to the further studies of the biological functions of PprI protein in vivo.

  7. λ N gene expression regulated by translation termination in ribosome L24 mutant

    Institute of Scientific and Technical Information of China (English)

    LI; Muyang; (李沐阳); HU; Qirui; (胡其锐); XUAN; Jinsong; (宣劲松); DENG; Daiyong; (邓代永); WENG; Manli; (翁曼丽)

    2003-01-01

    Besides transcription regulation, gene expression is also regulated at translation level. Although translation regulation is mainly mediated by translation initiation, an abundance of evidence shows that the termination phase of translation is also important for gene expression. The expression of λN gene is down regulated at translation level in L24 mutant, however the precise mechanism still remains unknown. We report here that in an L24 mutant strain, the expression of lac-λN and GST-λN is decreased to 25% and 50% of that in wild type T83 strain respectively. Strikingly, the yield of GST-λN fusion protein in L24 mutant can be restored to the level as in T83 wild type strain by changing the two codons upstream λN stop codon. These findings imply that the stop codon and its context are involved in the translation regulation. The possible reason is that the translation termination complex containing L24 mutant ribosome may not dissociate properly in stop code region. This failure of disengagement from mRNA will slow down the process of following ribosomes, and consequently decrease the efficiency of λN gene expression.

  8. A Sorghum Mutant Resource as an Efficient Platform for Gene Discovery in Grasses.

    Science.gov (United States)

    Jiao, Yinping; Burke, John; Chopra, Ratan; Burow, Gloria; Chen, Junping; Wang, Bo; Hayes, Chad; Emendack, Yves; Ware, Doreen; Xin, Zhanguo

    2016-07-01

    Sorghum (Sorghum bicolor) is a versatile C4 crop and a model for research in family Poaceae. High-quality genome sequence is available for the elite inbred line BTx623, but functional validation of genes remains challenging due to the limited genomic and germplasm resources available for comprehensive analysis of induced mutations. In this study, we generated 6400 pedigreed M4 mutant pools from EMS-mutagenized BTx623 seeds through single-seed descent. Whole-genome sequencing of 256 phenotyped mutant lines revealed >1.8 million canonical EMS-induced mutations, affecting >95% of genes in the sorghum genome. The vast majority (97.5%) of the induced mutations were distinct from natural variations. To demonstrate the utility of the sequenced sorghum mutant resource, we performed reverse genetics to identify eight genes potentially affecting drought tolerance, three of which had allelic mutations and two of which exhibited exact cosegregation with the phenotype of interest. Our results establish that a large-scale resource of sequenced pedigreed mutants provides an efficient platform for functional validation of genes in sorghum, thereby accelerating sorghum breeding. Moreover, findings made in sorghum could be readily translated to other members of the Poaceae via integrated genomics approaches.

  9. Promoter deletions of Klebsiella pneumoniae carbapenemase (KPC)-encoding genes (blaKPC -2) and efflux pump (AcrAB) on β-lactam susceptibility in KPC-producing Enterobacteriaceae.

    Science.gov (United States)

    Seecoomar, Gomattie D; Marmol, Brenda C; Kwon, Dong H

    2013-11-01

    Klebsiella pneumoniae carbapenemase (KPC)-encoding genes containing promoter-deletions (bla(KPC-2a), bla(KPC-2b), and bla(KPC-2c) have disseminated in Enterobacteriaceae. The minimal inhibitory concentrations (MICs) to β-lactams in clinical KPC-producing Enterobacteriaceae range from susceptible to high-level resistant, resulting in diagnostic problems. To better understand the variability in β-lactam MICs among KPC-producing Enterobacteriaceae, three isoforms of bla(KPC-2) gene were used to transform Escherichia coli W4573 and its deletion mutant of an efflux pump (AcrAB) to examine the effects on β-lactam susceptibility. MICs to β-lactams in E. coli W4573 and its acrAB mutant strain increased 1- to 500-fold (MIC from 0.125 to 64 μg mL(-1) of aztreonam) in the bla(KPC-2a), bla(KPC-2b), and bla(KPC-2c) transformants compared with the cloning vector alone. However, transformants of the acrAB mutant strain remained susceptible to all β-lactams tested except for aztreonam and carbenicillin. Levels of the three promoters' length and carbapenemase activities in the transformants harboring the bla(KPC-2a), bla(KPC-2b), and bla(KPC-2c) were correlated to the levels of β-lactam MICs in both E. coli W4573 and its mutant of an efflux pump (AcrAB). Overall, these results suggest that promoter-deletions of bla(KPC-2) gene and AcrAB may be associated with the variability in β-lactam MICs in KPC-producing Enterobacteriaceae.

  10. Promoter Deletions of Klebsiella pneumoniae Carbapenemase (KPC)-Encoding Genes (blaKPC-2) and Efflux Pump (AcrAB) on β-Lactam Susceptibility in KPC-Producing Enterobacteriaceae

    Science.gov (United States)

    Seecoomar, Gomattie D.; Marmol, Brenda C.; Kwon, Dong H.

    2013-01-01

    Klebsiella pneumoniae carbapenemase (KPC)-encoding genes containing promoter-deletions (blaKPC-2a, blaKPC-2b, and blaKPC-2c) have disseminated in Enterobacteriaceae. The minimal inhibitory concentrations (MICs) to β-lactams in clinical KPC-producing Enterobacteriaceae are ranging from susceptible to high-level resistant resulting in diagnostic problems. In order to better understand the variability in β-lactam MICs among KPC-producing Enterobacteriaceae, three isoforms of blaKPC-2 gene were used to transform E. coli W4573 and its deletion mutant of an efflux pump (AcrAB) to examine the effects on β-lactam susceptibility. MICs to β-lactams in E. coli W4573 and its acrAB mutant strain increased 1- to 500-fold (MIC from 0.125 to 64 µg/mL of aztreonam) in the blaKPC-2a, blaKPC-2b, and blaKPC-2c transformants compared with the cloning vector alone. However, transformants of the acrAB mutant strain remained susceptible to all β-lactams tested except for aztreonam and carbenicillin. Levels of the three promoters’ length and carbapenemase activities in the transformants harboring the blaKPC-2a, blaKPC-2b, and blaKPC-2c were correlated to the levels of β-lactam MICs in both E. coli W4573 and its mutant of an efflux pump (AcrAB). Overall, these results suggest that promoter-deletions of blaKPC-2 gene and AcrAB may be associated with the variability in β-lactam MICs in KPC-producing Enterobacteriaceae. PMID:24033858

  11. An Alu-mediated large deletion of the FUT2 gene in individuals with the ABO-Bombay phenotype.

    Science.gov (United States)

    Koda, Y; Soejima, M; Johnson, P H; Smart, E; Kimura, H

    2000-01-01

    Recently, we have found an allelic deletion of the secretor alpha(1,2)fucosyltransferase (FUT2) gene in individuals with the classical Bombay phenotype of the ABO system. The FUT2 gene consists of two exons separated by an intron that spans approximately 7 kb. The first exon is noncoding, whereas exon 2 contains the complete coding sequence. Since the 5' breakpoint of the deletion has previously been mapped to the single intron of FUT2, we have cloned the junction region of the deletion in a Bombay individual by cassette-mediated polymerase chain reaction. In addition, the region from the 3' untranslated region of FUT2 to the 3' breakpoint sequence has been amplified from a control individual. DNA sequence analysis of this region indicates that the 5' breakpoint is within a free left Alu monomer (FLAM-C) sequence that lies 1.3 kb downstream of exon 1, and that the 3' breakpoint is within a complete Alu element (AluSx) that is positioned 1.5 kb downstream of exon 2. The size of the deletion is estimated to be about 10 kb. There is a 25-bp sequence identity between the reference DNA sequences surrounding the 5' and 3' breakpoints. This demonstrates that an Alu-mediated large gene deletion generated by unequal crossover is responsible for secretor alpha(1,2)fucosyltransferase deficiency in Indian Bombay individuals.

  12. SPC-Cre-ERT2 transgenic mouse for temporal gene deletion in alveolar epithelial cells.

    Directory of Open Access Journals (Sweden)

    Yao-Song Gui

    Full Text Available Although several Cre-loxP-based gene knockout mouse models have been generated for the study of gene function in alveolar epithelia in the lung, their applications are still limited. In this study, we developed a SPC-Cre-ER(T2 mouse model, in which a tamoxifen-inducible Cre recombinase (Cre-ER(T2 is under the control of the human surfactant protein C (SPC promoter. The specificity and efficiency of Cre-ER(T2 activity was first evaluated by crossing SPC-Cre-ER(T2 mouse with ROSA26R mouse, a β-galactosidase reporter strain. We found that Cre-ER(T2 was expressed in 30.7% type II alveolar epithelial cells of SPC-Cre-ER(T2/ROSA26R mouse lung tissues in the presence of tamoxifen. We then tested the tamoxifen-inducible recombinase activity of Cre-ER(T2 in a mouse strain bearing TSC1 conditional knockout alleles (TSC1(fx/fx. TSC1 deletion was detected in the lungs of tamoxifen treated SPC-Cre-ER(T2/TSC1(fx/fx mice. Therefore this SPC-Cre-ER(T2 mouse model may be a valuable tool to investigate functions of genes in lung development, physiology and disease.

  13. SPC-Cre-ERT2 transgenic mouse for temporal gene deletion in alveolar epithelial cells.

    Science.gov (United States)

    Gui, Yao-Song; Wang, Lianmei; Tian, Xinlun; Feng, Ruie; Ma, Aiping; Cai, Baiqiang; Zhang, Hongbing; Xu, Kai-Feng

    2012-01-01

    Although several Cre-loxP-based gene knockout mouse models have been generated for the study of gene function in alveolar epithelia in the lung, their applications are still limited. In this study, we developed a SPC-Cre-ER(T2) mouse model, in which a tamoxifen-inducible Cre recombinase (Cre-ER(T2)) is under the control of the human surfactant protein C (SPC) promoter. The specificity and efficiency of Cre-ER(T2) activity was first evaluated by crossing SPC-Cre-ER(T2) mouse with ROSA26R mouse, a β-galactosidase reporter strain. We found that Cre-ER(T2) was expressed in 30.7% type II alveolar epithelial cells of SPC-Cre-ER(T2)/ROSA26R mouse lung tissues in the presence of tamoxifen. We then tested the tamoxifen-inducible recombinase activity of Cre-ER(T2) in a mouse strain bearing TSC1 conditional knockout alleles (TSC1(fx/fx)). TSC1 deletion was detected in the lungs of tamoxifen treated SPC-Cre-ER(T2)/TSC1(fx/fx) mice. Therefore this SPC-Cre-ER(T2) mouse model may be a valuable tool to investigate functions of genes in lung development, physiology and disease.

  14. Induction of deletion mutation onompR gene ofSalmonella enterica serovar Typhi isolates from asymptomatic typhoid carriers to evolve attenuated strains for vaccine development

    Institute of Scientific and Technical Information of China (English)

    Senthilkumar B; Anbarasu K; Senbagam D; Rajasekarapandian M

    2014-01-01

    Objective:To develop attenuated strains ofSalmonella enterica serovar Typhi(S. typhi) for the candidate vaccine by osmolar stress.Methods:S. typhiSS3 andSS5 strains were isolated from asymptomatic typhoid carriers inNamakkal,TamilNadu,India.Both strains were grown inLB (LuriaBertani) medium supplemented with various concentration ofNaCl(0.1-0.7M) respectively. The effect of osmolar stress was determined at molecular level byPCR usingMGR06 andMGR 07 primers corresponding to ompR with chromosomalDNA of S. typhiSS3 andSS5 strains. Attenuation by osmolar stress results in deletion mutation of theS. typhi strains was determined by agglutination assays, precipitation method,SDSPAGE analysis and by animal models. Results:The799 bp amplifiedompRgene product from wild typeS. typhiSS3 andSS5 illustrate the presence of virulent gene.Interestingly, there was only a282 bp amplified product fromS. typhiSS3 andSS5 grown in the presence of0.5,0.6 and0.7MNaCl.This illustrates the occurrence of deletion mutation inompRgene at high concentration ofNaCl.Furthermore, both the wild-type and mutantS. typhi outer membraneSDS-PAGE profile reveals the differences in the expression ofompF,ompC andompA proteins.In mice, wild type and mutant strains lethal dose (LD50) were determined.The mice died within72 h when both the wild type strains were injected intraperitoneally with3 logCFU.mL-1.When the mice were injected with the mutants in same dosage, no clinical symptoms were observed; whereas the serum antibody titre was elicited within two weeks indicated that the mutants have the ability to induce protective humoral immune response.These results suggest thatS. typhiSS3 andSS5 may be used as good candidate strains for the development of live attenuated vaccine against salmonellosis.Conclusions:This study demonstrates that theS. typhistrains were attenuated and could be good vaccine candidates in future.

  15. Genetic analysis and gene mapping of a narrow leaf mutant in rice (Oryza sativa L.)

    Institute of Scientific and Technical Information of China (English)

    WANG DeKai; LIU HeQin; LI KeLei; LI SuJuan; TAO YueZhi

    2009-01-01

    A narrow leaf mutant was obtained after T-DNA transformation conducted on a rice variety Zhonghua 11. Several abnormal morphological characteristics, including semi-dwarf, delayed flowering time, narrow and inward rolling leaves, and lower seed-setting, were observed. The rate of net photosynthesis (un-der saturate light) of flag leaves in the mutant was significantly lower than that of the wild type. More-over, the leaf transpiration rate and stomatal conductance in the mutant flag leaf were lower than those of the wild type at the grain filling stage. It was found that the mutant phenotype was not caused by the T-DNA insertion. Genetic analysis showed that the mutant was controlled by a single recessive gene,designated as nal3(t). A genetic linkage map was constructed using a large F2 mapping population de-rived from a cross between nal3(t) and an indica variety Longtefu B with 6 polymorphic markers on chromosome 12 identified from 366 SSR markers by the BAS method. Gene nal3(t) was mapped be-tween the markers RM7018 and RM3331. Fine mapping of nal3(t) locus was conducted with 22 newly developed STS markers based on the sequence diversity around the region harboring nal3(t) between Nipponbare and 93-11, and nal3(f) was finally mapped to a 136-kb region between the STS markers NS10 and RH12-8.

  16. Gene expression and biological processes influenced by deletion of Stat3 in pulmonary type II epithelial cells

    Directory of Open Access Journals (Sweden)

    Whitsett Jeffrey A

    2007-12-01

    Full Text Available Abstract Background The signal transducer and activator of transcription 3 (STAT3 mediates gene expression in response to numerous growth factors and cytokines, playing an important role in many cellular processes. To better understand the molecular mechanisms by which Stat3 influences gene expression in the lung, the effect of pulmonary epithelial cell specific deletion of Stat3 on genome wide mRNA expression profiling was assessed. Differentially expressed genes were identified from Affymetrix Murine GeneChips analysis and subjected to gene ontology classification, promoter analysis, pathway mapping and literature mining. Results Total of 791 mRNAs were significantly increased and 314 mRNAs were decreased in response to the deletion of Stat3Δ/Δ in the lung. STAT is the most enriched cis-elements in the promoter regions of those differentially expressed genes. Deletion of Stat3 induced genes influencing protein metabolism, transport, chemotaxis and apoptosis and decreased the expression of genes mediating lipid synthesis and metabolism. Expression of Srebf1 and 2, genes encoding key regulators of fatty acid and steroid biosynthesis, was decreased in type II cells from the Stat3Δ/Δ mice, consistent with the observation that lung surfactant phospholipids content was decreased. Stat3 influenced both pro- and anti-apoptotic pathways that determine cell death or survival. Akt, a potential transcriptional target of Stat3, was identified as an important participant in Stat3 mediated pathways including Jak-Stat signaling, apoptosis, Mapk signaling, cholesterol and fatty acid biosynthesis. Conclusion Deletion of Stat3 from type II epithelial cells altered the expression of genes regulating diverse cellular processes, including cell growth, apoptosis and lipid metabolism. Pathway analysis indicates that STAT3 regulates cellular homeostasis through a complex regulatory network that likely enhances alveolar epithelial cell survival and surfactant

  17. Analysis of crossover breakpoints yields new insights into the nature of the gene conversion events associated with large NF1 deletions mediated by nonallelic homologous recombination.

    Science.gov (United States)

    Bengesser, Kathrin; Vogt, Julia; Mussotter, Tanja; Mautner, Victor-Felix; Messiaen, Ludwine; Cooper, David N; Kehrer-Sawatzki, Hildegard

    2014-02-01

    Large NF1 deletions are mediated by nonallelic homologous recombination (NAHR). An in-depth analysis of gene conversion operating in the breakpoint-flanking regions of large NF1 deletions was performed to investigate whether the rate of discontinuous gene conversion during NAHR with crossover is increased, as has been previously noted in NAHR-mediated rearrangements. All 20 germline type-1 NF1 deletions analyzed were mediated by NAHR associated with continuous gene conversion within the breakpoint-flanking regions. Continuous gene conversion was also observed in 31/32 type-2 NF1 deletions investigated. In contrast to the meiotic type-1 NF1 deletions, type-2 NF1 deletions are predominantly of post-zygotic origin. Our findings therefore imply that the mitotic as well as the meiotic NAHR intermediates of large NF1 deletions are processed by long-patch mismatch repair (MMR), thereby ensuring gene conversion tract continuity instead of the discontinuous gene conversion that is characteristic of short-patch repair. However, the single type-2 NF1 deletion not exhibiting continuous gene conversion was processed without MMR, yielding two different deletion-bearing chromosomes, which were distinguishable in terms of their breakpoint positions. Our findings indicate that MMR failure during NAHR, followed by post-meiotic/mitotic segregation, has the potential to give rise to somatic mosaicism in human genomic rearrangements by generating breakpoint heterogeneity.

  18. A De Novo Whole GCK Gene Deletion Not Detected by Gene Sequencing, in a Boy with Phenotypic GCK Insufficiency

    Directory of Open Access Journals (Sweden)

    N. H. Birkebæk

    2011-01-01

    Full Text Available We report on a boy with diabetes mellitus and a phenotype indicating glucokinase (GCK insufficiency, but a normal GCK gene examination applying direct gene sequencing. The boy was referred for diabetes mellitus at 7.5 years old. His father, grandfather and great grandfather suffered type 2 DM. Several blood glucose profiles showed (BG of 6.5–10 mmol/L L. After three years on neutral insulin Hagedorn (NPH in a dose of 0.3 IU/kg/day haemoglobin A1c (HbA1c was 6.8%. Treatment was changed to sulphonylurea 750 mg a day, and after 4 years HbA1c was 7%. At that time a multiplex ligation-dependent amplification gene dosage assay (MLPA was done, revealing a whole GCK gene deletion. Medical treatment was ceased, and after one year HbA1c was 6.8%. This case underscores the importance of a MLPA examination if the phenotype of a patient is strongly indicative of GCK insufficiency and no mutation is identified using direct sequencing.

  19. [Production and study of Bacillus subtilis mutants for genes involved in nucleoside catabolism].

    Science.gov (United States)

    Rumiantseva, E V; Sukhodolets, V V; Smirnov, Iu V

    1979-01-01

    By means of selection for a low thymine requirement the mutants fo thymine auxotrophs for deoxyriboaldolase (dra) and phosphodeoxyribomutase (drm) genes were obtained. Besides the mutants for pyrimidinenucleoside phosphorylase gene (pdp) were olso isolated using selection on the fluorodeoxyuridine resistance. The latter enzyme provides for pyrimidine nucleosides catabolism (thymidine, uridine) in Bacilli, as well as the conversion of exogenous thymine to thymidine in thymine auxotrophs. The data obtained when studying the deo-enzymes activities in various types of the mutants and also under the condition of induction by thymidine and acetoaldehyde are in accordance with the assumption that deoxyriboso-5-phosphate is an inductor of the deo-enzymes in Bacillus subtilis. The genes dra and pdp were tightly linked as it had been shown by the transformation experiments; in contrast, no linkage was revealed between dra and drm or pdp and drm. A secondary mutation (adn), not linked with dra and blocking the ability of bacteria to catabolise adenosine (purine nucleoside phosphorylase activity remains constant) was found in some dra-mutants.

  20. Prenatal diagnosis of a 7p15-p21 deletion encompassing the TWIST1 gene involved in Saethre-Chotzen syndrome.

    Science.gov (United States)

    Spaggiari, Emmanuel; Aboura, Azzedine; Sinico, Martine; Mabboux, Philippe; Dupont, Céline; Delezoide, Anne-Lise; Guimiot, Fabien

    2012-01-01

    Saethre-Chotzen syndrome is a craniosynostosis syndrome that is rarely diagnosed prenatally. It is caused by cytogenetic deletions or mutations of the TWIST1 gene. We report here a de novo prenatal case with clinically and molecularly well defined Saethre-Chotzen syndrome due to a TWIST1 deletion. This is the first reported case of a deletion encompassing the TWIST1 gene to be diagnosed prenatally. We recommend screening for a deletion of the TWIST1 gene if signs of coronal craniosynostosis with no clear etiology are observed on ultrasound examination.

  1. Alu Sx repeat-induced homozygous deletion of the StAR gene causes lipoid congenital adrenal hyperplasia.

    Science.gov (United States)

    Eiden-Plach, Antje; Nguyen, Huy-Hoang; Schneider, Ursula; Hartmann, Michaela F; Bernhardt, Rita; Hannemann, Frank; Wudy, Stefan A

    2012-05-01

    Lipoid congenital adrenal hyperplasia (Lipoid CAH) is the most severe form of the autosomal recessive disorder CAH. A general loss of the steroid biosynthetic activity caused by defects in the StAR gene manifests as life-threatening primary adrenal insufficiency. We report a case of Lipoid CAH caused by a so far not described homozygous deletion of the complete StAR gene and provide diagnostic results based on a GC-MS steroid metabolomics and molecular genetic analysis. The patient presented with postnatal hypoglycemia, vomiting, adynamia, increasing pigmentation and hyponatremia. The constellation of urinary steroid metabolites suggested Lipoid CAH and ruled out all other forms of CAH or defects of aldosterone biosynthesis. After treatment with sodium supplementation, hydrocortisone and fludrocortisone the child fully recovered. Molecular genetic analysis demonstrated a homozygous 12.1 kb deletion in the StAR gene locus. The breakpoints of the deletion are embedded into two typical genomic repetitive Alu Sx elements upstream and downstream of the gene leading to the loss of all exons and regulatory elements. We established deletion-specific and intact allele-specific PCR methods and determined the StAR gene status of all available family members over three generations. This analysis revealed that one of the siblings, who died a few weeks after birth, carried the same genetic defect. Since several Alu repeats at the StAR gene locus increase the probability of deletions, patients with typical symptoms of lipoid CAH lacking evidence for the presence of both StAR alleles should be analyzed carefully for this kind of disorder.

  2. The somatic hypermutation activity of a follicular lymphoma links to large insertions and deletions of immunoglobulin genes.

    Science.gov (United States)

    Wu, H Y; Kaartinen, M

    1995-07-01

    A biopsy specimen from a patient with follicular lymphoma was divided into two fragments. DNA was extracted from one fragment and a 1.2 kb region of the functional heavy chain (IgH) gene was amplified, cloned and sequenced (eight clones). From the other fragment a cell line (HF-1) was started. The IgH gene region was amplified from the cell line, and sequenced without cloning. The nine sequences obtained could be arranged into a genealogical tree where the individual sequences differed from the deduced ancestor by 16-29 single nucleotide changes, some also by an insertion and/or a deletion. It is apparent that the sequence alterations were caused by somatic mutations during the growth of the lymphoma. The comparison of the sequences with two published (allelic) germline sequences of the human JH region showed approximately 20% non-homology. The differences included five additional multinucleotide insertion/deletion changes, the longest of them a 101-nucleotide insertion. Two long insertions were homologous to the adjacent germline sequences. We propose that most of the changes observed, including long deletions and insertions, represent or are linked to somatic hypermutation events of the Ig gene type. Although in a few cases large deletions and insertions (> 2 bp) have been found in mutated immunoglobulin genes, our results, for the first time, firmly link these deletions/insertions to somatic hypermutations; their frequency was found to be 2.2% of the observed mutational events in the non-translated gene regions. HF-1 is the first follicular lymphoma line successfully established from a lymphoma known to have hypermutated its Ig genes during the malignant growth. It is a candidate cell line to be studied for its ability to generate mutations of B cell type in cell cultures.

  3. Proinsulin misfolding and diabetes: mutant INS gene-induced diabetes of youth.

    Science.gov (United States)

    Liu, Ming; Hodish, Israel; Haataja, Leena; Lara-Lemus, Roberto; Rajpal, Gautam; Wright, Jordan; Arvan, Peter

    2010-11-01

    Type 1B diabetes (typically with early onset and without islet autoantibodies) has been described in patients bearing small coding sequence mutations in the INS gene. Not all mutations in the INS gene cause the autosomal dominant Mutant INS-gene Induced Diabetes of Youth (MIDY) syndrome, but most missense mutations affecting proinsulin folding produce MIDY. MIDY patients are heterozygotes, with the expressed mutant proinsulins exerting dominant-negative (toxic gain of function) behavior in pancreatic beta cells. Here we focus primarily on proinsulin folding in the endoplasmic reticulum, providing insight into perturbations of this folding pathway in MIDY. Accumulated evidence indicates that, in the molecular pathogenesis of the disease, misfolded proinsulin exerts dominant effects that initially inhibit insulin production, progressing to beta cell demise with diabetes.

  4. Gene Expression Proifling Related to Hyphal Growth in a Temperature- Sensitive Mutant ofMagnaporthe oryzae

    Institute of Scientific and Technical Information of China (English)

    LI Xue-song; XU Fei; WANG Hong-kai; and LIN Fu-cheng

    2013-01-01

    The rice blast, caused by fungusMagnaporthe oryzae, is a major constraint to the world food security. Hyphal growth is the foundation of fungal development and proliferation of fungi. To investigate genes involved in hyphal growth of this fungus, digital gene expression tag proifling was used to compare a previously generated temperature-sensitive mutant which defect at hyphae growth and reduction on pathogenicity, with its related wildtype strain. 416 genes were detected as differential expression, 178 of which were speciifcally expressed in Guy-11 but down-regulated expression in the mutant. Functional classiifcation analysis revealed the phenotype mutation may be mainly caused by a defection in translational and vacuole-related processes. The results and the protocol used will improve our knowledge on morphogenesis and promote the further study onM. oryzae pathogenesis.

  5. ALIX Rescues Budding of a Double PTAP/PPEY L-Domain Deletion Mutant of Ebola VP40: A Role for ALIX in Ebola Virus Egress.

    Science.gov (United States)

    Han, Ziying; Madara, Jonathan J; Liu, Yuliang; Liu, Wenbo; Ruthel, Gordon; Freedman, Bruce D; Harty, Ronald N

    2015-10-01

    Ebola (EBOV) is an enveloped, negative-sense RNA virus belonging to the family Filoviridae that causes hemorrhagic fever syndromes with high-mortality rates. To date, there are no licensed vaccines or therapeutics to control EBOV infection and prevent transmission. Consequently, the need to better understand the mechanisms that regulate virus transmission is critical to developing countermeasures. The EBOV VP40 matrix protein plays a central role in late stages of virion assembly and egress, and independent expression of VP40 leads to the production of virus-like particles (VLPs) by a mechanism that accurately mimics budding of live virus. VP40 late (L) budding domains mediate efficient virus-cell separation by recruiting host ESCRT and ESCRT-associated proteins to complete the membrane fission process. L-domains consist of core consensus amino acid motifs including PPxY, P(T/S)AP, and YPx(n)L/I, and EBOV VP40 contains overlapping PPxY and PTAP motifs whose interactions with Nedd4 and Tsg101, respectively, have been characterized extensively. Here, we present data demonstrating for the first time that EBOV VP40 possesses a third L-domain YPx(n)L/I consensus motif that interacts with the ESCRT-III protein Alix. We show that the YPx(n)L/I motif mapping to amino acids 18-26 of EBOV VP40 interacts with the Alix Bro1-V fragment, and that siRNA knockdown of endogenous Alix expression inhibits EBOV VP40 VLP egress. Furthermore, overexpression of Alix Bro1-V rescues VLP production of the budding deficient EBOV VP40 double PTAP/PPEY L-domain deletion mutant to wild-type levels. Together, these findings demonstrate that EBOV VP40 recruits host Alix via a YPx(n)L/I motif that can function as an alternative L-domain to promote virus egress.

  6. Demonstration of the presence of the "deleted" MIR122 gene in HepG2 cells.

    Science.gov (United States)

    Hamad, Ibrahim A Y; Fei, Yue; Kalea, Anastasia Z; Yin, Dan; Smith, Andrew J P; Palmen, Jutta; Humphries, Steve E; Talmud, Philippa J; Walker, Ann P

    2015-01-01

    MicroRNA 122 (miR-122) is highly expressed in the liver where it influences diverse biological processes and pathways, including hepatitis C virus replication and metabolism of iron and cholesterol. It is processed from a long non-coding primary transcript (~7.5 kb) and the gene has two evolutionarily-conserved regions containing the pri-mir-122 promoter and pre-mir-122 hairpin region. Several groups reported that the widely-used hepatocytic cell line HepG2 had deficient expression of miR-122, previously ascribed to deletion of the pre-mir-122 stem-loop region. We aimed to characterise this deletion by direct sequencing of 6078 bp containing the pri-mir-122 promoter and pre-mir-122 stem-loop region in HepG2 and Huh-7, a control hepatocytic cell line reported to express miR-122, supported by sequence analysis of cloned genomic DNA. In contrast to previous findings, the entire sequence was present in both cell lines. Ten SNPs were heterozygous in HepG2 indicating that DNA was present in two copies. Three validation isolates of HepG2 were sequenced, showing identical genotype to the original in two, whereas the third was different. Investigation of promoter chromatin status by FAIRE showed that Huh-7 cells had 6.2 ± 0.19- and 2.7 ± 0.01- fold more accessible chromatin at the proximal (HNF4α-binding) and distal DR1 transcription factor sites, compared to HepG2 cells (p=0.03 and 0.001, respectively). This was substantiated by ENCODE genome annotations, which showed a DNAse I hypersensitive site in the pri-mir-122 promoter in Huh-7 that was absent in HepG2 cells. While the origin of the reported deletion is unclear, cell lines should be obtained from a reputable source and used at low passage number to avoid discrepant results. Deficiency of miR-122 expression in HepG2 cells may be related to a relative deficiency of accessible promoter chromatin in HepG2 versus Huh-7 cells.

  7. Conditional gene deletion reveals functional redundancy of GABAB receptors in peripheral nociceptors in vivo

    Directory of Open Access Journals (Sweden)

    Bettler Bernhard

    2009-11-01

    Full Text Available Abstract Background γ-aminobutyric acid (GABA is an important inhibitory neurotransmitter which mainly mediates its effects on neurons via ionotropic (GABAA and metabotropic (GABAB receptors. GABAB receptors are widely expressed in the central and the peripheral nervous system. Although there is evidence for a key function of GABAB receptors in the modulation of pain, the relative contribution of peripherally- versus centrally-expressed GABAB receptors is unclear. Results In order to elucidate the functional relevance of GABAB receptors expressed in peripheral nociceptive neurons in pain modulation we generated and analyzed conditional mouse mutants lacking functional GABAB(1 subunit specifically in nociceptors, preserving expression in the spinal cord and brain (SNS-GABAB(1-/- mice. Lack of the GABAB(1 subunit precludes the assembly of functional GABAB receptor. We analyzed SNS-GABAB(1-/- mice and their control littermates in several models of acute and neuropathic pain. Electrophysiological studies on peripheral afferents revealed higher firing frequencies in SNS-GABAB(1-/- mice compared to corresponding control littermates. However no differences were seen in basal nociceptive sensitivity between these groups. The development of neuropathic and chronic inflammatory pain was similar across the two genotypes. The duration of nocifensive responses evoked by intraplantar formalin injection was prolonged in the SNS-GABAB(1-/- animals as compared to their control littermates. Pharmacological experiments revealed that systemic baclofen-induced inhibition of formalin-induced nociceptive behaviors was not dependent upon GABAB(1 expression in nociceptors. Conclusion This study addressed contribution of GABAB receptors expressed on primary afferent nociceptive fibers to the modulation of pain. We observed that neither the development of acute and chronic pain nor the analgesic effects of a systematically-delivered GABAB agonist was significantly

  8. Autoimmune lymphoproliferative syndrome in a patient with a new minimal deletion in the death domain of the FAS gene

    NARCIS (Netherlands)

    Gualco, Gabrieta; van den Berg, Anke; Koopmans, Sicco; Bacchi, Livia M.; Carneiro, Siderley S.; Ruiz, Everaldo; Vecchi, Ana Paula; Chan, John K. C.

    2008-01-01

    We present a case of autoimmune lymphoproliferative syndrome (ALPS) caused by a previously undescribed minimal deletion in the death domain of the FAS gene. ALPS is an uncommon disease associated with an impaired Fas-mediated apoptosis. The patient presented with a history of splenomegaly since 4 mo

  9. Lethal osteogenesis imperfecta congenita and a 300 base pair gene deletion for an α1(I)-like collagen.

    NARCIS (Netherlands)

    F.M. Pope; K.S.E. Cheah (Kathryn); A.C. Nicholls; A.B. Price; F.G. Grosveld (Frank)

    1984-01-01

    textabstractBroad boned lethal osteogenesis imperfecta is a severely crippling disease of unknown cause. By means of recombinant DNA technology a 300 base pair deletion in an alpha 1(I)-like collagen gene was detected in six patients and four complete parent-child groups including patients with this

  10. Robust Parameter Identification to Perform the Modeling of pta and poxB Genes Deletion Effect on Escherichia Coli.

    Science.gov (United States)

    Guerrero-Torres, V; Rios-Lozano, M; Badillo-Corona, J A; Chairez, I; Garibay-Orijel, C

    2016-08-01

    The aim of this study was to design a robust parameter identification algorithm to characterize the effect of gene deletion on Escherichia coli (E. coli) MG1655. Two genes (pta and poxB) in the competitive pathways were deleted from this microorganism to inhibit pyruvate consumption. This condition deviated the E. coli metabolism toward the Krebs cycle. As a consequence, the biomass, substrate (glucose), lactic, and acetate acids as well as ethanol concentrations were modified. A hybrid model was proposed to consider the effect of gene deletion on the metabolism of E. coli. The model parameters were estimated by the application of a least mean square method based on the instrument variable technique. To evaluate the parametric identifier method, a set of robust exact differentiators, based on the super-twisting algorithm, was implemented. The hybrid model was successfully characterized by the parameters obtained from experimental information of E. coli MG1655. The significant difference between parameters obtained with wild-type strain and the modified (with deleted genes) justifies the application of the parametric identification algorithm. This characterization can be used to optimize the production of different byproducts of commercial interest.

  11. Conditional beta1-integrin gene deletion in neural crest cells causes severe developmental alterations of the peripheral nervous system

    DEFF Research D