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Sample records for gene molecular cloning

  1. Gene Transfer and Molecular Cloning of the Human NGF Receptor

    NASA Astrophysics Data System (ADS)

    Chao, Moses V.; Bothwell, Mark A.; Ross, Alonzo H.; Koprowski, Hilary; Lanahan, Anthony A.; Buck, C. Randall; Sehgal, Amita

    1986-04-01

    Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

  2. Molecular cloning, characterization, and expression of sheep FGF5 gene.

    PubMed

    Zhang, Lihua; He, Sangang; Liu, Mingjun; Liu, Guosong; Yuan, Zheng; Liu, Chenxi; Zhang, Xumei; Zhang, Ning; Li, Wenrong

    2015-01-25

    The fibroblast growth factor 5 gene (FGF5) is a member of the FGF gene family, and represents a candidate gene for hair length because of its role in the regulation of the hair follicle growth cycle. In our current study, we cloned, sequenced, and characterized the full-length FGF5 cDNA of Chinese Merino sheep. We obtained the complete genomic sequence of the FGF5 gene from sheep blood samples, and compared it to other FGF5 sequences in GenBank. We found that the FGF5 gene spanned 21,743bp of genomic DNA, and consisted of 3 exons and 2 introns, both of which differed from those of a previously annotated FGF5 genomic sequence from sheep. We also identified a previously undescribed FGF5 mRNA splicing variant, FGF5S, and the western blot analysis showed that the molecular weights of the FGF5 (34kDa) and FGF5s (17kDa) proteins were consistent with the estimates based on the genomic and cDNA sequence data. We examined the expression of both FGF5 mRNAs in various tissues of sheep, and found that the expression of the FGF5S mRNA was restricted to the brain, spleen, and skin tissue. The single-nucleotide polymorphism analysis of the genomic sequence revealed 72 genetic variants of the FGF5 gene. Our findings provide insight into the functions of the FGF5 gene in Chinese Merino. PMID:25445274

  3. Molecular cloning of gluconobacter oxydans DSM 2003 xylitol dehydrogenase gene

    PubMed Central

    Sadeghi, H. Mir Mohammad; Ahmadi, R.; Aghaabdollahian, S.; Mofid, M.R.; Ghaemi, Y.; Abedi, D.

    2011-01-01

    Due to the widespread applications of xylitol dehydrogenase, an enzyme used for the production of xylitol, the present study was designed for the cloning of xylitol dehydrogenase gene from Glcunobacter oxydans DSM 2003. After extraction of genomic DNA from this bacterium, xylitol dehydrogenase gene was replicated using polymerase chain reaction (PCR). The amplified product was entered into pTZ57R cloning vector by T/A cloning method and transformation was performed by heat shocking of the E. coli XL1-blue competent cells. Following plasmid preparation, the cloned gene was digested out and ligated into the expression vector pET-22b(+). Electrophoresis of PCR product showed a 789 bp band. Recombinant plasmid (rpTZ57R) was then constructed. This plasmid was double digested with XhoI and EcoRI resulting in 800 bp and 2900 bp bands. The obtained insert was ligated into pET-22b(+) vector and its orientation was confirmed with XhoI and BamHI restriction enzymes. In conclusion, in the present study the recombinant expression vector containing xylitol dehydrogenase gene has been constructed and can be used for the production of this enzyme in high quantities. PMID:22110522

  4. Using "Pseudomonas Putida xylE" Gene to Teach Molecular Cloning Techniques for Undergraduates

    ERIC Educational Resources Information Center

    Dong, Xu; Xin, Yi; Ye, Li; Ma, Yufang

    2009-01-01

    We have developed and implemented a serial experiment in molecular cloning laboratory course for undergraduate students majored in biotechnology. "Pseudomonas putida xylE" gene, encoding catechol 2, 3-dioxygenase, was manipulated to learn molecular biology techniques. The integration of cloning, expression, and enzyme assay gave students a chance…

  5. A highly efficient molecular cloning platform that utilises a small bacterial toxin gene.

    PubMed

    Mok, Wendy W K; Li, Yingfu

    2013-04-15

    Molecular cloning technologies that have emerged in recent years are more efficient and simpler to use than traditional strategies, but many have the disadvantages of requiring multiple steps and expensive proprietary enzymes. We have engineered cloning vectors containing variants of IbsC, a 19-residue toxin from Escherichia coli K-12. These toxic peptides offer selectivity to minimise the background, labour, and cost associated with conventional molecular cloning. As demonstrated with the cloning of reporter genes, this "detox cloning" system consistently produced over 95 % positive clones. Purification steps between digestion and ligation are not necessary, and the total time between digestion and plating of transformants can be as little as three hours. Thus, these IbsC-based cloning vectors are as reliable and amenable to high-throughput cloning as commercially available systems, and have the advantage of being more time-efficient and cost-effective. PMID:23512843

  6. Molecular transformation, gene cloning, and gene expression systems for filamentous fungi

    USGS Publications Warehouse

    Gold, Scott E.; Duick, John W.; Redman, Regina S.; Rodriguez, Rusty J.

    2001-01-01

    This chapter discusses the molecular transformation, gene cloning, and gene expression systems for filamentous fungi. Molecular transformation involves the movement of discrete amounts of DNA into cells, the expression of genes on the transported DNA, and the sustainable replication of the transforming DNA. The ability to transform fungi is dependent on the stable replication and expression of genes located on the transforming DNA. Three phenomena observed in bacteria, that is, competence, plasmids, and restriction enzymes to facilitate cloning, were responsible for the development of molecular transformation in fungi. Initial transformation success with filamentous fungi, involving the complementation of auxotrophic mutants by exposure to sheared genomic DNA or RNA from wt isolates, occurred with low transformation efficiencies. In addition, it was difficult to retrieve complementing DNA fragments and isolate genes of interest. This prompted the development of transformation vectors and methods to increase efficiencies. The physiological studies performed with fungi indicated that the cell wall could be removed to generate protoplasts. It was evident that protoplasts could be transformed with significantly greater efficiencies than walled cells.

  7. Molecular Cloning and Physical Mapping of the Daptomycin Gene Cluster from Streptomyces roseosporus

    PubMed Central

    Mchenney, Margaret A.; Hosted, Thomas J.; Dehoff, Bradley S.; Rosteck, Paul R.; Baltz, Richard H.

    1998-01-01

    The daptomycin biosynthetic gene cluster of Streptomyces roseosporus was analyzed by Tn5099 mutagenesis, molecular cloning, partial DNA sequencing, and insertional mutagenesis with cloned segments of DNA. The daptomycin biosynthetic gene cluster spans at least 50 kb and is located about 400 to 500 kb from one end of the ∼7,100-kb linear chromosome. We identified two peptide synthetase coding regions interrupted by a 10- to 20-kb region that may encode other functions in lipopeptide biosynthesis. PMID:9422604

  8. Molecular cloning and bioinformatic analysis of SPATA4 gene.

    PubMed

    Liu, Shang-feng; Ai, Chao; Ge, Zhong-qi; Liu, Hai-luo; Liu, Bo-wen; He, Shan; Wang, Zhao

    2005-11-30

    Full-length cDNA sequences of four novel SPATA4 genes in chimpanzee, cow, chicken and ascidian were identified by bioinformatic analysis using mouse or human SPATA4 cDNA fragment as electronic probe. All these genes have 6 exons and have similar protein molecular weight and do not localize in sex chromosome. The mouse SPATA4 sequence is identified as significantly changed in cryptorchidism, which shares no significant homology with any known protein in swissprot databases except for the homologous genes in various vertebrates. Our searching results showed that all SPATA4 proteins have a putative conserved domain DUF1042. The percentages of putative SPATA4 protein sequence identity ranging from 30 % to 99 %. The high similarity was also found in 1 kb promoter regions of human, mouse and rat SPATA4 gene. The similarities of the sequences upstream of SPATA4 promoter also have a high proportion. The results of searching SymAtlas (http://symatlas.gnf.org/SymAtlas/) showed that human SPATA4 has a high expression in testis, especially in testis interstitial, leydig cell, seminiferous tubule and germ cell. Mouse SPATA4 was observed exclusively in adult mouse testis and almost no signal was detected in other tissues. The pI values of the protein are negative, ranging from 9.44 to 10.15. The subcellular location of the protein is usually in the nucleus. And the signal peptide possibilities for SPATA4 are always zero. Using the SNPs data in NCBI, we found 33 SNPs in human SPATA4 gene genomic DNA region, with the distribution of 29 SNPs in the introns. CpG island searching gives the data about CpG island, which shows that the regions of the CpG island have a high similarity with each other, though the length of the CpG island is different from each other. This research is a fundamental work in the fields of the bioinformational analysis, and also put forward a new way for the bioinformatic analysis of other genes. PMID:16336790

  9. Molecular cloning and expression of Corynebacterium glutamicum genes for amino acid synthesis in Escherichia coli cells

    SciTech Connect

    Beskrovnaya, O.Yu.; Fonshtein, M.Yu.; Kolibaba, L.G.; Yankovskii, N.K.; Debabov, V.G.

    1989-01-01

    Molecular cloning of Corynebacterium glutamicum genes for threonine and lysine synthesis has been done in Escherichia coli cells. The clonal library of EcoRI fragments of chromosomal DNA of C. glutamicum was constructed on the plasmid vector /lambda/pSL5. The genes for threonine and lysine synthesis were identified by complementation of E. coli mutations in thrB and lysA genes, respectively. Recombinant plasmids, isolated from independent ThrB/sup +/ clone have a common 4.1-kb long EcoRI DNA fragment. Hybrid plasmids isolated from LysA/sup +/ transductants of E. coli have common 2.2 and 3.3 kb long EcoRI fragments of C. glutamicum DNA. The hybrid plasmids consistently transduced the markers thrB/sup +/ and lysA/sup +/. The Southern hybridization analysis showed that the cloned DNA fragments hybridized with the fragments of identical length in C. glutamicum chromosomes.

  10. [Advances in Molecular Cloning].

    PubMed

    Ashwini, M; Murugan, S B; Balamurugan, S; Sathishkumar, R

    2016-01-01

    "Molecular cloning" meaning creation of recombinant DNA molecules has impelled advancement throughout life sciences. DNA manipulation has become easy due to powerful tools showing exponential growth in applications and sophistication of recombinant DNA technology. Cloning genes has become simple what led to an explosion in the understanding of gene function by seamlessly stitching together multiple DNA fragments or by the use of swappable gene cassettes, maximizing swiftness and litheness. A novel archetype might materialize in the near future with synthetic biology techniques that will facilitate quicker assembly and iteration of DNA clones, accelerating the progress of gene therapy vectors, recombinant protein production processes and new vaccines by in vitro chemical synthesis of any in silico-specified DNA construct. The advent of innovative cloning techniques has opened the door to more refined applications such as identification and mapping of epigenetic modifications and high-throughput assembly of combinatorial libraries. In this review, we will examine the major breakthroughs in cloning techniques and their applications in various areas of biological research that have evolved mainly due to easy construction of novel expression systems. PMID:27028806

  11. Molecular cloning of virulence genes from Erwinia stewartii.

    PubMed Central

    Coplin, D L; Frederick, R D; Majerczak, D R; Haas, E S

    1986-01-01

    A library of Erwinia stewartii DNA was constructed in cosmid pVK100 and used to complement spontaneous and Mu pf7701-induced (designated by the prefix MU) avirulent mutants. Plasmid pES4507 restored water-soaking ability and extracellular polysaccharide (EPS) synthesis to mutants MU14110 and MU2B70 (group I); pES1044 restored water-soaking ability to MU43, MU51, MU136, MU141, and RDF6011 (group II); and pES2144 complemented four spontaneous EPS- mutants (group III). Hybridization of labeled plasmid DNA to Southern blots of genomic DNA from the mutants revealed that a Mu pf7701 insertion was associated with the respective cloned region in all mutants except MU2B70 and MU223. In these strains, the plasmid may be suppressing the avirulent phenotype rather than complementing the mutation. Images PMID:3782017

  12. Molecular study on cloned endoglucanase gene from rumen bacterium.

    PubMed

    Ozkose, Emin; Akyol, Ismail; Ekinci, Mehmet Sait

    2004-01-01

    An endoglucanase gene was subcloned from anaerobic rumen bacterium Ruminococcus flavefaciens strain 17. To express endoglucanase gene in Escherichia coli and Streptococcus bovis JB1, an endoglucanase gene fragment was inserted into pVA838-based shuttle vectors. Removal of endoglucanase gene promoter and expression of endoglucanase by promoter of S. bovis JB1 alpha-amylase gene (pACMCS) was also achieved. Survival of constructs pVACMCI, pTACMC and pACMCS, which carry endoglucanase gene, and stability of endoglucanase gene in S. bovis JB1, were observed. Maximal endoglucanase activities from S. bovis JB1/pVACMCI were 2- to 3-fold higher than from E. coli/pVACMCI. Specific cell activity of E. coli/pACMCS was found to be approximately 2- to -3 fold higher than the both E. coli/pVACMCI and E. coli/pTACMC. Specific cell activity of S. bovis JB1/pACMCS was also found to be approximately 2-fold higher than the both S. bovis/pVACMCI and S. bovis JB1/pTACMC. PMID:15925902

  13. [Molecular cloning and expression of Nattokinase gene in Bacillus subtilis].

    PubMed

    Liu, B Y; Song, H Y

    2002-05-01

    In order to characterize biochemically the nattokinase,the nucleotide sequence of the nattokinase gene was amplified from the chromosomal DNA of B.subtilis (natto) by PCR. The expression plasmid pBL NK was constructed and was used to transform Bacillus subtilis containing a chromosomal deletion in its subtilisin gene. The supernatant of the culture was collected after 15 h culture. The target proteins were identified by SDS-PAGE. Nattokinase was purified by a method including ultrafiltration, Sephacryl S-100 gel filtration and S-Sepharose ion-exchange chromatography, and 100 mg of purified nattokinase was obtained from one liter of culture. The purity of the protein and the specific activity were 95% and 12 000 u/mg (compared to tPA), respectively. PMID:12019448

  14. Molecular cloning and comparison of avian preproghrelin genes.

    PubMed

    Yuan, Jing; Zhou, Jianjun; Hu, Xiaoxiang; Li, Ning

    2007-04-01

    We report cDNA sequences for the preproghrelin gene from goose, duck, and emu. This gene is involved in stimulating the release of growth hormone in mammals and may play a similar role in avian species. The complete coding sequence of avian preproghrelin encodes a 116 amino acid (aa) protein, which is organized into three parts: the N-terminal hydrophobic signal peptide, a 26 aa peptide for mature ghrelin, and a long C-terminal polypeptide. Domain/motif structures of preproghrelin protein are highly conserved among avian species. Although the avian and mammalian homologs are not highly similar for the whole 116 aa sequence, the identity of the highly conserved "active core" sequence and the n-octanoyl modification of the serine 3 residue avian ghrelin protein with its mammalian homologs implies conserved function of ghrelin protein during evolution. Information provided in this study will be useful in further studies to determine the role the preproghrelin gene plays in the regulation of growth hormone release and body weight gain in avian species. PMID:17347866

  15. Molecular cloning, characterization, and expression of pannexin genes in chicken.

    PubMed

    Kwon, Tae-Jun; Kim, Dong-Bin; Bae, Jae Woong; Sagong, Borum; Choi, Soo-Young; Cho, Hyun-Ju; Kim, Un-Kyung; Lee, Kyu-Yup

    2014-09-01

    Pannexins (Panx) are a family of proteins that share sequences with the invertebrate gap junction proteins, innexins, and have a similar structure to that of the vertebrate gap junction proteins, connexins. To date, the Panx family consists of 3 members, but their genetic sequences have only been completely determined in a few vertebrate species. Moreover, expression of the Panx family has been reported in several rodent tissues: Panx1 is ubiquitously expressed in mammals, whereas Panx2 and Panx3 expressions are more restricted. Although members of the Panx family have been detected in mammals, their genetic sequences in avian species have not yet been fully elucidated. Here, we obtained the full-length mRNA sequences of chicken PANX genes and evaluated the homology of the amino acids from these sequences with those of other species. Furthermore, PANX gene expression in several chicken tissues was investigated based on mRNA levels. PANX1 was detected in the brain, cochlea, chondrocytes, eye, lung, skin, and intestine, and PANX2 was expressed in the brain, eye, and intestine. PANX3 was observed in the cochlea, chondrocytes, and bone. In addition, expression of PANX3 was higher than PANX1 in the cochlea. Immunofluorescent staining revealed PANX1 in hair cells, as well as the supporting cells, ganglion neurons, and the tegmentum vasculosum in chickens, whereas PANX3 was only detected in the bone surrounding the cochlea. Overall, the results of this study provide the first identification and characterization of the sequence and expression of the PANX family in an avian species, and fundamental data for confirmation of Panx function. PMID:25002553

  16. Molecular cloning, expression, and sequence of the pilin gene from nontypeable Haemophilus influenzae M37.

    PubMed Central

    Coleman, T; Grass, S; Munson, R

    1991-01-01

    Nontypeable Haemophilus influenzae M37 adheres to human buccal epithelial cells and exhibits mannose-resistant hemagglutination of human erythrocytes. An isogenic variant of this strain which was deficient in hemagglutination was isolated. A protein with an apparent molecular weight of 22,000 was present in the sodium dodecyl sulfate-polyacrylamide gel profile of sarcosyl-insoluble proteins from the hemagglutination-proficient strain but was absent from the profile of the isogenic hemagglutination-deficient variant. A monoclonal antibody which reacts with the hemagglutination-proficient isolate but not with the hemagglutination-deficient isolate has been characterized. This monoclonal antibody was employed in an affinity column for purification of the protein as well as to screen a genomic library for recombinant clones expressing the gene. Several clones which contained overlapping genomic fragments were identified by reaction with the monoclonal antibody. The gene for the 22-kDa protein was subcloned and sequenced. The gene for the type b pilin from H. influenzae type b strain MinnA was also cloned and sequenced. The DNA sequence of the strain MinnA gene was identical to that reported previously for two other type b strains. The DNA sequence of the strain M37 gene is 77% identical to that of the type b pilin gene, and the derived amino acid sequence is 68% identical to that of the type b pilin. Images PMID:1673447

  17. Molecular cloning of tetracycline resistance genes from Streptomyces rimosus in Streptomyces griseus and characterization of the cloned genes.

    PubMed Central

    Ohnuki, T; Katoh, T; Imanaka, T; Aiba, S

    1985-01-01

    Two tetracycline resistance genes of Streptomyces rimosus, an oxytetracycline producer, were cloned in Streptomyces griseus by using pOA15 as a vector plasmid. Expression of the cloned genes, designated as tetA and tetB was inducible in S. griseus as well as in the donor strain. The tetracycline resistance directed by tetA and tetB was characterized by examining the uptake of tetracycline and in vitro polyphenylalanine synthesis by the sensitive host and transformants with the resultant hybrid plasmids. Polyphenylalanine synthesis with crude ribosomes and the S150 fraction from S. griseus carrying the tetA plasmid was resistant to tetracycline, and, by a cross-test of ribosomes and S150 fraction coming from both the sensitive host and the resistant transformant, the resistance directed by tetA was revealed to reside mainly in crude ribosomes and slightly in the S150 fraction. However, the resistance in the crude ribosomes disappeared when they were washed with 1 M ammonium chloride. These results suggest that tetA specified the tetracycline resistance of the machinery for protein synthesis not through ribosomal subunits, but via an unidentified cytoplasmic factor. In contrast, S. griseus carrying the tetB plasmid accumulated less intracellular tetracycline than did the host, and the protein synthesis by reconstituting the ribosomes and S150 fraction was sensitive to the drug. Therefore, it is conceivable that tetB coded a tetracycline resistance determinant responsible for the reduced accumulation of tetracycline. Images PMID:2982781

  18. Molecular cloning and expression of an Erwinia sp. gene encoding diphenyl ether cleavage in Escherichia coli.

    PubMed Central

    Liaw, H J; Srinivasan, V R

    1989-01-01

    A 2.1-kilobase fragment obtained by restriction enzyme HindIII digestion of Erwinia sp. genomic DNA was cloned into plasmid pUC19 and introduced into Escherichia coli by transformation. The transformants with diphenyl ether cleaving activity (Dpe+) were selected on agar plates with a specially designed medium (LTFN) containing 4-nitrodiphenyl ether. The positive clones showed a clear zone around the colonies. Analysis of mutants obtained by transposon mini-Mu dI(lacZ Kmr) mutagenesis indicated the coding region of the gene (dpe) and the utilization of a lacZ promoter of pUC19 for transcription of dpe. Clones with dpe in the opposite orientation in pUC19 were not expressed, confirming the need for a lacZ promoter. Utilization of a lacZ promoter in pUC19 was further confirmed by the observation that the degradation of 4-nitrodiphenyl ether was enhanced in the presence of isopropyl-beta-D-thiogalactoside. Expression of dpe was also found in pDPE7321, generated from cloning this gene into another plasmid, pSP73. Analysis of the plasmid-encoded proteins by the maxicell technique showed a polypeptide of 21,000 molecular weight as the product of dpe. Images PMID:2679381

  19. Molecular cloning and nucleotide sequence of a transforming gene detected by transfection of chicken B-cell lymphoma DNA

    NASA Astrophysics Data System (ADS)

    Goubin, Gerard; Goldman, Debra S.; Luce, Judith; Neiman, Paul E.; Cooper, Geoffrey M.

    1983-03-01

    A transforming gene detected by transfection of chicken B-cell lymphoma DNA has been isolated by molecular cloning. It is homologous to a conserved family of sequences present in normal chicken and human DNAs but is not related to transforming genes of acutely transforming retroviruses. The nucleotide sequence of the cloned transforming gene suggests that it encodes a protein that is partially homologous to the amino terminus of transferrin and related proteins although only about one tenth the size of transferrin.

  20. Cloning and molecular characterization of a putative voltage-gated sodium channel gene in the crayfish.

    PubMed

    Coskun, Cagil; Purali, Nuhan

    2016-06-01

    Voltage-gated sodium channel genes and associated proteins have been cloned and studied in many mammalian and invertebrate species. However, there is no data available about the sodium channel gene(s) in the crayfish, although the animal has frequently been used as a model to investigate various aspects of neural cellular and circuit function. In the present work, by using RNA extracts from crayfish abdominal ganglia samples, the complete open reading frame of a putative sodium channel gene has firstly been cloned and molecular properties of the associated peptide have been analyzed. The open reading frame of the gene has a length of 5793 bp that encodes for the synthesis of a peptide, with 1930 amino acids, that is 82% similar to the α-peptide of a sodium channel in a neighboring species, Cancer borealis. The transmembrane topology analysis of the crayfish peptide indicated a pattern of four folding domains with several transmembrane segments, as observed in other known voltage-gated sodium channels. Upon analysis of the obtained sequence, functional regions of the putative sodium channel responsible for the selectivity filter, inactivation gate, voltage sensor, and phosphorylation have been predicted. The expression level of the putative sodium channel gene, as defined by a qPCR method, was measured and found to be the highest in nervous tissue. PMID:27032955

  1. Molecular cloning, expression, and characterization of endoglucanase genes from Fibrobacter succinogenes AR1.

    PubMed Central

    Cavicchioli, R; Watson, K

    1991-01-01

    A cosmid gene library was constructed in Escherichia coli from genomic DNA isolated from the ruminal anaerobe Fibrobacter succinogenes AR1. Clones were screened on carboxymethyl cellulose, and 8 colonies that produced large clearing zones and 25 colonies that produced small clearing zones were identified. Southern blot hybridization revealed the existence of at least three separate genes encoding cellulase activity. pRC093, which is representative of cosmid clones that produce large clearing zones, was subcloned in pGem-1, and the resulting hybrid pRCEH directed synthesis of endoglucanase activity localized on a 2.1-kb EcoRI-HindIII insert. Activity was expressed from this fragment when it was cloned in both orientations in pGem-1 and pGem-2, indicating that F. succinogenes promoters functioned successfully in E. coli. A high level of endoglucanase activity was detected on acid-swollen cellulose, ball-milled cellulose, and carboxymethyl cellulose; and a moderate level was detected on filter paper, Avicel, lichenan, and xylan. Most activity (80%) was localized in the periplasm of E. coli, with low but significant levels (16%) being detected in the extracellular medium. The periplasmic endoglucanase had an estimated molecular weight of 46,500, had an optimum temperature of 39 degrees C, and exhibited activity over a broad pH range, with a maximum at pH 5.0. Images PMID:2014986

  2. Molecular cloning, sequence identification, and gene expression analysis of bovine ADCY2 gene.

    PubMed

    Li, Y X; Jin, H G; Yan, C G; Ren, C Y; Jiang, C J; Jin, C D; Seo, K S; Jin, X

    2014-06-01

    Adenylyl cyclase 2 (ADCY2), a class B member of adenylyl cyclases, is important in accelerating phosphor-acidification as well as glycogen synthesis and breakdown. Given its distinct role in flesh tenderization after butchering, we cloned and sequenced the ADCY2 gene from Yanbian cattle and assessed its expression in bovine tissues. A 2947 bp nucleotide sequence representing the full-length cDNA of bovine ADCY2 gene was obtained by 5' and 3' remote analysis computations for gene expression. Analyses of the putative protein sequence showed that ADCY2 had high homology among species, except with the non-mammal Oreochromis niloticus. Gene structural domain analyses in humans and rats indicated that the ADCY2 protein had no flaw; only the transmembrane domain was reduced and the CYCc structure domain was shortened. Assessment of ADCY2 expression in bovine tissues by real-time PCR showed that the highest expression was in the testes, followed by the longissimus dorsi, tensor fasciae latae, and latissimus dorsi. These data will serve as a foundation for further insight into the cattle ADCY2 gene. PMID:24797538

  3. Molecular cloning and characterization of two hypersensitive induced reaction genes from wheat infected by stripe rust pathogen

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A novel gene induced during hypersensitive reaction (HIR) in wheat was identified using in silico cloning and designated as TaHIR2. The TaHIR2 gene was deduced to encode a 284-amino acid protein, whose molecular mass and isoelectric point (pI) were 31.05 kD and 5.18, respectively. Amino acid sequenc...

  4. Molecular cloning and characterization of beta-expansin gene related to root hair formation in barley.

    PubMed

    Kwasniewski, Miroslaw; Szarejko, Iwona

    2006-07-01

    Root hairs are specialized epidermal cells that play a role in the uptake of water and nutrients from the rhizosphere and serve as a site of interaction with soil microorganisms. The process of root hair formation is well characterized in Arabidopsis (Arabidopsis thaliana); however, there is a very little information about the genetic and molecular basis of root hair development in monocots. Here, we report on isolation and cloning of the beta-expansin (EXPB) gene HvEXPB1, tightly related to root hair initiation in barley (Hordeum vulgare). Using root transcriptome differentiation in the wild-type/root-hairless mutant system, a cDNA fragment present in roots of wild-type plants only was identified. After cloning of full-length cDNA and genomic sequences flanking the identified fragment, the subsequent bioinformatics analyses revealed homology of the protein coded by the identified gene to the EXPB family. Reverse transcription-PCR showed that expression of HvEXPB1 cosegregated with the root hair phenotype in F2 progeny of the cross between the hairless mutant rhl1.a and the wild-type Karat parent variety. Expression of the HvEXPB1 gene was root specific; it was expressed in roots of wild-type forms, but not in coleoptiles, leaves, tillers, and spikes. The identified gene was active in roots of two other analyzed root hair mutants: rhp1.a developing root hair primordia only and rhs1.a with very short root hairs. Contrary to this, a complete lack of HvEXPB1 expression was observed in roots of the spontaneous root-hairless mutant bald root barley. All these observations suggest a role of the HvEXPB1 gene in the process of root hair formation in barley. PMID:16679418

  5. Molecular characterization of tobacco sulfite reductase: enzyme purification, gene cloning, and gene expression analysis.

    PubMed

    Yonekura-Sakakibara, K; Ashikari, T; Tanaka, Y; Kusumi, T a; Hase, T

    1998-09-01

    A cDNA clone, NtSiR1, that encodes the precursor of ferredoxin-dependent sulfite reductase (Fd-SiR) has been isolated from a cDNA library of tobacco (Nicotiana tabacum cv. SR1). The identity of the cDNA was established by comparison of the purified protein and the predicted structure with the nucleotide sequence. The amino terminus of the purified enzyme was Thr62 of the precursor protein, and the mature region of NtSiR1 consisted of 632 amino acids. Tobacco Fd-SiR is 82, 77, and 48% identical with Fd-SiRs from Zea mays, Arabidopsis thaliana, and a cyanobacterium, respectively. Significant similarity was also found with Escherichia coli NADPH-SiR in the region involved in ligation of siroheme and the [4Fe-4S] cluster. On Northern blot analysis, a transcript of NtSiR1 was detected in leaves, stems, roots, and petals in similar amounts. We also isolated a genomic SiR clone named gNtSiR1. It consists of 8 exons and 7 introns. Genomic Southern blot analysis indicated that at least two SiR genes are present in the tobacco genome. PMID:9722674

  6. Molecular cloning of the Escherichia coli B L-fucose-D-arabinose gene cluster.

    PubMed Central

    Elsinghorst, E A; Mortlock, R P

    1994-01-01

    To metabolize the uncommon pentose D-arabinose, enteric bacteria often recruit the enzymes of the L-fucose pathway by a regulatory mutation. However, Escherichia coli B can grow on D-arabinose without the requirement of a mutation, using some of the L-fucose enzymes and a D-ribulokinase that is distinct from the L-fuculokinase of the L-fucose pathway. To study this naturally occurring D-arabinose pathway, we cloned and partially characterized the E. coli B L-fucose-D-arabinose gene cluster and compared it with the L-fucose gene cluster of E. coli K-12. The order of the fucA, -P, -I, and -K genes was the same in the two E. coli strains. However, the E. coli B gene cluster contained a 5.2-kb segment located between the fucA and fucP genes that was not present in E. coli K-12. This segment carried the darK gene, which encodes the D-ribulokinase needed for growth on D-arabinose by E. coli B. The darK gene was not homologous with any of the L-fucose genes or with chromosomal DNA from other D-arabinose-utilizing bacteria. D-Ribulokinase and L-fuculokinase were purified to apparent homogeneity and partially characterized. The molecular weights, substrate specificities, and kinetic parameters of these two enzymes were very dissimilar, which together with DNA hybridization analysis, suggested that these enzymes are not related. D-Arabinose metabolism by E. coli B appears to be the result of acquisitive evolution, but the source of the darK gene has not been determined. Images PMID:7961494

  7. Molecular cloning, sequence characteristics, and tissue expression analysis of ECE1 gene in Tibetan pig.

    PubMed

    Wang, Yan-Dong; Zhang, Jian; Li, Chuan-Hao; Xu, Hai-Peng; Chen, Wei; Zeng, Yong-Qing; Wang, Hui

    2015-10-25

    Low air pressure and low oxygen partial pressure at high altitude seriously affect the survival and development of human beings and animals. ECE1 is a recently discovered gene that is involved in anti-hypoxia, but the full-length cDNA sequence has not been obtained. For a better understanding of the structure and function of the ECE1 gene and to study its effect in Tibetan pig, the cDNA of the ECE1 gene from the muscle of Tibetan pig was cloned, sequenced and characterized. The ECE1 full-length cDNA sequence consists of 2262 bp coding sequence (CDS) that encodes 753 amino acids with a molecular mass of 85,449 kD, 2 bp 5'UTR and 1507 bp 3'UTR. In addition, the phylogenetic tree analysis revealed that the Tibetan pig ECE1 has a closer genetic relationship and evolution distance with the land mammals ECE1. Furthermore, analysis by qPCR showed that the ECE1 transcript is constitutively expressed in the 10 tissues tested: the liver, subcutaneous fat, kidney, muscle, stomach, heart, brain, spleen, pancreas, and lung. These results serve as a foundation for further insight into the Tibetan pig ECE1 gene. PMID:26115769

  8. Molecular cloning and transcriptional analysis of the Aspergillus terreus gla1 gene encoding a glucoamylase.

    PubMed Central

    Ventura, L; González-Candelas, L; Pérez-González, J A; Ramón, D

    1995-01-01

    The Aspergillus terreus gla1 gene, coding for a glucoamylase, has been cloned by heterologous hybridization. The gene is interrupted by four introns and encodes a protein with an N-terminal catalytic domain and a C-terminal starch-binding domain. The expression of the gene is induced by starch and maltose and repressed by glucose. PMID:7534054

  9. Molecular cloning of genes related to aflatoxin biosynthesis by differential screening.

    PubMed Central

    Feng, G H; Chu, F S; Leonard, T J

    1992-01-01

    A differential hybridization strategy was used to clone genes associated with aflatoxin biosynthesis. A genomic library, formed between nuclear DNA and the pUC19 plasmid, was screened with three different cDNA probes by the colony hybridization procedure. Nineteen clones were selected; all were positively correlated with and presumably enriched with genes associated with aflatoxin production. Some of these clones were further characterized by using them as probes in Northern (RNA blot) hybridizations. Five clones hybridized strongly with some polyadenylated RNAs formed during the transition to or during idiophase when aflatoxin was produced. However, little or no corresponding hybridization occurred with polyadenylated RNAs formed in early and mid-log growth phase. Two of the clones were further used as probes to hybridize with polyadenylated RNAs formed under aflatoxin-permissive and nonpermissive temperatures. Hybridization occurred with RNA species formed under the permissive temperature only. Images PMID:1610169

  10. Molecular cloning and expression in Escherichia coli of a cellodextrinase gene from Bacteroides succinogenes S85.

    PubMed Central

    Gong, J H; Lo, R Y; Forsberg, C W

    1989-01-01

    A DNA fragment coding for a cellodextrinase of Bacteroides succinogenes S85 was isolated by screening of a pBR322 gene library in Escherichia coli HB101. Of 100,000 colonies screened on a complex medium with methylumbelliferyl-beta-D-cellobioside as the indicator substrate, two cellodextrinase-positive clones (CB1 and CB2) were isolated. The DNA inserts from the two recombinant plasmids were 7.7 kilobase pairs in size and had similar restriction maps. After subcloning from pCB2, a 2.5-kilobase-pair insert which coded for cellodextrinase activity was isolated. The enzyme was located in the cytoplasm of the E. coli host. It exhibited no activity on carboxymethyl cellulose, Avicel microcrystalline cellulose, acid-swollen cellulose, or cellobiose but hydrolyzed p-nitrophenyl-beta-D-cellobioside and p-nitrophenyl-beta-D-lactoside. The Km (0.1 mM) for the hydrolysis of p-nitrophenyl-cellobioside by the enzyme expressed in E. coli was similar to that reported for the purified enzyme from B. succinogenes. Expression of the cellodextrinase gene was subjected to catabolite repression by glucose and was not induced by cellobiose. The origin of the DNA insert from B. succinogenes was confirmed by Southern blot analysis. Western blotting (immunoblotting) using antibodies raised against the purified B. succinogenes cellodextrinase revealed a protein with a molecular weight of approximately 50,000 in E. coli clones which comigrated with the native enzyme isolated from B. succinogenes. These data indicate that the cellodextrinase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to those of the native enzyme. Images PMID:2650617

  11. Molecular cloning and characterization of a Bombyx mori gene encoding the transcription factor Atonal.

    PubMed

    Hu, Ping; Feng, Fan; Xia, Hengchuan; Chen, Liang; Yao, Qin; Chen, Keping

    2014-01-01

    The atonal genes are an evolutionarily conserved group of genes encoding regulatory basic helix-loop-helix (bHLH) transcription factors. These transcription factors have a critical antioncogenic function in the retina, and are necessary for cell fate determination through the regulation of the cell signal pathway. In this study, the atonal gene was cloned from Bombyx mori, and the transcription factor was named BmAtonal. Sequence analysis showed that the BmAtonal protein shares extensive homology with other invertebrate Atonal proteins with the bHLH motif. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses revealed that BmAtonal was expressed in all developmental stages of B. mori and various larval tissues. The BmAtonal protein was expressed in Escherichia coli, and polyclonal antibodies were raised against the purified protein. By immunofluorescence, the BmAtonal protein was localized to both the nucleus and cytoplasm of BmN cells. After knocking out nuclear localization signals (NLS), the BmAtonal protein was only detected in the cytoplasm. In addition, using the B. mori nuclear polyhedrosis virus (BmNPV) baculovirus expression system, the recombinant BmAtonal protein was successfully expressed in the B. mori cell line BmN. This work lays the foundation for exploring the biological functions of the BmAtonal protein, such as identifying its potential binding partners and understanding the molecular control of the formation of sensory organs. PMID:24873037

  12. Molecular Cloning and Sequencing of Hemoglobin-Beta Gene of Channel Catfish, Ictalurus Punctatus Rafinesque

    Technology Transfer Automated Retrieval System (TEKTRAN)

    : Hemoglobin-y gene of channel catfish , lctalurus punctatus, was cloned and sequenced . Total RNA from head kidneys was isolated, reverse transcribed and amplified . The sequence of the channel catfish hemoglobin-y gene consists of 600 nucleotides . Analysis of the nucleotide sequence reveals one o...

  13. Molecular characterization of a mouse prostaglandin D receptor and functional expression of the cloned gene.

    PubMed

    Hirata, M; Kakizuka, A; Aizawa, M; Ushikubi, F; Narumiya, S

    1994-11-01

    Prostanoid receptors belong to the family of G protein-coupled receptors with seven transmembrane domains. By taking advantage of nucleotide sequence homology among the prostanoid receptors, we have isolated and identified a cDNA fragment and its gene encoding a mouse prostaglandin (PG) D receptor by reverse transcription polymerase chain reaction and gene cloning. This gene codes for a polypeptide of 357 amino acids, with a calculated molecular weight of 40,012. The deduced amino acid sequence has a high degree of similarity with the mouse PGI receptor and the EP2 subtype of the PGE receptor, which together form a subgroup of the prostanoid receptors. Chinese hamster ovary cells stably expressing the gene showed a single class of binding sites for [#H]PGD2 with a Kd of 40 nM. This binding was displaced by unlabeled ligands in the following order: PGD2 > BW 245C (a PGD agonist) > BW A868C (a PGD antagonist) > STA2 (a thromboxane A2 agonist). PGE2, PGF2 alpha, and iloprost showed little displacement activity at concentrations up to 10 microM. PGD2 and BW 245C also increased cAMP levels in Chinese hamster ovary cells expressing the receptor, in a concentration-dependent manner. BW A868C showed a partial agonist activity in the cAMP assay. Northern blotting analysis with mouse poly(A)+ RNA identified a major mRNA species of 3.5 kb that was most abundantly expressed in the ileum, followed by lung, stomach, and uterus. PMID:7972033

  14. Molecular cloning in Escherichia coli of Erwinia chrysanthemi genes encoding multiple forms of pectate lyase.

    PubMed Central

    Collmer, A; Schoedel, C; Roeder, D L; Ried, J L; Rissler, J F

    1985-01-01

    The phytopathogenic enterobacterium Erwinia chrysanthemi excretes multiple isozymes of the plant tissue-disintegrating enzyme, pectate lyase (PL). Genes encoding PL were cloned from E. chrysanthemi CUCPB 1237 into Escherichia coli HB101 by inserting Sau3A-generated DNA fragments into the BamHI site of pBR322 and then screening recombinant transformants for the ability to sink into pectate semisolid agar. Restriction mapping of the cloned DNA in eight pectolytic transformants revealed overlapping portions of a 9.8-kilobase region of the E. chrysanthemi genome. Deletion derivatives of these plasmids were used to localize the pectolytic genotype to a 2.5-kilobase region of the cloned DNA. PL gene expression in E. coli was independent of vector promoters, repressed by glucose, and not induced by galacturonan. PL accumulated largely in the periplasmic space of E. coli. An activity stain used in conjunction with ultrathin-layer isoelectric focusing resolved the PL in E. chrysanthemi culture supernatants and shock fluids of E. coli clones into multiple forms. One isozyme with an apparent pI of 7.8 was produced at a far higher level in E. coli and was common to all of the pectolytic clones. Activity staining of renatured PL in sodium dodecyl sulfate-polyacrylamide gels revealed that this isozyme comigrated with the corresponding isozyme produced by E. chrysanthemi. The PL isozyme profiles produced by different clones and deletion derivative subclones suggest that the cloned region contains at least two PL isozyme structural genes. Pectolytic E. coli clones possessed a limited ability to macerate potato tuber tissues. Images PMID:2982794

  15. Molecular cloning and characterization of a Candida tsukubaensis alpha-glucosidase gene in the yeast Saccharomyces cerevisiae.

    PubMed

    Kinsella, B T; Larkin, A; Bolton, M; Cantwell, B A

    1991-07-01

    The molecular cloning of an alpha-glucosidase gene isolated from a Candida tsukubaensis (CBS 6389) genomic library in Saccharomyces cervisiae is reported. The cloned gene is contained within a 6.2 kb Sau3A DNA fragment and directs the synthesis and secretion of an amylolytic enzyme into the extracellular medium of the recombinant host, S. cerevisiae. The cloned enzyme was found to have an unusually broad substrate specificity and is capable of hydrolysing alpha-1,2, alpha-1,3, alpha-1,4 and alpha-1,6 linked, as well as aryl and alkyl, D-glucosides. On the basis of its substrate specificity profile, the cloned enzyme was classified as an alpha-glucosidase (E.C. 3.2.1.20). It has a pH optimum in the range 4.2-4.6, a temperature optimum of 58 degrees C and is readily inactivated at pasteurization temperature (60 degrees C). Southern blot analysis failed to reveal any homology between the cloned gene and genomic DNA isolated from other well characterized amylolytic yeasts. A rapid plate-assay, based on the utilization of a chromogenic substrate X-alpha-D-glucoside to detect the expression of the cloned alpha-glucosidase in S. cerevisiae transformants, was developed. PMID:1934116

  16. Molecular cloning and characterization of Izumo1 gene from sheep and cashmere goat reveal alternative splicing.

    PubMed

    Xing, Wan-Jin; Han, Bao-Da; Wu, Qi; Zhao, Li; Bao, Xiao-Hong; Bou, Shorgan

    2011-03-01

    We cloned the cDNA and genomic DNA encoding for Izumo1 of cashmere goat (Capra hircus) and sheep (Ovis aries). Analysis of 4.6 kb Izumo1 genomic sequences in sheep and goat revealed a canonical open reading frame (ORF) of 963 bp spliced by eight exons. Sheep and goat Izumo1 genes share >99% identity at both DNA and protein levels and are also highly homologous to the orthologues in cattle, mouse, rat and human. Extensive cloning and analysis of Izumo1 cDNA revealed three (del 69, del 182 and del 217) and two (del 69 and ins 30) alternative splicing isoforms in goat and sheep, respectively. All of the isoforms are derived from splicing at typical GT-AG sites leading to partial or complete truncation of the immunoglobulin (Ig)-like domain. Bioinformatics analysis showed that caprine and ovine Izumo1 proteins share similar structure with their murine orthologue. There are a signal peptide at the N-terminus (1-22 aa), a transmembrane domain at the C-terminus (302-319 aa), and an extracellular Ig-like region in the middle (161-252 aa) with a putative N-linked glycosylation site (N(205)-N-S). Alignment of Izumo1 protein sequences among 15 mammalian species displayed several highly conserved regions, including LDC and YRC motifs with cysteine residues for potential disulfide bridge formation, CPNKCG motif upstream of the Ig-like domain, GLTDYSFYRVW motif upstream of the putative N-linked glycosylation site, and a number of scattered cysteine residues. These distinctive features are very informative to pinpoint the important gene motifs and functions. The C-terminal regions, however, are more variable across species. Izumo1 cDNA sequences of goat, sheep, and cow were found to be largely homologous, and the molecular phylogenetic analysis is consistent with their morphological taxonomy. This implies the Izumo1 gene evolves from the same ancestor, and the mechanism of sperm-egg fusion in mammals may be under the same principle in which Izumo1 plays an important role. PMID

  17. Molecular cloning and bioinformatic analysis of the Streptococcus agalactiae neuA gene isolated from tilapia.

    PubMed

    Wang, E L; Wang, K Y; Chen, D F; Geng, Y; Huang, L Y; Wang, J; He, Y

    2015-01-01

    Cytidine monophosphate (CMP) N-acetylneuraminic acid (NeuNAc) synthetase, which is encoded by the neuA gene, can catalyze the activation of sialic acid with CMP, and plays an important role in Streptococcus agalactiae infection pathogenesis. To study the structure and function of the S. agalactiae neuA gene, we isolated it from diseased tilapia, amplified it using polymerase chain reaction (PCR) with specific primers, and cloned it into a pMD19-T vector. The recombinant plasmid was confirmed by PCR and restriction enzyme digestion, and identified by sequencing. Molecular characterization analyses of the neuA nucleotide amino acid sequence were performed using bioinformatic tools and an online server. The results showed that the neuA nucleotide sequence contained a complete coding region, which comprised 1242 bp, encoding 413 amino acids (aa). The aa sequence was highly conserved and contained a Glyco_tranf_GTA_type superfamily and an SGNH_hydrolase superfamily conserved domain, which are related to sialic acid activation catalysis. The NeuA protein possessed many important sites related to post-translational modification, including 28 potential phosphorylation sites and 2 potential N-glycosylation sites, had no signal peptides or transmembrane regions, and was predicted to reside in the cytoplasm. Moreover, the protein had some B-cell epitopes, which suggests its potential in development of a vaccine against S. agalactiae infection. The codon usage frequency of neuA differed greatly in Escherichia coli and Homo sapiens genes, and neuA may be more efficiently expressed in eukaryotes (yeast). S. agalactiae neuA from tilapia maintains high structural homology and sequence identity with CMP-NeuNAc synthetases from other bacteria. PMID:26125800

  18. Molecular cloning and structural characterization of the human histidase gene (HAL)

    SciTech Connect

    Suchi, Mariko; Sano, Hirofumi; Mizuno, Haruo; Wada, Yoshiro

    1995-09-01

    Histidase (EC 4.3.1.3) is a cytosolic enzyme that catalyzes the nonoxidative determination of histidine to urocanic acid. Histidinemia, resulting from reduced histidase activity as reported in Cambridge stock his/her mice and in humans, is the most frequent inborn metabolic error in Japan. The histidase chromosomal gene (HAL) was isolated from a {lambda}EMBL-3 human genomic library using the human histidase cDNA as a probe. Restriction mapping and Southern blot analysis of the isolated clones reveal a single-copy gene spanning approximately 25 kb and consisting of 21 exons. Exon 1 encodes only 5{prime} untranslated sequence of liver histidase mRNA, with protein coding beginning in exon 2. A rarely observed 5{prime}GC, similar to that reported in the human P-450(SCC) gene, is present in intron 20. All other splicing junctions adhere to the canonical GT/AG rule. A TATA box sequence is located 25 bp upstream of the liver histidase transcription initiation site determined by S1 nuclease protection analysis. Several liver- and epidermis-specific transcription factor binding sites, including C/EBP, NFIL6, HNF5, AP2/ KER1, MNF, and others, are also identified in the 5{prime} flanking region. Consistent with the hepatic and epidermal expression of histidase, this finding suggests that histidase transcription may be regulated by these factors. We further identify a polymorphism (A to G transition) in the histidase coding region of exon 16. The human histidase genomic structure presented here should facilitate the molecular investigation of symptomatic and asymptomatic forms of histidinemia. 69 refs., 4 figs., 1 tab.

  19. Molecular cloning and characterization of the translationally controlled tumor protein gene in Bombyx mori.

    PubMed

    Lee, Jae Man; Kusakabe, Takahiro; Kawaguchi, Yutaka; Miyagawa, Yoshitaka; Takahashi, Masateru; Mon, Hiroaki; Nho, Si-Kab; Koga, Katsumi

    2004-09-01

    Translationally controlled tumor protein (Tctp/p23) is known to be synthesized preferentially in cells during the early growth phase of tumors, but is also expressed in normal cells. To elucidate its molecular basis of the expression and physiological significance, a cDNA encoding for the Bombyx mori Tctp (BmTctp) was deduced by editing the partial cDNA sequences registered in a Bombyx EST database. RT-PCR analyses indicated that the BmTCTP mRNA was transcribed in all larval organs examined and was present constantly during the cell cycle of BmN4 cells. A genomic clone of 4255 nucloetide residues produced by inverse PCR contained the 5'-flanking region, two introns and three exons of the BmTCTP gene. Sequence analysis of the 5'-flanking region indicated that a putative promoter region contains several canonical transcription elements such as GATA box, CCAAT motif, MEF2, E4BP4.01 and AP-1, but lacks a TATA box element. Luciferase reporter assay of the deletion constructs of the 5'-flanking region revealed that the -676 to +66 region enhanced the promoter activity the most markedly. In addition to this, there were at least two enhancer-like elements and several repressor elements. PMID:15364286

  20. Novel glucose dehydrogenase from Mucor prainii: Purification, characterization, molecular cloning and gene expression in Aspergillus sojae.

    PubMed

    Satake, Ryoko; Ichiyanagi, Atsushi; Ichikawa, Keiichi; Hirokawa, Kozo; Araki, Yasuko; Yoshimura, Taro; Gomi, Keiko

    2015-11-01

    Glucose dehydrogenase (GDH) is of interest for its potential applications in the field of glucose sensors. To improve the performance of glucose sensors, GDH is required to have strict substrate specificity. A novel flavin adenine dinucleotide (FAD)-dependent GDH was isolated from Mucor prainii NISL0103 and its enzymatic properties were characterized. This FAD-dependent GDH (MpGDH) exhibited high specificity toward glucose. High specificity for glucose was also observed even in the presence of saccharides such as maltose, galactose and xylose. The molecular masses of the glycoforms of GDH ranged from 90 to 130 kDa. After deglycosylation, a single 80 kDa band was observed. The gene encoding MpGDH was cloned and expressed in Aspergillus sojae. The apparent kcat and Km values of recombinant enzyme for glucose were found to be 749.7 s(-1) and 28.3 mM, respectively. The results indicated that the characteristics of MpGDH were suitable for assaying blood glucose levels. PMID:25912449

  1. Molecular cloning and characterization of a chlorophyll degradation regulatory gene (ZjSGR) from Zoysia japonica.

    PubMed

    Teng, K; Chang, Z H; Xiao, G Z; Guo, W E; Xu, L X; Chao, Y H; Han, L B

    2016-01-01

    The stay-green gene (SGR) is a key regulatory factor for chlorophyll degradation and senescence. However, to date, little is known about SGR in Zoysia japonica. In this study, ZjSGR was cloned, using rapid amplification of cDNA ends-polymerase chain reaction (PCR). The target sequence is 831 bp in length, corresponding to 276 amino acids. Protein BLAST results showed that ZjSGR belongs to the stay-green superfamily. A phylogenetic analysis implied that ZjSGR is most closely related to ZmSGR1. The subcellular localization of ZjSGR was investigated, using an Agrobacterium-mediated transient expression assay in Nicotiana benthamiana. Our results demonstrated that ZjSGR protein is localized in the chloroplasts. Quantitative real time PCR was carried out to investigate the expression characteristics of ZjSGR. The expression level of ZjSGR was found to be highest in leaves, and could be strongly induced by natural senescence, darkness, abscisic acid (ABA), and methyl jasmonate treatment. Moreover, an in vivo function analysis indicated that transient overexpression of ZjSGR could accelerate chlorophyll degradation, up-regulate the expression of SAG113, and activate ABA biosynthesis. Taken together, these results provide evidence that ZjSGR could play an important regulatory role in leaf chlorophyll degradation and senescence in plants at the molecular level. PMID:27173268

  2. Molecular cloning, sequence analysis and expression of a novel gene induced by near-UV light in Bipolaris oryzae.

    PubMed

    Kihara, J; Sato, A; Okajima, S; Kumagai, T

    2001-09-01

    A cDNA clone derived from a novel gene (uvi-1) that is inducible by near-UV light was isolated by a differential screening procedure from a cDNA library of the fungus Bipolaris oryzae and characterized further. Sequence analysis of the clone revealed that uvi-1 encodes a protein with a putative molecular mass of 17 kDa; the UVI-1 protein shows significant similarity to a putative protein encoded by a cDNA which is expressed during appressorium formation in the rice blast fungus, Magnaporthe grisea. The corresponding genomic clone was also isolated, and Southern analysis of genomic DNA indicated the presence of a single copy of the uvi-1 gene in B. oryzae. Northern analysis showed that the uvi-1 transcripts are induced by exposure to near-UV light, but not by blue or red light. Furthermore, accumulation of uvi-1 transcripts is observed during differentiation of the appressorium. PMID:11589579

  3. Molecular Cloning, Nucleotide Sequence, and Expression of Genes Encoding a Polycyclic Aromatic Ring Dioxygenase from Mycobacterium sp. Strain PYR-1

    PubMed Central

    Khan, Ashraf A.; Wang, Rong-Fu; Cao, Wei-Wen; Doerge, Daniel R.; Wennerstrom, David; Cerniglia, Carl E.

    2001-01-01

    Mycobacterium sp. strain PYR-1 degrades high-molecular-weight polycyclic hydrocarbons (PAHs) primarily through the introduction of both atoms of molecular oxygen by a dioxygenase. To clone the dioxygenase genes involved in PAH degradation, two-dimensional (2D) gel electrophoresis of PAH-induced proteins from cultures of Mycobacterium sp. strain PYR-1 was used to detect proteins that increased after phenanthrene, dibenzothiophene, and pyrene exposure. Comparison of proteins from induced and uninduced cultures on 2D gels indicated that at least six major proteins were expressed (105, 81, 52, 50, 43, and 13 kDa). The N-terminal sequence of the 50-kDa protein was similar to those of other dioxygenases. A digoxigenin-labeled oligonucleotide probe designed from this protein sequence was used to screen dioxygenase-positive clones from a genomic library of Mycobacterium sp. strain PYR-1. Three clones, each containing a 5,288-bp DNA insert with three genes of the dioxygenase system, were obtained. The genes in the DNA insert, from the 5′ to the 3′ direction, were a dehydrogenase, the dioxygenase small (β)-subunit, and the dioxygenase large (α)-subunit genes, arranged in a sequence different from those of genes encoding other bacterial dioxygenase systems. Phylogenetic analysis showed that the large α subunit did not cluster with most of the known α-subunit sequences but rather with three newly described α subunits of dioxygenases from Rhodococcus spp. and Nocardioides spp. The genes from Mycobacterium sp. strain PYR-1 were subcloned and overexpressed in Escherichia coli with the pBAD/ThioFusion system. The functionality of the genes for PAH degradation was confirmed in a phagemid clone containing all three genes, as well as in plasmid subclones containing the two genes encoding the dioxygenase subunits. PMID:11472934

  4. Molecular cloning, genomic organization, and chromosomal localization of the human pancreatitis-associated protein (PAP) gene

    SciTech Connect

    Dusetti, N.J.; Frigerio, J.M.; Dagorn, J.C.; Iovanna, J.L. ); Fox, M.F.; Swallow, D.M. )

    1994-01-01

    Pancreatitis-associated protein (PAP) is a secretory pancreatic protein present in small amounts in normal pancreas and overexpressed during the acute phase of pancreatitis. In this paper, the authors describe the cloning, characterization, and chromosomal mapping of the human PAP gene. The gene spans 2748 bp and contains six exons interrupted by five introns. The gene has a typical promoter containing the sequences TATAAA and CCAAT 28 and 52 bp upstream of the cap site, respectively. They found striking similarities in genomic organization as well as in the promoter sequences between the human and rat PAP genes. The human PAP gene was mapped to chromosome 2p12 using rodent-human hybrid cells and in situ chromosomal hybridization. This localization coincides with that of the reg/lithostathine gene, which encodes a pancreatic secretory protein structurally related to PAP, suggesting that both genes derived from the same ancestral gene by duplication. 35 refs., 4 figs., 1 tab.

  5. Building a human kinase gene repository: Bioinformatics, molecular cloning, and functional validation

    PubMed Central

    Park, Jaehong; Hu, Yanhui; Murthy, T. V. S.; Vannberg, Fredrik; Shen, Binghua; Rolfs, Andreas; Hutti, Jessica E.; Cantley, Lewis C.; LaBaer, Joshua; Harlow, Ed; Brizuela, Leonardo

    2005-01-01

    Kinases catalyze the phosphorylation of proteins, lipids, sugars, nucleosides, and other important cellular metabolites and play key regulatory roles in all aspects of eukaryotic cell physiology. Here, we describe the mining of public databases to collect the sequence information of all identified human kinase genes and the cloning of the corresponding ORFs. We identified 663 genes, 511 encoding protein kinases, and 152 encoding nonprotein kinases. We describe the successful cloning and sequence verification of 270 of these genes. Subcloning of this gene set in mammalian expression vectors and their use in high-throughput cell-based screens allowed the validation of the clones at the level of expression and the identification of previously uncharacterized modulators of the survivin promoter. Moreover, expressions of the kinase genes in bacteria, followed by autophosphorylation assays, identified 21 protein kinases that showed autocatalytic activity. The work described here will facilitate the functional assaying of this important gene family in phenotypic screens and their use in biochemical and structural studies. PMID:15928075

  6. Molecular genetics of X-linked retinitis pigmentosa: Progress towards cloning the RP3 gene

    SciTech Connect

    Fujita, R.; Yan, D.; McHenry, C.

    1994-09-01

    Our goal is to identify the X-linked retinitis pigmentosa (XLRP) gene RP3. The location of RP3 is genetically delimited to a region of 1 Mb, distal to DXS140, CYBB and tctex-1-like gene and proximal to the gene OTC. It is currently thought that RP3 is within 40 kb of the proximal deletion breakpoint of a patient BB. However, a more proximal location of the gene, closer to OTC, is not ruled out. We initiated the isolation of the genomic region between DXS140 to OTC in YACs. One of the clones from DXS140 region (55B) is 460 kb and spans about 200 kb at each side of BB patient`s proximal breakpoint. It contains CYBB, tctex-1-like genes and two additional CpG islands. The 55B clone has been covered by cosmid and phage subclones. Another YAC clone from the OTC region (OTCC) spans about 1 Mb and contains at least 5 CpG islands. In situ hybridization performed with OTCC showed its location in Xp21; however, several derivative cosmids map to chromosome 7, indicating that it is a chimeric YAC. No overlap is evident between 55B and OTCC. We have isolated the YAC end-sequences and isolation of clones to close the gap is in progress. Cosmids are being used for screening eye tissue cDNA libraries, mainly from retina. Screening is done by hybridization to replica filters or by cDNA enrichment methods. Several cDNA clones have been isolated and are being characterized. Exon-amplification is also being used with the cosmids and phages. Genetic analysis is being performed to determine RP3 patients from clinically indistinguishable RP2, located in Xp11.23-p11.4, and to reduce the genetic distance of current flanking markers. For this we are analyzing a number of XLRP families with established markers in the region and with new microsatellites.

  7. Molecular cloning and characterization of the β-catenin gene from fine-wool sheep.

    PubMed

    Cui, Kai; Yang, Zu; Darwish, Hesham; Zhang, Yuanyuan; Ge, Yaqiong; Zhang, Xiyue; Li, Rongni; Deng, Xuemei

    2014-08-10

    β-Catenin is an evolutionarily conserved molecule that functions as a crucial effector in both cell-to-cell adhesion and Wnt signaling. To gain a better understanding of its role in the development of hair follicles, we cloned the cDNA sequence of the β-catenin gene from the skin of Aohan fine-wool sheep and performed a variety of bioinformatics analyses. We obtained the full-length sequence, which was 4573-bp long and contained a 2346-bp open reading frame encoding a protein of 781 amino acids. The protein had a predicted molecular weight of 85.4 kDa and a theoretical isoelectric point of 5.57. Domain architecture analysis of the β-catenin protein revealed an armadillo repeat region, which is a common feature of β-catenin in other species. The ovine β-catenin gene shares 97.91%, 94.25%, 94.59%, 83.89%, and 89.39% sequence identity with its homologs in Bos taurus, Homo sapiens, Sus scrofa, Gallus gallus, and Mus musculus, respectively, while the amino acid sequence is more than 99% identical with each of these species. The expression of β-catenin mRNA was detected in the heart, liver, spleen, lung, kidney, skin, muscle, and adipose tissue. Expression levels were maximal in the lung and minimal in the muscle, and the difference in expression in these tissues was significant (P<0.01). Western blot analysis revealed the presence of the β-catenin protein in all tissues examined; expression was lowest in the skin and adipose tissues. PMID:24881815

  8. Mole ghrelin: cDNA cloning, gene expression, and diverse molecular forms in Mogera imaizumii.

    PubMed

    Satou, Motoyasu; Kaiya, Hiroyuki; Nishi, Yoshihiro; Shinohara, Akio; Kawada, Shin-Ichiro; Miyazato, Mikiya; Kangawa, Kenji; Sugimoto, Hiroyuki

    2016-06-01

    Here, we describe cDNA cloning and purification of the ghrelin gene sequences and ghrelin peptides from the Japanese true mole, Mogera imaizumii. The gene spans >2.9kbp, has four exons and three introns, and shares structural similarity with those of terrestrial animals. Mature mole ghrelin peptide was predicted to be 28 amino acids long (GSSFLSPEHQKVQQRKESKKPPSKPQPR) and processed from a prepropeptide of 116 amino acids. To further elucidate molecular characteristics, we purified ghrelin peptides from mole stomach. By mass spectrometry, we found that the mole ghrelin peptides had higher ratios of the odd-number fatty acids (C9 and C11 as much as C8) attached to the third serine residue than other vertebrate ghrelin. Truncated forms of ghrelins such as [1-27], [1-19], [1-16] and [1-15], and that lacked the 14th glutamine residue (des-Gln14 ghrelin) were produced in the stomach. Marked expression of ghrelin mRNA in lung was observed as in stomach and brain. Phylogenetic analysis indicated that the branch of M. imaizumii has slightly higher dN/dS ratios (the nucleotide substitution rates at non-synonymous and synonymous sites) than did other eulipotyphlans. Peptide length was positively correlated with human ghrelin receptor activation, whereas the length of fatty-acyl chains showed no obvious functional correlation. The basal higher luciferase activities of the 5'-proximal promoter region of mole ghrelin were detected in ghrelin-negative C2C12 cells and hypoxic culture conditions impaired transcriptional activity. These results indicated that moles have acquired diverse species of ghrelin probably through distinctive fatty acid metabolism because of their food preferences. The results provide a gateway to understanding ghrelin metabolism in fossorial animals. PMID:27102942

  9. Molecular cloning of chitinase 33 (chit33) gene from Trichoderma atroviride

    PubMed Central

    Matroudi, S.; Zamani, M.R.; Motallebi, M.

    2008-01-01

    In this study Trichoderma atroviride was selected as over producer of chitinase enzyme among 30 different isolates of Trichoderma sp. on the basis of chitinase specific activity. From this isolate the genomic and cDNA clones encoding chit33 have been isolated and sequenced. Comparison of genomic and cDNA sequences for defining gene structure indicates that this gene contains three short introns and also an open reading frame coding for a protein of 321 amino acids. The deduced amino acid sequence includes a 19 aa putative signal peptide. Homology between this sequence and other reported Trichoderma Chit33 proteins are discussed. The coding sequence of chit33 gene was cloned in pEt26b(+) expression vector and expressed in E. coli. PMID:24031242

  10. Molecular cloning of the human CTP synthetase gene by functional complementation with purified human metaphase chromosomes.

    PubMed

    Yamauchi, M; Yamauchi, N; Meuth, M

    1990-07-01

    Successive rounds of chromosome-mediated gene transfer were used to complement a hamster cytidine auxotroph deficient in CTP synthetase activity and eventually to clone human genomic and cDNA fragments coding for the structural gene. Our approach was to isolate human Alu+ fragments from a tertiary transfectant and to utilize these fragments to screen a panel of primary transfectants. In this manner two DNA fragments, both mapping within the structural gene, were identified and used to clone a partial length cDNA. The remaining portion of the open reading frame was obtained through the RACE polymerase chain reaction technique. The open reading frame encodes 591 amino acids having a striking degree of similarity to the Escherichia coli structural gene (48% identical amino acids with 76% overall similarity including conservative substitutions) with the glutamine amide transfer domain being particularly conserved. As regulatory mutations of CTP synthetase confer both multi-drug resistance to agents widely used in cancer chemotherapy and a mutator phenotype, the cloning of the structural gene will be important in assessing the relevance of such phenotypes to the development of cellular drug resistance. PMID:2113467

  11. Molecular cloning and characterization of mutant and wild-type human. beta. -actin genes

    SciTech Connect

    Leavitt, J.; Gunning, P.; Porreca, P.; Ng, S.Y.; Lin, C.H.; Kedes, L.

    1984-10-01

    There are more than 20 ..beta..-actin-specific sequences in the human genome, many of which are pseudogenes. To facilitate the isolation of potentially functional ..beta..-actin genes, they used the new method of B. Seed for selecting genomic clones by homologous recombination. A derivative of the ..pi..VX miniplasmid, ..pi..AN7..beta..1, was constructed by insertion of the 600-base-pair 3' untranslated region of the ..beta..-actin mRNA expressed in human fibroblasts. Five clones containing ..beta..-actin sequences were selected from an amplified human fetal gene library by homologous recombination between library phage and the miniplasmid. One of these clones contained a complete ..beta..-actin gene with a coding sequence identical to that determined for the mRNA of human fibroblasts. A DNA fragment consisting of mostly intervening sequences from this gene was then use to identify 13 independent recombinant copies of the analogous gene from two specially constructed gene libraries, each containing one of the two types of mutant ..beta..-actin genes found in a line of neoplastic human fibroblasts. The amino acid and nucleotide sequences encoded by the unmutated gene predict that a guanine-to-adenine transition is responsible for the glycine-to-aspartic acid mutation at codon 244 and would also result in the loss of a HaeIII site. Detection of this HaeIII polymorphism among the fibroblast-derived closed verified the identity of the ..beta..-actin gene expressed in human fibroblasts.

  12. Molecular cloning and sequence analysis of the X-prolyl dipeptidyl aminopeptidase gene from Lactococcus lactis subsp. cremoris.

    PubMed Central

    Mayo, B; Kok, J; Venema, K; Bockelmann, W; Teuber, M; Reinke, H; Venema, G

    1991-01-01

    Lactococcus lactis subsp. cremoris P8-2-47 contains an X-prolyl dipeptidyl aminopeptidase (X-PDAP; EC 3.4.14.5). A mixed-oligonucleotide probe prepared on the basis of the N-terminal amino acid sequence of the purified protein was made and used to screen a partial chromosomal DNA bank in Escherichia coli. A partial XbaI fragment cloned in pUC18 specified X-PDAP activity in E. coli clones. The fragment was also able to confer X-PDAP activity on Bacillus subtilis. The fact that none of these organisms contain this enzymatic activity indicated that the structural gene for X-PDAP had been cloned. The cloned fragment fully restored X-PDAP activity in X-PDAP-deficient mutants of L. lactis. We have sequenced a 3.8-kb fragment that includes the X-PDAP gene and its expression signals. The X-PDAP gene, designated pepXP, comprises 2,289 nucleotide residues encoding a protein of 763 amino acids with a predicted molecular weight of 87,787. No homology was detected between pepXP and genes that had been previously sequenced. A second open reading frame, divergently transcribed, was present in the sequenced fragment; the function or relationship to pepXP of this open reading frame is unknown. Images PMID:1674655

  13. Molecular cloning and regulatory analysis of the arylsulfatase structural gene of Neurospora crassa.

    PubMed Central

    Paietta, J V

    1989-01-01

    The ars-1+ gene of Neurospora crassa encodes the enzyme arylsulfatase. ars-1+ is in a group of highly regulated sulfur-related structural genes that are expressed under conditions of sulfur limitation and are under coordinate control of the cys-3+ and scon+ regulatory genes. The ars-1+ gene was cloned by chromosome walking from the qa gene cluster, using a lambda library. Cotransformation of an N. crassa ars-1 mutant with the isolated lambda clones and the benomyl resistance gene, followed by assay for arylsulfatase activity, was used to screen for the ars-1+ gene. Further confirmation that the cloned segment mapped to the ars-1+ locus was obtained by restriction-fragment-length polymorphism analysis. Northern (RNA) blot analysis showed that the ars-1+ gene was transcribed to give an mRNA of 2.3 kilobases. In wild-type cells, the ars-1+ transcript was abundant under sulfur-derepressing conditions but absent under repressing conditions. Time course analysis showed that the appearance of ars-1+ message in sulfur-derepressed cultures paralleled the appearance of arylsulfatase enzyme activity. In addition, transcription of ars-1+ was detected only under derepressing conditions in a nuclear transcription assay. In a cys-3 regulatory mutant that was unable to synthesize arylsulfatase (or other sulfur-controlled enzymes), there was no ars-1+ transcript under repressing or derepressing conditions. In a temperature-sensitive cys-3 mutant, the ars-1+ transcript was present only at the permissive growth temperature and under sulfur derepression. A negative regulatory mutant, sconc, displayed both constitutive expression of arylsulfatase enzyme activity and content of ars-1+ message. Images PMID:2528685

  14. Molecular cloning of the human nucleotide-excision-repair gene ERCC4

    SciTech Connect

    Thompson, L.H.; Brookman, K.W.; Weber, C.A.; Salazar, E.P.; Reardon, J.T.; Sancar, A.; Deng, Z.; Siciliano, M.J.

    1994-07-19

    ERCC4 was previously identified in somatic cell hybrids as a human gene that corrects the nucleotide-excision-repair deficiency in mutant hamster cells. The cloning strategy for ERCC4 involved transfection of the repair-deficient hamster cell line UV41 with a human sCos-1 cosmid library derived from chromosome 16. Enhanced UV resistance was seen with one cosmid-library transformant and two secondary transformants of UV41. Cosmid clones carrying a functional ERCC4 gene were isolated from a library of a second transformant by selecting in Escherichia coli for expression of a linked neomycin-resistance gene that was present in the sCos-1 vector. The cosmids mapped to 16p13.13-p13.2, the location assigned to ERCC4 by using somatic cell hybrids. Upon transfection into UV41, six cosmid clones gave partial correction ranging from 30% to 64%, although all appeared to contain the complete gene. The capacity for in vitro excision of thymine dimers from a plasmid by transformant cell extracts correlated qualitatively with enhanced UV resistance.

  15. Molecular Cloning and Heterologous Expression of the Dehydrophos Biosynthetic Gene Cluster

    PubMed Central

    Circello, Benjamin T.; Eliot, Andrew C.; Lee, Jin-Hee; van der Donk, Wilfred A.; Metcalf, William W.

    2010-01-01

    Summary Dehydrophos is a vinyl phosphonate tripeptide produced by Streptomyces luridus with demonstrated broad spectrum antibiotic activity. To identify genes necessary for biosynthesis of this unusual compound we screened a fosmid library of S. luridus for the presence of the phosphoenolpyruvate mutase gene, which is required for biosynthesis of most phosphonates. Integration of one such fosmid clone into the chromosome of Streptomyces lividans led to heterologous production of dehydrophos. Deletion analysis of this clone allowed identification of the minimal contiguous dehydrophos cluster, which contained 17 open reading frames (ORFs). Bioinformatic analyses of these ORFs are consistent with a proposed biosynthetic pathway that generates dehydrophos from phosphoenolpyruvate. The early steps of this pathway are supported by analysis of intermediates accumulated by blocked mutants and in vitro biochemical experiments. PMID:20416511

  16. Molecular cloning, functional verification, and evolution of TmPm3, the powdery mildew resistance gene of Triticum monococcum L.

    PubMed

    Zhao, C Z; Li, Y H; Dong, H T; Geng, M M; Liu, W H; Li, F; Ni, Z F; Wang, X J; Xie, C J; Sun, Q X

    2016-01-01

    Powdery mildew (Pm) is one of the most harmful diseases in wheat. Three Pm-resistance genes, Pm3, Pm21, and Pm8, have been cloned but most Pm3/Pm8 alleles have lost their resistance to Pm in hexaploid wheat. In this study, a new Pm3 homolog gene (TmPm3) was isolated from Triticum monococcum L. using a homology-based cloning strategy, being the first report of a functional Pm3 homolog gene from a diploid wheat species. The transient expression of TmPm3 in leaf epidermal cells showed that over-expressed TmPm3 could significantly inhibit the penetration of Blumeria graminis f. sp tritici conidia spores and the formation of haustoria. Sequence analysis of Pm3 alleles shed new light on the evolution of Pm3 genes, providing a better understanding of the molecular basis of disease resistance. This study also suggested that homology-based cloning of resistance genes is a feasible method for the isolation of functional resistance genes from wheat germplasm. PMID:27173250

  17. Molecular cloning and expression of the ilvGEDAY genes from Salmonella typhimurium.

    PubMed

    Blazey, D L; Kim, R; Burns, R O

    1981-08-01

    The ilvGEDAY genes of Salmonella typhimurium were cloned in Escherichia coli K-12 by in vitro recombination techniques. A single species of recombinant plasmid, designated pDU1, was obtained by selecting for Valr Ampr transformants of strain SK1592. pDU1 was shown to contain a 14-kilobase EcoRI partial digestion product of the S. typhimurium chromosome inserted into the EcoRI site of the pVH2124 cloning vector. The ilvGEDAY genes were found to occupy a maximum length of 7.5 kilobases. Restriction endonuclease analysis of the S. typhimurium ilv gene cluster provided another demonstration of the gene order as well as established the location of ilv Y between ilvA and ilvC. The presence of a ribosomal ribonucleic acid operon on the pDU1 insert, about 3 kilobases from the 5' end of ilvG, was shown by Southern hybridization. The expression of the ilvGEDA operon from pDU1 was found to be elevated, reflecting the increased gene dosage of the multicopy plasmid. A polarity was observed with respect to ilvEDA expression which is discussed in terms of the possible translational effects of the two internal promoter sequences, one located proximal to ilvE and the other located proximal to ilvD. PMID:6167564

  18. Molecular cloning and high-level expression of a bromoperoxidase gene from Streptomyces aureofaciens Tü24.

    PubMed Central

    van Pée, K H

    1988-01-01

    A bromoperoxidase gene was cloned from Streptomyces aureofaciens Tü24 into Streptomyces lividans TK64 by using the promoter-probe vector pIJ486. Subcloning of DNA from the original, unstable clone allowed the gene to be localized to a 1.7-kilobase (kb) fragment of DNA. Southern blotting showed that the cloned 1.7-kb insert hybridized to a 4.3-kb fragment in an SstI digest of S. aureofaciens Tü24 total DNA. The 1.7-kb insert was shown to code for a protein with the electrophoretic properties of the subunits of the nonheme bromoperoxidase isolated from S. aureofaciens Tü24. The protein produced by S. lividans TK64 transformed with pHM621, which contained an 8.0-kb insert, was shown to be identical to the S. aureofaciens Tü24 bromoperoxidase in terms of its electrophoretic mobility on denaturing and nondenaturing polyacrylamide gels and its NH2-terminal amino acid sequence. The bromoperoxidase was overproduced (up to 180 times) by S. lividans TK64 containing pHM621. Based on the heat stability of the S. aureofaciens Tü24 bromoperoxidase, a new and simple purification procedure with very high yields was developed. Images PMID:3142859

  19. Cloning and molecular analysis of voraxin-α gene of Rhipicephalus (Boophilus) microplus.

    PubMed

    Kumar, Binod; Ghosh, Srikanta

    2016-03-01

    To identify suitable targets for development of cross-protective tick vaccine, in silico analysis was attempted and male tick derived molecule, voraxin-α was targeted. The voraxin-α homologue of Rhipicephalus (Boophilus) microplus was cloned, sequenced and analyzed employing standard methods. The deduced amino acids sequence analysis of the 419 bp cloned voraxin-α gene of R. (B.) microplus indicated very high (94.6 %) similarity with voraxin-α of the R. appendiculatus and moderate to low identity with Amblyomma hebraeum, Dermacentor silvarum and Haemaphysalis longicornis. The results suggest that recombinant voraxin-α might be a good candidate as cross-protective anti-tick vaccine. PMID:27065622

  20. Cloning and molecular analysis of poly(3-hydroxyalkanoate) biosynthesis genes in Pseudomonas aureofaciens.

    PubMed

    Nishikawa, Tomohiro; Ogawa, Keiko; Kohda, Ryoko; Zhixiong, Wang; Miyasaka, Hitoshi; Umeda, Fusako; Maeda, Isamu; Kawase, Masaya; Yagi, Kiyohito

    2002-02-01

    Pseudomonas aureofaciens grown on octanoate or gluconate synthesized medium-chain-length polyhydroxyalkanoates (mcl-PHAs). To clone the PHA synthase gene(s) (phaC), the genomic library of P. aureofaciens was constructed using a cosmid vector. The recombinant cosmids that clone phaC were detected by the complementation with a PHA-negative mutant, P. putida GPp104. The resulting recombinant cosmid, named pVK6, contained a 13-kbp DNA insert. Genetic analysis of the pha locus in pVK6 revealed the presence of six ORFs, genes encoding two PHA synthases, 1 and 2 (phaC1 and phaC2), PHA depolymerase (phaZ), two PHA granule-associated proteins (phaF and phaI), and an unknown protein (phaD). The heterologous expression of pha genes from P. aureofaciens was confirmed. P. putida GPp104 regained the ability to accumulate PHA on introduction of pVK6. Wild-type strains P. oleovorans and P. fluorescens, which were unable to accumulate PHA when grown on gluconate, acquired the ability to accumulate PHA from gluconate when they possessed pVK6. PMID:11815858

  1. Molecular cloning and expression of the calmodulin gene from guinea pig hearts

    PubMed Central

    FENG, RUI; LIU, YAN; SUN, XUEFEI; WANG, YAN; HU, HUIYUAN; GUO, FENG; ZHAO, JINSHENG; HAO, LIYING

    2015-01-01

    The aim of the present study was to isolate and characterize a complementary DNA (cDNA) clone encoding the calmodulin (CaM; GenBank accession no. FJ012165) gene from guinea pig hearts. The CaM gene was amplified from cDNA collected from guinea pig hearts and inserted into a pGEM®-T Easy vector. Subsequently, CaM nucleotide and protein sequence similarity analysis was conducted between guinea pigs and other species. In addition, reverse transcription-polymerase chain reaction (RT-PCR) was performed to investigate the CaM 3 expression patterns in different guinea pig tissues. Sequence analysis revealed that the CaM gene isolated from the guinea pig heart had ∼90% sequence identity with the CaM 3 genes in humans, mice and rats. Furthermore, the deduced peptide sequences of CaM 3 in the guinea pig showed 100% homology to the CaM proteins from other species. In addition, the RT-PCR results indicated that CaM 3 was widely and differentially expressed in guinea pigs. In conclusion, the current study provided valuable information with regard to the cloning and expression of CaM 3 in guinea pig hearts. These findings may be helpful for understanding the function of CaM3 and the possible role of CaM3 in cardiovascular diseases. PMID:26136979

  2. Molecular cloning and expression profiling of multiple Dof genes of Sorghum bicolor (L) Moench.

    PubMed

    Gupta, Shubhra; Arya, Gulab C; Malviya, Neha; Bisht, Naveen C; Yadav, Dinesh

    2016-08-01

    DNA binding with one finger (Dof) proteins represent a family of plant specific transcription factors associated with diverse biological processes, such as seed maturation and germination, phytohormone and light mediated regulation, and plant responses to biotic and abiotic stresses. In present study, a total of 21 Dof genes from Sorghum bicolor were cloned, sequenced and in silico characterized for homology search, revealing their identity to Dof like proteins. The expression profiling of SbDof genes using quantitative RT-PCR in different tissue types and also under drought and salt stresses was attempted. The SbDof genes displayed differential expression either in their transcript abundance or in their expression patterns under normal growth condition. Two of the SbDof genes namely SbDof8 and SbDof12 showed comparatively high level of transcript abundance in all the tissue types tested; whereas some of the SbDof genes showed a distinct tissue specific expression pattern. Further a total of 13 SbDof genes showed differential expression when subjected to either of the abiotic stress i.e. drought or salinity. Three of the SbDof genes namely SbDof12, SbDof19 and SbDof24 were found to be up-regulated in response to drought and salt stress. Comparative analysis of SbDof genes expression revealed existence of a complex transcriptional and functional diversity across plant growth and developmental stages. PMID:27230576

  3. Molecular Cloning and Expression of cor (Cold-Regulated) Genes in Arabidopsis thaliana1

    PubMed Central

    Hajela, Ravindra K.; Horvath, David P.; Gilmour, Sarah J.; Thomashow, Michael F.

    1990-01-01

    We have previously shown that changes in gene expression occur in Arabidopsis thaliana. L. (Heyn) during cold acclimation (SJ Gilmour, RK Hajela, MF Thomashow [1988] Plant Physiol 87: 745-750). Here we report the isolation of cDNA clones of four cold-regulated (cor) genes from Arabidopsis and examine their expression in response to low temperature, abscisic acid (ABA), water stress, and heat shock. The results of Northern analysis indicated that the transcript levels for the four cor genes, represented by clones pHH7.2, pHH28, pHH29, and pHH67, increased markedly between 1 and 4 hours of cold treatment, reached a maximum at about 8 to 12 hours, and remained at elevated levels for as long as the plants were kept in the cold (up to 2 weeks). Returning cold acclimated plants to control temperature resulted in the levels of the cor transcripts falling rapidly to those found in nonacclimated plants; this occurred within 4 hours for the transcripts represented by pHH7.2 and pHH28, and 8 hours for those represented by pHH29 and pHH67. Nuclear run-on transcription assays indicated that the temperature-regulated expression of the cor genes represented by pHH7.2, pHH28, and pHH29 was controlled primarily at the posttranscriptional level while the cor gene represented by pHH67 was regulated largely at the transcriptional level. Northern analysis also indicated that the levels of cor gene transcripts increased in response to both ABA application and water stress, but not to heat shock. The possible significance of cor genes being regulated by both low temperature and water stress is discussed. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:16667586

  4. Molecular cloning and analysis of the scon-2 negative regulatory gene of Neurospora crassa.

    PubMed Central

    Paietta, J V

    1990-01-01

    The sulfur regulatory system of Neurospora crassa is composed of a group of highly regulated structural genes (e.g., the gene encoding arylsulfatase) that are under coordinate control of scon+ (sulfur controller) negative and cys-3+ positive regulatory genes. In scon-1 (previously designated sconC) and scon-2 mutants, there is constitutive expression of sulfur structural genes regardless of the sulfur level available to the cells. The scon-2+ gene was cloned by sib selection screening of a cosmid-based gene library. The screening was based on the use of chromate, a toxic sulfate analog, which is transported into scon-2 cells grown on high sulfur but is not transported into cells that have regained normal sulfur regulation. Restriction fragment length polymorphism analysis was used to confirm that the cloned segment mapped to the proper chromosomal location. In wild-type cells, Northern (RNA) blot analysis showed that a 2.6-kilobase scon-2+ transcript was present at a substantial level only under sulfur-derepressing conditions. Kinetic analysis showed that scon-2+ mRNA content increased as the cells became sulfur starved. Further, scon-2+ RNA was detectable in a nuclear transcription assay only under derepressing conditions. In scon-1, the levels of scon-2+ mRNA were found to be constitutive. In the cys-3 regulatory mutant, there was a reduced level of scon-2+ transcript. cys-3+ and ars-1+ mRNAs were present under both derepressing and repressing conditions in the scon-2 mutant. Repeat-induced point mutation-generated scon-2 mutants were identical in phenotype to the known mutant. Images PMID:1975945

  5. Molecular cloning, expression, and evolution analysis of type II CHI gene from peanut (Arachis hypogaea L.).

    PubMed

    Liu, Yu; Zhao, Shuzhen; Wang, Jiangshan; Zhao, Chuanzhi; Guan, Hongshan; Hou, Lei; Li, Changsheng; Xia, Han; Wang, Xingjun

    2015-01-01

    Chalcone isomerase (CHI) plays critical roles in plant secondary metabolism, which is important for the interaction between plants and the environment. CHI genes are widely studied in various higher plants. However, little information about CHI genes is available in peanut. Based on conservation of CHI gene family, we cloned the peanut type II CHI gene (AhCHI II) cDNA and genome sequence. The amino acid sequence of peanut CHI II was highly homologous to type II CHI from other plant species. qRT-PCR results showed that peanut CHI II is mainly expressed in roots; however, peanut CHI I is mainly expressed in tissues with high content of anthocyanin. Gene duplication and gene cluster analysis indicated that CHI II was derived from CHI I 65 million years ago approximately. Our gene structure analysis results are not in agreement with the previous hypothesis that CHI II was derived from CHI I by the insertion of an intron into the first exon. Moreover, no positive selection pressure was found in CHIs, while, 32.1 % of sites were under neutral selection, which may lead to mutation accumulation and fixation during great changes of environment. PMID:25608978

  6. Molecular cloning and expression analysis of a novel BCCP subunit gene from Aleurites moluccana.

    PubMed

    Xuan, W Y; Zhang, Y; Liu, Z Q; Feng, D; Luo, M Y

    2015-01-01

    Aleurites moluccana L. is grown as a roadside tree in southern China and the oil content of its seed is higher than other oil plants, such as Jatropha curcas and Camellia oleifera. A. moluccana is considered a promising energy plant because its seed oil could be used to produce biodiesel and bio-jet fuel. In addition, the bark, leaves, and kernels of A. moluccana have various medical and commercial uses. Here, a novel gene coding the biotin carboxyl carrier protein subunit (BCCP) was cloned from A. moluccana L. using the homology cloning method combined with rapid amplification of cDNA end (RACE) technology. The isolated full-length cDNA sequence (designated AM-accB) was 1188 bp, containing a 795-bp open reading frame coding for 265 amino acids. The deduced amino acid sequence of AM-accB contained a biotinylated domain located between amino acids 190 and 263. A. moluccana BCCP shows high identity at the amino acid level to its homologues in other higher plants, such as Vernicia fordii, J. curcas, and Ricinus communis (86, 77, and 70%, respectively), which all contain conserved domains for ACCase activity. The expression of the AM-accB gene during the middle stage of development and maturation in A. moluccana seeds was higher than that in early and later stages. The expression pattern of the AM-accB gene is very similar to that of the oil accumulation rate. PMID:26345927

  7. Molecular cloning and characterization of the full-length Hsp90 gene from Matricaria recutita.

    PubMed

    Ling, S P; Su, S S; Zhang, H M; Zhang, X S; Liu, X Y; Pan, G F; Yuan, Y

    2014-01-01

    Heat shock protein 90 (Hsp90) is one of the most abundant and conserved chaperone proteins and plays important roles in plant growth and responses to environmental stimuli. However, little is known regarding the sequence and function of Hsp90s in Matricaria recutita. In the present study, we cloned the full-length cDNA sequence of the hsp90 gene from this species. Using rapid amplification of cDNA ends technologies with 2 degenerate primers that were designed based on the hsp90 gene sequence from other members of Asteraceae, we isolated and characterized an Hsp90 homolog gene from M. recutita (Mr-Hsp90). The full-length Mr-hsp90 cDNA sequence, containing 2097 base pairs, encodes a protein of 698 amino acids. Based on amino acid sequence identity, Mr-Hsp90 showed high similarity to other cloned Hsp90 proteins. The Mr-Hsp90 protein was closely clustered with the Lactuca sativa in a phylogenetic tree. These results indicate that the cloned sequence of Mr-Hsp90 is a member of the Hsp90 family, which is reported for the first time in M. recutita. Next, we conducted a salt stress experiment to determine the protein's function under salt stress conditions. Survival of chamomile seedlings subjected to heat-shock pretreatment was significantly increased compared with groups that had not undergone heat-shock pretreatment in a salt stress environment. This indicates that Mr-Hsp90 plays an important role in the salt resistance of chamomile seedlings. PMID:25526220

  8. Molecular cloning of allelopathy related genes and their relation to HHO in Eupatorium adenophorum.

    PubMed

    Guo, Huiming; Pei, Xixiang; Wan, Fanghao; Cheng, Hongmei

    2011-10-01

    In this study, conserved sequence regions of HMGR, DXR, and CHS (encoding 3-hydroxy-3-methylglutaryl-CoA reductase, 1-deoxyxylulose-5-phosphate reductoisomerase and chalcone synthase, respectively) were amplified by reverse transcriptase (RT)-PCR from Eupatorium adenophorum. Quantitative real-time PCR showed that the expression of CHS was related to the level of HHO, an allelochemical isolated from E. adenophorum. Semi-quantitative RT-PCR showed that there was no significant difference in expression of genes among three different tissues, except for CHS. Southern blotting indicated that at least three CHS genes are present in the E. adenophorum genome. A full-length cDNA from CHS genes (named EaCHS1, GenBank ID: FJ913888) was cloned. The 1,455 bp cDNA contained an open reading frame (1,206 bp) encoding a protein of 401 amino acids. Preliminary bioinformatics analysis of EaCHS1 revealed that EaCHS1 was a member of CHS family, the subcellular localization predicted that EaCHS1 was a cytoplasmic protein. To the best of our knowledge, this is the first report of conserved sequences of these genes and of a full-length EaCHS1 gene in E. adenophorum. The results indicated that CHS gene is related to allelopathy of E. adenophorum. PMID:21127986

  9. Molecular cloning and expression of an isomalto-dextranase gene from Arthrobacter globiformis T6.

    PubMed Central

    Iwai, A; Ito, H; Mizuno, T; Mori, H; Matsui, H; Honma, M; Okada, G; Chiba, S

    1994-01-01

    The gene encoding an extracellular isomalto-dextranase, designated imd, was isolated from the chromosomal DNA of Arthrobacter globiformis T6 and cloned and expressed in Escherichia coli. A single open reading frame consisting of 1,926 base pairs that encoded a polypeptide composed of a signal peptide of 39 amino acids and a mature protein of 602 amino acids (M(r), 65,900) was found. The primary structure had no significant homology with the structures of any other reported carbohydrases, including two other dextranases. Transformed E. coli cells carrying the 2.3-kb fragment overproduced isomalto-dextranase into the periplasmic space under control of the promoter of the imd gene itself. Images PMID:8002600

  10. Molecular cloning of a recA-like gene from the cyanobacterium Anabaena variabilis

    SciTech Connect

    Owttrim, G.W.; Coleman, J.R.

    1987-05-01

    A recA-like gene isolated from the cyanobacterium Anabaena variabilis was cloned and partially characterized. When introduced into Escherichia coli recA mutants, the 7.5-kilobase-pair plasmid-borne DNA insert restored resistance to methyl methanesulfonate and UV irradiation, as well as recombination proficiency when measured by Hfr-mediated conjugation. The cyanobacterial recA gene restored spontaneous but not mitomycin C-induced prophage production. Restriction analysis and subcloning yielded a 1.5-kilobase-pair Sau3A fragment which also restored methylmethane sulfonate resistance and coded for a 38- to 40-kilodalton polypeptide when expressed in an in vitro transcription-translation system.

  11. Molecular cloning and expression of the KIF3A gene in the frog brain and testis.

    PubMed

    Nakajima, T; Miura, I; Kashiwagi, A; Nakamura, M

    1997-12-01

    KIF3A is a member of the kinesin superfamily proteins (KIFs), but its gene has been cloned only in mouse and sea urchin. We have cloned a homolog of KIF3A from the frog, Rana rugosa (rrKIF3A). The sequence encoded a 699 amino acid protein that shares 93% similarity with mouse KIF3A (mKIF3A) and 69% with sea urchin kinesin-related protein (SpKRP85). The putative ATP-binding domain was completely identical to that of mKIF3A and SpKRP85. The level of rrKIF3A mRNA appeared to be high in the brain and testis of adult frogs, but low in the heart, lung and kidney. The results suggest that the rrKIF3A gene is expressed in the brain and testis more than other tissues of adult frogs examined, and that KIF3A is widely distributed in eukaryotic organisms. PMID:9520632

  12. Molecular cloning of the perilipin gene and its association with carcass and fat traits in Chinese ducks.

    PubMed

    Zhang, H L; Fan, H J; Liu, X L; Wu, Y; Hou, S S

    2013-01-01

    The perilipin (PLIN) gene is a candidate gene of carcass and fat traits in ducks. In order to study the molecular character of the PLIN gene and its function in different breeds of Chinese ducks, samples were obtained from the Chinese Academy of Agricultural Sciences Research Center for Birds, including 95 Peking ducks of the Z2 series, 91 Peking ducks of the Z4 series, 82 hybrid systems (Z2 x Z4), and 93 Cherry Valley ducks. We used RT-PCR and 3'-RACE to clone the duck PLIN gene, detect SNPs and analyze their associations with carcass and fat traits. A 2212-bp sequence was cloned with the complete coding region and a 3'-untranslated region. We found a nucleotide mutation (C → T) in exon 2 of the PLIN gene. There were no significant correlations between the 3 genotypes (CC, CT, TT) in breast muscle weight (BMW), leg muscle weight (LMW), subcutaneous fat weight (SFW), and intramuscular fat (IMF) in the Cherry Valley duck. The CC and CT genotypes had significant differences in carcass weight (CW), carcass net weight (CNW), and percentage of abdominal fat weight (AFW); there were significant differences in AFW and percentage of SFW. In Z4, there were no significant correlations between the 3 genotypes (TT, CC, and CT) in CW, BMW, LMW, SFW, AFW, the percentage of SFW and AFW, and IMF. CNW was significantly different between TT, CC, and CT genotypes. In Z2 x Z4, there were no significant correlations between the 3 genotypes in CW, BMW, LMW, SFW, AFW, the percentage of SFW and AFW, and IMF, while the CC and CT genotypes had significant differences in CNW. In Z2, there were no significant differences between the 3 genotypes in all traits. We deduced that the PLIN gene is a potential major gene. It is linked to a major gene affecting meat quality traits. This SNP has potential as a molecular marker for marker-assisted selection. PMID:23765965

  13. Molecular cloning and long terminal repeat sequences of human endogenous retrovirus genes related to types A and B retrovirus genes

    SciTech Connect

    Ono, M.

    1986-06-01

    By using a DNA fragment primarily encoding the reverse transcriptase (pol) region of the Syrian hamster intracisternal A particle (IAP; type A retrovirus) gene as a probe, human endogenous retrovirus genes, tentatively termed HERV-K genes, were cloned from a fetal human liver gene library. Typical HERV-K genes were 9.1 or 9.4 kilobases in length, having long terminal repeats (LTRs) of ca. 970 base pairs. Many structural features commonly observed on the retrovirus LTRs, such as the TATAA box, polyadenylation signal, and terminal inverted repeats, were present on each LTR, and a lysine (K) tRNA having a CUU anticodon was identified as a presumed primer tRNA. The HERV-K LTR, however, had little sequence homology to either the IAP LTR or other typical oncovirus LTRs. By filter hybridization, the number of HERV-K genes was estimated to be ca. 50 copies per haploid human genome. The cloned mouse mammary tumor virus (type B) gene was found to hybridize with both the HERV-K and IAP genes to essentially the same extent.

  14. Molecular cloning of the mouse CCK gene: expression in different brain regions and during cortical development.

    PubMed Central

    Vitale, M; Vashishtha, A; Linzer, E; Powell, D J; Friedman, J M

    1991-01-01

    In this paper we describe experiments that address specific issues concerning the regulation of the mouse cholecystokinin gene in brain and intestine. The mouse cholecystokinin gene was cloned and sequenced. Extensive homology among the mouse, man and rat genes was noted particularly in the three exons and the regions upstream of the RNA start site. RNAse protection assays for each of the three exons were used to demonstrate that CCK is expressed in only a subset of tissues and that the same cap site and splice choices are used in brain, intestine as well as in cerebellum, cortex, midbrain, hypothalamus and hippocampus. CCK RNA was also noted to be detectable in kidney. Thus the same gene using the same promoter is expressed in subsets of cells that differ in their biochemical, morphologic and functional characteristics. The level of expression of CCK was also monitored during mouse cortical development and the appearance of CCK RNA was compared to glutamate decarboxylase (GAD), enkephalin and somatostatin. It was noted that each of these cortical markers was first expressed at different times during cortical development. The appearance of CCK RNA during intestinal development was also measured and found to precede appearance in cortex by several days. Images PMID:2011497

  15. Molecular cloning, characterization and expression of the energy homeostasis-associated gene in piglet*

    PubMed Central

    Wang, Sheng-ping; Gao, Yun-ling; Liu, Gang; Deng, Dun; Chen, Rong-jun; Zhang, Yu-zhe; Li, Li-li; Wen, Qing-qi; Hou, Yong-qing; Feng, Ze-meng; Guo, Zhao-hui

    2015-01-01

    The energy homeostasis-associated (Enho) gene encodes a secreted protein, adropin, which regulates the expression of hepatic lipogenic genes and adipose tissue peroxisome proliferator-activated receptor γ, a major regulator of lipogenesis. In the present study, the porcine (Sus scrofa) homologue of the Enho gene, which was named pEnho, was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using oligonucleotide primers derived from in silico sequences. The gene sequence was submitted into the GenBank of NCBI, and the access number is GQ414763. The pEnho encodes a protein of 76 amino acids which shows 75% similarity to Homo sapiens adropin. The expression profile of pEnho in tissues (liver, muscle, anterior jejunum, posterior jejunum, and ileum) was determined by quantitative real-time RT-PCR. pEnho was localized on porcine chromosome 10 and no introns were found. In conclusion, pEnho was cloned and analysed with the aim of increasing knowledge about glucose and lipid metabolism in piglets and helping to promote the health and growth of piglets through adropin regulation. PMID:26055914

  16. Molecular cloning, characterization and expression of the energy homeostasis-associated gene in piglet.

    PubMed

    Wang, Sheng-ping; Gao, Yun-ling; Liu, Gang; Deng, Dun; Chen, Rong-jun; Zhang, Yu-zhe; Li, Li-li; Wen, Qing-qi; Hou, Yong-qing; Feng, Ze-meng; Guo, Zhao-hui

    2015-06-01

    The energy homeostasis-associated (Enho) gene encodes a secreted protein, adropin, which regulates the expression of hepatic lipogenic genes and adipose tissue peroxisome proliferator-activated receptor γ, a major regulator of lipogenesis. In the present study, the porcine (Sus scrofa) homologue of the Enho gene, which was named pEnho, was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using oligonucleotide primers derived from in silico sequences. The gene sequence was submitted into the GenBank of NCBI, and the access number is GQ414763. The pEnho encodes a protein of 76 amino acids which shows 75% similarity to Homo sapiens adropin. The expression profile of pEnho in tissues (liver, muscle, anterior jejunum, posterior jejunum, and ileum) was determined by quantitative real-time RT-PCR. pEnho was localized on porcine chromosome 10 and no introns were found. In conclusion, pEnho was cloned and analysed with the aim of increasing knowledge about glucose and lipid metabolism in piglets and helping to promote the health and growth of piglets through adropin regulation. PMID:26055914

  17. [Molecular cloning and characterization of a novel ice gene from Capsella bursapastoris].

    PubMed

    Wang, Xinglong; Sun, Xiaoqing; Liu, Sixiu; Liu, Li; Liu, Xiaojun; Sun, Xiaofen; Tang, Kexuan

    2005-01-01

    A new ice gene (designated as Cbice53, an inducer of CBF expression) was cloned from Capsella bursa-pastoris by rapid amplification of cDNA ends (RACE). The full-length cDNA of Cbice53 was 1811 bp long with a 1476 bp open reading frame (ORF) encoding a Myc-like protein of 492 amino acids. The predicted CbICE53 protein contained a potential basic helix-loop-helix, a nuclear localization signal (NLS), a RNA-binding regions RGG box, N-glycosylation and kinase phosphorylation sites. Bioinformatic analysis revealed that CbICE53 was highly homologous to ICE1 from Arabidopsis thaliana. Transcription of Cbice53 gene was induced transiently during salt and cold treatments, suggesting that it was involved in someway in cold-acclimation process. Our study implies that the Cbice53 gene is a new member of the ice gene family and may exert functions in cold- and salt-responsiveness in C. bursa-pastoris. PMID:15773544

  18. Molecular Cloning and Characterization of a New C-type Lysozyme Gene from Yak Mammary Tissue

    PubMed Central

    Jiang, Ming Feng; Hu, Ming Jun; Ren, Hong Hui; Wang, Li

    2015-01-01

    Milk lysozyme is the ubiquitous enzyme in milk of mammals. In this study, the cDNA sequence of a new chicken-type (c-type) milk lysozyme gene (YML), was cloned from yak mammary gland tissue. A 444 bp open reading frames, which encodes 148 amino acids (16.54 kDa) with a signal peptide of 18 amino acids, was sequenced. Further analysis indicated that the nucleic acid and amino acid sequences identities between yak and cow milk lysozyme were 89.04% and 80.41%, respectively. Recombinant yak milk lysozyme (rYML) was produced by Escherichia coli BL21 and Pichia pastoris X33. The highest lysozyme activity was detected for heterologous protein rYML5 (M = 1,864.24 U/mg, SD = 25.75) which was expressed in P. pastoris with expression vector pPICZαA and it clearly inhibited growth of Staphylococcus aureus. Result of the YML gene expression using quantitative polymerase chain reaction showed that the YML gene was up-regulated to maximum at 30 day postpartum, that is, comparatively high YML can be found in initial milk production. The phylogenetic tree indicated that the amino acid sequence was similar to cow kidney lysozyme, which implied that the YML may have diverged from a different ancestor gene such as cow mammary glands. In our study, we suggest that YML be a new c-type lysozyme expressed in yak mammary glands that plays a role as host immunity. PMID:26580446

  19. Molecular cloning of copper resistance genes from Pseudomonas syringae pv. tomato

    SciTech Connect

    Bender, C.L.; Cooksey, D.A.

    1987-02-01

    A cosmid library of copper-resistant (Cu/sup r/) Psuedomonas syringe pv. tomato PT23 plasmid DNA was constructed and mobilized into the copper-sensitive recipient P. syringae pv. syringae PS61. One resultant cosmid clone, pCOP1 (46 kilobases), conferred copper resistance. The PT23 Cu/sup r/ gene(s) was located on pCOP1 by subcloning PstI restriction endonuclease fragments of pCOP1 in the broad-host-range vector pRK404. A subclone containing a 4.4-kilobase PstI fragment conferred Cu/sup r/ on PS61. The Cu/sup r/ gene(s0 was further located by insertional inactivation with Tn5. A subcloned fragment internal to the Cu/sup r/ determinant on pCOP2 was probed to plasmid and chromosomal DNA of four copper-resistant and three copper-sensitive strains of P. syringae pv. tomato. The probe hybridized to plasmids in resistant strains, but showed no detectable homology to copper-sensitive strains.

  20. Molecular cloning, sequence analysis and tissue-specific expression of Akirin2 gene in Tianfu goat.

    PubMed

    Ma, Jisi; Xu, Gangyi; Wan, Lu; Wang, Nianlu

    2015-01-01

    The Akirin2 gene is a nuclear factor and is considered as a potential functional candidate gene for meat quality. To better understand the structures and functions of Akirin2 gene, the cDNA of the Tianfu goat Akirin2 gene was cloned. Sequence analysis showed that the Tianfu goat Akirin2 cDNA full coding sequence (CDS) contains 579bp nucleotides that encode 192 amino acids. A phylogenic tree of the Akirin2 protein sequence from the Tianfu goat and other species revealed that the Tianfu goat Akirin2 was closely related with cattle and sheep Akirin2. RT-qPCR analysis showed that Akirin2 was expressed in the myocardium, liver, spleen, lung, kidney, leg muscle, abdominal muscle and the longissimus dorsi muscle. Especially, high expression levels of Akirin2 were detected in the spleen, lung, and kidney whereas lower expression levels were seen in the liver, myocardium, leg muscle, abdominal muscle and longissimus dorsi muscle. Temporal mRNA expression showed that Akirin2 expression levels in the longissimus dorsi muscle, first increased then decreased from day 1 to month 12. Western blotting results showed that the Akirin2 protein was only detected in the lung and three skeletal muscle tissues. PMID:25239665

  1. Cloning and molecular characterization of the Chinese hamster ERCC2 nucleotide excision repair gene

    SciTech Connect

    Kirchner, J.M.; Salazar, E.P.; Lamerdin, J.E.

    1994-10-01

    The Chinese hamster ERCC2 nucleotide excision repair gene, encoding a presumed ATP-dependent DNA helicase, was cloned from the V79 cell line, and its nucleotide sequence was determined. The {approximately}15-kb gene comprises 23 exons with a 2283-base open reading frame. The predicted 760-amino-acid protein is 98% identical to the human ERCC2/EXP (760 amino acids), 51% identical to the Saccharomyces cerevisiae RAD3 (778 amino acids), and 54% identical to the Schizosaccharomyces pombe rad15 (772 amino acids) proteins. The promoter region of the hamster ERCC2 gene contains a pyrimidine-rich stretch (42 nucleotides, 88% C+T) similar to sequences found in the promoter regions of two other nucleotide excision repair genes, a GC box, a putative {alpha}-Pal transcription factor binding site, and two CAAT boxes. There is no apparent TAATA box. No consensus polyadenylation sequence (AATAAA or its variants) was found with 663 bases 3{prime} of the translation termination codon. 54 refs., 2 figs., 2 tabs.

  2. Analysis and molecular cloning of genes involved in thiophene and furan oxidation by Escherichia coli

    SciTech Connect

    Alam, K.Y.; Worland, M.J.; Clark, D.P.

    1989-01-01

    Alternative methods for the desulfurization of coal are currently needed. The microbial removal of organic sulfur from coal is addressed in this issue by attempting to construct by genetic means, strains of bacteria which can degrade thiophenes and related organic sulfur compounds. Our first attempts in this direction have resulted in the isolation of a series of mutant strains of Escherchia coli with successively increased oxidizing ability towards furan and thiophene compounds. Three novel genes involved in thiophene oxidation, thdA, thdC, and thdD, were identified and mapped on the E. coli chromosome. In addition, mutations in two previously known regulatory genes fadR and atoC were also required. Further work resulted in more accurate mapping of thdA and thdD relative to known chromosomal genes and the isolation of a further mutation, thdE, so far unmapped. This conference presentation reviews some more recent findings, including the cloning of several genes involved in thiophene metabolism. 23 refs., 2 figs., 5 tabs.

  3. Molecular cloning, sequencing and expression of a serine proteinase inhibitor gene from Toxoplasma gondii.

    PubMed

    Pszenny, V; Angel, S O; Duschak, V G; Paulino, M; Ledesma, B; Yabo, M I; Guarnera, E; Ruiz, A M; Bontempi, E J

    2000-04-15

    A cDNA clone from a Toxoplasma gondii tachyzoite cDNA library encoding a serine proteinase inhibitor (serpin) was isolated. The 1376 bp cDNA sequence encodes a 294 amino acid protein with a putative signal peptide of 23 amino acids resulting in a mature protein with a predicted mass of 30,190 Da and a pI of 4.86. This protein has internal sequence similarity of residues 30-66, 114-150, 181-217 and 247-283 indicating a four-domain structure. The four domains exhibit high identity to serine proteinase inhibitors belonging to the non-classical Kazal-type family. The gene is single copy in the tachyzoite haploid genome of RH strain and was amplified by polymerase chain reaction (PCR). Several introns were identified. The sequence encoding the mature protein was amplified by PCR, cloned into the pQE30 vector and expressed in Escherichia coli. Specific antiserum generated against the recombinant protein was used in immunoblot assay and two bands of 38 and 42 kDa were detected in a whole parasite homogenate. The recombinant protein showed trypsin-inhibitory activity, one of the two potential specificities. We discuss the possible roles that T. gondii serpin(s) may play in the survival of the tachyzoites in the host. PMID:10779600

  4. Molecular cloning and characterization of the Dicer-like 2 gene from Brassica rapa.

    PubMed

    Yan, Fei; Peng, Jiejun; Lu, Yuwen; Lin, Lin; Zheng, Hongying; Chen, Hairu; Chen, Jianping; Adams, Michael J

    2009-07-01

    Dicer-like proteins (DCLs) are involved in small RNA-mediated development and viral defense in plants. In model plants, at least four DCLs have been found and a number of studies have helped to understand their function. However, the function of the Dicer or DCLs in other plants is still unclear. Here, we report the full-length cDNA sequence of Brassica rapa ssp. chinensis DCL2 (BrDCL2) gene, which contains a 4,179 bp open reading frame (ORF) encoding a protein of 1,392 amino acids. At the 3' end of BrDCL2, clones with three different lengths of 3' untranslated region were found. An alternative splice variant of BrDCL2, BrDCL2sv, in which one intron was retained between exon9 and exon10, was also cloned. Because of a change in the coding sequence resulting in a premature terminal codon, BrDCL2sv was expected to translate a short peptide containing the whole DEXHc domain. PMID:18607769

  5. Arabidopsis genes essential for seedling viability: isolation of insertional mutants and molecular cloning.

    PubMed Central

    Budziszewski, G J; Lewis, S P; Glover, L W; Reineke, J; Jones, G; Ziemnik, L S; Lonowski, J; Nyfeler, B; Aux, G; Zhou, Q; McElver, J; Patton, D A; Martienssen, R; Grossniklaus, U; Ma, H; Law, M; Levin, J Z

    2001-01-01

    We have undertaken a large-scale genetic screen to identify genes with a seedling-lethal mutant phenotype. From screening approximately 38,000 insertional mutant lines, we identified >500 seedling-lethal mutants, completed cosegregation analysis of the insertion and the lethal phenotype for >200 mutants, molecularly characterized 54 mutants, and provided a detailed description for 22 of them. Most of the seedling-lethal mutants seem to affect chloroplast function because they display altered pigmentation and affect genes encoding proteins predicted to have chloroplast localization. Although a high level of functional redundancy in Arabidopsis might be expected because 65% of genes are members of gene families, we found that 41% of the essential genes found in this study are members of Arabidopsis gene families. In addition, we isolated several interesting classes of mutants and genes. We found three mutants in the recently discovered nonmevalonate isoprenoid biosynthetic pathway and mutants disrupting genes similar to Tic40 and tatC, which are likely to be involved in chloroplast protein translocation. Finally, we directly compared T-DNA and Ac/Ds transposon mutagenesis methods in Arabidopsis on a genome scale. In each population, we found only about one-third of the insertion mutations cosegregated with a mutant phenotype. PMID:11779813

  6. Molecular cloning, structural analysis, and expression in Escherichia coli of a chitinase gene from Enterobacter agglomerans.

    PubMed Central

    Chernin, L S; De la Fuente, L; Sobolev, V; Haran, S; Vorgias, C E; Oppenheim, A B; Chet, I

    1997-01-01

    The gene chiA, which codes for endochitinase, was cloned from a soilborne Enterobacter agglomerans. Its complete sequence was determined, and the deduced amino acid sequence of the enzyme designated Chia_Entag yielded an open reading frame coding for 562 amino acids of a 61-kDa precursor protein with a putative leader peptide at its N terminus. The nucleotide and polypeptide sequences of Chia_Entag showed 86.8 and 87.7% identity with the corresponding gene and enzyme, Chia_Serma, of Serratia marcescens, respectively. Homology modeling of Chia_Entag's three-dimensional structure demonstrated that most amino acid substitutions are at solvent-accessible sites. Escherichia coli JM109 carrying the E. agglomerans chiA gene produced and secreted Chia_Entag. The antifungal activity of the secreted endochitinase was demonstrated in vitro by inhibition of Fusarium oxysporum spore germination. The transformed strain inhibited Rhizoctonia solani growth on plates and the root rot disease caused by this fungus in cotton seedlings under greenhouse conditions. PMID:9055404

  7. Molecular cloning and characterization analysis of immunoglobulin M heavy chain gene in European eel (Anguilla anguilla).

    PubMed

    Feng, Jianjun; Guan, Ruizhang; Lin, Peng; Guo, Songlin

    2009-01-15

    In this study, the immunoglobulin M heavy chain gene of European eel (Anguilla anguilla) was cloned and analyzed. The full-length cDNA of the IgM heavy chain gene (GenBank accession no. EF062515) has 2089 nucleotides encoding a putative protein of 581 amino acids. The IgM heavy chain was composed of leader peptide (L), variable domain (VH), CH1, CH2, Hinge, CH3, CH4, and C-terminus and two novel continuous putative N-glycosylation sites were found close to the second cysteine of CH3 in A. anguilla-H1 and A. anguilla-H2. The deduced amino acid sequence of the European eel IgM heavy chain constant region shared similarities to that of the Ladyfish (Elops saurus), Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), Grass carp (Ctenopharingodon idella), Common carp (Cyprinus carpio), Channel catfish (Ictalurus punctatus), and the orange-spotted grouper (Epinephelus coioides) with the identity of 46.1%, 39.7%, 38.9%, 32.4%, 32.3%, 31.7%, and 30.7%, respectively. The highest level of IgM gene expression was observed in the kidney, followed by the spleen, gills, liver, muscle and heart in the apparently healthy European eels. PMID:19013650

  8. Molecular cloning of a pepper gene that is homologous to SELF-PRUNING.

    PubMed

    Kim, Dong Hwan; Han, Myeong Suk; Cho, Hyun Wooh; Jo, Yeong Deuk; Cho, Myeong Cheoul; Kim, Byung-Dong

    2006-08-31

    "Determinate" and "indeterminate" inflorescences in plants are controlled by a single recessive gene, for example, SELF-PRUNING (SP) in Solanum lycopersicum, TERMINAL FLOWER1 in Arabidopsis, CENTRORADI-ALIS in Antirrhinum, and CENTRORADIALIS-like gene in tobacco. Pepper (Capsicum annuum L.) is an indeterminate species in which shoots grow indefinitely. In this study, we cloned and characterized the pepper SP-like gene (CaSP). RT-PCR revealed that the CaSP transcript accumulates to higher levels in floral buds than in other organs. Comparison of genomic DNA and cDNA sequences from indeterminate and determinate pepper plants revealed the insertion of a single base in the first exon of CaSP in the determinate pepper plants. CaSP is annotated in linkage group 8 (chromosome 6) of the SNU2 pepper genetic map and showed similar synteny to SP in tomato. Transgenic tobacco plants overexpressing CaSP displayed late-flowering phenotypes similar to the phenotypes caused by overexpression of CaSP orthologs in other plants. Collectively, these results suggest that pepper CaSP is an ortholog of SP in tomato. PMID:16951555

  9. Molecular cloning, characterization and expression analysis of the complement component C6 gene in grass carp.

    PubMed

    Shen, Yu-Bang; Zhang, Jun-Bin; Xu, Xiao-Yan; Li, Jia-Le

    2011-05-15

    The complement system, as a representative of innate immunity, plays a key role in the host defense against infections. C6 is the member of complement components creating the membrane attack complex (MAC). In this study, we cloned and characterized the grass carp complement component C6 (gcC6) gene. Our data showed that gcC6 gene contained a 2724bp open reading frame (ORF), a 237bp 5'-untranslated region (UTR) and a 219bp 3'-UTR. The deduced amino acid sequence of gcC6 showed 77.6% and 58.9% identity to zebrafish C6 and rainbow trout C6, respectively. GcC6 gene was expressed in a wide range of grass carp tissues, and the highest expression level of gcC6 was detected in the spleen and liver. Upon challenge with Aeromonas hydrophila, its expression was significantly up-regulated in muscle, trunk kidney, liver, head kidney, spleen, heart and intestine, whereas it was down-regulated in the brain and skin. The expression level of gcC6 was high at the unfertilized egg stage. It was significantly increased at 1 day post-hatching, but it was decreased at 10 days post-hatching. This result suggested that the complement C6 transcripts in early embryos were of maternal origin. PMID:21353312

  10. Molecular cloning and functional analysis of Photobacterium damselae subsp. piscicida haem receptor gene.

    PubMed

    Naka, H; Hirono, I; Aoki, T

    2005-02-01

    A haem receptor gene from Photobacterium damselae subsp. piscicida (formerly known as Pasteurella piscicida) has been cloned, sequenced and analysed for its function. The gene, designated as pph, has an open reading frame consisting of 2154 bp, a predicted 718 amino acid residues and exists as a single copy. It is homologous with the haem receptors of Vibrio anguillarum hupA, V. cholerae hutA, V. mimicus mhuA and V. vulnificus hupA at 32.7, 32.7, 45.6 and 30.9%, respectively, and is highly conserved, consisting of a Phe-Arg-Ala-Pro sequence (FRAP), an iron transport related molecule (TonB) and a Asn-Pron-Asn-Leu sequence (NPNL), binding motifs associated with haem receptors. As a single gene knockout mutant P. damselae subsp. piscicida was able to bind haem in the absence of pph, suggesting that other receptors may be involved in its iron transport system. This study shows that the P. damselae subsp. piscicida pph belongs to the haem receptor family, is conserved and that its iron-binding system may involve more than one receptor. PMID:15705153

  11. Molecular cloning and expression analysis of the STAT1 gene from olive flounder, Paralichthys olivaceus

    PubMed Central

    Park, Eun-Mi; Kang, Jung-Ha; Seo, Jung Soo; Kim, GunDo; Chung, Jongkyeong; Choi, Tae-Jin

    2008-01-01

    Background Signal transducer and activator of transcription 1 (STAT1) is a critical component of interferon (IFN)-alpha/beta and IFN-gamma signaling. Although seven isoforms of STAT proteins have been reported from mammals, limited information is available for the STAT genes in fish. We isolated complementary DNA with high similarity to mammalian STAT1 from the olive flounder, Paralichthys olivaceus. Results A DNA fragment containing the conserved SH2 domain was amplified by RT-PCR using degenerate primers designed based on the highly conserved sequences in the SH2 domains of the zebrafish and mammalian STAT1. The complete cDNA sequence was obtained by 5' and 3' RACE. The flounder STAT1 transcript consisted of 2,909 bp that encoded a polypeptide of 749 amino acids. The overall similarity between flounder STAT1 and other STATs was very high, with the highest amino acid sequence identity to snakehead (89%). Phylogenetic analyses reveal that flounder STAT1 is in the same monophyletic group with snakehead STAT1. Quantitative real time RT-PCR and in situ hybridization revealed that STAT1 was expressed in almost all examined organs and tissues, with high expression in gill, spleen, kidney, and heart. The accumulation of STAT1 mRNA in different developmental stages, as determined by real time RT-PCR, increased with development. Conclusion Recent cloning of various cytokine genes and the STAT1 gene of olive flounder here suggest that fish also use the highly specialized JAK-STAT pathway for cytokine signaling. Identification of other STAT genes will elucidate in detail the signal transduction system in this fish. PMID:18578892

  12. Molecular cloning and characterization of an S-adenosylmethionine synthetase gene from Chorispora bungeana.

    PubMed

    Ding, Chenchen; Chen, Tao; Yang, Yu; Liu, Sha; Yan, Kan; Yue, Xiule; Zhang, Hua; Xiang, Yun; An, Lizhe; Chen, Shuyan

    2015-11-10

    S-adenosylmethionine synthetase (SAMS) catalyzes the formation of S-adenosylmethionine (SAM) which is a molecule essential for polyamines and ethylene biosynthesis, methylation modifications of protein, DNA and lipids. SAMS also plays an important role in abiotic stress response. Chorispora bungeana (C. bungeana) is an alpine subnival plant species which possesses strong tolerance to cold stress. Here, we cloned and characterized an S-adenosylmethionine synthetase gene, CbSAMS (C. bungeana S-adenosylmethionine synthetase), from C. bungeana, which encodes a protein of 393 amino acids containing a methionine binding motif GHPDK, an ATP binding motif GAGDQG and a phosphate binding motif GGGAFSGDK. Furthermore, an NES (nuclear export signal) peptide was identified through bioinformatics analysis. To explore the CbSAMS gene expression regulation, we isolated the promoter region of CbSAMS gene 1919bp upstream the ATG start codon, CbSAMSp, and analyzed its cis-acting elements by bioinformatics method. It was revealed that a transcription start site located at 320 bp upstream the ATG start codon and cis-acting elements related to light, ABA, auxin, ethylene, MeJA, low temperature and drought had been found in the CbSAMSp sequence. The gene expression pattern of CbSAMS was then analyzed by TR-qPCR and GUS assay method. The result showed that CbSAMS is expressed in all examined tissues including callus, roots, petioles, leaves, and flowers with a significant higher expression level in roots and flowers. Furthermore, the expression level of CbSAMS was induced by low temperature, ethylene and NaCl. Subcellular localization revealed that CbSAMS was located in the cytoplasm and nucleus but has a significant higher level in the nucleus. These results indicated a potential role of CbSAMS in abiotic stresses and plant growth in C. bungeana. PMID:26205258

  13. Molecular cloning and mRNA expression analysis of sheep MYL3 and MYL4 genes.

    PubMed

    Zhang, Chunlan; Wang, Jianmin; Wang, Guizhi; Ji, Zhibin; Hou, Lei; Liu, Zhaohua; Chao, Tianle

    2016-02-15

    Using longissimus dorsi muscles of Dorper sheep as the experimental materials, the complete cDNAs of ovine MYL3 (Myosin light chain 3) and MYL4 (Myosin light chain 4) genes were cloned using RT-PCR, 5' RACE and 3' RACE. We obtained 925-bp and 869-bp full-length cDNAs and submitted their sequences to GenBank as accession numbers of KJ710703 and KJ768855, respectively. The cDNAs contained 600-bp and 582-bp open reading frames (ORFs) and encoded proteins comprising 199 and 193 amino acid residues, respectively. Neither protein was predicted to have a signal peptide, but both were predicted to have several N-glycosylation, O-glycosylation, and phosphorylation sites. The secondary structures of MYL3 and MYL4 were predicted to be 40.70% and 48.70% α- helical, respectively. Sequence alignment showed that the MYL3 and MYL4 proteins of Ovis aries both shared more than 91% amino acid sequence similarity with those of Mus musculus, Homo sapiens, Rattus norvegicus, Bos taurus, and Sus scrofa. The levels of MYL3 and MYL4 mRNA in various sheep tissues were determined using qRT-PCR. The results showed that both mRNAs were highly expressed in the heart. This study has established a foundation for further investigation of the ovine MYL3 and MYL4 genes. PMID:26656596

  14. Molecular cloning and characterization of alpha - galactosidase gene from Glaciozyma antarctica

    NASA Astrophysics Data System (ADS)

    Moheer, Reyad Qaed Al; Bakar, Farah Diba Abu; Murad, Abdul Munir Abdul

    2015-09-01

    Psychrophilic enzymes are proteins produced by psychrophilic organisms which recently are the limelight for industrial applications. A gene encoding α-galactosidase from a psychrophilic yeast, Glaciozyma antarctica PI12 which belongs to glycoside hydrolase family 27, was isolated and analyzed using several bioinformatic tools. The cDNA of the gene with the size of 1,404-bp encodes a protein with 467 amino acid residues. Predicted molecular weight of protein was 48.59 kDa and hence we name the gene encoding α-galactosidase as GAL48. We found that the predicted protein sequences possessed signal peptide sequence and are highly conserved among other fungal α-galactosidase.

  15. Gene cloning and molecular characterization of the Talaromyces thermophilus lipase catalyzed efficient hydrolysis and synthesis of esters.

    PubMed

    Romdhane, Ines Belhaj-Ben; Frikha, Fakher; Maalej-Achouri, Inès; Gargouri, Ali; Belghith, Hafedh

    2012-02-15

    A genomic bank from Talaromyces thermophilus fungus was constructed and screened using a previously isolated fragment lipase gene as probe. From several clones isolated, the nucleotide sequence of the lipase gene (TTL gene) was completed and sequenced. The TTL coding gene consists of an open reading frame (ORF) of 1083bp encoding a protein of 269 Aa with an estimated molecular mass of 30kDa. The TTL belongs to the same gene family as Thermomyces lanuginosus lipase (TLL, Lipolase®), a well known lipase with multiple applications. The promoter sequence of the TTL gene showed the conservation of known consensus sequences PacC, CreA, Hap2-3-4 and the existence of a particular sequence like the binding sites of Oleate Response Element (ORE) and Fatty acids Responsis Element (FARE) which are similar to that already found to be specific of lipolytic genes in Candida and Fusarium, respectively. Northern blot analysis showed that the TTL expression was much higher on wheat bran than on olive oil as sole carbon source. Compared to the Lipolase®, this enzyme was found to be more efficient for the hydrolysis and the synthesis of esters; and its synthetic efficiency even reached 91.6% from Waste Cooking Oil triglycerides. PMID:22178764

  16. Molecular cloning and expression analysis of a F-type lectin gene from Japanese sea perch (Lateolabrax japonicus).

    PubMed

    Qiu, Lihua; Lin, Liansheng; Yang, Keng; Zhang, Hanhua; Li, Jianzhu; Zou, Falin; Jiang, Shigui

    2011-08-01

    The techniques of homology cloning and anchored PCR were used to clone the fucose-binding lectin (F-type lectin) gene from Japanese sea perch (Lateolabrax Japonicus). The full-length cDNA of sea perch F-lectin (JspFL) contained a 5' untranslated region (UTR) of 39 bp, an ORF of 933 bp encoding a polypeptide of 310 amino acids with an estimated molecular mass of 10.82 kDa and a 3' UTR of 332 bp. The searches for nucleotides and protein sequence similarities with BLAST analysis indicated that the deduced amino acid sequence of JspFL was homological to the Fucose-binding lectin in other fish species. In the JspFL deduced amino acid sequence, two tandem domains that exhibit the eel carbohydrate-recognition sequence motif were found. The temporal expressions of gene in the different tissues were measured by real-time PCR. And the mRNA expressions of the gene were constitutively expressed in tissues including spleen, head-kidney, liver, gill, and heart. The JspFL expression in spleen was different during the stimulated time point, 2 h later the expression level became up-regulated, and 6 h later the expression level became down-regulated. The result indicated that JspFL was constitutive and inducible expressed and could play a critical role in the host-pathogen interaction. PMID:21104013

  17. Molecular cloning of the human eosinophil-derived neurotoxin: A member of the ribonuclease gene family

    SciTech Connect

    Rosenberg, H.F.; Tenen, D.G.; Ackerman, S.J. )

    1989-06-01

    The authors have isolated a 725-base-pair cDNA clone for human eosinophil-derived neurotoxin (EDN). EDN is a distinct cationic protein of the eosinophil's large specific granule known primarily for its ability to induce ataxia, paralysis, and central nervous system cellular degeneration in experimental animals (Gordon phenomenon). The open reading frame encodes a 134-amino acid mature polypeptide with a molecular mass of 15.5 kDa and a 27-residue amino-terminal hydrophobic leader sequence. The sequence of the mature polypeptide is identical to that reported for human urinary ribonuclease, and to the amino-terminal sequence of human liver ribonuclease; the cDNA encodes a tryptophan in position 7. Both EDN and the related granule protein, eosinophil cationic protein, have ribonucleolytic activity; sequence similarities among EDN, eosinophil cationic protein, ribonucleases from liver, urine, and pancreas, and angiogenin define a ribonuclease multigene family. mRNA encoding EDN was detected in uninduced HL-60 cells and was up-regulated in cells induced toward eosinophilic differentiation with B-cell growth factor 2/interleukin 5 and toward neutrophilic differentiation with dimethyl sulfoxide. EDN mRNA was detected in mature neutrophils even though EDN-like neurotoxic activity is not found neutrophil extracts. These results suggest that neutrophils contain a protein that is closely related or identical to EDN.

  18. Purification, characterization, and molecular gene cloning of an antifungal protein from Ginkgo biloba seeds.

    PubMed

    Sawano, Yoriko; Miyakawa, Takuya; Yamazaki, Hiroshi; Tanokura, Masaru; Hatano, Ken-ichi

    2007-03-01

    A novel basic protein with antifungal activity was isolated from the seeds of Ginkgo biloba and purified to homogeneity. The protein inhibited the growth of some fungi (Fusarium oxysporum, Trichoderma reesei, and Candida albicans) but did not exhibit antibacterial action against Escherichia coli. Furthermore, this protein showed weak inhibitory activity against the aspartic protease pepsin. To design primers for gene amplification, the NH(2)-terminal and partial internal amino acid sequences were determined using peptides obtained from a tryptic digest of the oxidized protein. The full-length cDNA of the antifungal protein was cloned and sequenced by RT-PCR and rapid amplification of cDNA ends (RACE). The cDNA contained a 402-bp open reading frame encoding a 134-aa protein with a potential signal peptide (26 residues), suggesting that this protein is synthesized as a preprotein and secreted outside the cells. The antifungal protein shows approximately 85% identity with embryo-abundant proteins from Picea abies and Picea glauca at the amino acid level; however, there is no homology between this protein and other plant antifungal proteins, such as defensin, and cyclophilin-, miraculin- and thaumatin-like proteins. PMID:17338634

  19. Molecular cloning and functional analysis of duck ubiquitin-specific protease 18 (USP18) gene.

    PubMed

    Qian, Wei; Wei, Xiaoqin; Zhou, Hongbo; Jin, Meilin

    2016-09-01

    In mammals, ubiquitin-specific protease 18 (USP18) is an interferon (IFN)-inducible gene and is a negative regulator of Toll-like receptor-mediated nuclear factor kappa B (NF-κB) activation. The role of USP18 in ducks (duUSP18) remains poorly understood. In the present study, we cloned and characterized the full-length coding sequence of duUSP18 from duck embryo fibroblasts (DEFs). In healthy ducks, duUSP18 transcripts were broadly expressed in different tissues, with higher expression levels in the spleen, lung and kidney. Quantitative real-time PCR (qRT-PCR) analysis revealed that duUSP18 could be induced by treatment with Poly(I:C) or LPS. Overexpression of duUSP18 inhibited NF-κB and IFN-β expression. Furthermore, deletion mutant analysis revealed that the duUSP18 region between aa 75 and 304 was essential for inhibiting NF-κB. In addition, overexpression of duUSP18 also suppressed the secretion of NF-κB-dependent proinflammatory cytokines. Taken together, these results suggest that duUSP18 regulates duck innate immune responses. PMID:27133094

  20. Molecular cloning and characterization of a Mlo gene in rubber tree (Hevea brasiliensis).

    PubMed

    Qin, Bi; Zheng, Fucong; Zhang, Yu

    2015-03-01

    Mlo gene encodes a plant-specific seven-transmembrane domain protein involved in a variety of cellular processes. In this study, a novel Mlo gene from rubber tree (Hevea brasiliensis), designated HbMlo1, was cloned by RT-PCR in rubber tree. The ORF of HbMlo1 was 1551bp in length, encoding a putative protein of 516 amino acids. HbMlo1 was a typical Mlo protein with seven-transmembrane domain. Sequence comparison between HbMlo1 and other Mlo proteins demonstrated that HbMlo1 shared the highest similarity with the Cucumis melo CmMlo1 and Arabidopsis thaliana AtMlo1 with 75.1% and 71.3% sequence identity, respectively. Phylogenetic analysis revealed that HbMlo1, CmMlo1, AtMlo1, AtMlo13, and AtMlo15 formed into the phylogenetic clade II with 100% bootstrap support value. HbMlo1 transcript exhibited tissue specificity, and it was preferentially expressed in leaf. Furthermore, the amount of HbMlo1 transcript was significantly induced by various phytohormones (including ethephon, methyl jasmonate, salicylic acid, abscisic acid, indole-3-acetic acid, and gibberellic acid), H2O2, and wounding treatments. Under drought stress, HbMlo1 exhibited a complex pattern of regulation. However, HbMlo1 expression did not significantly change during powdery mildew infection. These results suggested that HbMlo1 might play a role in phytohormone signaling and abiotic stress response processes in rubber tree. PMID:25506769

  1. Molecular cloning and gene expression of mummichog (Fundulus heteroclitus) Runx2 during embryogenesis.

    PubMed

    Amano, Haruna; Mochida, Kazuhiko; Onduka, Toshimitsu; Fujii, Kazunori

    2013-12-01

    In our previous study, we clarified the toxicity of 2,2'-dipyridyldisulfide [(PS)2], one of photodegradation products of a metal pyrithione that is used as an alternative antifouling paint biocides to organotin compounds in Japan. In early life stage toxicity tests, we exposed the mummichog, (Fundulus heteroclitus) to (PS)2, and the hatched larvae subsequently displayed notochord undulations and skeletal deformities ( Mochida et al., 2012 ). Runx2, a transcription factor of the runt family, is a key regulator in skeletal development in mammals. It is possible that (PS)2 inhibits Runx2 gene expression, inducing the skeletal deformities in mummichog. In the present study, we cloned two Runx2 cDNAs (type I and type II) from mummichog embryos. The deduced amino acid sequences of type I and type II contain an open reading frame encoding 450 and 464 amino acid residues, respectively. The derived amino acid sequence of Fundulus Runx2 type I showed the highest identity (93.8%) with Takifugu Runx2 type I, and Fundulus Runx2 type II showed 94.6% homology with medaka Runx2. The expression level of Runx2 mRNA in the early stage series was measured using a real-time quantitative PCR assay. Expression levels tended to increase in both the blastula-gastrula and the retinal pigmentation stage. To examine the effect of toxic compounds on skeletal formation, mummichog embryos in the late blastula to retinal pigmentation stage were exposed to (PS)2. After exposure to (PS)2 for one week, the expression level of Runx2 mRNA was unchanged. These results suggest that there is no inhibition of Runx2 gene expression due to (PS)2 exposure. PMID:24320183

  2. Molecular cloning and characterization of the light-harvesting chlorophyll a/b gene from the pigeon pea (Cajanus cajan).

    PubMed

    Qiao, Guang; Wen, Xiao-Peng; Zhang, Ting

    2015-12-01

    Light-harvesting chlorophyll a/b-binding proteins (LHCB) have been implicated in the stress response. In this study, a gene encoding LHCB in the pigeon pea was cloned and characterized. Based on the sequence of a previously obtained 327 bp Est, a full-length 793 bp cDNA was cloned using the rapid amplification of cDNA ends (RACE) method. It was designated CcLHCB1 and encoded a 262 amino acid protein. The calculated molecular weight of the CcLHCB1 protein was 27.89 kDa, and the theoretical isoelectric point was 5.29. Homology search and sequence multi-alignment demonstrated that the CcLHCB1 protein sequence shared a high identity with LHCB from other plants. Bioinformatics analysis revealed that CcLHCB1 was a hydrophobic protein with three transmembrane domains. By fluorescent quantitative real-time polymerase chain reaction (PCR), CcLHCB1 mRNA transcripts were detectable in different tissues (leaf, stem, and root), with the highest level found in the leaf. The expression of CcLHCB1 mRNA in the leaves was up-regulated by drought stimulation and AM inoculation. Our results provide the basis for a better understanding of the molecular organization of LCHB and might be useful for understanding the interaction between plants and microbes in the future. PMID:26329890

  3. Immersing Undergraduate Students in the Research Experience: A Practical Laboratory Module on Molecular Cloning of Microbial Genes

    ERIC Educational Resources Information Center

    Wang, Jack T. H.; Schembri, Mark A.; Ramakrishna, Mathitha; Sagulenko, Evgeny; Fuerst, John A.

    2012-01-01

    Molecular cloning skills are an essential component of biological research, yet students often do not receive this training during their undergraduate studies. This can be attributed to the complexities of the cloning process, which may require many weeks of progressive design and experimentation. To address this issue, we incorporated an…

  4. Molecular cloning and characterization, and prokaryotic expression of the GnRH1 gene obtained from Jinghai yellow chicken.

    PubMed

    Zhang, T; Zhang, G X; Han, K P; Tang, Y; Wang, J Y; Fan, Q C; Chen, X S; Wei, Y; Wang, Y J

    2015-01-01

    The gonadotropin-releasing hormone (GnRH) plays an important role in the control of reproductive functions. Recent studies have reported the occurrence of GnRH molecular variants in numerous species. In this study, the GnRH1 gene from Jinghai yellow chicken was cloned by reverse transcriptase-polymerase chain reaction and transformed into BL21 (DE3) competent cells. The GnRH1 gene and amino acid sequences were subjected to bioinformatic analyses. The GnRH1 gene nucleotide sequence was discovered to be 352 bp long, containing a coding, promoter, and section of the 3'-regions. The GnRH1 gene shared 93, 81, 54, 58, 61, 76, 76, 59, 76, and 66% sequence identity with Meleagris gallopavo, Columba livia, Homo sapiens, Bos taurus, swines, Capra hircus, Ovis aries, Pantholops hodgsonii, Equus caballus, and Rattus norvegicus, respectively. The GnRH1 gene showed conserved domains. The GnRH1 protein was a secreted protein comprising 92 amino acids, with a molecular weight of 10205.6 Da and a theoretical pI of 5.67. Most of the amino acid residues were observed to be hydrophilic, indicating water solubility. The predicted secondary structures of proteins included α-helices (h; 23.08%), β-extensions (e; 10.92%), and random coils (c; 66.0%). The successful construction of prokaryotic expression vector pET32a-GnRH1 was confirmed by restriction and sequence analysis. SDS-PAGE analysis showed the successful expression of recombinant plasmid in Escherichia coli BL21 (molecular weight = 25-28 kDa). Larger quantities of protein were expressed in supernatant, indicating greater expression in soluble form. Western blot analysis confirmed the expression of the target protein. PMID:25867433

  5. Agouti signalling protein (ASIP) gene: molecular cloning, sequence characterisation and tissue distribution in domestic goose.

    PubMed

    Zhang, J; Wang, C; Liu, Y; Liu, J; Wang, H Y; Liu, A F; He, D Q

    2016-06-01

    Agouti signalling protein (ASIP) is an endogenous antagonist of melanocortin-1 receptor (MC1R) and is involved in the regulation of pigmentation in mammals. The objective of this study was to identify and characterise the ASIP gene in domestic goose. The goose ASIP cDNA consisted of a 44-nucleotide 5'-terminal untranslated region (UTR), a 390-nucleotide open-reading frame (ORF) and a 45-nucleotide 3'-UTR. The length of goose ASIP genomic DNA was 6176 bp, including three coding exons and two introns. Bioinformatic analysis indicated that the ORF encodes a protein of 130 amino-acid residues with a molecular weight of 14.88 kDa and an isoelectric point of 9.73. Multiple sequence alignments and phylogenetic analysis showed that the amino-acid sequence of ASIP was conserved in vertebrates, especially in the avian species. RT-qPCR showed that the goose ASIP mRNA was differentially expressed in the pigment deposition tissues, including eye, foot, feather follicle, skin of the back, as well as in skin of the abdomen. The expression level of the ASIP gene in skin of the abdomen was higher than that in skin of the back. Those findings will contribute to further understanding the functions of the ASIP gene in geese plumage colouring. PMID:26750999

  6. Molecular cloning, characterization and expression profiles of thioredoxin 1 and thioredoxin 2 genes in Mytilus galloprovincialis

    NASA Astrophysics Data System (ADS)

    Wang, Qing; Ning, Xuanxuan; Pei, Dong; Zhao, Jianmin; You, Liping; Wang, Chunyan; Wu, Huifeng

    2013-05-01

    Thioredoxin (Trx) proteins are involved in many biological processes especially the regulation of cellular redox homeostasis. In this study, two Trx cDNAs were cloned from the mussel Mytilus galloprovincialis using rapid amplifi cation of cDNA ends-polymerase chain reaction (RACE-PCR). The two cDNAs were named MgTrx1 and MgTrx2, respectively. The open reading frames of MgTrx1 and MgTrx2 were 318 and 507 base pairs (bp) and they encoded proteins of 105 and 168 amino acids with estimated molecular masses of 11.45 and 18.93 kDa, respectively. Sequence analysis revealed that both proteins possessed the conserved active site dithiol motif Cys-Gly-Pro-Cys. In addition, MgTrx2 also possessed a putative mitochondrial targeting signal suggesting that it is located in the mitochondria. Quantitative real-time polymerase chain reaction (qPCR) revealed that both MgTrx1 and MgTrx2 were constitutively expressed in all tissues examined. The MgTrx1 transcript was most abundant in hemocytes and gills, whereas the MgTrx2 transcript was most abundant in gonad, hepatopancreas, gill and hemocytes. Following Vibrio anguillarum challenge, the expression of MgTrx1 was up-regulated and reached its peak, at a value 10-fold the initial value, at 24 h. Subsequently, expression returned back to the original level. In contrast, the expression level of MgTrx2 was down-regulated following bacterial stimulation, with one fi fth of the control level evident at 12 h post challenge. These results suggest that MgTrx1 and MgTrx2 may play important roles in the response of M. galloprovincialis to bacterial challenge.

  7. Molecular cloning and characterization of SoxB2 gene from Zhikong scallop Chlamys farreri

    NASA Astrophysics Data System (ADS)

    He, Yan; Bao, Zhenmin; Guo, Huihui; Zhang, Yueyue; Zhang, Lingling; Wang, Shi; Hu, Jingjie; Hu, Xiaoli

    2013-11-01

    The Sox proteins play critical roles during the development of animals, including sex determination and central nervous system development. In this study, the SoxB2 gene was cloned from a mollusk, the Zhikong scallop ( Chlamys farreri), and characterized with respect to phylogeny and tissue distribution. The full-length cDNA and genomic DNA sequences of C. farreri SoxB2 ( Cf SoxB2) were obtained by rapid amplification of cDNA ends and genome walking, respectively, using a partial cDNA fragment from the highly conserved DNA-binding domain, i.e., the High Mobility Group (HMG) box. The full-length cDNA sequence of Cf SoxB2 was 2 048 bp and encoded 268 amino acids protein. The genomic sequence was 5 551 bp in length with only one exon. Several conserved elements, such as the TATA-box, GC-box, CAAT-box, GATA-box, and Sox/sry-sex/testis-determining and related HMG box factors, were found in the promoter region. Furthermore, real-time quantitative reverse transcription PCR assays were carried out to assess the mRNA expression of Cf SoxB 2 in different tissues. SoxB2 was highly expressed in the mantle, moderately in the digestive gland and gill, and weakly expressed in the gonad, kidney and adductor muscle. In male and female gonads at different developmental stages of reproduction, the expression levels of Cf SoxB2 were similar. Considering the specific expression and roles of SoxB 2 in other animals, in particular vertebrates, and the fact that there are many pallial nerves in the mantle, cerebral ganglia in the digestive gland and gill nerves in gill, we propose a possible essential role in nervous tissue function for Sox B 2 in C. farreri.

  8. Molecular cloning and sexually dimorphic expression of DMRT4 gene in Oreochromis aureus.

    PubMed

    Cao, Jinling; Chen, Jianjie; Wu, Tingting; Gan, Xi; Luo, Yongju

    2010-07-01

    The DM-domain gene family has at least eight members with conserved DNA-binding DM-domain, which encodes putative transcription factors related to the sexual regulator Dsx of Drosophila and Mab-3 of C. elegans. Although some of the DM genes are involved in sexual development, the function of most of these genes remains unclear. In this study, rapid amplification cDNA ends (RACE) was used for the isolation of DMRT4 full-length cDNA from the ovary of the blue tilapia Oreochromis aureus. The full-length of DMRT4 cDNA was 1,571 bp, containing the 148 bp 5'-untranslated region, 193 bp 3'-untranslated region and 1,230 bp open reading frame. The deduced amino acid sequence of the open reading frame (ORF) encoded a protein of 409 amino acids with a theoretical pI of 8.492 and a calculated molecular weight of 44.12 kDa. One conserved functional domain, DM-domain was identified in blue tilapia DMRT4. The DMRT4 full-length gene obtained from the blood was 1,741 bp, containing a 156 bp intron. Phylogenetic analysis indicated that the amino acid sequences encoded by DMRT4 genes from different species had a high degree of sequence identity as revealed in phylogenetic tree constructed. Real-time quantitative Reverse-Transcription Polymerase Chain Reaction (RT-PCR) was used to analyze the expression patterns of DMRT4 in different developmental stages and different tissues in Oreochromis aureus. DMRT4 mRNA was detected from early gastrulae stage during embryonic development, and maintained a considerable high level until 1 day post hatching. With the increase of age, enhanced DMRT4 mRNA was observed in ovary and brain. After 15 and 30 days, fries treated with 17beta-estradiol had a significant increase in DMRT4 mRNA levels compared with the control fries (P < 0.05). DMRT4 was found to be expressed in the ovary and endbrain, thalamencephalon, pituitary, not detected in the liver, kidney, spleen, heart and muscle of adult fish. These results showed that the DMRT4 gene have

  9. Molecular cloning and characterization of a glycine-like receptor gene from the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

    PubMed Central

    Flores-Fernández, José Miguel; Gutiérrez-Ortega, Abel; Padilla-Camberos, Eduardo; Rosario-Cruz, Rodrigo; Hernández-Gutiérrez, Rodolfo; Martínez-Velázquez, Moisés

    2014-01-01

    The cattle tick Rhipicephalus (Boophilus) microplus is the most economically important ectoparasite affecting the cattle industry in tropical and subtropical areas around the world. The principal method of tick control has relied mainly on the use of chemical acaricides, including ivermectin; however, cattle tick populations resistant to ivermectin have recently been reported in Brazil, Mexico, and Uruguay. Currently, the molecular basis for ivermectin susceptibility and resistance are not well understood in R. microplus. This prompted us to search for potential molecular targets for ivermectin. Here, we report the cloning and molecular characterization of a R. microplus glycine-like receptor (RmGlyR) gene. The characterized mRNA encodes for a 464-amino acid polypeptide, which contains features common to ligand-gated ion channels, such as a large N-terminal extracellular domain, four transmembrane domains, a large intracellular loop and a short C-terminal extracellular domain. The deduced amino acid sequence showed around 30% identity to GlyRs from some invertebrate and vertebrate organisms. The polypeptide also contains the PAR motif, which is important for forming anion channels, and a conserved glycine residue at the third transmembrane domain, which is essential for high ivermectin sensitivity. PCR analyses showed that RmGlyR is expressed at egg, larval and adult developmental stages. Our findings suggest that the deduced receptor is an additional molecular target to ivermectin and it might be involved in ivermectin resistance in R. microplus. PMID:25174962

  10. Molecular cloning and characterization of a glycine-like receptor gene from the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

    PubMed

    Flores-Fernández, José Miguel; Gutiérrez-Ortega, Abel; Padilla-Camberos, Eduardo; Rosario-Cruz, Rodrigo; Hernández-Gutiérrez, Rodolfo; Martínez-Velázquez, Moisés

    2014-01-01

    The cattle tick Rhipicephalus (Boophilus) microplus is the most economically important ectoparasite affecting the cattle industry in tropical and subtropical areas around the world. The principal method of tick control has relied mainly on the use of chemical acaricides, including ivermectin; however, cattle tick populations resistant to ivermectin have recently been reported in Brazil, Mexico, and Uruguay. Currently, the molecular basis for ivermectin susceptibility and resistance are not well understood in R. microplus. This prompted us to search for potential molecular targets for ivermectin. Here, we report the cloning and molecular characterization of a R. microplus glycine-like receptor (RmGlyR) gene. The characterized mRNA encodes for a 464-amino acid polypeptide, which contains features common to ligand-gated ion channels, such as a large N-terminal extracellular domain, four transmembrane domains, a large intracellular loop and a short C-terminal extracellular domain. The deduced amino acid sequence showed around 30% identity to GlyRs from some invertebrate and vertebrate organisms. The polypeptide also contains the PAR motif, which is important for forming anion channels, and a conserved glycine residue at the third transmembrane domain, which is essential for high ivermectin sensitivity. PCR analyses showed that RmGlyR is expressed at egg, larval and adult developmental stages. Our findings suggest that the deduced receptor is an additional molecular target to ivermectin and it might be involved in ivermectin resistance in R. microplus. PMID:25174962

  11. Molecular cloning of the structural gene for exopolygalacturonate lyase from Erwinia chrysanthemi EC16 and characterization of the enzyme product.

    PubMed Central

    Brooks, A D; He, S Y; Gold, S; Keen, N T; Collmer, A; Hutcheson, S W

    1990-01-01

    The ability of Erwinia chrysanthemi to cause soft-rot diseases involving tissue maceration in many plants has been linked to the production of endo-pectate lyase E. chrysanthemi EC16 mutant UM1005, however, contains deletions in the pel genes that encode the known endopectate lyases, yet still macerates plant tissues. In an attempt to identify the remaining macerating factor(s), a gene library of UM1005 was constructed in Escherichia coli and screened for pectolytic activity. A clone (pPNL5) was identified in this library that contained the structural gene for an exopolygalacturonate lyase (ExoPL). The gene for ExoPL was localized on a 3.3-kb EcoRV fragment which contained an open reading frame for a 79,500-Da polypeptide. ExoPL was purified to apparent homogeneity from Escherichia coli DH5 alpha (pPNL5) and found to have an apparent molecular weight of 76,000 with an isoelectric point of 8.6. Purified ExoPL had optimal activity between pH 7.5 and 8.0 and could utilize pectate, citrus pectin, and highly methyl-esterified Link pectin as substrates. A PL- ExoPL- mutant of EC16 was constructed that exhibited reduced growth on pectate, but retained pathogenicity on chrysanthemum equivalent to that of UM1005. The results indicate that ExoPL does not contribute to the residual macerating activity of UM1005. Images PMID:2254266

  12. Molecular cloning and expression of novel metallothionein (MT) gene in the polychaete Perinereis nuntia exposed to metals.

    PubMed

    Won, Eun-Ji; Rhee, Jae-Sung; Ra, Kongtae; Kim, Kyung-Tae; Au, Doris W T; Shin, Kyung-Hoon; Lee, Jae-Seong

    2011-08-01

    To report a novel metallothionein (MT) gene and evaluate its potency as a biomarker, we clone this MT gene and measured the expression levels in the metal-exposed polychaete Perinereis nuntia. Accumulated metal contents and metallothionein-like proteins (MTLPs), which have been recognized as potential biomarkers, were compared with the relative mRNA expressions of the MT gene of P. nuntia (Pn-MT). In addition, the metal-binding affinity was estimated by recombinant Pn-MT protein. Pn-MT having high cysteine residues with three metal response elements in the promoter region closely clusters with those of other invertebrates. The accumulation patterns of metals were dependent on the exposure times in lead (Pb), cadmium (Cd), and copper (Cu) exposure. Particularly, both MTLP levels and relative mRNA expressions of MT were increased with accumulated metal contents and exposure time in P. nuntia exposed to Pb and Cd. There was no significant modulation of the Pn-MT gene in polychaetes exposed to Zn and As. However, the metal-binding ability of the recombinant Pn-MT protein provides a clear evidence for a high affinity of MT to several metal elements. These results suggest that Pn-MT would play an important role in the detoxification and/or sequestration of specific metals (e.g., Pb and Cd) in P. nuntia and have potential as a molecular biomarker in the monitoring of the marine environment using a polychaete. PMID:22828888

  13. Molecular cloning, expression and characterization of 100K gene of fowl adenovirus-4 for prevention and control of hydropericardium syndrome.

    PubMed

    Shah, M S; Ashraf, A; Khan, M I; Rahman, M; Habib, M; Qureshi, J A

    2016-01-01

    Fowl adenovirus-4 is an infectious agent causing Hydropericardium syndrome in chickens. Adenovirus are non-enveloped virions having linear, double stranded DNA. Viral genome codes for few structural and non structural proteins. 100K is an important non-structural viral protein. Open reading frame for coding sequence of 100K protein was cloned with oligo histidine tag and expressed in Escherichia coli as a fusion protein. Nucleotide sequence of the gene revealed that 100K gene of FAdV-4 has high homology (98%) with the respective gene of FAdV-10. Recombinant 100K protein was expressed in E. coli and purified by nickel affinity chromatography. Immunization of chickens with recombinant 100K protein elicited significant serum antibody titers. However challenge protection test revealed that 100K protein conferred little protection (40%) to the immunized chicken against pathogenic viral challenge. So it was concluded that 100K gene has 2397 bp length and recombinant 100K protein has molecular weight of 95 kDa. It was also found that the recombinant protein has little capacity to affect the immune response because in-spite of having an important role in intracellular transport & folding of viral capsid proteins during viral replication, it is not exposed on the surface of the virus at any stage. PMID:26558992

  14. Molecular cloning and characterization of human WINS1 and mouse Wins2, homologous to Drosophila segment polarity gene Lines (Lin).

    PubMed

    Katoh, Masaru

    2002-08-01

    WNT signaling molecules play key roles in carcinogenesis and embryogenesis. Drosophila segment polarity gene Lines (Lin) is essential for Wnt/Wingless-dependent patterning in dorsal epidermis and also for hindgut development. With Wnt signaling, Lin accumulates in the nucleus to modulate transcription of Wnt target genes through association with beta-catenin/Armadillo and TCF/Pangolin. Here, human WINS1 and mouse Wins2, encoding proteins with Drosophila Lin homologous domain, were isolated using bioinformatics and cDNA-PCR. Human WINS1 encoded 757-amino-acid protein, and mouse Wins2 encoded 498-amino-acid protein. Human WINS1 and mouse Wins2 showed 60.0% total-amino-acid identity. Lin homologous domain of WINS1 and Wins2 showed 29.4% and 27.2% amino-acid identity with that of Drosphila Lin, respectively. In the human chromosome 15q26 region, WINS1 gene was clustered with ASB7 gene encoding ankyrin repeat and SOCS box-containing protein 7. Human WINS1 mRNA of 2.8-kb in size was expressed in adult testis, prostate, spleen, thymus, skeletal muscle, fetal kidney and brain. This is the first report on molecular cloning and initial characterization of human WINS1 and mouse Wins2 PMID:12119551

  15. Molecular cloning, structural analysis, and tissue expression of the TNNT3 gene in Guizhou black goat.

    PubMed

    Chen, Haolin; Zhang, Jinhua; Yu, Bo; Li, Liang; Shang, Yishun

    2015-11-15

    The vertebrate fast skeletal troponin T (TNNT3) protein is an important regulatory and structural component of thin filaments in skeletal muscle, which improves meat quality traits of livestock and poultry. In this study, the troponin T isoforms from adult goat (skeletal muscle mRNA) were identified. We isolated the full-length coding sequence of the goat TNNT3 gene (GenBank: KM042888), analyzed its structure, and investigated its expression in different tissues from different aged goats (10, 30, 90, 180, and 360 days old). Real-time quantitative reverse transcription-polymerase chain reaction analyses revealed that Guizhou black goat TNNT3 was highly expressed in the biceps femoris muscle, abdominal muscle, and longissimus dorsi muscle (P<0.01), and lowly expressed in the cardiac muscle, masseter muscle, and rumen tissue (P>0.05). Western blotting confirmed that the TNNT3 protein was expressed in the muscle tissues listed above, with the highest level found in the longissimus dorsi muscle, and the lowest level in the masseter muscle. In the 10 to 360day study period the TNNT3 protein expression level was the highest when the goats were 30 days old. A peptide, ASPPPAEVPEVHEEVH that may contribute to improved goat meat tenderness was identified. This study provides an insight into the molecular structure of the vertebrate TNNT3 gene. PMID:26187066

  16. Molecular cloning, tissue expression and association of porcine NR4A1 gene with reproductive traits.

    PubMed

    Liu, L Q; Li, F E; Deng, C Y; Xiong, Y Z

    2011-01-01

    Nuclear receptor subfamily 4, group A, member 1 (NR4A1), other aliase NGFI-B, is an immediate-early gene that encodes an orphan nuclear receptor, which play a potential role in the ovulatory process. In this study, a 4,870 bp fragment covered the complete coding region (CDS) and its unique intron sequences of porcine NR4A1 gene was obtained. The reverse transcriptase-polymerase chain reaction (RT-PCR) indicated that NR4A1 was highly expressed in ovary, uterus, kidney, heart but at very low level in oviduct and not expressed in other tissues. Compared the sequence of CDS and its unique intron of Large White and Meishan pigs, a A/G mutation in intron 5 was found and a PCR-Dde1-RFLP genotyping assay was developed. Association of the SNP and litter size was assessed in two populations [purebred Large White and an experimental synthetic Line (DIV) sows]. Statistical analysis demonstrated that, in the first parity, AG animals in experimental synthetic Line (DIV) sows had 1.805 more piglets born compared to the GG animals (P<0.05). For all parities, in the purebred Large White pigs, those with the GG genotype had an additional 0.877 piglets born and 0.780 piglets born alive compared to the AA animals (P<0.05), those with the AG genotype had additional 0.780 piglets born compared to the AA animals (P<0.05). In addition, significant additive effect of 0.438±0.182 piglets/litter and 0.368±0.165 piglets/litter on piglets born and piglets born alive were detected in the purebred Large White lines (P<0.05), respectively. Therefore, NR4A1 gene was significantly associated with litter size in two populations and could be a useful molecular marker in selection for increasing litter size in pigs. PMID:20333549

  17. Molecular cloning and characterization of annexin genes in peanut (Arachis hypogaea L.).

    PubMed

    He, MeiJing; Yang, XinLei; Cui, ShunLi; Mu, GuoJun; Hou, MingYu; Chen, HuanYing; Liu, LiFeng

    2015-08-15

    Annexin, Ca(2+) or phospholipid binding proteins, with many family members are distributed throughout all tissues during plant growth and development. Annexins participate in a number of physiological processes, such as exocytosis, cell elongation, nodule formation in legumes, maturation and stress response. Six different full-length cDNAs and two partial-length cDNAs of peanut, (AnnAh1, AnnAh2, AnnAh3, AnnAh5, AnnAh6, AnnAh7, AnnAh4 and AnnAh8) encoding annexin proteins, were isolated and characterized using a RT-PCR/RACE-PCR based strategy. The predicted molecular masses of these annexins were 36.0kDa with acidic pIs of 5.97-8.81. ANNAh1, ANNAh2, ANNAh3, ANNAh5, ANNAh6 and ANNAh7 shared sequence similarity from 35.76 to 66.35% at amino acid level. Phylogenetic analysis revealed their evolutionary relationships with corresponding orthologous sequences in soybean and deduced proteins in various plant species. Real-time quantitative assays indicated that these genes were differentially expressed in various organs. Transcript level analysis for six annexin genes under stress conditions showed that these genes were regulated by drought, salinity, heavy metal stress, low temperature and hormone. Additionally, the prediction of cis-regulatory element suggested that different cis-responsive elements including stress- and hormone-responsive-related elements could respond to various stress conditions. These results indicated that members of AnnAhs family may play important roles in the adaptation of peanut to various environmental stresses. PMID:25958350

  18. Methods in molecular cardiology: in silico cloning

    PubMed Central

    Passier, R.; Doevendans, P.A.

    2004-01-01

    Advancements in sequencing technology have made it possible to obtain more information about the DNA sequence, structure and the transcript products of the genome from different species. This information is collected in DNA databases. These databases contain many genes of which the functions have not yet been discovered. By using online biotechnology tools novel genes and their transcripts can be identified. The identification of novel genes using DNA database analysis is referred to as in silico cloning. In silico cloning may not only provide new information on genes and their biological function, it may also lead to identification of molecular targets for drug discovery activities. In this review we describe the process of in silico cloning and its application in biomedical research. ImagesFigure 1Figure 3 PMID:25696371

  19. The gene controlling marijuana psychoactivity: molecular cloning and heterologous expression of Delta1-tetrahydrocannabinolic acid synthase from Cannabis sativa L.

    PubMed

    Sirikantaramas, Supaart; Morimoto, Satoshi; Shoyama, Yoshinari; Ishikawa, Yu; Wada, Yoshiko; Shoyama, Yukihiro; Taura, Futoshi

    2004-09-17

    Delta(1)-tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic acid into THCA, the precursor of Delta(1)-tetrahydrocannabinol. We cloned a novel cDNA (GenBank trade mark accession number AB057805) encoding THCA synthase by reverse transcription and polymerase chain reactions from rapidly expanding leaves of Cannabis sativa. This gene consists of a 1635-nucleotide open reading frame, encoding a 545-amino acid polypeptide of which the first 28 amino acid residues constitute the signal peptide. The predicted molecular weight of the 517-amino acid mature polypeptide is 58,597 Da. Interestingly, the deduced amino acid sequence exhibited high homology to berberine bridge enzyme from Eschscholtzia californica, which is involved in alkaloid biosynthesis. The liquid culture of transgenic tobacco hairy roots harboring the cDNA produced THCA upon feeding of cannabigerolic acid, demonstrating unequivocally that this gene encodes an active THCA synthase. Overexpression of the recombinant THCA synthase was achieved using a baculovirus-insect expression system. The purified recombinant enzyme contained covalently attached FAD cofactor at a molar ratio of FAD to protein of 1:1. The mutant enzyme constructed by changing His-114 of the wild-type enzyme to Ala-114 exhibited neither absorption characteristics of flavoproteins nor THCA synthase activity. Thus, we concluded that the FAD binding residue is His-114 and that the THCA synthase reaction is FAD-dependent. This is the first report on molecular characterization of an enzyme specific to cannabinoid biosynthesis. PMID:15190053

  20. Cloning, sequencing, and expression of the gene encoding the high-molecular-weight cytochrome c from Desulfovibrio vulgaris Hildenborough.

    PubMed Central

    Pollock, W B; Loutfi, M; Bruschi, M; Rapp-Giles, B J; Wall, J D; Voordouw, G

    1991-01-01

    By using a synthetic deoxyoligonucleotide probe designed to recognize the structural gene for cytochrome cc3 from Desulfovibrio vulgaris Hildenborough, a 3.7-kb XhoI genomic DNA fragment containing the cc3 gene was isolated. The gene encodes a precursor polypeptide of 58.9 kDa, with an NH2-terminal signal sequence of 31 residues. The mature polypeptide (55.7 kDa) has 16 heme binding sites of the form C-X-X-C-H. Covalent binding of heme to these 16 sites gives a holoprotein of 65.5 kDa with properties similar to those of the high-molecular-weight cytochrome c (Hmc) isolated from the same strain by Higuchi et al. (Y. Higuchi, K. Inaka, N. Yasuoka, and T. Yagi, Biochim. Biophys. Acta 911:341-348, 1987). Since the data indicate that cytochrome cc3 and Hmc are the same protein, the gene has been named hmc. The Hmc polypeptide contains 31 histidinyl residues, 16 of which are integral to heme binding sites. Thus, only 15 of the 16 hemes can have bis-histidinyl coordination. A comparison of the arrangement of heme binding sites and coordinated histidines in the amino acid sequences of cytochrome c3 and Hmc from D. vulgaris Hildenborough suggests that the latter contains three cytochrome c3-like domains. Cloning of the D. vulgaris Hildenborough hmc gene into the broad-host-range vector pJRD215 and subsequent conjugational transfer of the recombinant plasmid into D. desulfuricans G200 led to expression of a periplasmic Hmc gene product with covalently bound hemes. Images PMID:1846136

  1. Molecular cloning and characterization of GbDXS and GbGGPPS gene promoters from Ginkgo biloba.

    PubMed

    Xu, F; Huang, X H; Li, L L; Deng, G; Cheng, H; Rong, X F; Li, J B; Cheng, S Y

    2013-01-01

    Ginkgolides are key pharmaceutical components in Ginkgo biloba leaves. 1-Deoxy-D-xylulose-5-phosphate synthase (GbDXS) and geranylgeranyl pyrophosphate (GbGGPPS) genes are critical genes involved in ginkgolide biosynthesis. In this study, the promoters of GbDXS and GGPPS, with 676 and 570 bp in length, respectively, were cloned by chromosome walking. The cis-elements of GbDXS and GbGGPPS promoters were predicted and analyzed by the plant cis-acting regulatory element (CARE) database. We found some major cis-elements in the sequence of GbDXS and GbGGPPS promoters. The GbDXS promoter has 3 TATA boxes, 10 CAAT boxes, 6 GATA boxes, and 1 I box. The GbGGPPS promoter has 1 TATA box, 6 CAAT boxes, 6 GATA boxes, and 4 I boxes. Furthermore, some stress-related cis-elements in the promoters of GbDXS and GbGGPPS were found to be light-regulated elements, including sequences over-represented in light-induced promoters (SORLIP1- AT), GATA box, and I box, a gibberellin-responsive element (WRKY), salicylic acid-induced (GT-1), cold- and dehydration-responsive (MYC-Core), and copper-inducible (CURE-Core). Further analyses of these cis-elements will aid in elucidating the molecular mechanisms regulating the expression of the GbDXS and GbGGPPS genes during ginkgolide accumulation in G. biloba. PMID:23408416

  2. Molecular Cloning and Characterization of Four Genes Encoding Ethylene Receptors Associated with Pineapple (Ananas comosus L.) Flowering.

    PubMed

    Li, Yun-He; Wu, Qing-Song; Huang, Xia; Liu, Sheng-Hui; Zhang, Hong-Na; Zhang, Zhi; Sun, Guang-Ming

    2016-01-01

    Exogenous ethylene, or ethephon, has been widely used to induce pineapple flowering, but the molecular mechanism behind ethephon induction is still unclear. In this study, we cloned four genes encoding ethylene receptors (designated AcERS1a, AcERS1b, AcETR2a, and AcETR2b). The 5' flanking sequences of these four genes were also cloned by self-formed adaptor PCR and SiteFinding-PCR, and a group of putative cis-acting elements was identified. Phylogenetic tree analysis indicated that AcERS1a, AcERS1b, AcETR2a, and AcETR2b belonged to the plant ERS1s and ETR2/EIN4-like groups. Quantitative real-time PCR showed that AcETR2a and AcETR2b (subfamily 2) were more sensitive to ethylene treatment compared with AcERS1a and AcERS1b (subfamily 1). The relative expression of AcERS1b, AcETR2a, and AcETR2b was significantly increased during the earlier period of pineapple inflorescence formation, especially at 1-9 days after ethylene treatment (DAET), whereas AcERS1a expression changed less than these three genes. In situ hybridization results showed that bract primordia (BP) and flower primordia (FP) appeared at 9 and 21 DAET, respectively, and flowers were formed at 37 DAET. AcERS1a, AcERS1b, AcETR2a, and AcETR2b were mainly expressed in the shoot apex at 1-4 DAET; thereafter, with the appearance of BP and FP, higher expression of these genes was found in these new structures. Finally, at 37 DAET, the expression of these genes was mainly focused in the flower but was also low in other structures. These findings indicate that these four ethylene receptor genes, especially AcERS1b, AcETR2a, and AcETR2b, play important roles during pineapple flowering induced by exogenous ethephon. PMID:27252725

  3. Molecular Cloning and Characterization of Four Genes Encoding Ethylene Receptors Associated with Pineapple (Ananas comosus L.) Flowering

    PubMed Central

    Li, Yun-He; Wu, Qing-Song; Huang, Xia; Liu, Sheng-Hui; Zhang, Hong-Na; Zhang, Zhi; Sun, Guang-Ming

    2016-01-01

    Exogenous ethylene, or ethephon, has been widely used to induce pineapple flowering, but the molecular mechanism behind ethephon induction is still unclear. In this study, we cloned four genes encoding ethylene receptors (designated AcERS1a, AcERS1b, AcETR2a, and AcETR2b). The 5′ flanking sequences of these four genes were also cloned by self-formed adaptor PCR and SiteFinding-PCR, and a group of putative cis-acting elements was identified. Phylogenetic tree analysis indicated that AcERS1a, AcERS1b, AcETR2a, and AcETR2b belonged to the plant ERS1s and ETR2/EIN4-like groups. Quantitative real-time PCR showed that AcETR2a and AcETR2b (subfamily 2) were more sensitive to ethylene treatment compared with AcERS1a and AcERS1b (subfamily 1). The relative expression of AcERS1b, AcETR2a, and AcETR2b was significantly increased during the earlier period of pineapple inflorescence formation, especially at 1–9 days after ethylene treatment (DAET), whereas AcERS1a expression changed less than these three genes. In situ hybridization results showed that bract primordia (BP) and flower primordia (FP) appeared at 9 and 21 DAET, respectively, and flowers were formed at 37 DAET. AcERS1a, AcERS1b, AcETR2a, and AcETR2b were mainly expressed in the shoot apex at 1–4 DAET; thereafter, with the appearance of BP and FP, higher expression of these genes was found in these new structures. Finally, at 37 DAET, the expression of these genes was mainly focused in the flower but was also low in other structures. These findings indicate that these four ethylene receptor genes, especially AcERS1b, AcETR2a, and AcETR2b, play important roles during pineapple flowering induced by exogenous ethephon. PMID:27252725

  4. Molecular cloning and chromosomal mapping of the mouse cyclin-dependent kinase 5 gene

    SciTech Connect

    Ohshima, Toshio; Nagle, J.W.; Brady, R.O.; Kozak, C.A.

    1995-08-10

    Cyclin-dependent kinase 5 (Cdk5) is predominantly expressed in neurons. In vitro, Cdk5 purified from the nervous tissue phosphorylates both high-molecular-weight neurofilament and microtubule-associated tau. The mouse gene encoding Cdk5 (Cdk5) was found to be 5 kb in length and divided into 12 exons. All of the exon-intron junctions matched the expected consensus sequence with the exception of the splice junction for intron 9, which has AT and AC dinucleotides instead of the usual GT and AG bordering sequence. In the 5{prime}-flanking region of mouse Cdk5, several putative promoter elements were present, including AP1, Sp1, PuF, and TATA motifs. A metal regulatory element was also identified at position -207 to -201. Nucleotide sequence analysis of mouse Cdk5 showed high identity to the homologues of other vertebrate species, indicating that this kinase is highly conserved during evolution. Mouse Cdk5 was mapped to the centromeric region of mouse chromosome 5. 20 refs., 2 figs., 1 tab.

  5. Molecular cloning and expression analysis of the gene encoding proline dehydrogenase from Jatropha curcas L.

    PubMed

    Wang, Haibo; Ao, Pingxing; Yang, Shuanglong; Zou, Zhurong; Wang, Shasha; Gong, Ming

    2015-03-01

    Proline dehydrogenase (ProDH) (EC 1.5.99.8) is a key enzyme in the catabolism of proline. The enzyme JcProDH and its complementary DNA (cDNA) were isolated from Jatropha curcas L., an important woody oil plant used as a raw material for biodiesels. It has been classified as a member of the Pro_dh superfamily based on multiple sequence alignment, phylogenetic characterization, and its role in proline catabolism. Its cDNA is 1674 bp in length with a complete open reading frame of 1485 bp, which encodes a polypeptide chain of 494 amino acids with a predicted molecular mass of 54 kD and a pI of 8.27. Phylogenetic analysis indicated that JcProDH showed high similarity with ProDH from other plants. Reverse transcription PCR (RT-PCR) analysis revealed that JcProDH was especially abundant in the seeds and flowers but scarcely present in the stems, roots, and leaves. In addition, the expression of JcProDH increased in leaves experiencing environmental stress such as cold (5 °C), heat (42 °C), salt (300 mM), and drought (30 % PEG6000). The JcProDH protein was successfully expressed in the yeast strain INVSc1 and showed high enzyme activity in proline catabolism. This result confirmed that the JcProDH gene negatively participated in the stress response. PMID:25502926

  6. Molecular cloning and gene expression analysis of cystatin C-like proteins in spinyhead croaker Collichthys lucidus.

    PubMed

    Song, W; Jiang, K J; Zhang, F Y; Zhao, M; Ma, L B

    2016-01-01

    Cystatins are natural tight-binding reversible inhibitors of cysteine proteases. In this study, a cDNA library was constructed from Collichthys lucidus using the SMART technique. A complete cDNA sequence with high identity to the conserved sequence of the cystatin C gene was cloned from the library using EST analysis and rapid amplification of cDNA ends (RACE), then subjected to further investigation. The full-length cDNA of cystatin C from C. lucidus (Clcys) was 699 bp long, including a 5'-terminal untranslated region (5'-UTR) of 52 bp, a 3'-UTR of 290 bp, and an open-reading frame of 357 bp. The gene encoded a polypeptide of 118 amino acids, constituting a predicted molecular weight of 12.875 kDa and a theoretical isoelectric point of 8.81. The amino acid sequence of Clcys possessed typical features of type II cystatins and had the highest identity with cystatin C of Pseudosciaena crocea (89%); therefore, it clustered with the cystatin C group in the UPGMA phylogenetic tree. Quantitative real-time reverse transcription analysis revealed that the highest expression was found in the kidney, followed by the liver, heart, and testis, with the lowest expression in muscle. Interestingly, Clcys had relatively low identity with cystatin C genes from other fish and mammals, and its expression pattern did not possess features of a housekeeping gene. Based on these findings, we suspect that the classification of cystatins in fish is somewhat confusing, and the identification of more cystatin gene sequences is needed before a definite conclusion can be drawn. PMID:27050996

  7. Molecular cloning and expression of the human deoxythymidylate kinase gene in yeast.

    PubMed Central

    Su, J Y; Sclafani, R A

    1991-01-01

    (Deoxy)thymidylate (dTMP) kinase is an enzyme which phosphorylates dTMP to dTDP in the presence of ATP and magnesium. This enzyme is important in cellular DNA synthesis because the synthesis of dTTP, either via the de novo pathway or through the exogenous supply of thymidine, requires the activity of this enzyme. It has been suggested that the activities of the enzymes involved in DNA precursor biosynthesis, such as thymidine kinase, thymidylate synthase, thymidylate kinase, and dihydrofolate reductase, are subjected to cell cycle regulation. Here we describe the cloning of a human dTMP kinase cDNA by functional complementation of a yeast dTMP kinase temperature-sensitive mutant at the non-permissive temperature. The nucleotide sequence of the cloned human cDNA is predicted to encode a 24 KD protein that shows considerable homology with the yeast and vaccinia virus dTMP kinase enzymes. The human enzyme activity has been investigated by expressing it in yeast. In this work, we demonstrate that the cloned human cDNA, when expressed in yeast, produces dTMP kinase activity. Images PMID:2017365

  8. High-resolution mapping, cloning and molecular characterization of the Pi-k ( h ) gene of rice, which confers resistance to Magnaporthe grisea.

    PubMed

    Sharma, T R; Madhav, M S; Singh, B K; Shanker, P; Jana, T K; Dalal, V; Pandit, A; Singh, A; Gaikwad, K; Upreti, H C; Singh, N K

    2005-12-01

    In order to understand the molecular mechanisms involved in the gene-for-gene type of pathogen resistance, high-resolution genetic and physical mapping of resistance loci is required to facilitate map-based cloning of resistance genes. Here, we report the molecular mapping and cloning of a dominant gene (Pi-k ( h )) present in the rice line Tetep, which is associated with resistance to rice blast disease caused by Magnaporthe grisea. This gene is effective against M. grisea populations prevalent in the Northwestern Himalayan region of India. Using 178 sequence tagged microsatellite, sequence-tagged site, expressed sequence tag and simple sequence repeat (SSR) markers to genotype a population of 208 F(2) individuals, we mapped the Pi-k ( h ) gene between two SSR markers (TRS26 and TRS33) which are 0.7 and 0.5 cM away, respectively, and can be used in marker-assisted-selection for blast-resistant rice cultivars. We used the markers to identify the homologous region in the genomic sequence of Oryza sativa cv. Nipponbare, and a physical map consisting of two overlapping bacterial artificial chromosome and P1 artificial chromosome clones was assembled, spanning a region of 143,537 bp on the long arm of chromosome 11. Using bioinformatic analyses, we then identified a candidate blast-resistance gene in the region, and cloned the homologous sequence from Tetep. The putative Pi-k ( h ) gene cloned from Tetep is 1.5 kbp long with a single ORF, and belongs to the nucleotide binding site-leucine rich repeat class of disease resistance genes. Structural and expression analysis of the Pi-k ( h ) gene revealed that its expression is pathogen inducible. PMID:16228246

  9. Molecular cloning with a pMEA300-derived shuttle vector and characterization of the Amycolatopsis methanolica prephenate dehydratase gene.

    PubMed Central

    Vrijbloed, J W; van Hylckama Vlieg, J; van der Put, N M; Hessels, G I; Dijkhuizen, L

    1995-01-01

    An efficient restriction barrier for methylated DNA in the actinomycete Amycolatopsis methanolica could be avoided by using a nonmethylating Escherichia coli strain for DNA isolations. The A. methanolica prephenate dehydratase gene was cloned from a gene bank in a pMEA300-derived shuttle vector in E. coli and characterized. PMID:7592448

  10. Molecular cloning and characterization of the ABA-specific glucosyltransferase gene from bean (Phaseolus vulgaris L.).

    PubMed

    Palaniyandi, Sasikumar Arunachalam; Chung, Gyuhwa; Kim, Sang Hyon; Yang, Seung Hwan

    2015-04-15

    Levels of the plant hormone abscisic acid (ABA) are maintained in homeostasis by a balance of its biosynthesis, catabolism and conjugation. The detailed molecular and signaling events leading to strict homeostasis are not completely understood in crop plants. In this study, we obtained cDNA of an ABA-inducible, ABA-specific UDP-glucosyltransferase (ABAGT) from the bean plant (Phaseolus vulgaris L.) involved in conjugation of a glucose residue to ABA to form inactive ABA-glucose ester (ABA-GE) to examine its role during development and abiotic stress in bean. The bacterially expressed PvABAGTase enzyme showed ABA-specific glucosylation activity in vitro. A higher level of the PvABAGT transcript was observed in mature leaves, mature flowers, roots, seed coats and embryos as well as upon rehydration following a period of dehydration. Overexpression of 35S::PvABAGT in Arabidopsis showed reduced sensitivity to ABA compared with WT. The transgenic plants showed a high level of ABA-GE without significant decrease in the level of ABA compared with the wild type (WT) during dehydration stress. Upon rehydration, the levels of ABA and phaseic acid (PA) decreased in the WT and the PvABAGT-overexpressing lines with high levels of ABA-GE only in the transgenic plants. Our findings suggest that the PvABAGT gene could play a role in ABA homeostasis during development and stress responses in bean and its overexpression in Arabidopsis did not alter ABA homeostasis during dehydration stress. PMID:25747288

  11. Molecular cloning, characterization and expression analysis of woodchuck retinoic acid-inducible gene I.

    PubMed

    Yan, Qi; Liu, Qin; Li, Meng-Meng; Li, Fang-Hui; Zhu, Bin; Wang, Jun-Zhong; Lu, Yin-Ping; Liu, Jia; Wu, Jun; Zheng, Xin; Lu, Meng-Ji; Wang, Bao-Ju; Yang, Dong-Liang

    2016-06-01

    Cytosolic retinoic acid-inducible gene I (RIG-I) is an important innate immune RNA sensor and can induce antiviral cytokines, e.g., interferon-β (IFN-β). Innate immune response to hepatitis B virus (HBV) plays a pivotal role in viral clearance and persistence. However, knowledge of the role that RIG-I plays in HBV infection is limited. The woodchuck is a valuable model for studying HBV infection. To characterize the molecular basis of woodchuck RIG-I (wRIG-I), we analyzed the complete coding sequences (CDSs) of wRIG-I, containing 2778 base pairs that encode 925 amino acids. The deduced wRIG-I protein was 106.847 kD with a theoretical isoelectric point (pI) of 6.07, and contained three important functional structures [caspase activation and recruitment domains (CARDs), DExD/H-box helicases, and a repressor domain (RD)]. In woodchuck fibroblastoma cell line (WH12/6), wRIG-I-targeted small interfering RNA (siRNA) down-regulated RIG-I and its downstrean effector-IFN-β transcripts under RIG-I' ligand, 5'-ppp double stranded RNA (dsRNA) stimulation. We also measured mRNA levels of wRIG-I in different tissues from healthy woodchucks and in the livers from woodchuck hepatitis virus (WHV)-infected woodchucks. The basal expression levels of wRIG-I were abundant in the kidney and liver. Importantly, wRIG-I was significantly up-regulated in acutely infected woodchuck livers, suggesting that RIG-I might be involved in WHV infection. These results may characterize RIG-I in the woodchuck model, providing a strong basis for further study on RIG-I-mediated innate immunity in HBV infection. PMID:27376800

  12. Molecular cloning and characterization of a cassava translationally controlled tumor protein gene potentially related to salt stress response.

    PubMed

    Santa Brígida, Ailton Borges; dos Reis, Sávio Pinho; Costa, Carinne de Nazaré Monteirou; Cardoso, Cristina Michiko Yokoyama; Lima, Aline Medeiros; de Souza, Cláudia Regina Batista

    2014-03-01

    Cassava (Manihot esculenta Crantz) is one of the most important tropical crops showing tolerance to abiotic stress and adaptations to a wide range of environmental conditions. Here, we aimed to isolate and characterize the full-length cDNA and genomic sequences of a cassava translationally controlled tumor protein gene (MeTCTP), and evaluate its potential role in response to salt stress. The MeTCTP full-length cDNA sequence encodes for a deduced protein with 168 amino acid residues, with theoretical isoelectric point and molecular weight of 4.53 and 19 kDa, respectively, containing two putative signatures of TCTP family and one site for myristoylation. The MeTCTP genomic sequence includes four introns and five exons within a 1,643 bp coding region, and a 264 bp partial promoter sequence containing several putative cis-acting regulatory elements, among them, two putative GT-1 motifs, which may be related to response to sodium chloride (NaCl) and pathogen infection. Semi-quantitative RT-PCR assays showed that MeTCTP transcripts were higher in roots than leaves, and were significantly increased in detached leaves treated with NaCl. Furthermore, the recombinant MeTCTP conferred a protective function against salt stress in bacterial cells. We report for the first time the molecular cloning and characterization of a cassava TCTP with potential role in salt-stress response. Since salinity is one the most important abiotic factors affecting the production of crops worldwide, the MeTCTP gene could be a candidate gene for generation of salt tolerant crops. PMID:24413992

  13. Molecular cloning, sequencing, and expression of a L -glutamine D-fructose 6-phosphate amidotransferase gene from Volvariella volvacea.

    PubMed

    Luo, Chuping; Shao, Weilan; Li, Xun; Chen, Zhiyi; Liu, Yongfeng

    2009-01-01

    Using 3'-RACE and 5'-RACE, we have cloned and sequenced the genomic gene and complete cDNA encoding L: -glutamine D: -fructose 6-phosphate amidotransferase (GFAT) from the edible straw mushroom, Volvariella volvacea. Gfat contains five introns, and encodes a predicted protein of 697 amino acids that is homologous to other reported GFAT sequences. Southern hybridization indicated that a single gfat gene locus exists in the V. volvacea genome. Recombinant native V. volvacea GFAT enzyme, over-expressed using Escherichia coli and partially purified, had an estimated molecular mass of 306 kDa and consisted of four equal-sized subunits of 77 kD. Reciprocal plots revealed K (m) values of 0.55 and 0.75 mM for fructose 6-phosphate and L: -glutamine, respectively. V. volvacea GFAT activity was inhibited by the end-product of the hexosamine pathway, UDP-GlcNAc, and by the glutamine analogues N (3)-(4-methoxyfumaroyl)-L: -2,3-diaminopropanoic acid and 2-amino-2-deoxy-D: -glucitol-6-phosphate. PMID:19165584

  14. Characterization of the env gene and long terminal repeat of molecularly cloned Friend mink cell focus-inducing virus DNA.

    PubMed Central

    Adachi, A; Sakai, K; Kitamura, N; Nakanishi, S; Niwa, O; Matsuyama, M; Ishimoto, A

    1984-01-01

    The highly oncogenic erythroleukemia-inducing Friend mink cell focus-inducing (MCF) virus was molecularly cloned in phage lambda gtWES.lambda B, and the DNA sequences of the env gene and the long terminal repeat were determined. The nucleotide sequences of Friend MCF virus and Friend spleen focus-forming virus were quite homologous, supporting the hypothesis that Friend spleen focus-forming virus might be generated via Friend MCF virus from an ecotropic Friend virus mainly by some deletions. Despite their different pathogenicity, the nucleotide sequences of the env gene of Friend MCF virus and Moloney MCF virus were quite homologous, suggesting that the putative parent sequence for the generation of both MCF viruses and the recombinational mechanism for their generation might be the same. We compare the amino acid sequences in lymphoid leukemia-inducing ecotropic Moloney virus and Moloney MCF virus, and erythroblastic leukemia-inducing ecotropic Friend virus, Friend-MCF virus, and Friend spleen focus-forming virus. The Friend MCF virus long terminal repeat was found to be 550 base pairs long. This contained two copies of the 39-base-pair tandem repeat, whereas the spleen focus-forming virus genome contained a single copy of the same sequence. PMID:6328011

  15. Characterization of a cypermethrin-degrading Methylobacterium sp. strain A-1 and molecular cloning of its carboxylesterase gene.

    PubMed

    Diegelmann, Corinna; Weber, Joachim; Heinzel-Wieland, Regina; Kemme, Michael

    2015-11-01

    A novel mesophilic bacterial strain, designated A-1, was isolated from microbially contaminated biopolymer microcapsules. The bacterium was able to withstand and grow in liquid cultures supplemented with the pyrethroid cypermethrin in concentrations up to 400 mg L(-1) . Furthermore, strain A-1 could use cypermethrin as sole carbon source and could degrade >50% of it in 12 h. Based on phenotypic and chemotaxonomic characterization, and phylogenetic analysis of 16S rRNA gene sequence, the strain A-1 was identified as Methylobacterium sp., which is the first reported cypermethrin degrader of methylotrophic bacteria. A role for esterase activity in cypermethrin biodegradation was presumed. Therefore, the carboxylesterase gene mse1 was amplified from the Methylobacterium sp. strain A-1 genome and the resulting 1 kb amplicon cloned into E. coli. Sequence analysis of the mse1-DNA insert revealed an open reading frame of 633 bp encoding for a putative carboxylesterase of 210 amino acid residues with a predicted molecular mass of 22 kDa. The amino acid sequence of the deduced enzyme MsE1 with the catalytic triad Ser106 , Asp156 , and His187 was found to be similar to that of α/β-hydrolase fold proteins. The active site Ser106 residue is located in the consensus pentapeptide motif Gly-X-Ser-X-Gly that is typical of esterases. PMID:26131623

  16. Molecular Cloning, Expression Analysis, and Preliminarily Functional Characterization of the Gene Encoding Protein Disulfide Isomerase from Jatropha curcas.

    PubMed

    Wang, Haibo; Zou, Zhurong; Gong, Ming

    2015-05-01

    Reactive oxygen species (ROS) in plants, arising from various environmental stresses, impair the thiol-contained proteins that are susceptible to irregular oxidative formation of disulfide bonds, which might be alleviated by a relatively specific modifier called protein disulfide isomerase (PDI). From our previous data of the transcriptome and digital gene expression of cold-hardened Jatropha curcas, a PDI gene was proposed to be cold-relevant. In this study, its full-length cDNA (JcPDI) was cloned, with the size of 1649 bp containing the entire open reading frame (ORF) of 1515 bp. This ORF encodes a polypeptide of 504 amino acids with theoretical molecular weight of 56.6 kDa and pI value of 4.85. One N-terminal signal peptide (-MASKGSIWSCMFLFSLI VAISAGEG-) and the C-terminal anchoring sequence motif (-KDEL-) specific to the endoplasmic reticulum, as well as two thioredoxin domains (-CGHC-), are also found by predictions. Through semi-quantitative RT-PCR, the expression of JcPDI was characterized to be tissue-differential strongly in leaves and roots, but weakly in stems, and of cold-induced alternations. Furthermore, JcPDI overexpression in yeast could notably enhance the cold resistance of host cells. Conclusively, these results explicitly suggested a considerable association of JcPDI to cold response and a putative application potential for its correlated genetic engineering. PMID:25825250

  17. Molecular cloning of the human XRCC1 gene, which corrects defective DNA strand break repair and sister chromatid exchange.

    PubMed Central

    Thompson, L H; Brookman, K W; Jones, N J; Allen, S A; Carrano, A V

    1990-01-01

    We describe the cloning and function of the human XRCC1 gene, which is the first mammalian gene isolated that affects cellular sensitivity to ionizing radiation. The CHO mutant EM9 has 10-fold-higher sensitivity to ethyl methanesulfonate, 1.8-fold-higher sensitivity to ionizing radiation, a reduced capacity to rejoin single-strand DNA breaks, and a 10-fold-elevated level of sister chromatid exchange compared with the CHO parental cells. The complementing human gene was cloned from a cosmid library of a tertiary transformant. Two cosmid clones produced transformants that showed approximately 100% correction of the repair defect in EM9 cells, as determined by the kinetics of strand break repair, cell survival, and the level of sister chromatid exchange. A nearly full-length clone obtained from the pcD2 human cDNA expression library gave approximately 80% correction of EM9, as determined by the level of sister chromatid exchange. Based on an analysis of the nucleotide sequence of the cDNA insert compared with that of the 5' end of the gene from a cosmid clone, the cDNA clone appeared to be missing approximately 100 bp of transcribed sequence, including 26 nucleotides of coding sequence. The cDNA probe detected a single transcript of approximately 2.2 kb in HeLa polyadenylated RNA by Northern (RNA) blot hybridization. From the open reading frame and the positions of likely start sites for transcription and translation, the size of the putative XRCC1 protein is 633 amino acids (69.5 kDa). The size of the XRCC1 gene is 33 kb, as determined by localizing the endpoints on a restriction endonuclease site map of one cosmid clone. The deduced amino acid sequence did not show significant homology with any protein in the protein sequence data bases examined. Images PMID:2247054

  18. Molecular cloning and functional analysis of a novel oncogene, cancer-upregulated gene 2 (CUG2)

    SciTech Connect

    Lee, Soojin . E-mail: leesoojin@cnu.ac.kr; Gang, Jingu; Jeon, Sun Bok; Jung, Jinyoung; Song, Si Young; Koh, Sang Seok . E-mail: sskoh@kribb.re.kr

    2007-08-31

    We examined genome-wide differences in gene expression between tumor biopsies and normal tissues in order to identify differentially regulated genes in tumors. Cancer-upregulated gene 2 (CUG2) was identified as an expressed sequence tag (EST) that exhibits significant differential expression in multiple human cancer types. CUG2 showed weak sequence homology with the down-regulator of transcription 1 (DR1) gene, a human transcription repressor. We found that EGFP-CUG2 fusion proteins were predominantly localized in the nucleus, suggesting their putative role in gene regulation. In addition, CUG2-overexpressing mouse fibroblast cells exhibited distinct cancer-specific phenotypes in vitro and developed into tumors in nude mice. Taken together, these findings suggest that CUG2 is a novel tumor-associated gene that is commonly activated in various human cancers and exhibits high transforming activities; it possibly belongs to a transcription regulator family that is involved in tumor biogenesis.

  19. Molecular cloning and characterization of the structural gene for protein I, the major outer membrane protein of Neisseria gonorrhoeae.

    PubMed Central

    Carbonetti, N H; Sparling, P F

    1987-01-01

    Protein I (P.I) is the major outer membrane protein of Neisseria gonorrhoeae and serves as a porin. By using oligonucleotide probes derived from the known amino-terminal sequence of the mature protein, we have cloned the gene encoding the P.I of gonococcal strain FA19 in three overlapping fragments and determined the DNA sequence. The gene sequence predicts a protein with characteristics typical of the porins of other Gram-negative bacteria. A clone expressing P.I in Escherichia coli was obtained by removing a portion of the P.I gene promoter and reconstructing the entire P.I gene in a position just downstream from a phage T7 promoter. Expression of P.I was then achieved by introducing this recombinant plasmid into an E. coli strain containing an inducible T7 polymerase gene. The clone produced a protein that was identical in size to native P.I and reacted with anti-P.I monoclonal antibodies. Prolonged expression of the protein apparently was lethal for E. coli, possibly explaining failures to clone an intact P.I gene with its own promoter. Images PMID:3122212

  20. Molecular cloning and differential expression of three GnRH genes during ovarian maturation of spotted halibut, Verasper variegatus.

    PubMed

    Xu, Yong-Jiang; Liu, Xue-Zhou; Liao, Mei-Jie; Wang, Han-Ping; Wang, Qing-Yin

    2012-08-01

    In this study, the gonadotropin-releasing hormone (GnRH) genes in spotted halibut were cloned and sequenced by isolating their cDNAs. The species expressed three molecular forms of GnRH in the brain: chicken-type GnRH-II (cGnRH-II), seabream-type GnRH (sbGnRH), and salmon-type GnRH (sGnRH). Phylogenetic analysis divided the molecular forms of GnRHs into three branches: cGnRH-II branch, sGnRH branch, and fish-specific GnRH branch. The spatial expression showed that they had the highest expression levels in the brain. cGnRH-II was exclusively detected in the brain, while sbGnRH had a global expression pattern in all examined organs. sGnRH was detected in the brain, pituitary, and ovary. The temporal changes of brain GnRH mRNA expression levels were examined during ovarian maturation and postspawning, and the serum steroid hormones and gonadosomatic index (GSI) were recorded. Amounts of sbGnRH mRNA substantially elevated (P < 0.05) during ovarian maturation, which concomitant with considerable elevation of GSI and serum steroids levels. On the contrary, neither sGnRH nor cGnRH-II mRNA levels showed significant changes during ovarian maturation in this study. These results suggested that these three GnRH genes are the important regulators for the differential expression of GnRH in spotted halibut, and would help us better understand the reproductive endocrine mechanism of spotted halibut. PMID:22674773

  1. Molecular Cloning and Gene Expression of Canine Apoptosis Inhibitor of Macrophage

    PubMed Central

    TOMURA, Shintaro; UCHIDA, Mona; YONEZAWA, Tomohiro; KOBAYASHI, Masato; BONKOBARA, Makoto; ARAI, Satoko; MIYAZAKI, Toru; TAMAHARA, Satoshi; MATSUKI, Naoaki

    2014-01-01

    Apoptosis inhibitor of macrophage (AIM) plays roles in survival of macrophages. In this study, we cloned canine AIM cDNA and observed its transcriptional expression levels in various tissues. The coding sequence of canine AIM was 1,023 bp encoding 340 amino acid residues, which had around 65% homology with those of the human, mouse and rat. Transcriptional expression of AIM was observed in the spleen, lung, liver and lymph node, which confirmed the expression of canine AIM in tissue macrophages. Moreover, AIM was highly expressed in one of the canine histiocytic sarcoma cell lines. CD36, the receptor of AIM, was also expressed in various tissues and these cell lines. These findings are useful to reveal the actual functions of canine AIM. PMID:25649949

  2. Molecular cloning and sequence analysis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene.

    PubMed Central

    Bzik, D J; Li, W B; Horii, T; Inselburg, J

    1987-01-01

    Genomic DNA clones that coded for the bifunctional dihydrofolate reductase (DHFR) and thymidylate synthase (TS) (DHFR-TS) activities from a pyrimethamine-sensitive strain of Plasmodium falciparum were isolated and sequenced. The deduced DHFR-TS protein contained 608 amino acids (71,682 Da). The coding region for DHFR-TS contained no intervening sequences and had a high A + T content (75%). The DHFR domain, in the amino-terminal portion of the protein, was joined by a 94-amino acid junction sequence to the TS domain in the carboxyl-terminal portion of the protein. The TS domain was more conserved than the DHFR domain and both P. falciparum domains were more homologous to eukaryotic than to prokaryotic forms of the enzymes. Predicted secondary structures of the DHFR and TS domains were nearly identical to the structures identified in other DHFR and TS enzymes. PMID:2825189

  3. Molecular cloning and sequence analysis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene.

    PubMed

    Bzik, D J; Li, W B; Horii, T; Inselburg, J

    1987-12-01

    Genomic DNA clones that coded for the bifunctional dihydrofolate reductase (DHFR) and thymidylate synthase (TS) (DHFR-TS) activities from a pyrimethamine-sensitive strain of Plasmodium falciparum were isolated and sequenced. The deduced DHFR-TS protein contained 608 amino acids (71,682 Da). The coding region for DHFR-TS contained no intervening sequences and had a high A + T content (75%). The DHFR domain, in the amino-terminal portion of the protein, was joined by a 94-amino acid junction sequence to the TS domain in the carboxyl-terminal portion of the protein. The TS domain was more conserved than the DHFR domain and both P. falciparum domains were more homologous to eukaryotic than to prokaryotic forms of the enzymes. Predicted secondary structures of the DHFR and TS domains were nearly identical to the structures identified in other DHFR and TS enzymes. PMID:2825189

  4. Molecular cloning, polymorphisms, and expression analysis of the RERG gene in indigenous Chinese goats.

    PubMed

    Sui, M X; Wang, H H; Wang, Z W

    2015-01-01

    The current study aimed to investigate the coding sequence, polymorphisms, and expression of the RERG gene in indigenous Chinese goats. cDNA of RERG, obtained through reverse transcription PCR was analyzed using bioinformatic techniques. Polymorphisms in the exon regions of the RERG gene were identified and their associations with growth traits in three varieties of indigenous Chinese goats were investigated. Expression of the RERG gene in three goat breeds of the same age was detected using real-time quantitative PCR. The results revealed that the cDNA of RERG, which contained a complete open reading frame of 20-620 bp, was 629 bp in length. The associated accession numbers in GenBank are JN672576, JQ917222, and JN580309 for the QianBei Ma goat, the GuiZhou white goat, and the GuiZhou black goat, respectively. Four consistent SNP sites were found in the exon regions of the RERG gene for the three goat breeds. mRNA expression of the RERG gene differed between different tissues in adult goats of same age. The highest expression was observed in lung and spleen tissues, while the lowest expression was recorded in thymus gland tissue. In addition, the expression of the RERG gene in the muscle of Guizhou white goat, GuiZhou black goat, and QianBei Ma goat decreased sequentially. Our results lay the foundations for further investigation into the role of the RERG gene in goat growth traits. PMID:26634455

  5. Molecular cloning, expression analysis and subcellular localization of four DELLA genes from hybrid poplar.

    PubMed

    Liu, Sian; Xuan, Lei; Xu, Li-An; Huang, Minren; Xu, Meng

    2016-01-01

    Gibberellic acid (GA) signaling regulates diverse aspects of plant growth and developmental processes. The DELLA repressors of GA signaling are named for an N-terminal conserved DELLA domain. In this study, four genes encoding DELLA proteins, PeRGA1, PeRGA2, PeGAI1 and PeGAI2, were isolated and characterized in poplar. A gene structural analysis revealed that the DELLA genes were all intron-free. Multiple protein sequence alignments revealed that these proteins contained seven highly conserved domains: the DELLA domain, the TVHYNP domain, leucine heptad repeat I (LHR I), the VHIID domain, leucine heptad repeat II (LHR II), the PFYRE domain, and the SAM domain. Temporal expression patterns of these genes were profiled during the adventitious root development of poplar. The four DELLA genes were expressed in root, stem and leaf in a dynamic manner. The subcellular localization demonstrated that these DELLA genes were mainly localized to the nucleus. These results suggest that the four DELLA genes may play diverse regulatory roles in the adventitious root, stem and leaf development of poplar, and contribute to improving our understanding of conserved and divergent aspects of DELLA proteins that restrain GA signaling in various species. PMID:27478746

  6. Molecular cloning, sequence characterization and expression pattern of Rab18 gene from watermelon (Citrullus lanatus)

    PubMed Central

    Xinli, Xiao; Lei, Peng

    2015-01-01

    The complete mRNA sequence of watermelon Rab18 gene was amplified through the rapid amplification of cDNA ends (RACE) method. The full-length mRNA was 1010 bp containing a 645 bp open reading frame, which encodes a protein of 214 amino acids. Sequence analysis revealed that watermelon Rab18 protein shares high homology with the Rab18 of cucumber (99%), muskmelon (98%), Morus notabilis (90%), tomato (89%), wine grape (89%) and potato (88%). Phylogenetic analysis revealed that watermelon Rab18 gene has a closer genetic relationship with Rab18 gene of cucumber and muskmelon. Tissue expression profile analysis indicated that watermelon Rab18 gene was highly expressed in root, stem and leaf, moderately expressed in flower and weakly expressed in fruit. PMID:26019638

  7. Molecular cloning and characterization of the pgm gene encoding phosphoglucomutase of Escherichia coli.

    PubMed Central

    Lu, M; Kleckner, N

    1994-01-01

    We report here the identification and characterization of pgm, a gene in Escherichia coli that encodes the enzyme phosphoglucomutase, specifically required for the catalysis of the interconversion of glucose 1-phosphate and glucose 6-phosphate. The predicted amino acid sequence of the pgm gene is highly conserved in E. coli, Acetobacter xylinum, Saccharomyces cerevisiae, rabbits, and humans. pgm deletion mutant strains are deficient in phosphoglucomutase activity. Images PMID:8083177

  8. Molecular cloning and characterization of the promoter region of the porcine apolipoprotein E gene.

    PubMed

    Xia, Jihan; Hu, Bingjun; Mu, Yulian; Xin, Leilei; Yang, Shulin; Li, Kui

    2014-05-01

    Apolipoprotein E (APOE), a component of lipoproteins plays an important role in the transport and metabolism of cholesterol, and is associated with hyperlipoproteinemia and Alzheimer's disease. In order to further understand the characterization of APOE gene, the promoter of APOE gene of Landrace pigs was analyzed in the present study. The genomic structure and amino acid sequence in pigs were analyzed and found to share high similarity in those of human but low similarity in promoter region. Real-time PCR revealed the APOE gene expression pattern of pigs in diverse tissues. The highest expression level was observed in liver, relatively low expression in other tissues, especially in stomach and muscle. Furthermore, the promoter expressing in Hepa 1-6 was significantly better at driving luciferase expression compared with C2C12 cell. After analysis of porcine APOE gene promoter regions, potential transcription factor binding sites were predicted and two GC signals, a TATA box were indicated. Results of promoter activity analysis indicated that one of potential regulatory elements was located in the region -669 to -259, which was essential for a high expression of the APOE gene. Promoter mutation and deletion analysis further suggested that the C/EBPA binding site within the APOE promoter was responsible for the regulation of APOE transcription. Electrophoretic mobility shift assays also showed the binding site of the transcription factor C/EBPA. This study advances our knowledge of the promoter of the porcine APOE gene. PMID:24464129

  9. Molecular Cloning, Characterization, and Differential Expression of a Glucoamylase Gene from the Basidiomycetous Fungus Lentinula edodes

    PubMed Central

    Zhao, J.; Chen, Y. H.; Kwan, H. S.

    2000-01-01

    The complete nucleotide sequence of putative glucoamylase gene gla1 from the basidiomycetous fungus Lentinula edodes strain L54 is reported. The coding region of the genomic glucoamylase sequence, which is preceded by eukaryotic promoter elements CAAT and TATA, spans 2,076 bp. The gla1 gene sequence codes for a putative polypeptide of 571 amino acids and is interrupted by seven introns. The open reading frame sequence of the gla1 gene shows strong homology with those of other fungal glucoamylase genes and encodes a protein with an N-terminal catalytic domain and a C-terminal starch-binding domain. The similarity between the Gla1 protein and other fungal glucoamylases is from 45 to 61%, with the region of highest conservation found in catalytic domains and starch-binding domains. We compared the kinetics of glucoamylase activity and levels of gene expression in L. edodes strain L54 grown on different carbon sources (glucose, starch, cellulose, and potato extract) and in various developmental stages (mycelium growth, primordium appearance, and fruiting body formation). Quantitative reverse transcription PCR utilizing pairs of primers specific for gla1 gene expression shows that expression of gla1 was induced by starch and increased during the process of fruiting body formation, which indicates that glucoamylases may play an important role in the morphogenesis of the basidiomycetous fungus. PMID:10831434

  10. Insertional Mutagenesis in Neurospora Crassa: Cloning and Molecular Analysis of the Preg(+) Gene Controlling the Activity of the Transcriptional Activator Nuc-1

    PubMed Central

    Kang, S.; Metzenberg, R. L.

    1993-01-01

    The transcriptional activator NUC-1 controls the transcription of the genes for phosphorus acquisition enzymes, and its activity is regulated by the negative regulatory factors, PREG and PGOV. In this report, we describe the cloning and molecular analysis of the preg(+) gene. In Neurospora crassa, as in higher eukaryotes, transformation frequently results in nonhomologous integration of transforming DNA. Insertion of transforming DNA into host genes mutates the gene and provides a molecular tag for cloning it. We obtained two mutants that have an insertion in the preg(+) and pgov(+) genes, respectively, among 2 X 10(5) transformants. The preg(+) gene was cloned by screening a Neurospora genomic DNA library with DNA sequences flanking the transforming DNA of the rescued plasmid. Northern analysis showed that the transcription of the preg(+) gene is not regulated by phosphate. The carboxy-terminal half of PREG shows strong homology with Saccharomyces cerevisiae PHO80 whose function is analogous to that of PREG. The preg(c) mutations are located in the well conserved residues which may directly interact with the residues in the regulatory domain of NUC-1. PMID:8436269

  11. Molecular cloning, mRNA expression, and characterization of HSP90 gene from Chinese mitten crab Eriocheir japonica sinensis.

    PubMed

    Li, Peng; Zha, Jie; Zhang, Zhenhua; Huang, Hua; Sun, Hongying; Song, Daxiang; Zhou, Kaiya

    2009-07-01

    HSP90 is a highly conserved molecular chaperone important in the maturation of a broad spectrum of proteins. Using expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques, an HSP90 gene designated as EjsHSP90 was cloned and characterized from the Chinese mitten crab Eriocheir japonica sinensis. The full-length cDNA of EjsHSP90 is 2,517 bp and contains an open reading frame of 2157 bp which encodes a 718 amino acid polypeptide (82.8 kDa) bearing characteristics of the HSP90 family and an ATP binding domain. Sequence alignment shows that EjsHSP90 shared 79%-96% identity with HSP90 sequences reported in other animals, and it shares identical structural features. Fluorescent real-time quantitative RT-PCR approach was performed to examine the expression profiles of EjsHSP90 mRNA by testing its relative level in three types of tissues at three different developmental stages, respectively. We found that EjsHSP90 is expressed throughout the three developmental stages but expression levels varied among different body parts of crabs. EjsHSP90 mRNA expression in the abdomen of the first crab stage is consistently higher than that of the other two stages, suggesting that EjsHSP90 gene is involved in the crabs' early developmental process, especially in the crab brachyurization process. Results from quantitative RT-PCR excluded the possibility that the expression of EjsHSP90 mRNA is induced primarily by osmotic stress. Phylogenetic analyses reveal that HSP90 gene is informative and complementary for reconstruction of arthropod phylogenetic relationships. PMID:19166961

  12. Molecular cloning, sequence characterization, and gene expression profiling of a novel water buffalo (Bubalus bubalis) gene, AGPAT6.

    PubMed

    Song, S; Huo, J L; Li, D L; Yuan, Y Y; Yuan, F; Miao, Y W

    2013-01-01

    Several 1-acylglycerol-3-phosphate-O-acyltransferases (AGPATs) can acylate lysophosphatidic acid to produce phosphatidic acid. Of the eight AGPAT isoforms, AGPAT6 is a crucial enzyme for glycerolipids and triacylglycerol biosynthesis in some mammalian tissues. We amplified and identified the complete coding sequence (CDS) of the water buffalo AGPAT6 gene by using the reverse transcription-polymerase chain reaction, based on the conversed sequence information of the cattle or expressed sequence tags of other Bovidae species. This novel gene was deposited in the NCBI database (accession No. JX518941). Sequence analysis revealed that the CDS of this AGPAT6 encodes a 456-amino acid enzyme (molecular mass = 52 kDa; pI = 9.34). Water buffalo AGPAT6 contains three hydrophobic transmembrane regions and a signal 37-amino acid peptide, localized in the cytoplasm. The deduced amino acid sequences share 99, 98, 98, 97, 98, 98, 97 and 95% identity with their homologous sequences from cattle, horse, human, mouse, orangutan, pig, rat, and chicken, respectively. The phylogenetic tree analysis based on the AGPAT6 CDS showed that water buffalo has a closer genetic relationship with cattle than with other species. Tissue expression profile analysis shows that this gene is highly expressed in the mammary gland, moderately expressed in the heart, muscle, liver, and brain; weakly expressed in the pituitary gland, spleen, and lung; and almost silently expressed in the small intestine, skin, kidney, and adipose tissues. Four predicted microRNA target sites are found in the water buffalo AGPAT6 CDS. These results will establish a foundation for further insights into this novel water buffalo gene. PMID:24114207

  13. Molecular cloning and characterization of the glyceraldehyde-3-phosphate dehydrogenase gene from Penicillium expansum PE-12.

    PubMed

    Zhang, T; Qi, Z; Yu, Q S; Tang, K X

    2013-01-01

    Penicillium expansum produces large amounts of lipase, which is widely used in laundry detergent and leather industry. We isolated the glyceraldehyde-3-phosphate dehydrogenase gene (PeGPD) from P. expansum PE-12 through reverse transcriptase PCR and 5'-3' rapid amplification of cDNA ends (RACE-PCR). The gene is 1266 bp long, including an ORF of 1014 bp, encoding a polypeptide chain of 337 amino acids. A phylogenetic tree based on GPD proteins showed that P. expansum is close to Aspergillus species, but comparatively distant from P. marneffei. Southern blot results revealed a single copy of PeGPD, and expression analysis gave evidence of high expression levels. PeGPD genes have potential for genetic engineering of P. expansum for industrial lipase production. PMID:23420404

  14. Molecular Cloning and Expression Analysis of hyp-1 Type PR-10 Family Genes in Hypericum perforatum.

    PubMed

    Karppinen, Katja; Derzsó, Emese; Jaakola, Laura; Hohtola, Anja

    2016-01-01

    Hypericum perforatum L. is an important medicinal plant for the treatment of depression. The plant contains bioactive hypericins that accumulate in dark glands present especially in reproductive parts of the plant. In this study, pathogenesis-related class 10 (PR-10) family genes were identified in H. perforatum, including three previously unidentified members with sequence homology to hyp-1, a phenolic coupling protein that has earlier been suggested to participate in biosynthesis and binding/transportation of hypericin. The PR-10 genes showed constitutive but variable expression patterns in different H. perforatum tissues. They were all expressed at relatively high levels in leaves, variably in roots and low levels in stem and reproductive parts of the plant with no specific association with dark glands. The gene expression was up-regulated in leaves after salicylic acid, abscisic acid and wounding treatments but with variable levels. To study exact location of the gene expression, in situ hybridization of hyp-1 transcripts was performed and the accumulation of the Hyp-1 protein was examined in various tissues. The presence of Hyp-1 protein in H. perforatum tissues mostly paralleled with the mRNA levels. In situ RNA hybridization localized the hyp-1 transcripts predominantly in vascular tissues in root and stem, while in leaf the mRNA levels were high also in mesophyll cells in addition to vasculature. Our results indicate that the studied PR-10 genes are likely to contribute to the defense responses in H. perforatum. Furthermore, despite the location of the hyp-1 transcripts in vasculature, no support for the transportation of the Hyp-1 protein to dark glands was found in the current study. The present results together with earlier data question the role of the hyp-1 as a key gene responsible for the hypericin biosynthesis in dark glands of H. perforatum. PMID:27148343

  15. Molecular Cloning and Expression Analysis of hyp-1 Type PR-10 Family Genes in Hypericum perforatum

    PubMed Central

    Karppinen, Katja; Derzsó, Emese; Jaakola, Laura; Hohtola, Anja

    2016-01-01

    Hypericum perforatum L. is an important medicinal plant for the treatment of depression. The plant contains bioactive hypericins that accumulate in dark glands present especially in reproductive parts of the plant. In this study, pathogenesis-related class 10 (PR-10) family genes were identified in H. perforatum, including three previously unidentified members with sequence homology to hyp-1, a phenolic coupling protein that has earlier been suggested to participate in biosynthesis and binding/transportation of hypericin. The PR-10 genes showed constitutive but variable expression patterns in different H. perforatum tissues. They were all expressed at relatively high levels in leaves, variably in roots and low levels in stem and reproductive parts of the plant with no specific association with dark glands. The gene expression was up-regulated in leaves after salicylic acid, abscisic acid and wounding treatments but with variable levels. To study exact location of the gene expression, in situ hybridization of hyp-1 transcripts was performed and the accumulation of the Hyp-1 protein was examined in various tissues. The presence of Hyp-1 protein in H. perforatum tissues mostly paralleled with the mRNA levels. In situ RNA hybridization localized the hyp-1 transcripts predominantly in vascular tissues in root and stem, while in leaf the mRNA levels were high also in mesophyll cells in addition to vasculature. Our results indicate that the studied PR-10 genes are likely to contribute to the defense responses in H. perforatum. Furthermore, despite the location of the hyp-1 transcripts in vasculature, no support for the transportation of the Hyp-1 protein to dark glands was found in the current study. The present results together with earlier data question the role of the hyp-1 as a key gene responsible for the hypericin biosynthesis in dark glands of H. perforatum. PMID:27148343

  16. Molecular cloning and characterization of lactate dehydrogenase gene from Eimeria tenella.

    PubMed

    Dong, Hui; Wang, Yange; Zhao, Qiping; Han, Hongyu; Zhu, Shunhai; Li, Liujia; Wu, Youling; Huang, Bing

    2014-08-01

    Lactate dehydrogenase (LDH) is a key enzyme in the glycolytic pathway and is crucial for parasite survival. In this study, we cloned and expressed the LDH of Eimeria tenella (EtLDH). Real-time polymerase chain reaction and Western blot analysis revealed that the expression of EtLDH was developmentally regulated at the messenger RNA (mRNA) and protein levels. EtLDH mRNA levels were higher in second-generation merozoites than in other developmental stages (unsporulated oocysts, sporulated oocysts, and sporozoites). EtLDH protein expression levels were most prominent in second-generation merozoites, moderately expressed in unsporulated oocysts and sporulated oocysts, and weakly detected in sporozoites. Immunostaining with anti-recombinant EtLDH (rEtLDH) antibody indicated that EtLDH was mainly located in the anterior region in free sporozoites and became concentrated in the anterior region of intracellular sporozoites except for the apex after invasion into DF-1 cells. Specific staining of EtLDH protein was more intense in trophozoites and immature first-generation schizonts, but decreased in mature first-generation schizonts. Inhibition of EtLDH function using specific antibodies cannot efficiently reduce the ability of E. tenella sporozoites to invade host cells. These results suggest that EtLDH may be involved in glycolysis during the first-generation merogony stage in E. tenella and has little role in host invasion. PMID:24906988

  17. Molecular cloning and expression of an additional epidermal growth factor receptor-related gene.

    PubMed Central

    Plowman, G D; Whitney, G S; Neubauer, M G; Green, J M; McDonald, V L; Todaro, G J; Shoyab, M

    1990-01-01

    Epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), and amphiregulin are structurally and functionally related growth regulatory proteins. These secreted polypeptides all bind to the 170-kDa cell-surface EGF receptor, activating its intrinsic kinase activity. However, amphiregulin exhibits different activities than EGF and TGF-alpha in a number of biological assays. Amphiregulin only partially competes with EGF for binding EGF receptor, and amphiregulin does not induce anchorage-independent growth of normal rat kidney cells (NRK) in the presence of TGF-beta. Amphiregulin also appears to abrogate the stimulatory effect of TGF-alpha on the growth of several aggressive epithelial carcinomas that overexpress EGF receptor. These findings suggest that amphiregulin may interact with a separate receptor in certain cell types. Here we report the cloning of another member of the human EGF receptor (HER) family of receptor tyrosine kinases, which we have named "HER3/ERRB3." The cDNA was isolated from a human carcinoma cell line, and its 6-kilobase transcript was identified in various human tissues. We have generated peptide-specific antisera that recognizes the 160-kDa HER3 protein when transiently expressed in COS cells. These reagents will allow us to determine whether HER3 binds amphiregulin or other growth regulatory proteins and what role HER3 protein plays in the regulation of cell growth. Images PMID:2164210

  18. Cloning and characterization of resistance gene candidate sequences and molecular marker development in gerbera (Gerbera hybrida)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Improving disease resistance has become an important breeding objective in gerbera, one of the most important floricultural crops in the world. Development and application of molecular markers are expected to assist selection of gerberas with improved disease resistance. The availability of resistan...

  19. Molecular Cloning and Characterization of AltSB, A Major Aluminum Tolerance Gene in Sorghum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aluminum (Al) toxicity on acid soils represents a major constraint for crop production as ~ 50% of the potentially arable soils worldwide are acidic. Therefore, understanding the genetic and molecular mechanisms underlying plant Al tolerance has been a major focus for a number of laboratories aroun...

  20. Molecular cloning and expression analyses of RPS3a gene from mulberry under abiotic stresses and among different mulberry varieties.

    PubMed

    Qian, J; Zhou, H; Zhao, M D; Wang, H; Li, F; Wang, Y H; Fang, R J; Zhao, W G; Kim, H J

    2016-01-01

    A full-length cDNA sequence coding ribosomal protein S3a of mulberry tree, which we designated MmRPS3a (GenBank accession No. KR610331), was cloned based on mulberry expressed sequence tags. Sequence analysis showed that the MmRPS3a is 1089 bp long and contains a 80-bp 5'-UTR (untranslated region) and a 220-bp 3'-UTR. Its open reading frame consists of a 789-bp encoding 262 amino acids with a predicted molecular weight of 30.053 kDa and an isoelectric point of 9.84. Homology analysis revealed that MmRPS3a gene is highly conservative in mulberry and other species including Morus notabilis, Theobroma cacao, and Ricinus communis. Phylogenetic analysis based on MmRPS3a of other species showed that mulberry had a closer relationship with Prunus persica, Arabidopsis thaliana, Solanum tuberosum, Solanum lycopersicum, and Vitis vinifera. The results of quantitative PCR analysis showed that the transcriptional level of MmRPS3a mRNA changed significantly under the conditions of hypothermia, aridity, salt stress, and varieties of differing resistances. PMID:27173298

  1. Molecular cloning and expression analysis of an 1-aminocyclopropane-1-carboxylate synthase gene from Oncidium Gower Ramsey.

    PubMed

    Shi, Le-Song; Liu, Jin-Ping

    2016-01-01

    1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) is a rate-limiting enzyme in the biosynthesis of ethylene which regulates many aspects of the plant development and responses to biotic and abiotic stresses. In this study, a full-length cDNA of ACC synthase, OnACS2, was cloned from the senescing flower of Oncidium Gower Ramsey by RACE. The full-length cDNA of OnACS2 (GenBank accession no. JQ822087) was 1557 bp in length with an open reading frame (ORF) of 1308 bp encoding for a protein of 435 amino acid residues. The predicted OnACS2 protein had a molecular mass of 49.1 kDa with pI value of 7.51. Phylogenetic analysis indicated its evolutionary relationships with corresponding orthologous sequences in orchids, Hosta ventricosa and monocots. Real-time PCR assay demonstrated that OnACS2 was constitutively expressed in all tested organs with the highest transcript level in the gynandria. Differential expression pattern of OnACS2 gene correlated to the ethylene production and the subsequent occurrence of senescent symptoms in flower suggested that OnACS2 probably played an important role in the initiation of flower senescence. PMID:26631967

  2. Molecular cloning and polymorphism of the porcine H2AFZ gene.

    PubMed

    Zhang, Y H; Mei, S Q; Peng, X W; Zuo, B; Lei, M G; Xiong, Y Z; Li, F E

    2009-06-01

    H2A histone family, member Z (H2A.Z) is required for early mammalian development. In the present study, the 932 bp of full-length cDNA encoding a 128 amino-acid protein and the sequences of intron 2 to 4 of the porcine H2A histone family, member Z (pH2AFZ) gene were obtained. By comparative sequencing of pH2AFZ gene in Large White and Meishan pigs, a 4 bp deletion/insertion in intron 2 was detected and a PCR-Bsu15I-RFLP was established to detect this variation. In DIV (4th Dam line of Chinese lean-type new lines) pigs, the first-parity females with AA genotype had fewer piglets born alive (-2.64 and -1.83 piglets per litter) than those with AB (P < 0.01) and BB (P < 0.05) genotype. The additive allelic and dominance effect were estimated to be 0.92 (P < 0.05) and -0.87 piglets per litter (P < 0.01) for number of piglets born alive, respectively. This result suggests that the pH2AFZ gene might be a good candidate gene of litter-size trait and provides some marker information for marker-assisted selection. PMID:22444763

  3. Molecular cloning and expression analysis of multiple polyphenol oxidase genes in developing wheat (Triticum aestivum) kernels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyphenol oxidase (PPO, EC 1.10.31) is a major cause of discoloring in raw dough containing wheat flour. Minimization of PPO activity has proven difficult because bread wheat is genetically complex, composed of the genomes of three grass species. The PPO-A1 and PPO-D1 genes, on chromosomes 2A and...

  4. Molecular cloning of extensive sequences of the in vitro synthesized chicken ovalbumin structural gene.

    PubMed Central

    Humphries, P; Cochet, M; Krust, A; Gerlinger, P; Kourilsky, P; Chambon, P

    1977-01-01

    Double-stranded DNA molecules complementary to ovalbumin chicken messenger RNA were synthesized in vitro and integrated into the E. coli plasmid pCR1 using an oligodG-dc tailing procedure. The resultant hybrid plasmids, amplified by transfection of E. coli, were shown by hybridization and gel electrophoresis to contain extensive DNA sequences of the ovalbumin structural gene. Images PMID:333389

  5. MOLECULAR CLONING AND CHARACTERIZATION OF CHROMOSOME-ENCODED CITRATE SYNTHASE GENE FROM SINORHIZOBIUM FREDII USDA257

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Citrate synthase, a key metabolic enzyme that condenses acetyl-CoA and oxaloacetate to citrate, plays an important role in nodulation and nitrogen fixation. We have isolated a citrate synthase gene by screening a Sinorhizobium fredii USDA257 cosmid library with a heterologous probe from S. meliloti....

  6. Molecular cloning and functional analysis of an ethylene receptor gene from sugarcane (Saccharum spp.) by hormone and environmental stresses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ethylene receptor (ethylene response sensor, ERS) is the primary component involving in the ethylene biosynthesis and ethylene signal transduction pathway. In the present study, a GZ-ERS gene encoding ERS was cloned from a sugarcane cv. YL17 (Saccharum spp.) using RT-PCR and ligation-mediated PCR wi...

  7. Molecular cloning of a human gene that is a member of the nerve growth factor family.

    PubMed Central

    Jones, K R; Reichardt, L F

    1990-01-01

    Cell death within the developing vertebrate nervous system is regulated in part by interactions between neurons and their innervation targets that are mediated by neurotrophic factors. These factors also appear to have a role in the maintenance of the adult nervous system. Two neurotrophic factors, nerve growth factor and brain-derived neurotrophic factor, share substantial amino acid sequence identity. We have used a screen that combines polymerase chain reaction amplification of genomic DNA and low-stringency hybridization with degenerate oligonucleotides to isolate human BDNF and a human gene, neurotrophin-3, that is closely related to both nerve growth factor and brain-derived neurotrophic factor. mRNA products of the brain-derived neurotrophic factor and neurotrophin-3 genes were detected in the adult human brain, suggesting that these proteins are involved in the maintenance of the adult nervous system. Neurotrophin-3 is also expected to function in embryonic neural development. Images PMID:2236018

  8. Molecular cloning, characterization and expression analysis of a CC chemokine gene from miiuy croaker (Miichthys miiuy).

    PubMed

    Cheng, Yuanzhi; Sun, Yuena; Shi, Ge; Wang, Rixin; Xu, Tianjun

    2012-12-01

    Chemokines are a family of structurally related chemotactic cytokines that regulate the migration of leukocytes, under both physiological and inflammatory conditions. A partial cDNA of CC chemokine gene designed as Mimi-CC3 was isolated from miiuy croaker (Miichthys miiuy) spleen cDNA library. Unknown 3' part of the cDNA was amplified by 3'-RACE. The complete cDNA of Mimi-CC3 contains an 89-nt 5'-UTR, a 303-nt open reading frame and a 441-nt 3'-UTR. Three exons and two introns were identified in Mimi-CC3. The deduced Mimi-CC3 protein sequences contain a 22 amino acids signal peptide and a 78 amino acids mature polypeptide, which possesses the typical arrangement of four cysteines as found in other known CC chemokines. It shares low amino acid sequence identities with most other fish and mammalian CC chemokines (less than 54.1 %), but shares very high identities with large yellow croaker CC chemokine (94.6 %). Phylogenetic analysis showed that Mimi-CC3 gene may have an orthologous relationship with mammalian/amphibian CCL25 gene. Tissue expression distributed analysis showed that Mimi-CC3 gene was constitutively expressed in all nine tissues examined, although at different levels. Upon stimulated with Vibrio anguillarum, the time-course analysis using a real-time PCR showed that Mimi-CC3 transcript in kidney and liver was obviously up-regulated and reached the peak levels, followed by a recovery. Mimi-CC3 expression in kidney was more strongly increased than in liver. However, down-regulation was observed in spleen. These results indicated that Mimi-CC3 plays important roles in miiuy croaker immune response as well as in homeostatic mechanisms. PMID:22736236

  9. Molecular cloning and expression analysis of interferon stimulated gene 15 (ISG15) in turbot, Scophthalmus maximus.

    PubMed

    Lin, Jing-Yun; Hu, Guo-Bin; Liu, Da-Hai; Li, Song; Liu, Qiu-Ming; Zhang, Shi-Cui

    2015-08-01

    The interferon stimulated gene 15 (ISG15) is strongly induced in many cell types by double-stranded RNA (polyinosinic: polycytidylic acid, poly I:C) and viral infection. In this study, we described the nucleotide, mRNA tissue distribution and regulation of an ISG15 gene from turbot, Scophthalmus maximus (SmISG15). SmISG15 gene is 862 bp in length, composed of two exons and one intron, and encodes 158 amino acids. The deduced protein exhibits the highest homology (44.7-71.2% identity) with ISG15s from other fishes and possesses two conserved tandem ubiquitin-like (UBL) domains and a C-terminal RLRGG conjugating motif known to be important for the functions of ISG15s in vertebrates. Phylogenetic analysis grouped SmISG15 into fish ISG15. SmISG15 mRNA was constitutively expressed in all tissues examined, with higher levels observed in immune organs. Gene expression analysis was performed for SmISG15 in the spleen, head kidney, gills and muscle of turbots challenged with poly I:C or turbot reddish body iridovirus (TRBIV) over a 7-day time course. The result showed that SmISG15 was upregulated by both stimuli in all four tissues, with induction by poly I:C apparently stronger and initiated more quickly. A two-wave induced expression of SmISG15 was seen in the spleen, head kidney and gills, suggesting an induction of SmISG15 either by IFN-dependent or -independent pathway. These results provide insights into the roles of fish ISG15 in antiviral immunity. PMID:26095010

  10. Molecular cloning of the gene for the allatostatin family of neuropeptides from the cockroach Diploptera punctata.

    PubMed Central

    Donly, B C; Ding, Q; Tobe, S S; Bendena, W G

    1993-01-01

    Allatostatins (ASTs) are insect neuropeptides that inhibit juvenile hormone biosynthesis by the corpora allata. We have isolated a cDNA from the cockroach Diploptera punctata that encodes a 41.5-kDa precursor polypeptide containing the AST family of peptides. Translation of the cDNA revealed a 370-amino acid pre-pro-peptide consisting of 13 AST-type peptides and appropriate processing sites for endoproteolytic cleavage and amidation. The 13 potential AST sequences are characterized by the C-terminal AST corestructure Phe-Gly-Leu-NH2, with only one exception. Separating the clustered ASTs in the precursor, three acidic spacer regions are found. Contained within the largest of these are two potentially related peptides that may also be processed. Southern blot analysis revealed the presence of a single copy of the AST gene per haploid genome, as well as the probability that the gene may be present in at least two allelic forms. In situ hybridization indicated the AST-encoding gene is expressed in neurosecretory cells of D. punctata brain. Images Fig. 2 Fig. 4 Fig. 5 PMID:8415611

  11. Molecular cloning of the gene for the allatostatin family of neuropeptides from the cockroach Diploptera punctata.

    PubMed

    Donly, B C; Ding, Q; Tobe, S S; Bendena, W G

    1993-10-01

    Allatostatins (ASTs) are insect neuropeptides that inhibit juvenile hormone biosynthesis by the corpora allata. We have isolated a cDNA from the cockroach Diploptera punctata that encodes a 41.5-kDa precursor polypeptide containing the AST family of peptides. Translation of the cDNA revealed a 370-amino acid pre-pro-peptide consisting of 13 AST-type peptides and appropriate processing sites for endoproteolytic cleavage and amidation. The 13 potential AST sequences are characterized by the C-terminal AST corestructure Phe-Gly-Leu-NH2, with only one exception. Separating the clustered ASTs in the precursor, three acidic spacer regions are found. Contained within the largest of these are two potentially related peptides that may also be processed. Southern blot analysis revealed the presence of a single copy of the AST gene per haploid genome, as well as the probability that the gene may be present in at least two allelic forms. In situ hybridization indicated the AST-encoding gene is expressed in neurosecretory cells of D. punctata brain. PMID:8415611

  12. Molecular cloning, genetic mapping, and expression analysis of four zebrafish c/ebp genes.

    PubMed

    Lyons, S E; Shue, B C; Lei, L; Oates, A C; Zon, L I; Liu, P P

    2001-12-27

    The CCAAT/enhancer binding protein family (C/EBP) are transcription factors that play integral roles in the development and function of many organ systems, including hematopoietic cells, adipose tissues, and liver. We have identified and characterized putative zebrafish orthologs of mammalian C/EBP alpha, beta, gamma, and delta using low-stringency hybridization screening and computer searches of the GenBank EST database. c/ebpa and g were mapped within 1 cM of each other on linkage group (LG) 7, syntenic with human CEBPA and G genes on chromosome 19. c/ebpb was mapped to LG8, and c/ebpd was mapped to LG24, on the same LG as a recently identified unique c/ebp in zebrafish, c/ebp1. The mapping of these genes established new syntenic relationships between LG8 and human chromosome 20, extended existing synteny between LG7 and human chromosome 19, and confirmed the synteny between LG24 and human chromosome 8. In addition, these syntenies between zebrafish and human chromosomes are also conserved in the mouse genome. To characterize the expression of these genes, RNA in situ hybridization in embryos of wild type and a hematopoietic mutant, cloche, was performed. The results showed that zebrafish c/ebpa, b, g, and d were expressed in many embryonic tissues. c/ebpa and b were expressed in a subset of hematopoietic cells in a region consistent with myeloid expression. In addition, there was expression of c/ebpa and b in the liver and c/ebpa, b, and d in regions of the gastrointestinal tract. The expression of the c/ebps may serve as important markers for analysis of myelopoiesis, hepatic development, and other developmental processes in the future. PMID:11750126

  13. Molecular cloning and characterization of two nicotinic acetylcholine receptor β subunit genes from Apis cerana cerana.

    PubMed

    Yu, Xiaoli; Wang, Mian; Kang, Mingjiang; Liu, Li; Guo, Xingqi; Xu, Baohua

    2011-08-01

    Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission in the insect nervous system and are important targets for insecticides. In this study, we identified and characterized two novel β subunit genes (Accβ1 and Accβ2) from Apis cerana cerana. Homology analysis indicated that Accβ1 and Accβ2 possess characteristics that are typical of nAChR subunits although Accβ2 was distinct from Accβ1 and the other nAChR subunits, due to its unusual transmembrane structure and uncommon exon-intron boundary within the genomic region encoding the TM1 transmembrane domain. Analysis of the 5' flanking regions indicated that Accβ1 and Accβ2 possess different regulatory elements, suggesting that the genes might exhibit various expression and regulatory patterns. RT-PCR analysis demonstrated that Accβ2 was expressed at a much higher level than Accβ1 in the tissues of adult bees. During development, Accβ1 was highly expressed at the pupal stages, whereas Accβ2 was abundantly expressed at the larval stages. Furthermore, Accβ1 and Accβ2 were both induced by exposure to various insecticides and environmental stresses although Accβ2 was more responsive than Accβ1. These results indicate that Accβ1 and Accβ2 may have distinct roles in insect growth and development and that they may belong to separate regulatory pathways involved in the response to insecticides and environmental stresses. This report is the first description of the differences between the nAChR β subunit genes in the Chinese honey bee and establishes an initial foundation for further study. PMID:21618599

  14. Molecular cloning and characterization of a Toll receptor gene from Macrobrachium rosenbergii.

    PubMed

    Srisuk, Chutima; Longyant, Siwaporn; Senapin, Saengchan; Sithigorngul, Paisarn; Chaivisuthangkura, Parin

    2014-02-01

    Toll receptors are cell surface molecules acting as pattern recognition receptors (PRRs) that have been implicated in the signaling pathway of innate immune responses. In this study, the full-length cDNA of a Toll receptor gene of Macrobrachium rosenbergii, designated MrToll, was successfully isolated using designed degenerate primers and the rapid amplification of cDNA ends (RACE). The MrToll gene sequence contained an open reading frame (ORF) of 2799 nucleotides encoding a protein of 932 amino acid residues. The protein contained distinct structural motifs of the Toll-like receptor (TLR) family, including an extracellular domain containing 15 leucine-rich repeats (LRRs), a transmembrane segment of 23 amino acids, and a cytoplasmic Toll/interleukin-1R (TIR) domain of 139 residues. Phylogenetic analysis revealed that MrToll and Toll receptor of Marsupenaeus japonicus (MjToll) evolved closely. However, the MrToll ORF demonstrated only 48-49% identity with shrimp Toll1, suggesting that MrToll isolated from a palaemonid shrimp might belong to a novel class of Toll receptors in shrimp. The transcripts of the MrToll gene were constitutively expressed in various tissues, with high levels in hemocytes, the stomach and muscle. A reverse transcriptase PCR assay demonstrated that the expression patterns of MrToll were distinctly modulated after Aeromonas caviae stimulation, with significant enhancement at 3-12 h post-challenge and a decline to basal levels at 24 h post-challenge. In addition, when MrToll-silenced shrimp were challenged with A. caviae, there was a significant increase in mortality and bacterial CFU counts. These results suggest that MrToll might be involved in host innate defense, especially against the pathogen A. caviae. PMID:24398262

  15. Molecular cloning of L-methylmalonyl-CoA mutase: gene transfer and analysis of mut cell lines.

    PubMed Central

    Ledley, F D; Lumetta, M; Nguyen, P N; Kolhouse, J F; Allen, R H

    1988-01-01

    L-Methylmalonyl-CoA mutase (MCM, EC 5.4.99.2) is a mitochondrial adenosylcobalamin-requiring enzyme that catalyzes the isomerization of L-methylmalonyl-CoA to succinyl-CoA. This enzyme is deficient in methylmalonic acidemia, an often fatal disorder of organic acid metabolism. Antibody against human placental MCM was used to screen human placenta and liver cDNA expression libraries for MCM cDNA clones. One clone expressed epitopes that could affinity-purify antibodies against MCM. A cDNA corresponding in length to the mRNA was obtained and introduced into COS cells by DNA-mediated gene transfer. Cells transformed with this clone expressed increased levels of MCM enzymatic activity. RNA blot analysis of cells genetically deficient in MCM indicates that several deficient cell lines have a specific decrease in the amount of hybridizable mRNA. These data confirm the authenticity of the MCM cDNA clone, establish the feasibility of constituting MCM activity by gene transfer for biochemical analysis and gene therapy, and provide a preliminary picture of the genotypic spectrum underlying MCM deficiency. Images PMID:2453061

  16. Molecular cloning of L-methylmalonyl-CoA mutase: Gene transfer and analysis of mut cell lines

    SciTech Connect

    Ledley, F.D.; Lumetta, M.; Nguyen, P.N.; Kolhouse, J.F.; Allen, R.H. )

    1988-05-01

    L-Methylmalonyl-CoA mutase (MCM, EC 5.4.99.2) is a mitochondrial adenosylcobalamin-requiring enzyme that catalyzes the isomerization of L-methylmalonyl-CoA to succinyl-CoA. This enzyme is deficient in methylmalonic acidemia, an often fatal disorder of organic acid metabolism. Antibody against human placental MCM was used to screen human placenta and liver cDNA expression libraries for MCM cDNA clones. One clone expressed epitopes that could affinity-purify antibodies against MCM. A cDNA corresponding in length to the mRNA was obtained and introduced into COS cells by DNA-mediated gene transfer. Cells transformed with this clone expressed increased levels of MCM enzymatic activity. RNA blot analysis of cells genetically deficient in MCM indicates that several deficient cell lines have a specific decrease in the amount of hybridizable mRNA. These data confirm the authenticity of the MCM cDNA clone, establish the feasibility of constituting MCM activity by gene transfer for biochemical analysis and gene therapy, and provide a preliminary picture of the genotypic spectrum underlying MCM deficiency.

  17. Molecular cloning and expression analysis of MAT1 gene in black tiger shrimp (Penaeus monodon).

    PubMed

    Wang, Y; Fu, M J; Zhao, C; Bao, W Y; Zhou, F L; Yang, Q B; Jiang, S G; Qiu, L H

    2016-01-01

    MAT1 (ménage à trois 1), an assembly factor and targeting subunit of the CDK-dependent kinase (CAK), can regulate the cell cycle, transcription, and DNA repair. This study was intended to investigate the role of MAT1 in the reproductive maturation of black tiger shrimp (Penaeus monodon). In this study, the P. monodon MAT1 (PmMAT1) gene was identified and characterized. The full-length cDNA of PmMAT1 was 1490 bp in length with an open-reading frame of 993 bp corresponding to 330 amino acids. The temporal expression of PmMAT1 in various tissues was measured by quantitative real-time PCR with the highest expression observed in ovaries. In the ovaries, the PmMAT1 gene was continuously but differentially expressed during the maturation stages. Comparative analyses of MAT1, CDK7, and cyclin H in the CAK complex of P. monodon indicated that the expression of CDK7 and cyclin H coincided with that of MAT1 during the ovary maturation stages. Serotonin (5-HT) injection promoted the expression level of PmMAT1 in the ovaries of shrimp at 6-48 h post-injection. These results indicate that PmMat1 plays a prominent role in the process of ovarian maturation. PMID:26909956

  18. Molecular cloning, mapping, and polymorphism of the porcine SCG2 gene.

    PubMed

    Du, Hong-Li; Chen, Jing; Zhang, Yu-Shan; Zhang, Xi-Quan

    2008-06-01

    The secretogranin II (SCG2) gene is associated with the synthesis and secretion of follicle-stimulating hormone and luteinizing hormone. In the present study, we have determined the complete cDNA sequence of pig SCG2, which was submitted to GenBank with accession no. AY870646. Its complete open reading frame of 1,851 nucleotides encodes 616 amino acids. The predicted protein shares 80-87% identity with mouse, human, and bovine SCG2 proteins, and all four species share almost complete identity in the secretoneurin and EM66 domains. Pig SCG2 is a protein of 589 amino acids and 68,132 Da, preceded by a signal peptide of 27 residues. It contains nine pairs of dibasic residues, which are used as potential cleavage sites for generation of physiologically active peptides. Analysis of the SCG2 gene across the INRA-Minnesota porcine radiation hybrid panel indicates close linkage with microsatellite marker SW2608, located on Sus scrofa chromosome 15 (SSC15) q25, which harbors several QTL for ovulation rate and meat quality. Comparative sequencing and EST analysis revealed nine SNPs in porcine SCG2 cDNA, including seven SNPs in the coding region and two SNPs in the 3' UTR. Four nonsynonymous SNPs (G622A, G1671T, C1718T, and A1790C) resulted in amino acid substitutions of Ala-->Thr, Glu-->Asp, Pro-->Leu, and Asn-->Thr, respectively. PMID:18278550

  19. Molecular Cloning and Expression Analysis of a Catalase Gene (NnCAT) from Nelumbo nucifera.

    PubMed

    Dong, Chen; Zheng, Xingfei; Diao, Ying; Wang, Youwei; Zhou, Mingquan; Hu, Zhongli

    2015-11-01

    Rapid amplification cDNA end (RACE) assay was established to achieve the complete cDNA sequence of a catalase gene (NnCAT) from Nelumbo nucifera. The obtained full-length cDNA was 1666 bp in size and contained a 1476-bp open reading frame. The 3D structural model of NnCAT was constructed by homology modeling. The putative NnCAT possessed all the main characteristic amino acid residues and motifs of catalase (CAT) protein family, and the phylogenetic analysis revealed that NnCAT grouped together with high plants. Moreover, recombinant NnCAT showed the CAT activity (758 U/mg) at room temperature, holding high activity during temperature range of 20-50 °C, then the optimal pH of recombinant protein was assessed from pH 4 to pH 11. Additionally, real-time PCR assay demonstrated that NnCAT mRNA was expressed in various tissues of N. nucifera, with the highest expression in young leaf and lowest level in the root, and mRNA level of NnCAT was significantly augmented in response to short-time mechanical wounding. Different expression pattern of NnCAT gene suggested that NnCAT probably played a defensive role in the initial stages of oxidative stress, regulating the level of reactive oxygen species (ROS) by extracellular stimuli such as short-time mechanical wounding. PMID:26299377

  20. Molecular cloning and expression analysis of peptidase genes in the fish-pathogenic scuticociliate Miamiensis avidus

    PubMed Central

    2013-01-01

    Background Parasite peptidases have been actively studied as vaccine candidates or drug targets for prevention or treatment of parasitic diseases because of their important roles for survival and/or invasion in the host. Like other parasites, the facultative histophagous ciliate Miamiensis avidus would possess peptidases that are closely associated with the invasion into the host tissue and survival in the host. Results The 17 genes encoding peptidases, including seven cathepsin-like cysteine peptidases, four serine carboxypeptidases, a eukaryotic aspartyl protease family protein, an ATP-dependent metalloprotease FtsH family protein, three leishmanolysin family proteins and a peptidase family M49 protein were identified from a Miamiensis avidus cDNA library by BLAST X search. Expression of genes encoding two cysteine peptidases, three leishmanolysin-like peptidases and a peptidase family M49 protein was up-regulated in the cell-fed ciliates compared to the starved ciliates. Especially, one cysteine peptidase (MaPro 4) and one leishmanolysin-like peptidase (MaPro 14) were transcribed more than 100-folds in the cell-fed ciliates. Conclusions The genetic information and transcriptional characteristics of the peptidases in the present results would be helpful to elucidate the role of peptidases in the invasion of scuticociliates into their hosts. PMID:23311870

  1. Molecular cloning and functional characterization of a mouse gene upregulated by lipopolysaccharide treatment reveals alternative splicing

    SciTech Connect

    Du, Kejun; Chen, Yaoming; Dai, Zongming; Bi, Yuan; Cai, Tongjian; Hou, Lichao; Chai, Yubo; Song, Qinghe; Chen, Sumin; Luo, Wenjing; Chen, Jingyuan

    2010-01-01

    Treatment of mouse cells with lipopolysaccharide (LPS) potently initiates an inflammatory response, but the underlying mechanisms are unclear. We therefore sought to characterize cDNA sequences of a new mouse LPS-responsive gene, and to evaluate the effects of MLrg. Full-length cDNAs were obtained from LPS-treated NIH3T3 cells. We report that the MLrg gene produces two alternative splice products (GenBank Accession Nos. (DQ316984) and (DQ320011)), respectively, encoding MLrgW and MLrgS polypeptides. Both proteins contain zinc finger and leucine zipper domains and are thus potential regulators of transcription. Expression of MLrgW and MLrgS were robustly upregulated following LPS treatment, and the proteins were localized predominantly in the nuclear membrane and cytoplasm. In stable transfectants over-expressing MLrgW the proportion of cells in G1 phase was significantly reduced, while in cells over-expressing MLrgS the proportion of cells in G2 was significantly increased; both proteins are thus potential regulators of cell cycle progression. Upregulation of MLrgW and MLrgS may be an important component of the LPS inflammatory pathway and of the host response to infection with GNB.

  2. Molecular cloning and functional analysis of an ERF gene from cotton (Gossypium hirsutum).

    PubMed

    Qiao, Zhi-Xin; Huang, Bo; Liu, Jin-Yuan

    2008-02-01

    Ethylene response factors (ERFs) play important roles in regulating plant biotic and abiotic stress tolerance. In this paper, a new ethylene response factor gene GhERF1 was isolated from cotton. The deduced amino acid sequence of GhERF1 contained an AP2/ERF domain, which shared high similarity with other reported AP2/ERF domains and was most closely related to the B3 subgroup of the ERF subfamily. The particle bombardment assay showed that GhERF1 functioned as an in vivo transcription activator in tobacco cells and was localized in the nuclei of onion epidermis cells. In addition, semi-quantitative RT-PCR revealed that GhERF1 accumulated highly and rapidly when plants were treated with exogenous ethylene, abscisic acid (ABA), high salinity, cold and drought. These results suggested that GhERF1 might be functionally important in acclimation of cotton to stress. PMID:18078841

  3. Molecular cloning, expression and association study with reproductive traits of the duck LRP8 gene.

    PubMed

    Wang, C; Li, S J; Li, C; Yu, G H; Feng, Y P; Peng, X L; Gong, Y Z

    2013-01-01

    1. Two splice variants of duck LRP8 were identified, one containing 8 ligand-binding repeats (LRP8-1) and the other containing only 7 repeats (LRP8-2). The two transcripts share ~71-91% nucleic acid identity and ~65-94% amino acid identity with their counterparts in other species. A phylogenetic tree based on amino acid sequences shows that duck LRP8 proteins are closely related to those of chicken, turkey and zebra finch. 2. The semi-quantitative reverse transcription polymerase chain reaction (RT-PCR )analysis indicates that the two transcripts are expressed in all the examined tissues, and the LRP8-1 transcript is more highly expressed in hypothalamus, ovary and pituitary gland than in other detected tissues. 3. Six single nucleotide polymorphisms (SNPs) were identified in the coding region. Association analysis demonstrated that the c.528C > T genotypes were associated with egg production (EP) (EP210d, EP300d and EP360d), age at laying the first egg (AFE) and body weight at sexual maturity (BWSM). The c.1371A > G genotypes were associated with egg production (EP210d, EP300d and EP360d). 4. The haplotypes of c.528C > T and c.1371A > G were associated with EP (EP210d, EP300d and EP360d), yolk weight (YW), albumen weight (AW), egg weight (EW), BWSM and the first egg weight (FEW). 5. Duck LRP8 gene was associated with some reproductive traits and is an important candidate gene for the genetic selection of improved reproductive traits. PMID:24286503

  4. Molecular cloning and characterization of three isoprenyl diphosphate synthase genes from alfalfa.

    PubMed

    Sun, Yan; Long, Ruicai; Kang, Junmei; Zhang, Tiejun; Zhang, Ze; Zhou, He; Yang, Qingchuan

    2013-02-01

    Isoprenoid is the precursor for the biosynthesis of saponins, abscisic acid, gibberellins, chlorophylls and many other products in plants. Saponins are an important group of bioactive plant natural products. The alfalfa (Medicago sativa L.) saponins are glycosides of different triterpene aglycones and possess many biological activities. We isolated three genes (MsFPPS, MsGPPS and MsGGPPS) encoding isoprenyl diphosphate synthases (IDS) from alfalfa via a homology-based PCR approach. The enzyme activity assay of purified recombined MsFPPS and MsGGPPS expressed in Escherichia coli indicated that they all had IDS activity. Expression analysis of the three genes in different alfalfa tissues using real time PCR displayed that they were expressed in all tissues although they had a different expression patterns. MsFPPS and MsGPS displayed a significant increase in transcript level in response to methyl jasmonate, but the transcript level of MsGGPPS decreased obviously. To elucidate the functions of the three IDSs, their overexpression driven by a constitutive cauliflower mosaic virus-35S promoter in tobacco plants was applied and analyzed. The T(0) transgenic plants of MsFPPS showed high levels of squalene content when compared with control. However, no differences were detected in T(0) transgenic plants of MsGPPS and MsGGPPS. In addition, the overexpression of MsFPPS induced senescence response in transgenic plant leaves. This result may indicate that MsFPPS performs a role not only in phytosterol and triterpene biosynthesis, but also in growth regulation. PMID:23238915

  5. Molecular cloning of an endoglucanase gene from an alkalophilic bacillus sp. and its expression in escherichia coli

    SciTech Connect

    Kim, J.M.; Kong, I.S.; Yu, J.H.

    1987-11-01

    One of the cellulase genes from alkalophilic Bacillus sp. strain N-4 was cloned in pBR322. A recombinant plasmid, pYBC107, expressing carboxymethyl cellulase (CMCase) was isolated, and the size of the cloned HindIII fragment was found to be 5.5 kilobases. The restriction map of pYBC107 showed a different pattern from those of pNKI and pNKII. When the HindIII fragment from pYBC107 was subcloned into pYEJ001, there was a 3.8-fold increase in CMCase activity over that observed with pYBC107. Plasmid pYBC108 constructed by treatment of pYBC107 with HindIII and ECORI expressed the CMCase activity, although to a limited extend. To verify the originality of cloned pYBC107 from Bacillus sp;, the authors analyzed the restriction digest by Southern blotting.

  6. Molecular cloning of the Robl gene from Bombyx mori and studies of its developmental and physicochemical regulation.

    PubMed

    Wei, Hao; Xuling, He; Yusong, Xu

    2012-06-01

    Dynein light chains function as motor acceptor to recruit cargos, which play vital roles in many cellular processes such as intracellular transport and mitosis. In this study, we cloned and expressed the dynein light chain LC7 gene BmRobl in silkworm. The full-length cDNA of the dynein light chain LC7 gene BmRobl is 757 bp and encoded 97 aa polypeptide. Its molecular weight was ~11 kDa confirmed by western blotting. The tissue and stage expression profile of BmRobl drafted by real time PCR revealed that presence of BmRobl transcript was examined in all tissue but prominent expression level was found in brain, wing disc, ovary and testis. In metamorphosis wing disc, BmRobl reached to peak during the prepupae stage compared with the larval and pupal stages. This indicated BmRobl might involve in wing discs development during metamorphosis. Besides, in vitro wing discs 20E cultivation was performed and BmRobl expression profile was detected. The results demonstrated that the BmRobl gene was significantly up-regulated with increase of 20E concentration; the mRNA level peaked at 2 μg/ml of 20E. However, the BmRobl expression nearly has no change cultivated by 20 μg/ml 20E compared with 0.02 μg/ml 20E. These indicated that BmRobl expression might directly or indirectly induced by 20E, besides, high concentration 20E was far too inducible, suggesting that low concentrations of ecdysteroid induce cell proliferation, whereas high concentrations inhibit cell proliferation. Moreover, the transport role of BmRobl was clarified by UV challenge and vanadate cultivation. Both the real time PCR and western blotting results showed that the BmRobl gene was degraded with increase in the concentration of sodium vanadate combined with elongation in the time of UV challenge. Interestingly, compared with the single treatment group and non-treatment group, the group treated by both sodium vanadate and UV have severe degradation. This indicated that UV and vanadate might down

  7. Molecular cloning of the yeast mitochondrial aconitase gene (ACO1) and evidence of a synergistic regulation of expression by glucose plus glutamate.

    PubMed Central

    Gangloff, S P; Marguet, D; Lauquin, G J

    1990-01-01

    We have isolated genomic clones complementing the aconitase-deficient strain (glu1-1) of Saccharomyces cerevisiae. Identification of the aconitase gene was established by enzymatic assays and molecular analyses. The corresponding mRNA has been characterized, and its direction of transcription has been determined. The complete nucleotide sequence revealed strong amino acid homologies with the sequences of some peptides isolated from the mammalian protein. Disruption of the gene by deletion-insertion led to glutamate auxotrophy. Expression of the aconitase gene was sensitive to glucose repression and was synergistically down regulated by glucose and glutamate. Images PMID:1972545

  8. Molecular Cloning and Characterization of Novel Phytocystatin Gene from Turmeric, Curcuma longa

    PubMed Central

    Chan, Seow-Neng; Abu Bakar, Norliza; Mahmood, Maziah; Ho, Chai-Ling

    2014-01-01

    Phytocystatin, a type of protease inhibitor (PI), plays major roles in plant defense mechanisms and has been reported to show antipathogenic properties and plant stress tolerance. Recombinant plant PIs are gaining popularity as potential candidates in engineering of crop protection and in synthesizing medicine. It is therefore crucial to identify PI from novel sources like Curcuma longa as it is more effective in combating against pathogens due to its novelty. In this study, a novel cDNA fragment encoding phytocystatin was isolated using degenerate PCR primers, designed from consensus regions of phytocystatin from other plant species. A full-length cDNA of the phytocystatin gene, designated CypCl, was acquired using 5′/3′ rapid amplification of cDNA ends method and it has been deposited in NCBI database (accession number KF545954.1). It has a 687 bp long open reading frame (ORF) which encodes 228 amino acids. BLAST result indicated that CypCl is similar to cystatin protease inhibitor from Cucumis sativus with 74% max identity. Sequence analysis showed that CypCl contains most of the motifs found in a cystatin, including a G residue, LARFAV-, QxVxG sequence, PW dipeptide, and SNSL sequence at C-terminal extension. Phylogenetic studies also showed that CypCl is related to phytocystatin from Elaeis guineensis. PMID:25853138

  9. Molecular Cloning and Characterization of Two Genes Encoding Dihydroflavonol-4-Reductase from Populus trichocarpa

    PubMed Central

    Jia, Zhichun; Yang, Li; Sun, Yimin; Xiao, Xunyan; Song, Feng; Luo, Keming

    2012-01-01

    Dihydroflavonol 4-reductase (DFR, EC 1.1.1.219) is a rate-limited enzyme in the biosynthesis of anthocyanins and condensed tannins (proanthocyanidins) that catalyzes the reduction of dihydroflavonols to leucoanthocyanins. In this study, two full-length transcripts encoding for PtrDFR1 and PtrDFR2 were isolated from Populus trichocarpa. Sequence alignment of the two PtrDFRs with other known DFRs reveals the homology of these genes. The expression profile of PtrDFRs was investigated in various tissues of P. trichocarpa. To determine their functions, two PtrDFRs were overexpressed in tobacco (Nicotiana tabacum) via Agrobacterium-mediated transformation. The associated color change in the flowers was observed in all 35S:PtrDFR1 lines, but not in 35S:PtrDFR2 lines. Compared to the wild-type control, a significantly higher accumulation of anthocyanins was detected in transgenic plants harboring the PtrDFR1. Furthermore, overexpressing PtrDFR1 in Chinese white poplar (P. tomentosa Carr.) resulted in a higher accumulation of both anthocyanins and condensed tannins, whereas constitutively expressing PtrDFR2 only improved condensed tannin accumulation, indicating the potential regulation of condensed tannins by PtrDFR2 in the biosynthetic pathway in poplars. PMID:22363429

  10. Molecular cloning and expression of a novel MYB transcription factor gene in rubber tree.

    PubMed

    Qin, Bi; Zhang, Yu; Wang, Meng

    2014-12-01

    MYB family proteins regulate a variety of cellular processes in plants. Tapping panel dryness (TPD) in rubber tree (Hevea brasiliensis Muell. Arg.) affects latex biosynthesis and causes serious losses to rubber producers. In this study, a novel SANT/MYB transcription factor gene down-regulated in TPD rubber tree, named as HbSM1, was isolated from rubber tree. The complete HbSM1 open reading frame (ORF) was 948 bp in length. The deduced HbSM1 protein is 315 amino acids. HbSM1 belonged to 1RMYB subfamily with a single SANT domain. Sequence alignment revealed that HbSM1 had high homology with MYB members from Ricinus communis and Manihot esculenta, with 72 and 78 % identity, respectively. Moreover, HbSM1 shared 56 % identity with Glycine max GmMYB176. Phylogenetic analysis revealed that HbSM1, GmMYB176, rice OsMYBS2, and OsMYBS3 fell into the same cluster with 93 % bootstrap support value. Comparing expression among different tissues demonstrated that HbSM1 was ubiquitously expressed in all tissues, but it appeared to be preferentially expressed in leaf and latex. Furthermore, HbSM1 transcripts were significantly induced by various phytohormones (including gibberellic acid, ethephon, methyl jasmonate, salicylic acid, and abscisic acid) and wounding treatments. These results suggested that HbSM1 might play multiple roles in plant development via different phytohormones signaling pathways. PMID:25195053

  11. Molecular cloning, expression analysis and cellular localization of an LFRFamide gene in the cuttlefish Sepiella japonica.

    PubMed

    Cao, Zi-Hao; Sun, Lian-Lian; Chi, Chang-Feng; Liu, Hui-Hui; Zhou, Li-Qing; Lv, Zhen-Ming; Wu, Chang-Wen

    2016-06-01

    Neuropeptides are important regulators of physiological processes in metazoans, such as feeding, reproduction, and heart activities. In this study, an LFRFamide gene was identified from the cuttlefish Sepiella japonica (designated as SjLFRFamide). The full-length sequence of SjLFRFamide cDNA has 841bp, and the open reading frame contains 567bp encoding 188 amino acids, which shared high similarity with precursor SOFaRP2 from Sepia officinalis. The deduced SjLFRFamdie precursor protein contains a signal peptide and four different FLPs (FMRFamide-like peptides): one pentapeptide (TIFRFamide), two hexapeptides (NSLFRFamide and GNLFRFamide) and one heptapeptide (PHTPFRFamide). Multiple sequence alignment showed that SjLFRFamide contains rather conserved mature peptides, which all ended in FRF. The phylogenetic analysis suggests that SjLFRFamide belongs to the LFRFamide subfamily. The tissue distribution analysis through quantitative real-time PCR method showed that SjLFRFamide mRNA is significantly expressed in the brain, and slight trace are detected in female nidamental gland and accessory nidamental gland. In situ hybridization assay of the brain indicated that SjLFRFamide is transcribed in several different functional lobes, suggesting SjLFRFamide might associate with multiple physiological regulations, such as feeding, chromatophore regulation and reproduction. This is the first study describing LFRFamide in S. japonica, which might have great importance for cuttlefish artificial breeding. PMID:26494614

  12. Molecular cloning and characterization of a novel Y-box gene from Sepiella maindroni.

    PubMed

    Si, H P; Wang, C L; Zhang, Y Y; Mu, C K; Zhan, P P; Li, R H; Song, W W

    2015-01-01

    Y-box proteins are a family of highly conserved nucleic acid binding proteins that interact with genome and transcription product to modulate the transcriptional and translational processes. In the present study, a complete mRNA of Y-box binding protein (designated SmYB) was obtained from Sepiella maindroni by amplification of flanking sequences. The full size of SmYB cDNA was 1502 bp, including 99 bp at the 5ꞌ untranslated region (UTR), a 3ꞌ UTR of 821 bp with a poly (A) tail, and an open reading frame of 582 bp, encoding a polypeptide of 193 amino acids with the predicted molecular weight of 16.48 kDa. The conserved cold-shock domain and two known RNA binding motifs identified in SmYB strongly suggested that SmYB was a new member of Y-box proteins. Quantitative real-time PCR was performed to examine the expression of SmYB mRNA in various tissues, embryos, and its temporal expression in liver after cold shock. The mRNA transcript of SmYB was detected in all examined tissues, with the highest expression level in testis and ovary. SmYB was abundant in early developmental stages of S. maindroni embryos but diminished in the late post-embryonic development. In addition, cold-shock treatment upregulated the transcription of SmYB mRNA in liver. These results demonstrated that SmYB is involved in embryonic development of S. maindroni and its tolerance to acute low temperatures. PMID:26125774

  13. Molecular cloning and characterization of a flavanone 3-Hydroxylase gene from Artemisia annua L.

    PubMed

    Xiong, Shuo; Tian, Na; Long, Jinhua; Chen, Yuhong; Qin, Yu; Feng, Jinyu; Xiao, Wenjun; Liu, Shuoqian

    2016-08-01

    Flavonoids were found to synergize anti-malaria and anti-cancer compounds in Artemisia annua, a very important economic crop in China. In order to discover the regulation mechanism of flavonoids in Artemisia annua, the full length cDNA of flavanone 3-hydroxylase (F3H) were isolated from Artemisia annua for the first time by using RACE (rapid amplification of cDNA ends). The completed open read frame of AaF3H was 1095 bp and it encoded a 364-amino acid protein with a predicted molecular mass of 41.18 kDa and a pI of 5.67. The recombinant protein of AaF3H was expressed in E. coli BL21(DE3) as His-tagged protein, purified by Ni-NTA agrose affinity chromatography, and functionally characterized in vitro. The results showed that the His-tagged protein (AaF3H) catalyzed naringenin to dihydrokaempferol in the present of Fe(2+). The Km for naringenin was 218.03 μM. The optimum pH for AaF3H reaction was determined to be pH 8.5, and the optimum temperature was determined to be 35 °C. The AaF3H transcripts were found to be accumulated in the cultivar with higher level of flavonoids than that with lower level of flavonoids, which implied that AaF3H was a potential target for regulation of flavonoids biosynthesis in Artemisia annua through metabolic engineering. PMID:27070290

  14. Molecular cloning of a mouse DNA repair gene that complements the defect of group-A xeroderma pigmentosum.

    PubMed Central

    Tanaka, K; Satokata, I; Ogita, Z; Uchida, T; Okada, Y

    1989-01-01

    For isolation of the gene responsible for xeroderma pigmentosum (XP) complementation group A, plasmid pSV2gpt and genomic DNA from a mouse embryo were cotransfected into XP2OSSV cells, a group-A XP cell line. Two primary UV-resistant XP transfectants were isolated from about 1.6 X 10(5) pSV2gpt-transformed XP colonies. pSV2gpt and genomic DNA from the primary transfectants were again cotransfected into XP2OSSV cells and a secondary UV-resistant XP transfectant was obtained by screening about 4.8 X 10(5) pSV2gpt-transformed XP colonies. The secondary transfectant retained fewer mouse repetitive sequences. A mouse gene that complements the defect of XP2OSSV cells was cloned into an EMBL3 vector from the genome of a secondary transfectant. Transfections of the cloned DNA also conferred UV resistance on another group-A XP cell line but not on XP cell lines of group C, D, F, or G. Northern blot analysis of poly(A)+ RNA with a subfragment of cloned mouse DNA repair gene as the probe revealed that an approximately 1.0 kilobase mRNA was transcribed in the donor mouse embryo and secondary transfectant, and approximately 1.0- and approximately 1.3-kilobase mRNAs were transcribed in normal human cells, but none of these mRNAs was detected in three strains of group-A XP cells. These results suggest that the cloned DNA repair gene is specific for group-A XP and may be the mouse homologue of the group-A XP human gene. Images PMID:2748601

  15. Molecular cloning of a mouse DNA repair gene that complements the defect of group-A xeroderma pigmentosum

    SciTech Connect

    Tanaka, K.; Satokata, I.; Ogita, Z.; Uchida, T.; Okada, Y.

    1989-07-01

    For isolation of the gene responsible for xeroderma pigmentosum (XP) complementation group A, plasmid pSV2gpt and genomic DNA from a mouse embryo were cotransfected into XP2OSSV cells, a group-A XP cell line. Two primary UV-resistant XP transfectants were isolated from about 1.6 X 10(5) pSV2gpt-transformed XP colonies. pSV2gpt and genomic DNA from the primary transfectants were again cotransfected into XP2OSSV cells and a secondary UV-resistant XP transfectant was obtained by screening about 4.8 X 10(5) pSV2gpt-transformed XP colonies. The secondary transfectant retained fewer mouse repetitive sequences. A mouse gene that complements the defect of XP2OSSV cells was cloned into an EMBL3 vector from the genome of a secondary transfectant. Transfections of the cloned DNA also conferred UV resistance on another group-A XP cell line but not on XP cell lines of group C, D, F, or G. Northern blot analysis of poly(A)+ RNA with a subfragment of cloned mouse DNA repair gene as the probe revealed that an approximately 1.0 kilobase mRNA was transcribed in the donor mouse embryo and secondary transfectant, and approximately 1.0- and approximately 1.3-kilobase mRNAs were transcribed in normal human cells, but none of these mRNAs was detected in three strains of group-A XP cells. These results suggest that the cloned DNA repair gene is specific for group-A XP and may be the mouse homologue of the group-A XP human gene.

  16. Molecular Cloning and Expression Analysis of Eight PgWRKY Genes in Panax ginseng Responsive to Salt and Hormones

    PubMed Central

    Xiu, Hao; Nuruzzaman, Mohammed; Guo, Xiangqian; Cao, Hongzhe; Huang, Jingjia; Chen, Xianghui; Wu, Kunlu; Zhang, Ru; Huang, Yuzhao; Luo, Junli; Luo, Zhiyong

    2016-01-01

    Despite the importance of WRKY genes in plant physiological processes, little is known about their roles in Panax ginseng C.A. Meyer. Forty-eight unigenes on this species were previously reported as WRKY transcripts using the next-generation sequencing (NGS) technology. Subsequently, one gene that encodes PgWRKY1 protein belonging to subgroup II-d was cloned and functionally characterized. In this study, eight WRKY genes from the NGS-based transcriptome sequencing dataset designated as PgWRKY2-9 have been cloned and characterized. The genes encoding WRKY proteins were assigned to WRKY Group II (one subgroup II-c, four subgroup II-d, and three subgroup II-e) based on phylogenetic analysis. The cDNAs of the cloned PgWRKYs encode putative proteins ranging from 194 to 358 amino acid residues, each of which includes one WRKYGQK sequence motif and one C2H2-type zinc-finger motif. Quantitative real-time PCR (qRT-PCR) analysis demonstrated that the eight analyzed PgWRKY genes were expressed at different levels in various organs including leaves, roots, adventitious roots, stems, and seeds. Importantly, the transcription responses of these PgWRKYs to methyl jasmonate (MeJA) showed that PgWRKY2, PgWRKY3, PgWRKY4, PgWRKY5, PgWRKY6, and PgWRKY7 were downregulated by MeJA treatment, while PgWRKY8 and PgWRKY9 were upregulated to varying degrees. Moreover, the PgWRKY genes increased or decreased by salicylic acid (SA), abscisic acid (ABA), and NaCl treatments. The results suggest that the PgWRKYs may be multiple stress–inducible genes responding to both salt and hormones. PMID:26959011

  17. Molecular Cloning and Expression Analysis of Eight PgWRKY Genes in Panax ginseng Responsive to Salt and Hormones.

    PubMed

    Xiu, Hao; Nuruzzaman, Mohammed; Guo, Xiangqian; Cao, Hongzhe; Huang, Jingjia; Chen, Xianghui; Wu, Kunlu; Zhang, Ru; Huang, Yuzhao; Luo, Junli; Luo, Zhiyong

    2016-01-01

    Despite the importance of WRKY genes in plant physiological processes, little is known about their roles in Panax ginseng C.A. Meyer. Forty-eight unigenes on this species were previously reported as WRKY transcripts using the next-generation sequencing (NGS) technology. Subsequently, one gene that encodes PgWRKY1 protein belonging to subgroup II-d was cloned and functionally characterized. In this study, eight WRKY genes from the NGS-based transcriptome sequencing dataset designated as PgWRKY2-9 have been cloned and characterized. The genes encoding WRKY proteins were assigned to WRKY Group II (one subgroup II-c, four subgroup II-d, and three subgroup II-e) based on phylogenetic analysis. The cDNAs of the cloned PgWRKYs encode putative proteins ranging from 194 to 358 amino acid residues, each of which includes one WRKYGQK sequence motif and one C₂H₂-type zinc-finger motif. Quantitative real-time PCR (qRT-PCR) analysis demonstrated that the eight analyzed PgWRKY genes were expressed at different levels in various organs including leaves, roots, adventitious roots, stems, and seeds. Importantly, the transcription responses of these PgWRKYs to methyl jasmonate (MeJA) showed that PgWRKY2, PgWRKY3, PgWRKY4, PgWRKY5, PgWRKY6, and PgWRKY7 were downregulated by MeJA treatment, while PgWRKY8 and PgWRKY9 were upregulated to varying degrees. Moreover, the PgWRKY genes increased or decreased by salicylic acid (SA), abscisic acid (ABA), and NaCl treatments. The results suggest that the PgWRKYs may be multiple stress-inducible genes responding to both salt and hormones. PMID:26959011

  18. Molecular cloning and gene expression of the gonadotropin-releasing hormone receptor in the orange-spotted grouper, Epinephelus coioides.

    PubMed

    Hsieh, S L; Chuang, H C; Nan, F H; Ruan, Y H; Kuo, C M

    2007-06-01

    The objective of this study was to investigate the molecular mechanisms of gonadotropin-releasing hormone receptor (GnRH-R) involved in the endocrine regulation of reproduction in the orange-spotted grouper, Epinephelus coioides. The full-length cDNA encoding GnRH-R type I was successfully cloned from the pituitary by reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) methods in the grouper. The complete GnRH-R type I cDNA is 1607 bp, which includes an open reading frame of 1092 bp encoding a protein of 364 amino acids, a seven-alpha helix transmembrane domain, a N-terminal extracellular domain, and a C-terminal cytoplasmic domain. The expression of GnRH-R type I was found to be highest in the pituitary. An intramuscular injection of various GnRH types in vivo was attempted. The expression of GnRH-R type I was stimulated by a single injection of salmon GnRH, while in the case of chicken GnRH II treatment, the expression of GnRH-R type I was inhibited. This suggests that the action of chick GnRH II is probably enhanced through the GnRH receptor of different forms. Furthermore, none of them were expressed by an injection of seabream GnRH, and this is likely attributed to the injection dose being below the threshold level, and this remains to be further examined. In conclusion, GnRHs of various types are effective in stimulating the expression of gonadotropins through various forms of the GnRH-R, and multiple forms of the receptor gene likely exist in teleosts. PMID:17329139

  19. Molecular cloning and characterization of an ADP-ribosylation factor 6 gene (ptARF6) from Pisolithus tinctorius.

    PubMed

    Wang, Liling; Li, Haibo; Zhou, Yifeng; Qin, Yuchuan; Wang, Yanbin; Liu, Bentong; Qian, Hua

    2016-05-01

    ADP-ribosylation factor 6 (ARF6) is an evolutionarily conserved molecule that has an essential function in intracellular trafficking and organelle structure. To better understand its role during presymbiosis between plant roots and compatible filamentous fungi, the full-length cDNA sequence of ARF6 from Pisolithus tinctorius was cloned and a variety of bioinformatics analyses performed. The full-length sequence was 849 bp long and contained a 549 bp open reading frame encoding a protein of 182 amino acids. A phylogenetic analysis showed that ptARF6 was the ortholog of the ADP ribosylation factor 6/GTPase SAR1 gene from the white-rot basidiomycete Trametes versicolor. A domain architecture analysis of the ARF6 protein revealed a repeat region, which is a common feature of ARF6 in other species. Recombinant ARF6 protein was expressed with an N-terminal 6×His tag and purified using Ni(2+)-NTA affinity chromatography. The molecular mass of the recombinant protein was estimated by SDS-PAGE to be 25 kDa. The recombinant ARF6 protein bound strongly to 18:1 and 18:2 phosphatidic acids. Thus, ARF6 may participate in the signaling pathways involved in membrane phospholipid composition. The intracellular distribution of ptADP6 in HEK239T cells also indicates that ptADP6 may function not only in plasma membrane events but also in endosomal membranes events. Real-time quantitative PCR revealed that the differential expression of ptARF6 was associated with the presymbiotic stage. ptARF6 may be induced by presymbiosis during the regulation of mycorrhizal formation. PMID:26928195

  20. Molecular cloning and heterologous expression of the gene encoding dihydrogeodin oxidase, a multicopper blue enzyme from Aspergillus terreus.

    PubMed

    Huang, K X; Fujii, I; Ebizuka, Y; Gomi, K; Sankawa, U

    1995-09-15

    Aspergillus terreus dihydrogeodin oxidase (DHGO) is an enzyme catalyzing the stereospecific phenol oxidative coupling reaction converting dihydrogeodin to (+)- geodin. We previously reported the purification of DHGO from A. terreus and raised polyclonal antibody against DHGO. From the first cDNA library constructed in lambda gt11 using mRNA from 3-day-old mycelium of A. terreus, four clones were identified using anti-DHGO antibody, but all contained partial cDNA inserts around 280 base pairs. This cDNA fragment was used as a probe to clone the genomic DNA and cDNA for dihydrogeodin oxidase from A. terreus. The sequence of the cloned DHGO genomic DNA and cDNA predicted that the DHGO polypeptide consists of 605 amino acids showing significant homology with multicopper blue proteins such as laccase and ascorbate oxidase. Four potential copper binding domains exist in DHGO polypeptide. The DHGO gene consists of seven exons separated by six short introns. Expression of the DHGO gene in Aspergillus nidulans under the starch or maltose-inducible Taka-amylase A promoter as an active enzyme established the functional identity of the gene. Also, introduction of the genomic DNA for DHGO into Penicillium frequentans led to the production of DHGO polypeptide as judged by Western blot analysis. PMID:7665560

  1. Cloning, sequencing, and use as a molecular probe of a gene encoding an aminoglycoside 6'-N-acetyltransferase of broad substrate profile.

    PubMed Central

    Terán, F J; Suárez, J E; Mendoza, M C

    1991-01-01

    A gene coding for an aminoglycoside 6'-N-acetyltransferase that was able to modify amikacin was cloned from a plasmid isolated from a clinical strain of Enterobacter cloacae. Sequencing of a 955-bp segment which mediates the modifying activity revealed a single open reading frame of 432 nucleotides that predicted a polypeptide of 144 amino acid residues with a molecular weight of 16,021. Putative ribosomal binding sites and -10 and -35 sequences were located at the 5' end of the gene. The size of the polypeptide was confirmed through minicell analysis of the expression products of plasmids containing the sequence. The use of the gene as a molecular probe revealed its specificity toward strains harboring genes coding for related enzymes. This probe is therefore useful for epidemiological studies. Images PMID:2069376

  2. Three slow skeletal muscle troponin genes in small-tailed Han sheep (Ovis aries): molecular cloning, characterization and expression analysis.

    PubMed

    Sun, Yan; Wang, Guizhi; Ji, Zhibin; Chao, Tianle; Liu, Zhaohua; Wang, Xiaolong; Liu, Guanqing; Wu, Changhao; Wang, Jianmin

    2016-09-01

    To explore the basic characteristics and expressing profile of the three slow skeletal muscle troponin genes TNNC1 (Troponin C type 1), TNNI1 (troponin I type 1) and TNNT1 (troponin T type 1). Three purebred Dorper sheep and another three purebred small-tailed Han sheep were selected. The sequence of the genes from the small-tailed Han sheep was cloned using rapid amplification of cDNA ends and reverse transcription-polymerase chain reaction; The characteristics of the predicted amino acids sequences were analyzed using bioinformatics analysis software; Gene expression analyses were performed using quantitative reverse transcription PCR. The full-length cDNA sequences of the genes were 707, 898, and 1001 bp, respectively, and were submitted to GenBank under accession numbers KR153938, KT218688 and KT218690. The three predicted proteins were predicted to be hydrophilic, non-secretory proteins and contain several phosphorylation sites. Multiple alignments and phylogenetic tree analyses showed that the predicted proteins were relatively conserved in mammals. The expression results of the three genes in eight tissues of Dorper and small-tailed Han sheep revealed that the three genes had a similar mRNA expression pattern, whereas distinct differences were observed among the eight tissues of the two sheep species. We cloned the full-length cDNA of the three genes, analyzed the amino acid sequences, and determined the expression levels of the genes. These results might play important roles in facilitating the future research of the three genes. PMID:27295221

  3. Molecular cloning and expression analysis of small ubiquitin-like modifier (SUMO) genes from grouper (Epinephelus coioides).

    PubMed

    Xu, Meng; Wei, Jingguang; Chen, Xiuli; Gao, Pin; Zhou, Yongcan; Qin, Qiwei

    2016-01-01

    Small ubiquitin-like modifier (SUMO) is a group of proteins binding to lysine residues of target proteins and thereby modifying their stability, activity and subcellular localization. In the present study, two SUMO homolog genes (EcSUMO1 and EcSUMO2) from grouper (Epinephelus coioides) were cloned and characterized. The full-length sequence of EcSUMO1 was 749 bp in length and contained a predicted open reading frame of 306 bp encoding 101 amino acids with a molecular mass of 11.34 kDa. The full-length sequence of EcSUMO2 was 822 bp in length and contained a predicted open reading frame of 291 bp encoding 96 amino acids with a molecular mass of 10.88 kDa EcSUMO1 shares 44.55% identity with EcSUMO2. EcSUMO1 shares 99%, 90%, and 88% identity with those from Oreochromis niloticus, Danio rerio, and Homo sapiens, respectively. EcSUMO2 shares 98%, 93%, and 96% identity with those from Anoplopoma fimbria, D.rerio, and H. sapiens, respectively. Quantitative real-time PCR analysis indicated that EcSUMO1 and EcSUMO2 were constitutively expressed in all of the analyzed tissues in healthy grouper, but the expression of EcSUMO2 was higher than that of EcSUMO1. EcSUMO1 and EcSUMO2 were identified as a remarkably (P < 0.01) up-regulated responding to poly(I:C) and Singapore grouper iridovirus (SGIV) stimulation in head kidney of groupers. EcSUMO1 and EcSUMO2 were distributed in both cytoplasm and nucleus in GS cells. Over-expressed EcSUMO1 and EcSUMO2 enhanced SGIV and Red-spotted grouper nervous necrosis virus (RGNNV) replication during viral infection in vitro. Our study was an important attempt to understand the SUMO pathway in fish, which may provide insights into the regulatory mechanism of viral infection in E.coioides under farmed conditions. PMID:26616235

  4. Molecular cloning of functional genes for high growth-temperature and salt tolerance of the basidiomycete Fomitopsis pinicola isolated in a mangrove forest in Micronesia.

    PubMed

    Miyazaki, Yasumasa; Hiraide, Masakazu; Shibuya, Hajime

    2007-01-01

    Several functional genes encoding putative proteins, heat shock protein 70, sphingosine phosphate lyase, and Na+/H+ antiporter, were cloned from the basidiomycete Fomitopsis pinicola, a wood-rotting fungus isolated in the tropical mangrove forest of Pohnpei Island of the Federated States of Micronesia. The deduced amino acid sequences of the obtained genes involved in heat shock resistance, lipid synthesis, and salt tolerance showed diverse similarities to other homologous proteins. Molecular phylogenetic trees of these proteins suggested that encoded proteins of the cloned genes of F. pinicola differed remarkably from other homologs in various organisms, even fungal proteins. Putative candidates for other genes related to several cellular metabolisms were also amplified, implying the possible existence of those genes in F. pinicola. This is the first report of possibly functional genes derived from a basidiomycetous mushroom growing in tropical islands such as Micronesia. The genes found in this study might play important roles in the cellular survival of the basidiomycete F. pinicola under severe environmental conditions. PMID:17213639

  5. Molecular cloning and in situ localization of the human contactin gene (CNTN1) on chromosome 12q11-q12

    SciTech Connect

    Berglund, E.O.; Ranscht, B.

    1994-06-01

    Chick contactin/F11 (also known as F3 in mouse) is a neuronal cell adhesion molecule of the immunoglobulin (Ig) gene family that is implicated in playing a role in the formation of axon connections in the developing nervous system. In human brain, contactin was first identified by amino terminal and peptide sequencing of the lentil-lectin-binding glycoprotein Gp135. The authors now report the isolation and characterization of cDNA clones encoding human contactin. Human contactin is composed of six C2 Ig-domains and four fibronectin type III (FNIII) repeats and is anchored to the membrane via a glycosyl phosphatidylinositol moiety, as shown by PI-PLC treatment of cells transfected with contactin cDNA and metabolic labeling with [{sup 3}H]-ethanolamine. At the amino acid level, h-contactin is 78% identical to chick contactin/F11 and 94% to mouse F3. Independent cDNAs encoding two putative contactin 1 cDNA encodes a protein with the amino-terminal sequence of purified Gp135, while the putative h-contactin 2 gene has a deletion of 33 nucleotides that predicts a protein with a shortened amino terminus. Northern analysis with a probe common for both isoforms detects one mRNA species of approximately 6.6 kb in adult human brain. Fluorescence in situ hybridization maps the gene for human contactin to human chromosome 12q11-q12. The h-contactin gene locus is thus in close proximity to homeobox 3, integrin subunit {alpha}5, several proto-oncogene genes, a chromosomal breakpoint associated with various tumors, and the gene locus for Stickler syndrome. The cloning of human contactin now permits the study of its role in disorder of the human nervous system. 56 refs., 6 figs., 1 tab.

  6. Molecular cloning and expression of two alpha-amylase genes from Streptococcus bovis 148 in Escherichia coli.

    PubMed Central

    Satoh, E; Niimura, Y; Uchimura, T; Kozaki, M; Komagata, K

    1993-01-01

    The alpha-amylase genes of Streptococcus bovis 148 were cloned in Escherichia coli MC1061, using pBR322. The recombinant plasmids were classified into two groups on the basis of their restriction maps. Southern blot analysis did not show homology between the two types of alpha-amylase genes, and the two alpha-amylase genes existed on the chromosomal DNA of S. bovis 148. The enzymatic properties and N-terminal amino acid sequences of the two purified enzymes produced by the cloned E. coli strains were quite different from each other. Particularly, one alpha-amylase (Amy I) was adsorbed on raw corn starch and hydrolyzed raw corn starch, and another (Amy II) was not adsorbed on raw corn starch and did not hydrolyze raw corn starch. Amy I was considered to be the same as the extracellular alpha-amylase of S. bovis 148 in raw starch absorbability, ability to hydrolyze raw corn starch, enzymatic characteristics, N-terminal amino acid sequence, and mode of action on soluble starch. Amy II showed a unique pattern of oligosaccharide production from soluble starch compared with the extracellular alpha-amylase of S. bovis 148. Amy II was suggested to be an intracellular alpha-amylase of S. bovis 148. Images PMID:8285674

  7. Molecular cloning and expression of two alpha-amylase genes from Streptococcus bovis 148 in Escherichia coli.

    PubMed

    Satoh, E; Niimura, Y; Uchimura, T; Kozaki, M; Komagata, K

    1993-11-01

    The alpha-amylase genes of Streptococcus bovis 148 were cloned in Escherichia coli MC1061, using pBR322. The recombinant plasmids were classified into two groups on the basis of their restriction maps. Southern blot analysis did not show homology between the two types of alpha-amylase genes, and the two alpha-amylase genes existed on the chromosomal DNA of S. bovis 148. The enzymatic properties and N-terminal amino acid sequences of the two purified enzymes produced by the cloned E. coli strains were quite different from each other. Particularly, one alpha-amylase (Amy I) was adsorbed on raw corn starch and hydrolyzed raw corn starch, and another (Amy II) was not adsorbed on raw corn starch and did not hydrolyze raw corn starch. Amy I was considered to be the same as the extracellular alpha-amylase of S. bovis 148 in raw starch absorbability, ability to hydrolyze raw corn starch, enzymatic characteristics, N-terminal amino acid sequence, and mode of action on soluble starch. Amy II showed a unique pattern of oligosaccharide production from soluble starch compared with the extracellular alpha-amylase of S. bovis 148. Amy II was suggested to be an intracellular alpha-amylase of S. bovis 148. PMID:8285674

  8. Molecular Cloning and Characterization of Spermine Synthesis Gene Associated with Cold Tolerance in Tea Plant (Camellia sinensis).

    PubMed

    Zhu, Xujun; Li, Qinghui; Hu, Jingyan; Wang, Mingle; Li, Xinghui

    2015-11-01

    Spermine synthase (SPMS, EC 2.5.1.22), enzyme of spermine (Spm) biosynthesis, has been shown to be related to stress response. In this study, attempts were made to clone and characterize a gene encoding SPMS from tea plant (Camellia sinensis). The effect of exogenous application of Spm in C. sinensis subjected to low-temperature stress was also investigated. A full-length SPMS complementary DNA (cDNA) (CsSPMS) with an open reading frame of 1113 bp was cloned using reverse transcription-PCR and rapid amplification of cDNA ends (RACE) techniques from cultivar "Yingshuang". The CsSPMS gene, which encoded a 371 amino acid polypeptide, in four cultivars is highly homologous. Quantitative real-time PCR indicated that the CsSPMS gene shows tissue-specific expression, mainly in the leaf and root of tea plant. The expression analysis demonstrated that the CsSPMS gene is quickly induced by cold stress and had similar trends in four cultivars. Spm-supplemented "Baicha" cultivar contains higher endogenous polyamines compared to the control, coupling with higher expression levels of ADC and SPMS. In addition, activities of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), as well as free proline content in the Spm-supplemented samples were higher than the control during the experiment course or at a given time point, indicating that Spm exerted a positive effect on antioxidant systems. Moreover, Agrobacterium-mediated expression of CsSPMS in tobacco leaves showed relatively higher cold tolerance. Taken together, these findings will enhance the understanding of the relationships among CsSPMS gene regulatory, polyamines accumulation, and cold tolerance in tea plant. PMID:26276446

  9. Molecular cloning, characterization and differential expression of novel phytocystatin gene during tropospheric ozone stress in maize (Zea mays) leaves.

    PubMed

    Ahmad, Rafiq; Zuily-Fodil, Yasmine; Passaquet, Chantal; Ali Khan, Sabaz; Repellin, Anne

    2015-03-01

    In this study, a full-length cDNA encoding a novel phytocystatin gene, designated CC14, was identified in maize leaves. The CC14 gene sequence reported in this study has been deposited in the GenBank database (accession number JF290478). The CC14 gene was cloned into an expression vector pET30 EK/LIC and was then transformed into Escherichia coli strain BL21 (DE3) pLysS to produce a recombinant CC14 protein. The recombinant protein was purified by nickel nitrilotriacetic acid affinity chromatography after induction with 1 mM IPTG. The purified CC14 protein was electrophoresed on SDS-PAGE and a protein 25 kDa in size was observed. Antiprotease activities of the purified recombinant CC14 protein against cysteine proteases and commercially available papain were tested. The results showed that CC14 purified protein suppressed 100% activity of papain and 57-86% plant cysteine protease activity. Moreover, an upregulation of CC14 gene expression was observed after 20 days of ozone stress in maize leaves. Together, these observations concurred to conclude that CC14 gene could potentially be used as a basis for the development of transgenic crops and natural pesticides that resist biotic and abiotic stresses. PMID:25613048

  10. Molecular cloning and expression analysis of dihydroflavonol 4-reductase gene in flower organs of Forsythia x intermedia.

    PubMed

    Rosati, C; Cadic, A; Duron, M; Renou, J P; Simoneau, P

    1997-10-01

    The expression, during flower development, of the gene encoding the anthocyanin pathway key enzyme dihydroflavonol 4-reductase (DFR) was investigated in floral organs of Forsythia x intermedia cv. 'Spring Glory'. Full-length DFR and partial chalcone synthase (CHS) cDNAs, the gene of interest and a flavonoid pathway control gene respectively, were obtained from petal RNA by reverse transcription PCR. Whereas for CHS northern blot analysis enabled the study of its expression pattern, competitive PCR assays were necessary to quantify DFR mRNA levels in wild-type plants and in petals of 2 transgenic clones containing a CaMV 35S promoter-driven DFR gene of Antirrhinum majus. Results indicated a peak of CHS and DFR transcript levels in petals at the very early stages of anthesis, and different expression patterns in anthers and sepals. In comparison to wild-type plants, transformants showed a more intense anthocyanin pigmentation of some vegetative organs, and a dramatic increase in DFR transcript concentration and enzymatic activity in petals. However, petals of transformed plants did not accumulate any anthocyanins. These results indicate that other genes and/or regulatory factors should be considered responsible for the lack of anthocyanin production in Forsythia petals. PMID:9349254

  11. Molecular cloning, SNP detection and association analysis of 5' flanking region of the goat IGF1 gene with prolificacy.

    PubMed

    Thomas, Naicy; Venkatachalapathy, Thirupathy; Aravindakshan, Thazhathuveettil; Raghavan, K C

    2016-04-01

    The insulin-like growth factor 1 has an important role in reproduction, foetal development and growth. It regulates the secretion of gonadotrophin releasing hormone, stimulates ovarian function and steroidogenesis. The present study was conducted to characterise the 5' flanking region of goat IGF 1 gene, ascertain ovarian expression of the IGF1 gene, detect SNPs and assess the association with prolificacy in the two indigenous goat breeds of South India viz., low prolific Attappady Black and high prolific Malabari. The 5' flanking region of IGF1 gene was PCR amplified, cloned and sequenced from both breeds. Genotyping was performed in 277 goats from the two genetic groups using the PCR-Single Strand Conformational Polymorphism (SSCP) and the expression of the IGF1 gene in the ovary was analysed by quantitative real time PCR. The 5' flanking region of the IGF1 gene was 601 bp long and located at 450 bp upstream of the start codon. Sequence exhibited 97-99% similarity with that of the sheep, cattle and sika deer IGF1 genes. Three genotypes, PP, PQ and QR were observed at this locus with the frequency of 0.62, 0.30 and 0.08, respectively. Sequencing of the representative PCR products from each genotype revealed two SNPs, g.224A>G and g.227C>T. The population was found to be in Hardy-Weinberg disequilibrium at both loci. Statistical results indicated that these loci were associated with litter size (P ≤ 0.05). However, no significant difference was found in the expression of the IGF1 gene in the ovaries of the two goat breeds. These results suggest the significant influence of the IGF1 gene on prolificacy in goats and identified SNPs would benefit the selection of prolific animals in future breeding programs. PMID:26852275

  12. Molecular cloning of the mouse gene coding for {alpha}{sub 2}-macroglobulin and targeting of the gene in embryonic stem cells

    SciTech Connect

    Umans, L.; Serneels, L.; Hilliker, C.

    1994-08-01

    The authors have cloned the mouse gene coding for {alpha}{sub 2}-macroglobulin in overlapping {lambda} clones and have analyzed its structure. The gene contains 36 exons, coding for the 4.8-kb cDNA that we cloned previously. Including putative control elements in the 5{prime} flanking region, the gene covers about 45 kb. A region of 3.8 kb, stretching from 835 bases upstream of the cDNA start site to exon 4, including all intervening sequences, was sequenced completely. The analysis demonstrated that the putative promoter region of the mouse A2M gene differed considerably from the known promoter sequences of the human A2M gene and of the rat acute-phas A2M gene. Comparison of the exon-intron structure of all known genes of the A2M family confirmed that the rat acute phase A2M gene is more closely related to the human gene than to the mouse A2M gene. To generate mice with the A2M gene inactivated, an insertion type of construct containing 7.5 kb of genomic DNA of the mouse strain 129/J, encompassing exons 16 to 19, was synthesized. A hygromycin marker gene was embedded in intron 17. After electroporation, 198 hygromycin-resistant ES cell lines were isolated and analyzed by Southern blotting. Five ES cell lines were obtained with one allele of the mouse A2M gene targeted by this insertion construct, demonstrating that the position and the characteristics of the vector served the intended goal.

  13. Molecular cloning of the human leukotriene C4 synthase gene and assignment to chromosome 5q35.

    PubMed Central

    Bigby, T. D.; Hodulik, C. R.; Arden, K. C.; Fu, L.

    1996-01-01

    BACKGROUND: Cysteinyl leukotrienes (LT) are mediators involved in inflammatory and allergic disorders LTC4 synthase catalyzes the first committed step in the synthesis of these inflammatory mediators, and its cellular distribution appears to be unique. MATERIALS AND METHODS: A human genomic library was screened by polymerase chain reaction (PCR) with primers that were designed based on the reported cDNA sequence for the LTC4 synthase gene. The gene was identified in one clone by Southern blotting of restriction enzyme digests, subcloning of fragments containing regions of interest, and DNA sequencing of these subclones. The transcription initiation site was determined by primer extension analysis. Chromosome location was determined by fluorescent in situ hybridization and screening of somatic cell hybrids by PCR. RESULTS: The LTC4 synthase gene is approximately 2.5 kb in length, consisting of five exons (136, 100, 71, 82, and 257 bp, respectively) and four introns (1,447, 102, 84, and 230 bp, respectively). Transcription initiation occurs at a single site 78 bp upstream of the coding region. The 5'-flanking region contains neither a TATA nor a CAAT box. The first 1 kb of the 5'-flanking region, however, contains putative DNA binding motifs for SP-1, AP-1, AP-2, ets factors, and CREB/ATF. A STAT binding motif is present in the first intron. The LTC4 synthase gene is located in the distal region of the long arm of chromosome 5 in 5q35. CONCLUSIONS: The LTC4 synthase gene does not contain elements of a typical regulated gene and may therefore contain novel regulatory elements. This gene is also located in a region on chromosome 5 that appears to play a role in allergic and inflammatory disorders, such as asthma. Images FIG. 1 FIG. 5 FIG. 4 FIG. 6 PMID:8898379

  14. Molecular cloning and expression analysis of KIN10 and cold-acclimation related genes in wild banana 'Huanxi' (Musa itinerans).

    PubMed

    Liu, Weihua; Cheng, Chunzhen; Lai, Gongti; Lin, Yuling; Lai, Zhongxiong

    2015-01-01

    Banana cultivars may experience chilling or freezing injury in some of their cultivated regions, where wild banana can still grow very well. The clarification of the cold-resistant mechanism of wild banana is vital for cold-resistant banana breeding. In this study, the central stress integrator gene KIN10 and some cold-acclimation related genes (HOS1 and ICE1s) from the cold-resistant wild banana 'Huanxi' (Musa itinerans) were cloned and their expression patterns under different temperature treatments were analyzed. Thirteen full-length cDNA transcripts including 6 KIN10s, 1 HOS1 and 6 ICE1s were successfully cloned. Quantitative real-time PCR (qRT-PCR) results showed that all these genes had the highest expression levels at the critical temperature of banana (13 °C). Under chilling temperature (4 °C), the expression level of KIN10 reduced significantly but the expression of HOS1 was still higher than that at the optimal temperature (28 °C, control). Both KIN10 and HOS1 showed the lowest expression levels at 0 °C, the expression level of ICE1, however, was higher than control. As sucrose plays role in plant cold-acclimation and in regulation of KIN10 and HOS1 bioactivities, the sucrose contents of wild banana under different temperatures were detected. Results showed that the sucrose content increased as temperature lowered. Our result suggested that KIN10 may participate in cold stress response via regulating sucrose biosynthesis, which is helpful in regulating cold acclimation pathway in wild banana. PMID:26753116

  15. Molecular cloning and analysis of a receptor-like promoter of Gbvdr3 gene in sea island cotton.

    PubMed

    Zhang, B-J; Zhang, H-P; Chen, Q-Z; Tang, N; Wang, L-K; Wang, R-F; Zhang, B-L

    2016-01-01

    Verticillium wilt caused by soil borne fungus Verticillium dahliae could significantly reduce cotton yield. The Ve1 homologous gene Gbvdr3 is resistant to Verticillium wilt. In order to understand of the function of the promoter Gbvdr3 in Gossypium barbadense, the promoter region of the receptor-like gene Gbvdr3 was obtained by genome walking, and the cis-element in the promoter was identified using the PLACE software in this study. The sequence analysis showed that the promoter contained elements related to stress resistance and light regulation. The cloned promoter was fused to the GUS reporter gene and transformed into Arabidopsis. GUS expression was specifically detected in roots, flowers, and seeds, suggesting that the expression of Gbvdr3 is tissue-specific. Separation and characterization analysis of the promoter of Gbvdr3 provides a platform for further research and application of this gene. Thorough understanding of the function of the Gbvdr3 promoter is important for better understanding of Gbvdr3 function. These results indicated that the promoter of Gbvdr3 was a tissue-specific promoter. PMID:27323087

  16. Molecular cloning and functional analysis of the FSH receptor gene promoter from the volcano mouse (Neotomodon alstoni alstoni).

    PubMed

    Pérez-Solis, Marco Allán; Macías, Héctor; Acosta-MontesdeOca, Adriana; Pasapera, Ana María; Fierro, Reyna; Ulloa-Aguirre, Alfredo; Gutiérrez-Sagal, Rubén

    2010-02-01

    To gain further insights on the genetic divergence and the species-specific characteristics of the follicle-stimulating hormone receptor (FSHR), we cloned 946 bp of the 5'-flanking region of the FSHR gene from the volcano mouse (Neotomodon alstoni alstoni), and compared its features with those from other mammalian species. The sequence of neotomodon FSHR (nFSHR) gene from the translation initiation site to -946 is 74, 71, 64, and 59% homologous to rat, mouse (129/J), human, and sheep, respectively. The nFSHR 5'-flanking region exhibits new interesting putative cis-regulatory elements including those for the SRY transcription factor, which had not been previously related to the FSHR gene. The transcriptional regulation properties of nFSHR gene were studied in mouse Sertoli (MSC-1) and non-Sertoli (H441) cell lines, and compared with those obtained with similar 129/J constructs. All constructs tested were more active in H441 than in MSC-1 cells. The low transcription levels detected in MSC-1 cells probably reflect the recruitment of Sertoli cells-specific nuclear factors that repress transcription of the FSHR gene. In H441 cells, 129/J constructs were more active than their neotomodon counterparts, indicating important species-specific differences in their transcription pattern. Functional analysis of a series of progressive 5'-deletion mutants identified regions involved in positive and negative transcriptional regulation as well as the strongest minimal promoter spanning 260 bp upstream the translation initiation site. The identification of inhibitory nuclear transcription factors, which are apparently expressed in MSC-1 cells, may contribute to a better understanding of the transcriptional regulation of the FSHR gene. PMID:19862645

  17. Molecular analysis of the M protein of Streptococcus equi and cloning and expression of the M protein gene in Escherichia coli.

    PubMed Central

    Galán, J E; Timoney, J F

    1987-01-01

    A Streptococcus equi gene bank was constructed in the bacteriophage lambda gt11 cloning vector, and hybrid phage plaques were screened with S. equi M protein antiserum. A hybrid phage expressing the S. equi M protein (lambda gt11/SEM7) was identified and lysogenized into Escherichia coli Y1089. The cloned M protein appeared in immunoblots as three polypeptides with relative molecular weights of 58,000, 53,000, and 50,000. When reacted with S. equi M protein antiserum in an agar double-diffusion assay, the cloned M protein formed a line of identity with a protein in an acid extract of S. equi. Furthermore, lambda gt11/SEM7 protein inhibited opsonization of S. equi by antiserum to S. equi M protein. In addition, the recombinant protein expressed determinants of the antigen in the immune complexes of purpura hemorrhagica. Native M protein obtained from S. equi and recombinant M protein showed very similar molecular weight distributions on immunoblots, appearing as multiple closely spaced bands with molecular weights ranging from 52,000 to 60,000. Antisera prepared separately against each of the acid-extracted polypeptides shown to be important in serum bactericidal responses (molecular weight, 29,000) and nasopharyngeal local antibody responses (molecular weights, 41,000 and 46,000) of the horse each reacted with all three polypeptides in an acid extract. Moreover, antisera against protoplasts and against recombinant M protein of S. equi also reacted with these polypeptides. These results suggest that the entire M protein molecule of S. equi is present in these preparations and that the fragments in acid extracts carry overlapping segments. Images PMID:3316035

  18. Molecular analysis of the M protein of Streptococcus equi and cloning and expression of the M protein gene in Escherichia coli.

    PubMed

    Galán, J E; Timoney, J F

    1987-12-01

    A Streptococcus equi gene bank was constructed in the bacteriophage lambda gt11 cloning vector, and hybrid phage plaques were screened with S. equi M protein antiserum. A hybrid phage expressing the S. equi M protein (lambda gt11/SEM7) was identified and lysogenized into Escherichia coli Y1089. The cloned M protein appeared in immunoblots as three polypeptides with relative molecular weights of 58,000, 53,000, and 50,000. When reacted with S. equi M protein antiserum in an agar double-diffusion assay, the cloned M protein formed a line of identity with a protein in an acid extract of S. equi. Furthermore, lambda gt11/SEM7 protein inhibited opsonization of S. equi by antiserum to S. equi M protein. In addition, the recombinant protein expressed determinants of the antigen in the immune complexes of purpura hemorrhagica. Native M protein obtained from S. equi and recombinant M protein showed very similar molecular weight distributions on immunoblots, appearing as multiple closely spaced bands with molecular weights ranging from 52,000 to 60,000. Antisera prepared separately against each of the acid-extracted polypeptides shown to be important in serum bactericidal responses (molecular weight, 29,000) and nasopharyngeal local antibody responses (molecular weights, 41,000 and 46,000) of the horse each reacted with all three polypeptides in an acid extract. Moreover, antisera against protoplasts and against recombinant M protein of S. equi also reacted with these polypeptides. These results suggest that the entire M protein molecule of S. equi is present in these preparations and that the fragments in acid extracts carry overlapping segments. PMID:3316035

  19. Molecular Cloning and Characterization of DXS and DXR Genes in the Terpenoid Biosynthetic Pathway of Tripterygium wilfordii

    PubMed Central

    Tong, Yuru; Su, Ping; Zhao, Yujun; Zhang, Meng; Wang, Xiujuan; Liu, Yujia; Zhang, Xianan; Gao, Wei; Huang, Luqi

    2015-01-01

    1-Deoxy-d-xylulose-5-phosphate synthase (DXS) and 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) genes are the key enzyme genes of terpenoid biosynthesis but still unknown in Tripterygium wilfordii Hook. f. Here, three full-length cDNA encoding DXS1, DXS2 and DXR were cloned from suspension cells of T. wilfordii with ORF sizes of 2154 bp (TwDXS1, GenBank accession no.KM879187), 2148 bp (TwDXS2, GenBank accession no.KM879186), 1410 bp (TwDXR, GenBank accession no.KM879185). And, the TwDXS1, TwDXS2 and TwDXR were characterized by color complementation in lycopene accumulating strains of Escherichia coli, which indicated that they encoded functional proteins and promoted lycopene pathway flux. TwDXS1 and TwDXS2 are constitutively expressed in the roots, stems and leaves and the expression level showed an order of roots > stems > leaves. After the suspension cells were induced by methyl jasmonate, the mRNA expression level of TwDXS1, TwDXS2, and TwDXR increased, and triptophenolide was rapidly accumulated to 149.52 µg·g−1, a 5.88-fold increase compared with the control. So the TwDXS1, TwDXS2, and TwDXR could be important genes involved in terpenoid biosynthesis in Tripterygium wilfordii Hook. f. PMID:26512659

  20. Molecular cloning of cDNA for the B beta subunit of Xenopus fibrinogen, the product of a coordinately-regulated gene family.

    PubMed

    Bhattacharya, A; Shepard, A R; Moser, D R; Roberts, L R; Holland, L J

    1991-02-01

    Fibrinogen, the principal blood-clotting protein, is made up of three different subunits synthesized in the liver. In vitro administration of glucocorticoids to liver cells from the frog Xenopus laevis causes a dramatic increase in fibrinogen synthesis. Investigations of molecular mechanisms underlying this hormonal stimulation at the mRNA level require cDNA clones complementary to the mRNAs coding for the three fibrinogen subunits, called A alpha, B beta, and gamma. We describe here the isolation and characterization of cDNA clones for the B beta subunit of Xenopus fibrinogen. cDNA libraries in both plasmid (pBR322) and phage (lambda gt10) cloning vectors were constructed from frog liver mRNA and screened with a rat B beta cDNA. Clones thus isolated hybridized to two Xenopus liver mRNAs 2500 and 1800 bases long, the previously-determined sizes for B beta mRNAs. The identity of the plasmid clone B beta-27 was confirmed by hybridization-selection of complementary mRNA which translated in vitro into the B beta polypeptide, as determined by size and susceptibility to thrombin cleavage. lambda/B beta 10, a clone representing nearly all of the 2500-base B beta mRNA, was isolated from the phage cDNA library. The 3'-end of this clone includes a polyadenylation signal about 20 residues upstream of a stretch of 34 adenosine residues, which probably represents the 3'-poly(A) tail of the messenger RNA. lambda/B beta 10 lacks only 20 nucleotides of full-length B beta mRNA at the 5'-end and there is one major start site of transcription. The 2500-base B beta mRNA has a 700-base extension at the 3'-end that is not present in the 1800-base mRNA. The Xenopus laevis genome contains two or three genes for the B beta fibrinogen subunit. Using the cDNA clone as a probe, B beta mRNA was shown to be induced at least 20-fold by glucocorticoid treatment of purified parenchymal cells of Xenopus liver maintained in primary culture. PMID:2050271

  1. Molecular cloning and complementation analysis of nifV gene from Frankia EuIK1 strain.

    PubMed

    Oh, Chang Jae; Kim, Ho Bang; An, Chung Sun

    2003-02-28

    The nifV gene from the Frankia EuIK1 strain, a symbiont of Elaeagnus umbellata, was cloned and a complementation test using the Klebsiella pneumoniae nifV mutant was performed to verify its function. The nifV ORF consists of 1245 bp, which encodes 414 amino acids. However, the putative promoter and Shine-Dalgarno sequences were not found in the 5' region of the ORF. The Frankia EuIK1 nifV ORF showed about a 70% nucleotide identity and 80% amino acid similarity with that of Frankia sp. FaC1. In the upstream region of the nifV, a putative ORF that showed a 51% nucleotide identity with the afcD gene from Burkholderia cepacia BC11 was found. The other partial ORF that showed a 59% identity with the pkaD gene from Streptomyces coelicolor A(3) was found in the downstream region. In this respect, Frankia EuIK1 nifV has an unusual location on the genome, considering the nif gene organization. A phylogenetic analysis revealed that the NifV from Frankia EuIK1 was close to those from two Alnus-infective Frankia species, and they were grouped with those of the alpha-class proteobacteria, supporting the vertical descent of nifV. The transcription and function of Frankia EuIK1 nifV were verified by a RT-PCR analysis and complementation test with the K. pneumoniae mutant, respectively. These results suggested that Frankia EuIK1 nifV is a functional gene. PMID:12661757

  2. Molecular cloning and expression of chitin deacetylase 1 gene from the gills of Penaeus monodon (black tiger shrimp).

    PubMed

    Sarmiento, Katreena P; Panes, Vivian A; Santos, Mudjekeewis D

    2016-08-01

    Chitin deacetylases have been identified and studied in several fungi and insects but not in crustaceans. These glycoproteins function in catalyzing the conversion of chitin to chitosan by the hydrolysis of N-acetamido bonds of chitin. Here, for the first time, the full length cDNA of chitin deacetylase (CDA) gene from crustaceans was fully cloned using a partial fragment obtained from a transcriptome database of the gills of black tiger shrimp Penaeus monodon that survived White Spot Syndrome Virus (WSSV) infection employing Rapid Amplification of cDNA Ends (RACE) PCR. The shrimp CDA, named PmCDA1, was further characterized by in silico analysis, and its constitutive expression determined in apparently healthy shrimp through reverse transcription PCR (RT-PCR). Results revealed that the P. monodon chitin deacetylase (PmCDA1) is 2176 bp-long gene with an open reading frame (ORF) of 1596 bp encoding for 532 amino acids. Phylogenetic analysis revealed that PmCDA1 belongs to Group I CDAs together with CDA1 and CDA2 proteins found in insects. Moreover, PmCDA1 is composed of a conserved chitin-binding peritrophin-A domain (CBD), a low-density lipoprotein receptor class A domain (LDL-A) and a catalytic domain that is part of CE4 superfamily, all found in group I CDAs, which are known to serve critical immune function against WSSV. Finally, high expression of PmCDA1 gene in the gills of apparently healthy P. monodon was observed suggesting important basal function of the gene in this tissue. Taken together, this is a first report of the full chitin deacetylase 1 (CDA1) gene in crustaceans particularly in shrimp that exhibits putative immune function against WSSV and is distinctly highly expressed in the gills of shrimp. PMID:27335260

  3. Molecular cloning of the human homeobox gene goosecoid (GSC) and mapping of the gene to human chromosome 14q32. 1

    SciTech Connect

    Blum, M.; De Robertis, E.M.; Geissert, D. ); Kojis, T.; Heinzmann, C.; Klisak, I.; Sparkes, R.S. )

    1994-05-15

    Goosecoid is a homeobox gene first isolated from a Xenopus dorsal lip cDNA library. Homologous genes have been isolated from mouse, zebrafish, and chick. In all species examined, the gene is expressed and plays an important role during the process of gastrulation in early embryonic development. The authors report here the cloning of the human goosecoid (GSC) from a genomic library and the sequence of its encoded protein. The genomic organization and protein sequence of the human gene are highly conserved with respect to those of its Xenopus and mouse counterparts: all three genes consist of three exons, with conserved exon-intron boundaries. The sequence of the homeo-domain is 100% conserved in most vertebrates. Using somatic cell hybrid and chromosomal in situ hybridization, the gene was mapped to chromosome 14q32.1. 30 refs., 3 figs., 2 tabs.

  4. Construction of an infectious molecular clone of Japanese encephalitis virus genotype V and its derivative subgenomic replicon capable of expressing a foreign gene.

    PubMed

    Ishikawa, Tomohiro; Abe, Makoto; Masuda, Michiaki

    2015-01-01

    Japanese encephalitis virus (JEV) genotype V was originally isolated in Malaysia in 1952 and has long been restricted to the area. In 2009, sudden emergence of the genotype V in China and Korea was reported, suggesting expansion of its geographical distribution. Although studies on the genotype V are becoming more important, they have been limited partly due to lack of its infectious molecular clone. In this study, a plasmid carrying cDNA corresponding to the entire genome of JEV Muar strain, which belongs to genotype V, in the downstream of T7 promoter was constructed. Electroporation of viral RNA transcribed by T7 RNA polymerase (T7RNAP) in vitro from the plasmid led to production of progeny viruses both in mammalian and mosquito cells. Also, transfection of the infectious clone plasmid into mammalian cells expressing T7RNAP transiently or stably was demonstrated to generate infectious progenies. When the viral structural protein genes were partially deleted from the full-length cDNA, the subgenomic RNA transcribed in vitro from the modified plasmid was shown to replicate itself in mammalian cells as a replicon. The replicon carrying the firefly luciferase gene in place of the deleted structural protein genes was also shown to efficiently replicate itself and express luciferase in mammalian cells. Compared with the replicon derived from JEV genotype III (Nakayama strain), the genotype V-derived replicon appeared to be more tolerant to introduction of a foreign gene. The infectious clone and the replicons constructed in this study may serve as useful tools for characterizing JEV genotype V. PMID:25451067

  5. Molecular Cloning, Expression Pattern and Genotypic Effects on Glucoraphanin Biosynthetic Related Genes in Chinese Kale (Brassica oleracea var. alboglabra Bailey).

    PubMed

    Yin, Ling; Chen, Changming; Chen, Guoju; Cao, Bihao; Lei, Jianjun

    2015-01-01

    Glucoraphanin is a plant secondary metabolite that is involved in plant defense and imparts health-promoting properties to cruciferous vegetables. In this study, three genes involved in glucoraphanin metabolism, branched-chain aminotransferase 4 (BCAT4), methylthioalkylmalate synthase 1 (MAM1) and dihomomethionine N-hydroxylase (CYP79F1), were cloned from Chinese kale (Brassica oleracea var. alboglabra Bailey). Sequence homology and phylogenetic analysis identified these genes and confirmed the evolutionary status of Chinese kale. The transcript levels of BCAT4, MAM1 and CYP79F1 were higher in cotyledon, leaf and stem compared with flower and silique. BCAT4, MAM1 and CYP79F1 were expressed throughout leaf development with lower transcript levels during the younger stages. Glucoraphanin content varied extensively among different varieties, which ranged from 0.25 to 2.73 µmol·g(-1) DW (dry weight). Expression levels of BCAT4 and MAM1 were high at vegetative-reproductive transition phase, while CYP79F1 was expressed high at reproductive phase. BCAT4, MAM1 and CYP79F1 were expressed significantly high in genotypes with high glucoraphanin content. All the results provided a better understanding of the roles of BCAT4, MAM1 and CYP79F1 in the glucoraphanin biosynthesis of Chinese kale. PMID:26569208

  6. Molecular gene cloning and nucleotide sequencing and construction of an aroA mutant of Pasteurella haemolytica serotype A1.

    PubMed Central

    Tatum, F M; Briggs, R E; Halling, S M

    1994-01-01

    The aroA gene of Pasteurella haemolytica serotype A1 was cloned by complementation of the aroA mutation in Escherichia coli K-12 strain AB2829. The nucleotide sequence of a 2.2-kb fragment encoding aroA predicted an open reading frame product 434 amino acids long that shows homology to other bacterial AroA proteins. Several strategies to inactivate aroA were unsuccessful. Gene replacement was finally achieved by constructing a replacement plasmid with aroA inactivated by insertion of a P. haemolytica ampicillin resistance fragment into a unique NdeI site in aroA. A hybrid plasmid was constructed by joining the aroA replacement plasmid with a 4.2-kb P. haemolytica plasmid which encodes streptomycin resistance. Following PhaI methylation, the replacement plasmid was introduced by electroporation into P. haemolytica NADC-D60, a plasmidless strain of serotype 1A. Allelic exchange between the replacement plasmid and the chromosome of P. haemolytica gave rise to an ampicillin-resistant mutant which grew on chemically defined P. haemolytica medium supplemented with aromatic amino acids but failed to grow on the same medium lacking tryptophan. Southern blot analysis confirmed that aroA of the mutant was inactivated and that the mutant was without a plasmid. Images PMID:8031095

  7. Molecular cloning and characterisation of metallothionein type 2a gene from Jatropha curcas L., a promising biofuel plant.

    PubMed

    Mudalkar, Shalini; Golla, Ramesh; Sengupta, Debashree; Ghatty, Sreenivas; Reddy, Attipalli Ramachandra

    2014-01-01

    In the present study, we have cloned a gene encoding JcMT2a protein from Jatropha curcas L., a promising biofuel tree species. Full length sequence of JcMT2a gene was isolated using RACE PCR. Heterologous expression of JcMT2a in Escherichia coli and its purification has shown distinct bands corresponding to the GST and GST-fused JcMT2a protein. Significant tolerance was observed in E. coli cells expressing recombinant GST-JcMT2a for zinc, copper and cadmium metals compared to cells expressing GST alone. JcMT2a also restored Cu and Cd tolerance in the metal sensitive yeast mutants. Quantitative real time PCR showed a significant increase in JcMT2a transcripts with Cu and Cd in the leaf compared to root tissue. Our Scanning electron microscopy and energy dispersive X-ray spectroscopy analysis clearly demonstrates that J. curcas L. could be a potential candidate for phytoremediation to clean heavy metals from the environment, in addition to its non-edible oil seed yields for biodiesel production. PMID:24190491

  8. Molecular cloning and comparative analysis of immunoglobulin heavy chain genes from Phasianus colchicus, Meleagris gallopavo, and Coturnix japonica.

    PubMed

    Choi, Jin Won; Kim, Jin-Kyoo; Seo, Hee Won; Cho, Byung Wook; Song, Gwonhwa; Han, Jae Yong

    2010-08-15

    To date, immunoglobulin (Ig) genes have only been fully characterized in a small number of aves, which pose a major obstacle to understanding Ig evolution. Thus, we cloned the cDNAs of three immunoglobulin classes, IgA, IgM, and IgY, from Phasianus colchicus, Coturnix japonica, and Meleagris gallopavo. Multiple sequence alignments revealed that the highest degree of sequence homology in all Ig classes was observed between pheasant and turkey whereas the degree of homology between the galliforms and non-galliforms was relatively low compared to that among the galliforms. When the constant region domains of the four human Ig classes were compared with the corresponding regions in aves, the average percent homology between human CH2 and avian CH3, and between human CH3 and avian CH4, was greater than between identical domains in IgA and IgY, which are in partial agreement with the hypothesis that the avian CH2 domain evolved to form the mammalian hinge via domain condensation. Phylogenetic analysis showed that the galliform Ig heavy chain constant regions were divided into quail and the common ancestor of chicken, turkey, and pheasant, and that chicken was separated from turkey and pheasant, which were grouped together. These results add to our knowledge of galliform Igs and the diversification of these genes. PMID:20398946

  9. Molecular cloning of the 130-kilodalton mosquitocidal delta-endotoxin gene of Bacillus thuringiensis subsp. israelensis in Bacillus sphaericus.

    PubMed Central

    Trisrisook, M; Pantuwatana, S; Bhumiratana, A; Panbangred, W

    1990-01-01

    A 3.7-kilobase (kb) XbaI fragment harboring the cryIVB gene (L. Thorne, F. Garduno, T. Thompson, D. Decker, M. A. Zounes, M. Wild, A. M. Walfield, and T. J. Pollock, J. Bacteriol. 166:801-811, 1986) which encoded a 130-kilodalton (kDa) mosquitocidal toxin from a 110-kb plasmid of Bacillus thuringiensis subsp. israelensis 4Q2-72 was cloned into pUC12 and transformed into Escherichia coli. The clone with a recombinant plasmid (designated pBT8) was toxic to Aedes aegypti larvae. The fragment (3.7 kb) was ligated into pBC16 (tetracycline resistant [Tcr]) and transformed by the method of protoplast transformation into Bacillus sphaericus 1593 and 2362, which were highly toxic to Anopheles and Culex mosquito larvae but less toxic to Aedes larvae. After cell regeneration on regeneration medium, the Tcr plasmids from transformants (pBTC1) of both strains of B. sphaericus were prepared and analyzed. The 3.7-kb XbaI fragment from the B. thuringiensis subsp. israelensis plasmid was shown to be present by agarose gel electrophoresis and Southern blot hybridization. In addition, B. sphaericus transformants produced a 130-kDa mosquitocidal toxin which was detected by Western (immuno-) blot analysis with antibody prepared against B. thuringiensis subsp. israelensis 130-kDa mosquitocidal toxin. The 50% lethal concentrations of the transformants of strains 1593 and 2362 against A. aegypti larvae were 2.7 X 10(2) and 5.7 X 10(2) cells per ml, respectively. This level of toxicity was comparable to the 50% lethal concentration of B. thuringiensis subsp. israelensis but much higher than that of B. sphaericus 1593 and 2362 (4.7 X 10(4) cells per ml) against A. aegypti larvae.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:2200339

  10. Molecular cloning, spatial and temporal expression analysis of CatSper genes in the Chinese Meishan pigs

    PubMed Central

    2011-01-01

    Background Sperm ion channel proteins (CatSpers) are essential for sperm hyperactivated motility, and then penetration through the zona pellucida. The CatSper class of proteins have well been characterized in the mouse and human. However, such data for pigs are not available. In the present study, we cloned the porcine CatSper 1-4 genes, analysed their spatial expression in various organs and temporal expression in the testes from birth until sexual maturity in Meishan boars. Methods Rapid amplification of cDNA ends (RACE) was performed to clone the full length cDNAs of porcine CatSper genes and bioinformatics analysis of inferred CatSper proteins was also determined. Various organs were collected from 150 day-old pigs to characterize the spatial expression of CatSper genes by qualitative reverse transcriptase polymerase chain reaction (RT-PCR), and testes from birth to 150 day-old boars were sampled to detect the temporal expression of CatSper genes by quantitative real-time RT-PCR. Results The mRNA sequences of CatSper1 (2452 bp), CatSper2 (2038 bp), CatSper3 (1408 bp), and CatSper4 (1799 bp), including full length of cDNAs, 5' and 3' flanks, were obtained. The bioinformatics analysis indicated that coding regions spanning the ion transport domains were conserved for different species analyzed. Among the four CatSpers, CatSper2, 3, and 4 were more conserved across species, compared with CatSper1. In addition, six conservative trans-membrane domains, a pore forming motif, and a coiled-coil motif were also identified. The spatial analysis from different organs showed that CatSper1 was detected in both testes and hypothalamus, CatSper2 was restricted in testes only, CatSper4 was expressed in testes and rete testes; whereas CatSper3 was more ubiquitously. CatSper3 and CatSper4 transcripts were also detected in ejaculated sperm. At Days 1 and 30 of age, CatSper mRNAs exhibited only sparse expression in the testes. However, these transcripts highly expressed at Day 60

  11. Positional Gene Cloning in Experimental Populations.

    PubMed

    Jagodic, Maja; Stridh, Pernilla

    2016-01-01

    Positional cloning is a technique that identifies a trait-associated gene based on its location in the genome and involves methods such as linkage analysis, association mapping, and bioinformatics. This approach can be used for gene identification even when little is known about the molecular basis of the trait. Vast majority of traits are regulated by multiple genomic loci called quantitative trait loci (QTL). We describe experimental populations and designs that can be used for positional cloning, including backcrosses, intercrosses, and heterogeneous stocks, and advantages and disadvantages of different approaches. Once the phenotype and genotype of each individual in an experimental population have been determined, QTL identification can be accomplished. We describe the statistical tools used to identify the existence, location, and significance of QTLs. These different methods have advantages and disadvantages to consider when selecting the appropriate model to be used, which is briefly discussed.Although the objective of QTL mapping is to identify genomic regions associated with a trait, the ultimate goal is to identify the gene and the genetic variation (which is often quantitative trait nucleotide, QTN) or haplotype that is responsible for the phenotype. By discovering the function of causative variants or haplotypes we can understand the molecular changes that lead to the phenotype. We briefly describe how the genomic sequences can be exploited to identify QTNs and how these can be validated in congenic strains and functionally tested to understand their influence on phenotype expression. PMID:25103675

  12. Molecular cloning of the heat shock protein 20 gene from Paphia textile and its expression in response to heat shock

    NASA Astrophysics Data System (ADS)

    Li, Jiakai; Wu, Xiangwei; Tan, Jing; Zhao, Ruixiang; Deng, Lingwei; Liu, Xiande

    2015-07-01

    P. textile is an important aquaculture species in China and is mainly distributed in Fujian, Guangdong, and Guangxi Provinces. In this study, an HSP20 cDNA designated PtHSP20 was cloned from P. textile. The full-length cDNA of PtHSP20 is 1 090 bp long and contains a 5' untranslated region (UTR) of 93 bp, a 3' UTR of 475 bp, and an open reading frame (ORF) of 522 bp. The PtHSP20 cDNA encodes 173 amino acid residues and has a molecular mass of 20.22 kDa and an isoelectric point of 6.2. Its predicted amino acid sequence shows that PtHSP20 contains a typical α-crystallin domain (residues 77-171) and three polyadenylation signal-sequences at the C-terminus. According to an amino acid sequence alignment, PtHSP20 shows moderate homology to other mollusk sHSPs. PtHSP20 mRNA was present in all of the test tissues including the heart, digestive gland, adductor muscle, gonad, gill, and mantle, with the highest concentration found in the gonad. Under the stress of high temperature, the expression of PtHSP20 mRNA was down-regulated in all of the tissues except the adductor muscle and gonad.

  13. Cloning, molecular characterization, and expression analysis of a nucleoporin gene (rgNUP98-96) from Rehmannia glutinosa.

    PubMed

    Yang, Y H; Li, M J

    2015-01-01

    Nucleoporin 98 (NUP98) and nucleoporin 96 (NUP96) are essential components of the nuclear pore complex (NPC) in eukaryote cells. However, there is a lack of available information about complete Rehmannia glutinosa NUP98-96 (rgNUP98-96) sequences. Here, the full-length cDNA sequence of rgNUP96-98 was isolated from R. glutinosa using rapid amplification of cDNA ends (RACE) technology, based on a cloned cDNA sequence (GenBank accession No. JZ483329). The identified rgNUP98-96 was 3476 bp, and it encoded a 1041-amino acid peptide. The BLAST search analysis of rgNUP98-96 showed an intermediate degree of similarity (60-79%) to the NUP98-96 protein sequences of 34 other plants, including the dicotyledons Erythranthe guttata, Genlisea aurea, Coffea canephora, Nicotiana benthamiana, Solanum lycopersicum, and Solanum tuberosum. The phylogenetic analysis of NUP96-98 sequences indicated that R. glutinosa and E. guttata sequences shared the closest homology. The calculated molecular mass and predicted isolectric point of the complex protein were 117.6 kDa and 4.99, respectively. The secondary and three-dimensional structure studies illustrated that the rgNUP96-98 protein folded into a channel motif comprised of 34 alpha-helices, nine beta-strands, and several long loops. Using quantitative real-time PCR, the spatio-temporal expression patterns of rgNUP98-96 were analyzed in R. glutinosa, and the results indicated that rgNUP98-96 was highly expressed at the early stage of R. glutinosa tuberous root expansion, which is associated with a higher expression pattern in roots. The study provides a valuable foundation for further investigation of rgNUP96-98 molecular functions in R. glutinosa. PMID:26505455

  14. Cloning

    MedlinePlus

    Cloning describes the processes used to create an exact genetic replica of another cell, tissue or organism. ... named Dolly. There are three different types of cloning: Gene cloning, which creates copies of genes or ...

  15. Molecular cloning and expression profile of an abiotic stress and hormone responsive MYB transcription factor gene from Panax ginseng.

    PubMed

    Afrin, Sadia; Zhu, Jie; Cao, Hongzhe; Huang, Jingjia; Xiu, Hao; Luo, Tiao; Luo, Zhiyong

    2015-04-01

    The v-myb avian myeloblastosis viral oncogene homolog (MYB) family constitutes one of the most abundant groups of transcription factors and plays vital roles in developmental processes and defense responses in plants. A ginseng (Panax ginseng C.A. Meyer) MYB gene was cloned and designated as PgMYB1. The cDNA of PgMYB1 is 762 base pairs long and encodes the R2R3-type protein consisting 238 amino acids. Subcellular localization showed that PgMYB1-mGFP5 fusion protein was specifically localized in the nucleus. To understand the functional roles of PgMYB1, we investigated the expression patterns of PgMYB1 in different tissues and under various conditions. Quantitative real-time polymerase chain reaction and western blot analysis showed that PgMYB1 was expressed at higher level in roots, leaves, and lateral roots than in stems and seeds. The expression of PgMYB1 was up-regulated by abscisic acid, salicylic acid, NaCl, and cold (chilling), and down-regulated by methyl jasmonate. These results suggest that PgMYB1 might be involved in responding to environmental stresses and hormones. PMID:25791525

  16. Molecular cloning and tissue expression of uncoupling protein 1, 2 and 3 genes in Chinese perch (Siniperca chuatsi).

    PubMed

    Wen, Zheng-Yong; Liang, Xu-Fang; He, Shan; Li, Ling; Shen, Dan; Tao, Ya-Xiong

    2015-07-01

    Uncoupling proteins (UCPs) are mitochondrial anion carrier proteins, which play important roles in several physiological processes, including thermogenesis, reactive oxygen species generation, growth, lipid metabolism and insulin secretion. Although the roles of UCPs are well understood in mammals, little is known in fish. To investigate the thermogenesis roles in Chinese perch (Siniperca chuatsi), we cloned the UCP1, 2 and 3. The UCP1 consisted of six exons and five introns, and the UCP2 consisted of eight exons and seven introns. The UCP1 was primarily expressed in liver, UCP2 was ubiquitously expressed, and UCP3 was primarily expressed in muscle. The mRNA levels of UCP1 and UCP2 in liver, and UCP3 in muscle were significantly increased after prolonged cold exposure, but did not change after prolonged heat exposure, suggesting that Chinese perch might have a mechanism of response to cold environment, but not to hot environment. The intestinal UCP1 mRNA level was significantly up-regulated after prolonged heat exposure, while the UCP2 mRNA level was significantly up-regulated after prolonged cold exposure, suggesting that the two paralogs might play different roles in intestine of Chinese perch. In addition, the phylogenetic analysis could shed new light on the evolutionary diversification of UCP gene family. PMID:25829150

  17. Molecular cloning of vasa gene and the effects of LHRH-A on its expression in blue tilapia Oreochromis aureus.

    PubMed

    Xiao, Jun; Luo, Yongju; Chen, Libing; Yang, Li; Huang, Yulin; Guo, Zhongbao; Guo, Enyan; Tang, Zhanyang; Zhang, Ming; Gan, Xi

    2013-08-01

    The full length of vasa cDNA in blue tilapia Oreochromis aureus was cloned and sequenced using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Nucleotide sequence analysis revealed that the cDNA contained 2,143 bp and was consisted of a 48-bp 5' untranslated terminal region (5'-UTR), a 157-bp 3' untranslated terminal region (3'-UTR) and a 1,938-bp open reading frame (ORF) which encoded 645 amino acids. Homological protein analysis showed that vasa in O. aureus was highly conserved with Nile tilapia Oreochromis niloticus. Tissue distribution expression analysis indicated that vasa was specifically expressed in the gonads. Using in situ hybridization, we found that vasa was expressed in spermatogonia and spermatocytes rather than spermatids and sperm. In order to examine the influence of luteinizing hormone releasing hormone analog (LHRH-A) on vasa, the in vivo injections were performed different concentrations of LHRH-A. Our results showed that LHRH-A induced meiosis and down-regulated vasa mRNA expression. In summary, our results showed that vasa was specifically expressed in gonads and LHRH-A inhibited vasa expression in the testis. Our results also suggested that LHRH-A could regulate vasa gene expression in O. aureus testis. PMID:23224831

  18. Molecular cloning and sequence of the thdF gene involved in the thiophene and furan oxidation by Escherichia coli

    SciTech Connect

    Alam, K.Y.; Clark, D.P.

    1990-01-01

    Since sulfur dioxide emission from burning high sulfur coals is a major contributor to acid rain, it is important to develop bacteria which are capable of efficiently removing the sulfur from coal before combustion. Inorganic sulfur can be removed from coal by certain strains of Thiobacillus or Sulfolobus; however the organic sulfur remains intransigent. Since high sulfur Illinois coals typically contain 60% to 70% of their sulfur in the form of the heterocyclic thiophene ring we have started to investigate the biodegradation of derivatives of thiophene and the corresponding oxygen heterocycle, furan. Our previous work resulted in the isolation of a triple mutant, NAR30, capable of oxidizing a range of furan and thiophene derivatives. However, NAR30 does not completely degrade thiophenes or furans and its oxidation of these compounds is slow and inefficient. We decided to clone the thd genes both in order to increase the efficiency of degradation and to investigate the nature of the reactions involved. 37 refs., 4 figs., 3 tabs.

  19. Purification, characterization, molecular cloning, and expression of two laccase genes from the white rot basidiomycete Trametes villosa.

    PubMed Central

    Yaver, D S; Xu, F; Golightly, E J; Brown, K M; Brown, S H; Rey, M W; Schneider, P; Halkier, T; Mondorf, K; Dalboge, H

    1996-01-01

    Two laccases have been purified to apparent electrophoretic homogeneity from the extracellular medium of a 2,5-xylidine-induced culture of the white rot basidiomycete Trametes villosa (Polyporus pinsitus or Coriolus pinsitus). These proteins are dimeric, consisting of two subunits of 63 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and have typical blue laccase spectral properties. Under nondenaturing conditions, the two purified laccases have different pIs; purified laccase forms 1 and 3 have pIs of 3.5 and 6 to 6.5, respectively. A third purified laccase form 2 has the same N terminus as that of laccase form 3, but its pI is in the range of 5 to 6. The laccases have optimal activity at pH 5 to 5.5 and pH < or = 2.7 with syringaldazine and ABTS [2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)] as substrates, respectively. The genes lcc1 and lcc2 coding for the two purified laccases (forms 1 and 3) have been cloned, and their nucleotide sequences have been determined. The genes for lcc1 and lcc2 have 8 and 10 introns, respectively. The predicted proteins are 79% identical at the amino acid level. From Northern (RNA) blots containing total RNA from both induced and uninduced cultures, expression of lcc1 is highly induced, while the expression of lcc2 appears to be constitutive. Lcc1 has been expressed in Aspergillus oryzae, and the purified recombinant protein has the same pI, spectral properties, stability, and pH profiles as the purified native protein. PMID:8975613

  20. Molecular cloning and expression of a novel CYP26 gene (cyp26d1) during zebrafish early development.

    PubMed

    Gu, Xingxing; Xu, Fang; Wang, Xiaolin; Gao, Xiang; Zhao, Qingshun

    2005-08-01

    Proper restriction of retinoid signaling by Cyp26s is essential for development of vertebrate embryos while inappropriate retinoid signaling can cause teratogenesis. Here, we report cloning and expression analysis of a novel cyp26 gene (cyp26d1) isolated from zebrafish. The predicted protein encoded by cyp26d1 consists of 554 amino acids. It exhibits 54% amino acid identity with human Cyp26C1, 50% with zebrafish Cyp26B1 and 38% with zebrafish Cyp26A1. Whole-mount in situ hybridization shows that cyp26d1 is first expressed in sphere stage, then disappears at 50% epiboly and resumes its expression at 75% epiboly. During segmentation period, cyp26d1 message is found at presumptive hindbrain. Double in situ hybridization with krox20 and cyp26d1 reveals that cyp26d1 is expressed in presumptive rhombomere 2-4 (r2-r4) at 2-somite stage. At 3-somite stage, cyp26d1 gene is expressed in r6 and pharyngeal arch (pa) one in addition to its expression at r2 and r4. At 6-somite stage, cyp26d1 message is present in continuous bands at r2-r6 and in pa1. This expression pattern is maintained from 10-somite stage through 21-somite stage except that the expression level is greatly reduced at r2 and r4. At 21-somite stage, cyp26d1 is also found in a group of cells in telencephalon and diencephalons. At 25-31h post-fertilization (hpf), the zebrafish cyp26d1 expression domain is extended to eyes, otic vesicles and midbrain in addition to its expression in hindbrain, telencephalon, diencephalons, and pharyngeal arches. At 35-48hpf, the expression of cyp26d1 is mainly restricted to otic vesicles, pharyngeal arches and pectoral fins and the expression level is greatly reduced. PMID:15979416

  1. Molecular cloning, tissue expression of gene Muc2 in blunt snout bream Megalobrama amblycephala and regulation after re-feeding

    NASA Astrophysics Data System (ADS)

    Xue, Chunyu; Xi, Bingwen; Ren, Mingchun; Dong, Jingjing; Xie, Jun; Xu, Pao

    2015-03-01

    Mucins are important components of mucus, which form a natural, physical, biochemical and semipermeable mucosal layer on the epidermis of fish gills, skin, and the gastrointestinal tract. As the first step towards characterizing the function of Muc2, we cloned a partial Megalobrama amblycephala Muc2 cDNA of 2 175 bp, and analyzed its tissue-specific expression pattern by quantitative real-time PCR (qPCR). The obtained sequence comprised 41 bp 5'-untranslated region (5'-UTR), 2 134 bp open reading frame encoding a protein of 711 amino acids. BLAST searching and phylogenetic analysis showed that the predicted protein contained several common secreted mucin-module domains (VWD-C8-TIL-VWD-C8) and had high homology with mucins from other vertebrates. Among four candidate reference genes ( β- Actin, RPI13α, RPII, 18S) for the qPCR, RPII was chosen as an appropriate reference gene because of its lowest variation in different tissues. M. amblycephala Muc2 was mainly expressed in the intestine, in the order (highest to lowest) middle-intestine > fore-intestine > hind-intestine. Muc2 was expressed relatively poorly in other organs (brain, liver, kidney, spleen, skin and gill). Furthermore, after 20-days of starvation, M. amblycephala Muc2 expressions after refeeding for 0 h, 3 h, 16 h, 3 d, and 10 d were significantly decreased in the three intestinal segments ( P<0.05) at 16 h, and were then upregulated to near the initial level at 10 d.

  2. Molecular cloning and characterization of the MsHSP17.7 gene from Medicago sativa L.

    PubMed

    Li, Zhen-Yi; Long, Rui-Cai; Zhang, Tie-Jun; Yang, Qing-Chuan; Kang, Jun-Mei

    2016-08-01

    Heat shock proteins (HSPs) are ubiquitous protective proteins that play crucial roles in plant development and adaptation to stress, and the aim of this study is to characterize the HSP gene in alfalfa. Here we isolated a small heat shock protein gene (MsHSP17.7) from alfalfa by homology-based cloning. MsHSP17.7 contains a 477-bp open reading frame and encodes a protein of 17.70-kDa. The amino acid sequence shares high identity with MtHSP (93.98 %), PsHSP17.1 (83.13 %), GmHSP17.9 (74.10 %) and SlHSP17.6 (79.25 %). Phylogenetic analysis revealed that MsHSP17.7 belongs to the group of cytosolic class II small heat shock proteins (sHSP), and likely localizes to the cytoplasm. Quantitative RT-PCR indicated that MsHSP17.7 was induced by heat shock, high salinity, peroxide and drought stress. Prokaryotic expression indicated that the salt and peroxide tolerance of Escherichia coli was remarkably enhanced. Transgenic Arabidopsis plants overexpressing MsHSP17.7 exhibited increased root length of transgenic Arabidopsis lines under salt stress compared to the wild-type line. The malondialdehyde (MDA) levels in the transgenic lines were significantly lower than in wild-type, although proline levels were similar between transgenic and wild-type lines. MsHSP17.7 was induced by heat shock, high salinity, oxidative stress and drought stress. Overexpression analysis suggests that MsHSP17.7 might play a key role in response to high salinity stress. PMID:27193169

  3. Molecular cloning and characterization of Siamese crocodile (Crocodylus siamensis) copper, zinc superoxide dismutase (CSI-Cu,Zn-SOD) gene.

    PubMed

    Sujiwattanarat, Penporn; Pongsanarakul, Parinya; Temsiripong, Yosapong; Temsiripong, Theeranan; Thawornkuno, Charin; Uno, Yoshinobu; Unajak, Sasimanas; Matsuda, Yoichi; Choowongkomon, Kiattawee; Srikulnath, Kornsorn

    2016-01-01

    Superoxide dismutase (SOD, EC 1.15.1.1) is an antioxidant enzyme found in all living cells. It regulates oxidative stress by breaking down superoxide radicals to oxygen and hydrogen peroxide. A gene coding for Cu,Zn-SOD was cloned and characterized from Siamese crocodile (Crocodylus siamensis; CSI). The full-length expressed sequence tag (EST) of this Cu,Zn-SOD gene (designated as CSI-Cu,Zn-SOD) contained 462bp encoding a protein of 154 amino acids without signal peptides, indicated as intracellular CSI-Cu,Zn-SOD. This agreed with the results from the phylogenetic tree, which indicated that CSI-Cu,Zn-SOD belonged to the intracellular Cu,Zn-SOD. Chromosomal location determined that the CSI-Cu,Zn-SOD was localized to the proximal region of the Siamese crocodile chromosome 1p. Several highly conserved motifs, two conserved signature sequences (GFHVHEFGDNT and GNAGGRLACGVI), and conserved amino acid residues for binding copper and zinc (His(47), His(49), His(64), His(72), His(81), Asp(84), and His(120)) were also identified in CSI-Cu,Zn-SOD. Real-time PCR analysis showed that CSI-Cu,Zn-SOD mRNA was expressed in all the tissues examined (liver, pancreas, lung, kidney, heart, and whole blood), which suggests a constitutively expressed gene in these tissues. Expression of the gene in Escherichia coli cells followed by purification yielded a recombinant CSI-Cu,Zn-SOD, with Km and Vmax values of 6.075mM xanthine and 1.4×10(-3)mmolmin(-1)mg(-1), respectively. This Vmax value was 40 times lower than native Cu,Zn-SOD (56×10(-3)mmolmin(-1)mg(-1)), extracted from crocodile erythrocytes. This suggests that cofactors, protein folding properties, or post-translational modifications were lost during the protein purification process, leading to a reduction in the rate of enzyme activity in bacterial expression of CSI-Cu,Zn-SOD. PMID:26523498

  4. The role of 5'-adenylylsulfate reductase in the sulfur assimilation pathway of soybean: molecular cloning, kinetic characterization, and gene expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean seeds are a major source of protein, but contain low levels of sulfur-containing amino acids. With the objective of studying the sulfur assimilation pathway of soybean, a full-length cDNA clone for 5’-adenylylsulfate reductase (APS reductase) was isolated and characterized. The cDNA clone ...

  5. Molecular cloning and heterologous expression of the isopullulanase gene from Aspergillus niger A.T.C.C. 9642.

    PubMed Central

    Aoki, H; Yopi; Sakano, Y

    1997-01-01

    Isopullulanase (IPU) from Aspergillus niger A.T.C.C. (American Type Culture Collection) 9642 hydrolyses pullulan to isopanose. IPU is important for the production of isopanose and is used in the structural analysis of oligosaccharides with alpha-1,4 and alpha-1,6 glucosidic linkages. We have isolated the ipuA gene encoding IPU from the filamentous fungi A. niger A.T.C.C. 9642. The ipuA gene encodes an open reading frame of 1695 bp (564 amino acids). IPU contained a signal sequence of 19 amino acids, and the molecular mass of the mature form was calculated to be 59 kDa. IPU has no amino-acid-sequence similarity with the other pullulan-hydrolysing enzymes, which are pullulanase, neopullulanase and glucoamylase. However, IPU showed a high amino-acid-sequence similarity with dextranases from Penicillium minioluteum (61%) and Arthrobacter sp. (56%). When the ipuA gene was expressed in Aspergillus oryzae, the expressed protein (recombinant IPU) had IPU activity and was immunologically reactive with antibodies raised against native IPU. The substrate specificity, thermostability and pH profile of recombinant IPU were identical with those of the native enzyme, but recombinant IPU (90 kDa) was larger than the native enzyme (69-71 kDa). After deglycosylation with peptide-N-glycosidase F, the deglycosylated recombinant IPU had the same molecular mass as deglycosylated native enzyme (59 kDa). This result suggests that the carbohydrate chain of recombinant IPU differed from that of the native enzyme. PMID:9169610

  6. Molecular cloning and expression analysis of the ethylene insensitive3 (EIN3) gene in cucumber (Cucumis sativus).

    PubMed

    Bie, B B; Pan, J S; He, H L; Yang, X Q; Zhao, J L; Cai, R

    2013-01-01

    The plant gaseous hormone ethylene regulates many aspects of plant growth, development, and responses to the environment. Ethylene insensitive3 (EIN3) is a key transcription factor involved in the ethylene signal transduction pathway. To gain a better understanding of this particular pathway in cucumber, the full-length cDNA encoding EIN3 (designated as CsEIN3) was cloned from cucumber for the first time by rapid amplification of cDNA ends. The full length of CsEIN3 was 2560 bp, with an open reading frame of 1908 bp encoding 635 amino acids. Sequence alignment and phylogenetic analyses revealed that CsEIN3 has high homology with other plant EIN3/EIL proteins that were derived from a common ancestor during evolution, and CsEIN3 was grouped into a cluster along with melon. Homology modeling demonstrated that CsEIN3 has a highly similar structure to the specific DNA-binding domain contained in EIN3/EIL proteins. Based on quantitative reverse transcription-polymerase chain reaction analysis, we found that CsEIN3 was constitutively expressed in all organs examined, and was increased during flower development and maturation in both male and female flowers. Our results suggest that CsEIN3 is involved in processes of flower development. In conclusion, this study will provide the basis for further study on the role of EIN3 in relevant biological processes of cucumber and on the molecular mechanism of the cucumber ethylene signaling pathway. PMID:24114213

  7. Molecular Cloning and Characterization of Three Genes Encoding Dihydroflavonol-4-Reductase from Ginkgo biloba in Anthocyanin Biosynthetic Pathway

    PubMed Central

    Hua, Cheng; Linling, Li; Shuiyuan, Cheng; Fuliang, Cao; Feng, Xu; Honghui, Yuan; Conghua, Wu

    2013-01-01

    Dihydroflavonol-4-reductase (DFR, EC1.1.1.219) catalyzes a key step late in the biosynthesis of anthocyanins, condensed tannins (proanthocyanidins), and other flavonoids important to plant survival and human nutrition. Three DFR cDNA clones (designated GbDFRs) were isolated from the gymnosperm Ginkgo biloba. The deduced GbDFR proteins showed high identities to other plant DFRs, which form three distinct DFR families. Southern blot analysis showed that the three GbDFRs each belong to a different DFR family. Phylogenetic tree analysis revealed that the GbDFRs share the same ancestor as other DFRs. The expression of the three recombinant GbDFRs in Escherichia coli showed that their actual protein sizes were in agreement with predictions from the cDNA sequences. The recombinant proteins were purified and their activity was analyzed; both GbDFR1 and GbDFR3 could catalyze dihydroquercetin conversion to leucocyanidin, while GbDFR2 catalyzed dihydrokaempferol conversion to leucopelargonidin. qRT-PCR showed that the GbDFRs were expressed in a tissue-specific manner, and transcript accumulation for the three genes was highest in young leaves and stamens. These transcription patterns were in good agreement with the pattern of anthocyanin accumulation in G.biloba. The expression profiles suggested that GbDFR1 and GbDFR2 are mainly involved in responses to plant hormones, environmental stress and damage. During the annual growth cycle, the GbDFRs were significantly correlated with anthocyanin accumulation in leaves. A fitted linear curve showed the best model for relating GbDFR2 and GbDFR3 with anthocyanin accumulation in leaves. GbDFR1 appears to be involved in environmental stress response, while GbDFR3 likely has primary functions in the synthesis of anthocyanins. These data revealed unexpected properties and differences in three DFR proteins from a single species. PMID:23991027

  8. Molecular cloning and expression of ptxA, the gene encoding the 120-kilodalton cytotoxin of Actinobacillus pleuropneumoniae serotype 2.

    PubMed

    MacDonald, J; Rycroft, A N

    1992-07-01

    The genetic determinants of the 120-kDa cytotoxin of Actinobacillus pleuropneumoniae serotype 2 were isolated from a lambda DNA library by a plaque immunoblot technique. Expression of the 120-kDa polypeptide was confirmed by Western immunoblot analysis of infected Escherichia coli cell lysates, which were shown to be toxic for porcine alveolar macrophages in vitro. The genetic determinants of the toxin were subcloned into the plasmid vector pUC18. This plasmid (pPTX1) directed the synthesis and secretion of the active 120-kDa cytotoxin in E. coli. The recombinant toxin was indistinguishable from native cytotoxin from A. pleuropneumoniae serotype 2 with respect to molecular size, reaction in Western blot analysis, heat lability, cytotoxic activity, and neutralization by serum antibody. A restriction endonuclease cleavage map of pPTX1 was prepared, and deletion mutants were used to locate the minimal region of DNA required for production of intracellular toxin; this gene was termed ptxA. Southern hybridization analysis with a 1.7-kb PvuII fragment located within the ptxA gene revealed sequences with a high degree of homology in serotype reference strains 2, 3, 4, 6, and 8. Other reference strains did not contain sequences that were recognized by this probe. However, related sequences (greater than 71% homology) were detected in Actinobacillus actinomycetemcomitans and A. equuli. Weak hybridization was observed between the ptxA probe and pLKT5, which carries the lktAC genes of Pasteurella haemolytica, and between the ptxA probe and pAPH1, which carries the structural gene for type II hemolysin from A. pleuropneumoniae. The isolation of the genetic determinants of this cytotoxin will enable investigations of the structure and organization of the ptx DNA region and further analysis of its role in the pathogenesis of pleuropneumonia. PMID:1612740

  9. Molecular cloning of a thermostable neutral protease gene from Bacillus stearothermophilus in a vector plasmid and its expression in Bacillus stearothermophilus and Bacillus subtilis.

    PubMed Central

    Fujii, M; Takagi, M; Imanaka, T; Aiba, S

    1983-01-01

    The structural gene for a thermostable protease from Bacillus stearothermophilus was cloned in plasmid pTB90. It is expressed in both B. stearothermophilus and Bacillus subtilis. B. stearothermophilus carrying the recombinant plasmid produced about 15-fold more protease (310 U/mg of cell dry weight) than did the wild-type strain of B. stearothermophilus. Some properties of the proteases that have been purified from the transformants of B. stearothermophilus and B. subtilis were examined. No significant difference was observed among the enzyme properties studied here despite the difference in host cells. We found that the protease, neutral in pH characteristics and with a molecular weight of 36,000, retained about 80% of its activity even after treatment of 65 degrees C for 30 min. Images PMID:6302083

  10. Molecular cloning of protein-based polymers.

    PubMed

    Mi, Lixin

    2006-07-01

    Protein-based biopolymers have become a promising class of materials for both biomedical and pharmaceutical applications, as they have well-defined molecular weights, monomer compositions, as well as tunable chemical, biological, and mechanical properties. Using standard molecular biology tools, it is possible to design and construct genes encoding artificial proteins or protein-based polymers containing multiple repeats of amino acid sequences. This article reviews some of the traditional methods used for constructing DNA duplexes encoding these repeat-containing genes, including monomer generation, concatemerization, iterative oligomerization, and seamless cloning. A facile and versatile method, called modules of degenerate codons (MDC), which uses PCR and codon degeneracy to overcome some of the disadvantages of traditional methods, is introduced. Re-engineering of the random coil spacer domain of a bioactive protein, WPT2-3R, is used to demonstrate the utility of the MDC method. MDC re-constructed coding sequences facilitate further manipulations, such as insertion, deletion, and swapping of various sequence modules. A summary of some promising emerging techniques for synthesizing repetitive sequence-containing artificial proteins is also provided. PMID:16827576

  11. Molecular cloning and evolutionary analysis of captive forest musk deer bitter taste receptor gene T2R16.

    PubMed

    Zhao, G J; Wu, N; Li, D Y; Zeng, D J; Chen, Q; Lu, L; Feng, X L; Zhang, C L; Zheng, C L; Jie, H

    2015-01-01

    Sensing bitter tastes is crucial for most animals because it can prevent them from ingesting harmful food. This process is mainly mediated by the bitter taste receptors (T2R) that are largely expressed in the taste buds. Previous studies have identified some T2R gene repertoires. Marked variation in repertoire size has been noted among species. However, research on T2Rs is still limited and the mechanisms underlying the evolution of vertebrate T2Rs remain poorly understood. In the present study, we analyzed the structure and features of the protein encoded by the forest musk deer (Moschus berezovskii) T2R16 and submitted the gene sequence to NCBI GenBank. The results showed that the full coding DNA sequence (CDS) of musk deer T2R16 (GenBank accession No. KP677279) was 906 bp, encoding 301 amino acids, which contained ATG start codon and TGA stop codon, with a calculated molecular weight of 35.03 kDa and an isoelectric point of 9.56. The T2R16 protein receptor had seven conserved transmembrane regions. Hydrophobicity analysis showed that most amino acid residues in T2R16 protein were hydrophobic, and the grand average of hydrophobicity (GRAVY) was 0.657. Phylogenetic analysis based on this gene revealed that forest musk deer had the closest association with sheep (Ovis aries), as compared to cow (Bos taurus), Tursiops truncatus, and other species, whereas it was genetically farthest from humans (Homo sapiens). We hope these results would complement the existing data on T2R16 and encourage further research in this respect. PMID:26662411

  12. Molecular cloning of a maize gene involved in photosynthetic membrane organization that is regulated by Robertson's Mutator.

    PubMed Central

    Martienssen, R A; Barkan, A; Freeling, M; Taylor, W C

    1989-01-01

    The maize photosynthetic mutant hcf106 has a distinctive and unusual thylakoid membrane organization, and fails to accumulate three of the five thylakoid membrane protein complexes. This mutant arose in a Robertson's Mutator background, and shows somatic instability typical of a transposon-induced allele. In addition, hcf106 is suppressed when Mu1 elements are inactive and modified in their terminal inverted repeats. Thus plants homozygous for the mutant allele adopt a mutant phenotype only when Mu1 elements are active and unmodified. DNA from the mutant allele has been cloned by 'transposon-tagging' using the transposon Mu1, and the identity of the clone confirmed by observing somatic excision of the transposon in a revertant sector. A 1.2 kb transcript homologous to the cloned DNA is found in wild-type and suppressed seedlings, but is not found in mutant seedlings, suggesting that suppression is mediated at the level of transcript accumulation. Images PMID:2548850

  13. Molecular cloning and characterization of a human cDNA and gene encoding a novel acid ceramidase-like protein.

    PubMed

    Hong, S B; Li, C M; Rhee, H J; Park, J H; He, X; Levy, B; Yoo, O J; Schuchman, E H

    1999-12-01

    Computer-assisted database analysis of sequences homologous to human acid ceramidase (ASAH) revealed a 1233-bp cDNA (previously designated cPj-LTR) whose 266-amino-acid open reading frame had approximately 36% identity with the ASAH polypeptide. Based on this high degree of homology, we undertook further molecular characterization of cPj-LTR and now report the full-length cDNA sequence, complete gene structure (renamed human ASAHL since it is a human acid ceramidase-like sequence), chromosomal location, primer extension and promoter analysis, and transient expression results. The full-length human ASAHL cDNA was 1825 bp and contained an open-reading frame encoding a 359-amino-acid polypeptide that was 33% identical and 69% similar to the ASAH polypeptide over its entire length. Numerous short regions of complete identity were observed between these two sequences and two sequences obtained from the Caenorhabditis elegans genome database. The 30-kb human ASAHL genomic sequence contained 11 exons, which ranged in size from 26 to 671 bp, and 10 introns, which ranged from 150 bp to 6.4 kb. The gene was localized to the chromosomal region 4q21.1 by fluorescence in situ hybridization analysis. Northern blotting experiments revealed a major 2.0-kb ASAHL transcript that was expressed at high levels in the liver and kidney, but at relatively low levels in other tissues such as the lung, heart, and brain. Sequence analysis of the 5'-flanking region of the human ASAHL gene revealed a putative promoter region that lacked a TATA box and was GC rich, typical features of a housekeeping gene promoter, as well as several tissue-specific and/or hormone-induced transcription regulatory sites. 5'-Deletion analysis localized the promoter activity to a 1. 1-kb fragment within this region. A major transcription start site also was located 72 bp upstream from the ATG translation initiation site by primer extension analysis. Expression analysis of a green fluorescence protein/ASAHL fusion

  14. Analysis of the ergosterol biosynthesis pathway cloning, molecular characterization and phylogeny of lanosterol 14 α-demethylase (ERG11) gene of Moniliophthora perniciosa

    PubMed Central

    de Oliveira Ceita, Geruza; Vilas-Boas, Laurival Antônio; Castilho, Marcelo Santos; Carazzolle, Marcelo Falsarella; Pirovani, Carlos Priminho; Selbach-Schnadelbach, Alessandra; Gramacho, Karina Peres; Ramos, Pablo Ivan Pereira; Barbosa, Luciana Veiga; Pereira, Gonçalo Amarante Guimarães; Góes-Neto, Aristóteles

    2014-01-01

    The phytopathogenic fungus Moniliophthora perniciosa (Stahel) Aime & Philips-Mora, causal agent of witches’ broom disease of cocoa, causes countless damage to cocoa production in Brazil. Molecular studies have attempted to identify genes that play important roles in fungal survival and virulence. In this study, sequences deposited in the M. perniciosa Genome Sequencing Project database were analyzed to identify potential biological targets. For the first time, the ergosterol biosynthetic pathway in M. perniciosa was studied and the lanosterol 14α-demethylase gene (ERG11) that encodes the main enzyme of this pathway and is a target for fungicides was cloned, characterized molecularly and its phylogeny analyzed. ERG11 genomic DNA and cDNA were characterized and sequence analysis of the ERG11 protein identified highly conserved domains typical of this enzyme, such as SRS1, SRS4, EXXR and the heme-binding region (HBR). Comparison of the protein sequences and phylogenetic analysis revealed that the M. perniciosa enzyme was most closely related to that of Coprinopsis cinerea. PMID:25505843

  15. Molecular cloning and characterization of two genes encoding sigma factors that direct transcription from a Bacillus thuringiensis crystal protein gene promoter.

    PubMed Central

    Adams, L F; Brown, K L; Whiteley, H R

    1991-01-01

    Two sigma factors, sigma 35 and sigma 28, direct transcription from the Bt I and Bt II promoters of the cryIA(a) gene of Bacillus thuringiensis; this gene encodes a lepidopteran-specific crystal protoxin. These sigma factors were biochemically characterized in previous work (K. L. Brown and H. R. Whiteley, Proc. Natl. Acad. Sci. USA 85:4166-4170, 1988; K. L. Brown and H. R. Whiteley, J. Bacteriol. 172:6682-6688, 1990). In this paper, we describe the cloning of the genes encoding these two sigma factors, as well as their nucleotide and deduced amino acid sequences. The deduced amino acid sequences of the sigma 35 and sigma 28 genes show 88 and 85% identity, respectively, to the sporulation-specific sigma E and sigma K polypeptides of Bacillus subtilis. Transformation of the sigma 35 and sigma 28 genes into B. subtilis shows that the respective B. thuringiensis sigma factor genes can complement spoIIG55 (sigma E) and spoIIIC94 (sigma K) defects. Further, B. thuringiensis core polymerase reconstituted with either the sigma 35 or sigma 28 polypeptide directs transcription from B. subtilis promoters recognized by B. subtilis RNA polymerase containing sigma E and sigma K, respectively. Thus, sigma 35 and sigma 28 of B. thuringiensis appear to be functionally equivalent to sigma E and sigma K of B. subtilis. However, unlike the situation for sigma K in B. subtilis, the homologous sigma 28 gene in B. thuringiensis does not result from a late-sporulation-phase chromosomal rearrangement of two separate, partial genes. Images PMID:1904859

  16. Molecular cloning and characterization of multidomain xylanase from manure library

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The gene (manf-x10) encoding xylanase from an environmental genomic DNA library was cloned and expressed in Escherichia coli. The encoded enzyme was predicted to be 467 amino acids with a molecular mass of 50.3 kD. The recombinant ManF-X10 was purified by HisTrap affinity column and showed activit...

  17. Cloning genes for non-syndromal hearing impairment.

    PubMed

    Smith, R J; Van Camp, G

    1999-10-01

    Over 45 genes that cause autosomal non-syndromic hearing impairment (NSHI) have been localized and many more are predicted to exist. To clone these genes, a number of different strategies can be used. This paper focuses on four general approaches: functional cloning, positional cloning, position-dependent candidate gene cloning, and position-independent candidate gene cloning. PMID:10890140

  18. Molecular cloning, modeling and differential expression of a gene encoding a silent information regulator-like protein from Sporothrix schenckii

    PubMed Central

    HOU, BINBIN; LIU, XIAOMING; ZHENG, FANGLIANG; XU, XUEZHU; ZHANG, ZHENYING

    2014-01-01

    Sporothrix schenckii (S. schenckii) is a dimorphic fungus that produces lymphocutaneous lesions. The signature characteristic of S. schenckii is a temperature-induced phase transition. Silent information regulator (Sir) has been proven to be involved in phenotypic switching in Saccharomyces cerevisiae (S. cerevisiae) and Candida albicans (C. albicans) by organizing chromatin structure. In this study, we isolated and characterized a Sir homologue gene, designated as SsSir2, from the yeast form of S. schenckii. The full-length SsSir2 cDNA sequence is 1753 bp in size and contains an open reading frame of 1329 bp encoding 442 amino acids. The predicted molecular mass of SsSir2 is 48.1 kDa with an estimated theoretical isoelectric point of 4.6. The SsSir2 kinase domain shows a 78% identity with that of Hst2, a Sir2 Ib gene from S. cerevisiae. Three exons and two introns were identified within the 1472-bp SsSir2 genomic DNA sequence of S. schenckii. A three-dimensional model of SsSir2 was constructed using a homology modeling method, and its reliability was evaluated. The active site of SsSir2 was identified by docking simulation, which indicated that several important residues, such as Asn127 and Asp129, play an important role in the histone deacetylase activity of Sir2 family proteins. The differential expression of the SsSir2 in two stages was demonstrated by real-time RT-PCR. The expression of SsSir2 was higher in the yeast stage compared with that in the mycelial one, which indicated that SsSir2 may be involved in the phenotypic switching and morphogenesis of the yeast phase in S. schenckii. PMID:24682409

  19. Molecular cloning and characterization of the human interleukin-11 receptor {alpha}-chain gene, IL11RA, located on chromosome 9p13

    SciTech Connect

    Van Leuven, F.; Stas, L.; Hilliker, C.

    1996-01-01

    The human gene coding for the interleukin-11 receptor (IL11RA) was cloned and its structure analyzed. The gene is composed of 13 exons comprising nearly 10 kb of DNA that was completely sequenced. The intron-exon boundaries were determined based on the mouse Etl2 and interleukin-11 receptor cDNAs that were recently cloned. The protein sequence predicted by the human gene was over 83% identical with its murine counterpart, with very strict conservation of functionally important domains and signatures. Fluorescence in situ hybridization showed the gene to be located on human chromosome 9p13, syntenic with the mouse etl2 gene on chromosome 4. The coding exons of the interleukin-11 gene were sequenced in a patient with the cartilage-hair hypoplasia syndrome, which has been linked to a gene on chromosome 9, but no functional mutations were detected. 26 refs., 4 figs., 2 tabs.

  20. Molecular cloning, sequencing analysis, and chromosomal localization of the human protease inhibitor 4 (Kallistatin) gene (P14)

    SciTech Connect

    Chai, K.X.; Chao, J.; Chao, L.; Ward, D.C.

    1994-09-15

    The gene encoding human protease inhibitor 4 (kallistatin; gene symbol PI4), a novel serine proteinase inhibitor (serpin), has been isolated and completely sequenced. The kallistatin gene is 9618 bp in length and contains five exons and four introns. The structure and organization of the kallistatin gene are similar to those of the genes encoding {alpha}{sub 1}-antichymotrypsin. The kallistatin gene is also similar to the genes encoding rat and mouse kallikrein-binding proteins. The first exon of the kallistatin gene is a noncoding 89-bp fragment, as determined by primer extension. The fifth exon, which contains 308 bp of noncoding sequence, encodes the reactive center of kallistatin. In the 5`-flanking region of the kallistatin gene, 1125 bp have been sequenced and a consensus promoter segment with potential transcription regulatory sites, including CAAT and TATA boxes, an AP-2 binding site, a GC-rich region, a cAMP response element, and an AP-1 binding site, has been identified within this region. The kallistatin gene was localized by in situ hybridization to human chromosome 14q31-132.1, close to the serpin genes encoding {alpha}{sub 1}-antichymotrypsin, protein C inhibitor, {alpha}{sub 1}-antitrypsin, and corticosteroid-binding globulin. In a genomic DNA Southern blot, kallistatin-related genes were identified in monkey, mouse, rat, bovine, dog, cat, and a ground mole. The patterns of hybridization revealed clues of human serpin evolution. 34 refs., 6 figs.

  1. Molecular Cloning and mRNA Expression of Heat Shock Protein Genes and Their Response to Cadmium Stress in the Grasshopper Oxya chinensis.

    PubMed

    Zhang, Yuping; Liu, Yaoming; Zhang, Jianzhen; Guo, Yaping; Ma, Enbo

    2015-01-01

    Heat shock proteins (Hsps) are highly conserved molecular chaperones that are synthesized in response to stress. In this study, we cloned the full-length sequences of the Grp78 (glucose-regulated protein 78), Hsp70, Hsp90, and Hsp40 genes from the Chinese rice grasshopper Oxya chinensis. The full-length cDNA sequences of OcGrp78, OcHsp70, OcHsp90, and OcHsp40 contain open reading frames of 1947, 1920, 2172, and 1042 bp that encode proteins of 649, 640, 724, and 347 amino acids, respectively. Fluorescent real-time quantitative PCR (RT-qPCR) was performed to quantify the relative transcript levels of these Hsp genes in different tissues and developmental stages. The mRNAs encoding these four Hsp genes were present at all developmental stages and in all tissues examined but were expressed at varying levels. Additionally, we investigated the mRNA expression profiles of these four Hsps in O. chinensis subjected to Cadmium (Cd) stress. OcGrp78, OcHsp70, OcHsp90, and OcHsp40 mRNA expression was induced under acute Cd stress; the levels reached a maximum within a short time (6 h), were reduced significantly at 12 h, and were lowered to or below control levels by 48 h. Regarding induction efficiency, OcHsp70 was the most sensitive gene to acute Cd stress. Chronic Cd exposure showed that dietary Cd treatment induced increased OcGrp78, OcHsp90, and OcHsp40 expression. However, dietary Cd induced a significant reduction of OcHsp70 expression. In the period tested, no significant difference in the mortality of the grasshoppers was observed. Our results suggest that these four Hsps genes, especially OcHsp70, are sensitive to acute Cd stress and could be used as molecular markers for toxicology studies. However, our results also indicate that OcHsp70 is not suitable for use as a molecular marker of chronic Cd contamination. PMID:26135744

  2. Molecular Cloning and mRNA Expression of Heat Shock Protein Genes and Their Response to Cadmium Stress in the Grasshopper Oxya chinensis

    PubMed Central

    Zhang, Yuping; Liu, Yaoming; Zhang, Jianzhen; Guo, Yaping; Ma, Enbo

    2015-01-01

    Heat shock proteins (Hsps) are highly conserved molecular chaperones that are synthesized in response to stress. In this study, we cloned the full-length sequences of the Grp78 (glucose-regulated protein 78), Hsp70, Hsp90, and Hsp40 genes from the Chinese rice grasshopper Oxya chinensis. The full-length cDNA sequences of OcGrp78, OcHsp70, OcHsp90, and OcHsp40 contain open reading frames of 1947, 1920, 2172, and 1042 bp that encode proteins of 649, 640, 724, and 347 amino acids, respectively. Fluorescent real-time quantitative PCR (RT-qPCR) was performed to quantify the relative transcript levels of these Hsp genes in different tissues and developmental stages. The mRNAs encoding these four Hsp genes were present at all developmental stages and in all tissues examined but were expressed at varying levels. Additionally, we investigated the mRNA expression profiles of these four Hsps in O. chinensis subjected to Cadmium (Cd) stress. OcGrp78, OcHsp70, OcHsp90, and OcHsp40 mRNA expression was induced under acute Cd stress; the levels reached a maximum within a short time (6 h), were reduced significantly at 12 h, and were lowered to or below control levels by 48 h. Regarding induction efficiency, OcHsp70 was the most sensitive gene to acute Cd stress. Chronic Cd exposure showed that dietary Cd treatment induced increased OcGrp78, OcHsp90, and OcHsp40 expression. However, dietary Cd induced a significant reduction of OcHsp70 expression. In the period tested, no significant difference in the mortality of the grasshoppers was observed. Our results suggest that these four Hsps genes, especially OcHsp70, are sensitive to acute Cd stress and could be used as molecular markers for toxicology studies. However, our results also indicate that OcHsp70 is not suitable for use as a molecular marker of chronic Cd contamination. PMID:26135744

  3. Molecular cloning and functional analysis of GbRVd, a gene in Gossypium barbadense that plays an important role in conferring resistance to Verticillium wilt.

    PubMed

    Yang, Jun; Ma, Qing; Zhang, Yan; Wang, Xingfen; Zhang, Guiyin; Ma, Zhiying

    2016-01-10

    Most of the disease resistance genes already characterized in plants encode nucleotide-binding site-leucine rich repeat (NBS-LRR) proteins that have key roles in resistance to Verticillium dahliae. Using a cDNA library and RACE protocols, we cloned a coiled-coil (CC)-NBS-LRR-type gene, GbRVd, from a resistant tetraploid cotton species, Gossypium barbadense (RVd=Resistance to V. dahliae). We also applied RT-qPCR and VIGS technologies to analyze how expression of GbRVd was induced upon attack by V. dahliae. Its 2862-bp ORF encodes a predicted protein containing 953 amino acid residues, with a predicted molecular weight of 110.17kDa and an isoelectric point of 5.87. GbRVd has three domains - CC, NBS, and LRR - and is most closely related to Gossypium raimondii RVd (88% amino acid identity). Profiling demonstrated that GbRVd is constitutively expressed in all tested tissues, and transcript levels are especially high in the leaves. In plants inoculated with V. dahliae, GbRVd was significantly up-regulated when compared with the control, with expression peaking at 48h post-inoculation. Silencing of GbRVd in cotton through VIGS dramatically down-regulated SA, NO, and H2O2 production, resulting in greater susceptibility to V. dahliae. Taken together, these results suggest that GbRVd has an important role in protecting G. barbadense against infection by V. dahliae. PMID:26407869

  4. Molecular cloning and expression of α-globin and β-globin genes from crocodile (Crocodylus siamensis).

    PubMed

    Anwised, Preeyanan; Kabbua, Thai; Temsiripong, Theeranan; Dhiravisit, Apisak; Jitrapakdee, Sarawut; Araki, Tomohiro; Yoneda, Kazunari; Thammasirirak, Sompong

    2013-03-01

    The first report of complete nucleotide sequences for α- and β-globin chains from the Siamese hemoglobin (Crocodylus siamensis) is given in this study. The cDNAs encoding α- and β-globins were cloned by RT-PCR using the degenerate primers and by the rapid amplification of cDNA ends method. The full-length α-globin cDNA contains an open reading frame of 423 nucleotides encoding 141 amino acid residues, whereas the β-globin cDNA contains an open reading frame of 438 nucleotides encoding 146 amino acid residues. The authenticity of both α- and β-globin cDNA clones were also confirmed by the heterologous expression in Escherichia coli (E. coli). This is the first time that the recombinant C. siamensis globins were produced in prokaryotic system. Additionally, the heme group was inserted into the recombinant proteins and purified heme-bound proteins were performed by affinity chromatography using Co(2+)-charged Talon resins. The heme-bound proteins appeared to have a maximum absorbance at 415 nm, indicated that the recombinant proteins bound to oxygen and formed active oxyhemoglobin (HbO2). The results indicated that recombinant C. siamensis globins were successfully expressed in prokaryotic system and possessed an activity as ligand binding protein. PMID:23463382

  5. The gene family encoding the Arabidopsis thaliana translation elongation factor EF-1 alpha: molecular cloning, characterization and expression.

    PubMed

    Axelos, M; Bardet, C; Liboz, T; Le Van Thai, A; Curie, C; Lescure, B

    1989-10-01

    The gene family encoding the Arabidopsis thaliana translation elongation factor (EF-1 alpha) was analysed. This family contains four genes (A1-A4) organized in a similar manner in different varieties of Arabidopsis. Based upon both their physical separation and a comparison of their sequences, it is suggested that the A4 gene and the A1, A2, and A3 genes constitute two distinct subfamilies within the genome. By introducing chimaeric gene constructs into Arabidopsis cells, we showed that the A1 gene promoter mediates a transient expression about twofold higher than that obtained using the CaMV 35 S promoter. This expression depends on a 348 bp DNA fragment extending from -982 to -634 bp upstream of the initiation codon. This element contains a characteristic telomeric sequence (AACCCTAA) which is also found in the promoters of the A2 and A4 genes as well as in the promoters of the Drosophila EF-1 alpha F1 gene and of several highly expressed plant genes. PMID:2615757

  6. Molecular cloning and characterization of genes for antibodies generated by orbital tissue-infiltrating B-cells in Graves` ophthalmopathy

    SciTech Connect

    Jaume, J.C.; Portolano, S.; Prummel, M.F.; McLachlan, S.M.; Rapoport, B.

    1994-02-01

    Graves` ophthalmopathy is a distressing autoimmune disease of unknown etiology. Analysis of the genes for antibodies secreted by orbital tissue-infiltrating plasma cells might provide insight into the pathogenesis of this disease. The authors, therefore, constructed an immunoglobulin heavy (H) chain and an immunoglobulin k light (L) chain cDNA library from the orbital tissue of a patient with active Graves` ophthalmopathy. Analysis of 15 H (IgG1) and 15 L (k) chains revealed a restricted spectrum of variable region genes. Fourteen of 15 variable k genes were about 94% homologous to the closest known germline gene, KL012. Thirteen of 15 H chain genes were 91% and 90% homologous to the closest germline genes, DP10 and hv1263, respectively. Remarkably, these germline genes also code for other autoantibodies to striated muscle (KL012) and thyroid peridase (KL012 and hv1263). These studies raise the possibility that particular germline genes may be associated with autoimmunity in humans. Further, the present study opens the way to identifying ocular autoantigens that may be the target of an humoral immune response. 29 refs., 4 figs., 1 tab.

  7. Identification and molecular cloning of three Halloween genes in the varroa mite, Varroa destructor (Anderson & Trueman) (Acari: Varroidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biosynthesis of 20-hydroxyecdysone (20E) in insects involves the action of five cytochrome P450s collectively known as Halloween genes. The complete transcripts of 3 Halloween genes [spook (Vdspo), disembodied (Vddib) and shade (Vdshd)] from the varroa mite were identified, sequenced and mapped to t...

  8. An Entry/Gateway® cloning system for general expression of genes with molecular tags in Drosophila melanogaster

    PubMed Central

    Akbari, Omar S; Oliver, Daniel; Eyer, Katie; Pai, Chi-Yun

    2009-01-01

    Background Tagged fusion proteins are priceless tools for monitoring the activities of biomolecules in living cells. However, over-expression of fusion proteins sometimes leads to the unwanted lethality or developmental defects. Therefore, vectors that can express tagged proteins at physiological levels are desirable tools for studying dosage-sensitive proteins. We developed a set of Entry/Gateway® vectors for expressing fluorescent fusion proteins in Drosophila melanogaster. The vectors were used to generate fluorescent CP190 which is a component of the gypsy chromatin insulator. We used the fluorescent CP190 to study the dynamic movement of related chromatin insulators in living cells. Results The Entry/Gateway® system is a timesaving technique for quickly generating expression constructs of tagged fusion proteins. We described in this study an Entry/Gateway® based system, which includes six P-element destination vectors (P-DEST) for expressing tagged proteins (eGFP, mRFP, or myc) in Drosophila melanogaster and a TA-based cloning vector for generating entry clones from unstable DNA sequences. We used the P-DEST vectors to express fluorecent CP190 at tolerable levels. Expression of CP190 using the UAS/Gal4 system, instead, led to either lethality or underdeveloped tissues. The expressed eGFP- or mRFP-tagged CP190 proteins are fully functional and rescued the lethality of the homozygous CP190 mutation. We visualized a wide range of CP190 distribution patterns in living cell nuclei, from thousands of tiny particles to less than ten giant ones, which likely reflects diverse organization of higher-order chromatin structures. We also visualized the fusion of multiple smaller insulator bodies into larger aggregates in living cells, which is likely reflective of the dynamic activities of reorganization of chromatin in living nuclei. Conclusion We have developed an efficient cloning system for expressing dosage-sensitive proteins in Drosophila melanogaster. This system

  9. Molecular cloning, sequence identification and tissue expression profile of three novel sheep (Ovis aries) genes - BCKDHA, NAGA and HEXA.

    PubMed

    Liu, G Y; Gao, S Z

    2009-01-01

    The complete coding sequences of three sheep genes- BCKDHA, NAGA and HEXA were amplified using the reverse transcriptase polymerase chain reaction (RT-PCR), based on the conserved sequence information of the mouse or other mammals. The nucleotide sequences of these three genes revealed that the sheep BCKDHA gene encodes a protein of 313 amino acids which has high homology with the BCKDHA gene that encodes a protein of 447 amino acids that has high homology with the Branched chain keto acid dehydrogenase El, alpha polypeptide (BCKDHA) of five species chimpanzee (93%), human (96%), crab-eating macaque (93%), bovine (98%) and mouse (91%). The sheep NAGA gene encodes a protein of 411 amino acids that has high homology with the alpha-N-acetylgalactosaminidase (NAGA) of five species human (85%), bovine (94%), mouse (91%), rat (83%) and chicken (74%). The sheep HEXA gene encodes a protein of 529 amino acids that has high homology with the hexosaminidase A(HEXA) of five species bovine (98%), human (84%), Bornean orangután (84%), rat (80%) and mouse (81%). Finally these three novel sheep genes were assigned to GenelDs: 100145857, 100145858 and 100145856. The phylogenetic tree analysis revealed that the sheep BCKDHA, NAGA, and HEXA all have closer genetic relationships to the BCKDHA, NAGA, and HEXA of bovine. Tissue expression profile analysis was also carried out and results revealed that sheep BCKDHA, NAGA and HEXA genes were differentially expressed in tissues including muscle, heart, liver, fat, kidney, lung, small and large intestine. Our experiment is the first to establish the primary foundation for further research on these three sheep genes. PMID:19621134

  10. Molecular cloning and functional characterization of the lycopene ε-cyclase gene via virus-induced gene silencing and its expression pattern in Nicotiana tabacum.

    PubMed

    Shi, Yanmei; Wang, Ran; Luo, Zhaopeng; Jin, Lifeng; Liu, Pingping; Chen, Qiansi; Li, Zefeng; Li, Feng; Wei, Chunyang; Wu, Mingzhu; Wei, Pan; Xie, He; Qu, Lingbo; Lin, Fucheng; Yang, Jun

    2014-01-01

    Lycopene ε-cyclase (ε-LCY) is a key enzyme that catalyzes the synthesis of α-branch carotenoids through the cyclization of lycopene. Two cDNA molecules encoding ε-LCY (designated Ntε-LCY1 and Ntε-LCY2) were cloned from Nicotiana tabacum. Ntε-LCY1 and Ntε-LCY2 are encoded by two distinct genes with different evolutionary origins, one originating from the tobacco progenitor, Nicotiana sylvestris, and the other originating from Nicotiana tomentosiformis. The two coding regions are 97% identical at the nucleotide level and 95% identical at the amino acid level. Transcripts of Ntε-LCY were detectable in both vegetative and reproductive organs, with a relatively higher level of expression in leaves than in other tissues. Subcellular localization experiments using an Ntε-LCY1-GFP fusion protein demonstrated that mature Ntε-LCY1 protein is localized within the chloroplast in Bright Yellow 2 suspension cells. Under low-temperature and low-irradiation stress, Ntε-LCY transcript levels substantially increased relative to control plants. Tobacco rattle virus (TRV)-mediated silencing of ε-LCY in Nicotiana benthamiana resulted in an increase of β-branch carotenoids and a reduction in the levels of α-branch carotenoids. Meanwhile, transcripts of related genes in the carotenoid biosynthetic pathway observably increased, with the exception of β-OHase in the TRV-ε-lcy line. Suppression of ε-LCY expression was also found to alleviate photoinhibition of Potosystem II in virus-induced gene silencing (VIGS) plants under low-temperature and low-irradiation stress. Our results provide insight into the regulatory role of ε-LCY in plant carotenoid biosynthesis and suggest a role for ε-LCY in positively modulating low temperature stress responses. PMID:25153631

  11. Molecular Cloning and Functional Characterization of the Lycopene ε-Cyclase Gene via Virus-Induced Gene Silencing and Its Expression Pattern in Nicotiana tabacum

    PubMed Central

    Shi, Yanmei; Wang, Ran; Luo, Zhaopeng; Jin, Lifeng; Liu, Pingping; Chen, Qiansi; Li, Zefeng; Li, Feng; Wei, Chunyang; Wu, Mingzhu; Wei, Pan; Xie, He; Qu, Lingbo; Lin, Fucheng; Yang, Jun

    2014-01-01

    Lycopene ε-cyclase (ε-LCY) is a key enzyme that catalyzes the synthesis of α-branch carotenoids through the cyclization of lycopene. Two cDNA molecules encoding ε-LCY (designated Ntε-LCY1 and Ntε-LCY2) were cloned from Nicotiana tabacum. Ntε-LCY1 and Ntε-LCY2 are encoded by two distinct genes with different evolutionary origins, one originating from the tobacco progenitor, Nicotiana sylvestris, and the other originating from Nicotiana tomentosiformis. The two coding regions are 97% identical at the nucleotide level and 95% identical at the amino acid level. Transcripts of Ntε-LCY were detectable in both vegetative and reproductive organs, with a relatively higher level of expression in leaves than in other tissues. Subcellular localization experiments using an Ntε-LCY1-GFP fusion protein demonstrated that mature Ntε-LCY1 protein is localized within the chloroplast in Bright Yellow 2 suspension cells. Under low-temperature and low-irradiation stress, Ntε-LCY transcript levels substantially increased relative to control plants. Tobacco rattle virus (TRV)-mediated silencing of ε-LCY in Nicotiana benthamiana resulted in an increase of β-branch carotenoids and a reduction in the levels of α-branch carotenoids. Meanwhile, transcripts of related genes in the carotenoid biosynthetic pathway observably increased, with the exception of β-OHase in the TRV-ε-lcy line. Suppression of ε-LCY expression was also found to alleviate photoinhibition of Potosystem II in virus-induced gene silencing (VIGS) plants under low-temperature and low-irradiation stress. Our results provide insight into the regulatory role of ε-LCY in plant carotenoid biosynthesis and suggest a role for ε-LCY in positively modulating low temperature stress responses. PMID:25153631

  12. Molecular cloning and expression of a novel catechol 2,3-dioxygenase gene from the benzoate meta-cleavage pathway in Azotobacter vinelandii.

    PubMed

    Keil, H

    1990-04-01

    Azotobacter vinelandii strain 206 degrades benzoate via the meta-cleavage pathway. In a genomic library derived from this organism a clone was obtained which carried and expressed the gene for the third enzyme in this pathway, catechol 2,3-dioxygenase (EC 1.13.11.2), on a 5.9 kb SalI restriction fragment. The structural gene was more precisely mapped on an internal 1.6 kb EcoRI fragment which, after insertion into expression vectors, directed the synthesis of a 33 kDa polypeptide. The gene showed very little or no homology with isofunctional genes derived from Pseudomonas. Comprehensive substrate specificity analysis showed significant differences between the specific activities obtained from the cloned gene product and extracts derived from Azotobacter itself. PMID:2398344

  13. Two beta-glycanase genes are clustered in Bacillus polymyxa: molecular cloning, expression, and sequence analysis of genes encoding a xylanase and an endo-beta-(1,3)-(1,4)-glucanase.

    PubMed Central

    Gosalbes, M J; Pérez-González, J A; González, R; Navarro, A

    1991-01-01

    Two genes, xynD and gluB, encoding a xylanase and an endo-beta-(1,3)-(1,4)-glucanase (lichenase) from Bacillus polymyxa have been cloned and expressed in Escherichia coli and Bacillus subtilis. A sequenced DNA fragment of 4,466 bp contains both genes, which are separated by 155 bp. The xynD and gluB genes encode proteins of 67.8 kDa (XYND) and 27 kDa (GLUB). Two peptides with molecular masses of 62 and 53 kDa appear in cell extracts of E. coli and culture supernatants of B. subtilis clones containing the xynD gene. Both peptides show xylanase activity in zymogram analysis. The XYND enzyme also shows alpha-L-arabinofuranosidase activity. The XYND peptide and the xylanase XYNZ from Clostridium thermocellum (O. Grépinet, M. C. Chebrou, and P. Béguin, J. Bacteriol. 170:4582-4588, 1988) show 64% homology in a stretch of about 280 amino acids. Images FIG. 3 PMID:1938968

  14. Two beta-glycanase genes are clustered in Bacillus polymyxa: molecular cloning, expression, and sequence analysis of genes encoding a xylanase and an endo-beta-(1,3)-(1,4)-glucanase.

    PubMed

    Gosalbes, M J; Pérez-González, J A; González, R; Navarro, A

    1991-12-01

    Two genes, xynD and gluB, encoding a xylanase and an endo-beta-(1,3)-(1,4)-glucanase (lichenase) from Bacillus polymyxa have been cloned and expressed in Escherichia coli and Bacillus subtilis. A sequenced DNA fragment of 4,466 bp contains both genes, which are separated by 155 bp. The xynD and gluB genes encode proteins of 67.8 kDa (XYND) and 27 kDa (GLUB). Two peptides with molecular masses of 62 and 53 kDa appear in cell extracts of E. coli and culture supernatants of B. subtilis clones containing the xynD gene. Both peptides show xylanase activity in zymogram analysis. The XYND enzyme also shows alpha-L-arabinofuranosidase activity. The XYND peptide and the xylanase XYNZ from Clostridium thermocellum (O. Grépinet, M. C. Chebrou, and P. Béguin, J. Bacteriol. 170:4582-4588, 1988) show 64% homology in a stretch of about 280 amino acids. PMID:1938968

  15. Molecular cloning and characterization of amh, dax1 and cyp19a1a genes and their response to 17α-methyltestosterone in Pengze crucian carp.

    PubMed

    Li, Meng; Wang, Lihong; Wang, Houpeng; Liang, Hongwei; Zheng, Yao; Qin, Fang; Liu, Shaozhen; Zhang, Yingying; Wang, Zaizhao

    2013-05-01

    The proteins encoded by amh, dax1 and cyp19a1a play important roles in gonad differentiation. Their functions have been far less studied in teleosts. In this study, the full-length cDNAs of amh, dax1 and cyp19a1a were cloned and characterized in a triploid gynogenic fish, the Pengze crucian carp. Their expression profilings in juvenile development, adult tissues and juveniles exposed to 100 ng/L 17α-methyltestosterone (MT) were investigated. Results showed that their putative proteins shared high identities to their counterparts in cyprinid fish species, respectively. The tissue distribution results indicated that amh and cyp19a1a were predominantly expressed in the ovary and dax1 was dominantly expressed in the liver. Gene profiling in the developmental stages showed that all the three target genes had a consistent highest expression at 48 days post hatching (dph). The period of 48 dph appeared to be a key time during the process of the gonad development of Pengze crucian carp. 100 ng/L MT significantly increased the mRNA expression of amh at 2- and 4-week exposures and enhanced dax1 and cyp19a1a at 6-week exposure. The present study indicated that MT could influence the gonad development in Pengze crucian carp by disturbing sex-differentiation associated gene expression. Furthermore, the present study will be of great significance to broaden the understanding of molecular mechanisms of the physiological processes of reproduction in fish. PMID:23528270

  16. Molecular cloning of a coiled-coil-nucleotide-binding-site-leucine-rich repeat gene from pearl millet and its expression pattern in response to the downy mildew pathogen.

    PubMed

    Veena, Mariswamy; Melvin, Prasad; Prabhu, Sreedhara Ashok; Shailasree, Sekhar; Shetty, Hunthrike Shekar; Kini, Kukkundoor Ramachandra

    2016-03-01

    Downy mildew caused by Sclerospora graminicola is a devastating disease of pearl millet. Based on candidate gene approach, a set of 22 resistance gene analogues were identified. The clone RGPM 301 (AY117410) containing a partial sequence shared 83% similarity to rice R-proteins. A full-length R-gene RGA RGPM 301 of 3552 bp with 2979 bp open reading frame encoding 992 amino acids was isolated by the degenerate primers and rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) approach. It had a molecular mass of 113.96 kDa and isoelectric point (pI) of 8.71. The sequence alignment and phylogenetic analysis grouped it to a non-TIR NBS LRR group. The quantitative real-time PCR (qRT-PCR) analysis revealed higher accumulation of the transcripts following inoculation with S. graminicola in the resistant cultivar (IP18296) compared to susceptible cultivar (7042S). Further, significant induction in the transcript levels were observed when treated with abiotic elicitor β-aminobutyric acid (BABA) and biotic elicitor Pseudomonas fluorescens. Exogenous application of phytohormones jasmonic acid or salicylic acid also up-regulated the expression levels of RGA RGPM 301. The treatment of cultivar IP18296 with mitogen-activated protein kinase (MPK) inhibitors (PD98059 and U0126) suppressed the levels of RGA RGPM 301. A 3.5 kb RGA RGPM 301 which is a non-TIR NBS-LRR protein was isolated from pearl millet and its up-regulation during downy mildew interaction was demonstrated by qRT-PCR. These studies indicate a role for this RGA in pearl millet downy mildew interaction. PMID:26842722

  17. Molecular cloning and sequencing of two phospho-beta-galactosidase I and II genes of Lactobacillus gasseri JCM1031 isolated from human intestine.

    PubMed

    Saito, T; Suzuki, M; Konno, K; Kitazawa, H; Kawai, Y; Itoh, T; Kamio, Y

    1998-12-01

    Lactobacillus (Lb.) gasseri JCM1031, which is classified into the B1 subgroup of the Lb. acidophilus group of lactic acid bacteria, characteristically produces two different phospho-beta-galactosidases (P-beta-gal) I and II in the same cytosol as reported in our previous papers [Biosci. Biotech. Biochem., 60, 139-141, 708-710 (1996)]. To clarify the functional and genetic properties of the two enzymes, the structural genes of P-beta-gal I and II were cloned and sequenced. The structural gene of P-beta-gal I had 1,446 bp, encoding a polypeptide of 482 amino acid residues. The structural gene of P-beta-gal II had 1,473 bp, encoding a polypeptide of 491 amino acid residues. The deduced relative molecular masses of 55,188 and 56,243 agreed well with the previous value obtained from the purified P-beta-gal I and II protein, respectively. Multiple alignment of the protein sequence of P-beta-gal I and II with those of P-beta-gals from 5 microorganisms had 30-35% identity on the amino acid level, but those with phospho-beta-glucosidases from 5 microorganisms had the relatively high identity of about 50%. Considering that this strain grows on lactose medium and shows no beta-galactosidase activity, and that purified P-beta-gal I and II can obviously hydrolyze o-nitrophenyl-beta-D-galactopyranoside 6-phosphate (substrate), and also the conservation of a cysteine residue in the molecule, the P-beta-gal I and II were each confirmed as a novel P-beta-gal enzyme. PMID:9972258

  18. Advances and applications of molecular cloning in clinical microbiology.

    PubMed

    Sharma, Kamal; Mishra, Ajay Kumar; Mehraj, Vikram; Duraisamy, Ganesh Selvaraj

    2014-10-01

    Molecular cloning is based on isolation of a DNA sequence of interest to obtain multiple copies of it in vitro. Application of this technique has become an increasingly important tool in clinical microbiology due to its simplicity, cost effectiveness, rapidity, and reliability. This review entails the recent advances in molecular cloning and its application in the clinical microbiology in the context of polymicrobial infections, recombinant antigens, recombinant vaccines, diagnostic probes, antimicrobial peptides, and recombinant cytokines. Culture-based methods in polymicrobial infection have many limitation, which has been overcome by cloning techniques and provide gold standard technique. Recombinant antigens produced by cloning technique are now being used for screening of HIV, HCV, HBV, CMV, Treponema pallidum, and other clinical infectious agents. Recombinant vaccines for hepatitis B, cholera, influenza A, and other diseases also use recombinant antigens which have replaced the use of live vaccines and thus reduce the risk for adverse effects. Gene probes developed by gene cloning have many applications including in early diagnosis of hereditary diseases, forensic investigations, and routine diagnosis. Industrial application of this technology produces new antibiotics in the form of antimicrobial peptides and recombinant cytokines that can be used as therapeutic agents. PMID:25023463

  19. Mismatch Repair in Schizosaccharomyces Pombe Requires the Mutl Homologous Gene Pms1: Molecular Cloning and Functional Analysis

    PubMed Central

    Schar, P.; Baur, M.; Schneider, C.; Kohli, J.

    1997-01-01

    Homologues of the bacterial mutS and mutL genes involved in DNA mismatch repair have been found in organisms from bacteria to humans. Here, we describe the structure and function of a newly identified Schizosaccharomyces pombe gene that encodes a predicted amino acid sequence of 794 residues with a high degree of homology to MutL related proteins. On the basis of its closer relationship to the eukaryotic ``PMS'' genes than to the ``MLH'' genes, we have designated the S. pombe homologue pms1. Disruption of the pms1 gene causes a significant increase of spontaneous mutagenesis as documented by reversion rate measurements. Tetrad analyses of crosses homozygous for the pms1 mutation reveal a reduction of spore viability from >92% to 80% associated with a low proportion (~50%) of meioses producing four viable spores and a significant, allele-dependent increase of the level of post-meiotic segregation of genetic marker allele pairs. The mutant phenotypes are consistent with a general function of pms1 in correction of mismatched base pairs arising as a consequence of DNA polymerase errors during DNA synthesis, or of hybrid DNA formation between homologous but not perfectly complementary DNA strands during meiotic recombination. PMID:9258673

  20. Molecular cloning, sequence characterization of a novel pepper gene NADP-ICDH and its effect on cytoplasmic male sterility.

    PubMed

    Deng, M H; Wen, J F; Huo, J L; Zhu, H S; Dai, X Z; Zhang, Z Q; Zhou, H; Zou, X X

    2012-01-01

    NADP-dependent isocitrate dehydrogenase (NADP-ICDH) is an important enzyme involved in energy metabolism. The complete coding sequence of the pepper (Capsicum annuum) NADP-ICDH gene was amplified using a reverse transcriptase PCR based on the conserved sequence information of the tomato and other Solanaceae plants and known highly homologous pepper ESTs. Nucleotide sequence analysis revealed that the pepper NADP-ICDH gene encodes a protein of 415 amino acids that has high homology with the proteins of seven species, Solanum tuberosum (100%), Citrus limon (98%), Daucus carota (98%), Nicotiana tabacum (98%), Vitis vinifera (99%), Arabidopsis thaliana (97%), and Oryza sativa (98%). Tissue expression analysis demonstrated that the pepper NADP-ICDH gene is over expressed in flower, pericarp and seed, moderately in placenta, weakly in stem and leaf, hardly expressed in root. At the abortion stages, activities and expression levels of NADP-ICDH in anthers of a sterile line were strongly reduced, while those in an F(1) hybrid remained normal. Activities and expression levels of NADP-ICDH were too low to maintain balanced energy metabolism in the sterile line, which indicated that stable transcripts of NADP-ICDH are necessary to maintain energy metabolism at a normal level. When the restorer gene was transferred to the cytoplasmic male sterile line, activities and expression level of NADP-ICDH were regulated by the restorer gene and became stable. The restorer gene likely plays an important role in keeping the balance of the energy metabolism within normal levels during microspore development. PMID:22653649

  1. Light and auxin responsive cytochrome P450s from Withania somnifera Dunal: cloning, expression and molecular modelling of two pairs of homologue genes with differential regulation.

    PubMed

    Srivastava, Sudhakar; Sangwan, Rajender Singh; Tripathi, Sandhya; Mishra, Bhawana; Narnoliya, L K; Misra, L N; Sangwan, Neelam S

    2015-11-01

    Cytochrome P450s (CYPs) catalyse a wide variety of oxygenation/hydroxylation reactions that facilitate diverse metabolic functions in plants. Specific CYP families are essential for the biosynthesis of species-specialized metabolites. Therefore, we investigated the role of different CYPs related to secondary metabolism in Withania somnifera, a medicinally important plant of the Indian subcontinent. In this study, complete complementary DNAs (cDNAs) of four different CYP genes were isolated and christened as WSCYP93Id, WSCYP93Sm, WSCYP734B and WSCYP734R. These cDNAs encoded polypeptides comprising of 498, 496, 522 and 550 amino acid residues with their deduced molecular mass of 56.7, 56.9, 59.4 and 62.2 kDa, respectively. Phylogenetic study and molecular modelling analysis of the four cloned WSCYPs revealed their categorization into two CYP families (CYP83B1 and CYP734A1) belonging to CYP71 and CYP72 clans, respectively. BLASTp searches showed similarity of 75 and 56 %, respectively, between the two CYP members of CYP83B1 and CYP734A1 with major variances exhibited in their N-terminal regions. The two pairs of homologues exhibited differential expression profiles in the leaf tissues of selected chemotypes of W. somnifera as well as in response to treatments such as methyl jasmonate, wounding, light and auxin. Light and auxin regulated two pairs of WSCYP homologues in a developing seedling in an interesting differential manner. Their lesser resemblance and homology with other CYP sequences suggested these genes to be more specialized and distinct ones. The results on chemotype-specific expression patterns of the four genes strongly suggested their key/specialized involvement of the CYPs in the biosynthesis of chemotype-specific metabolites, though their further biochemical characterization would reveal the specificity in more detail. It is revealed that WSCYP93Id and WSCYP93Sm may be broadly involved in the oxygenation reactions in the plant and, thereby, control

  2. Molecular cloning and functional characterization of the sex-determination gene doublesex in the sexually dimorphic broad-horned beetle Gnatocerus cornutus (Coleoptera, Tenebrionidae).

    PubMed

    Gotoh, Hiroki; Ishiguro, Mai; Nishikawa, Hideto; Morita, Shinichi; Okada, Kensuke; Miyatake, Takahisa; Yaginuma, Toshinobu; Niimi, Teruyuki

    2016-01-01

    Various types of weapon traits found in insect order Coleoptera are known as outstanding examples of sexually selected exaggerated characters. It is known that the sex determination gene doublesex (dsx) plays a significant role in sex-specific expression of weapon traits in various beetles belonging to the superfamily Scarabaeoidea. Although sex-specific weapon traits have evolved independently in various Coleopteran groups, developmental mechanisms of sex-specific expression have not been studied outside of the Scarabaeoidea. In order to test the hypothesis that dsx-dependent sex-specific expression of weapon traits is a general mechanism among the Coleoptera, we have characterized the dsx in the sexually dimorphic broad-horned beetle Gnatocerus cornutus (Tenebrionidea, Tenebirionidae). By using molecular cloning, we identified five splicing variants of Gnatocerus cornutus dsx (Gcdsx), which are predicted to code four different isoforms. We found one male-specific variant (GcDsx-M), two female-specific variants (GcDsx-FL and GcDsx-FS) and two non-sex-specific variants (correspond to a single isoform, GcDsx-C). Knockdown of all Dsx isoforms resulted in intersex phenotype both in male and female. Also, knockdown of all female-specific isoforms transformed females to intersex phenotype, while did not affect male phenotype. Our results clearly illustrate the important function of Gcdsx in determining sex-specific trait expression in both sexes. PMID:27404087

  3. Molecular cloning, mRNA expression and tissue distribution analysis of Slc7a11 gene in alpaca (Lama paco) skins associated with different coat colors.

    PubMed

    Tian, Xue; Meng, Xiaolin; Wang, Liangyan; Song, Yunfei; Zhang, Danli; Ji, Yuankai; Li, Xuejun; Dong, Changsheng

    2015-01-25

    Slc7a11 encoding solute carrier family 7 member 11 (amionic amino acid transporter light chain, xCT), has been identified to be a critical genetic regulator of pheomelanin synthesis in hair and melanocytes. To better understand the molecular characterization of Slc7a11 and the expression patterns in skin of white versus brown alpaca (lama paco), we cloned the full length coding sequence (CDS) of alpaca Slc7a11 gene and analyzed the expression patterns using Real Time PCR, Western blotting and immunohistochemistry. The full length CDS of 1512bp encodes a 503 amino acid polypeptide. Sequence analysis showed that alpaca xCT contains 12 transmembrane regions consistent with the highly conserved amino acid permease (AA_permease_2) domain similar to other vertebrates. Sequence alignment and phylogenetic analysis revealed that alpaca xCT had the highest identity and shared the same branch with Camelus ferus. Real Time PCR and Western blotting suggested that xCT was expressed at significantly high levels in brown alpaca skin, and transcripts and protein possessed the same expression pattern in white and brown alpaca skins. Additionally, immunohistochemical analysis further demonstrated that xCT staining was robustly increased in the matrix and root sheath of brown alpaca skin compared with that of white. These results suggest that Slc7a11 functions in alpaca coat color regulation and offer essential information for further exploration on the role of Slc7a11 in melanogenesis. PMID:25455099

  4. Molecular cloning and functional characterization of the sex-determination gene doublesex in the sexually dimorphic broad-horned beetle Gnatocerus cornutus (Coleoptera, Tenebrionidae)

    PubMed Central

    Gotoh, Hiroki; Ishiguro, Mai; Nishikawa, Hideto; Morita, Shinichi; Okada, Kensuke; Miyatake, Takahisa; Yaginuma, Toshinobu; Niimi, Teruyuki

    2016-01-01

    Various types of weapon traits found in insect order Coleoptera are known as outstanding examples of sexually selected exaggerated characters. It is known that the sex determination gene doublesex (dsx) plays a significant role in sex-specific expression of weapon traits in various beetles belonging to the superfamily Scarabaeoidea. Although sex-specific weapon traits have evolved independently in various Coleopteran groups, developmental mechanisms of sex-specific expression have not been studied outside of the Scarabaeoidea. In order to test the hypothesis that dsx-dependent sex-specific expression of weapon traits is a general mechanism among the Coleoptera, we have characterized the dsx in the sexually dimorphic broad-horned beetle Gnatocerus cornutus (Tenebrionidea, Tenebirionidae). By using molecular cloning, we identified five splicing variants of Gnatocerus cornutus dsx (Gcdsx), which are predicted to code four different isoforms. We found one male-specific variant (GcDsx-M), two female-specific variants (GcDsx-FL and GcDsx-FS) and two non-sex-specific variants (correspond to a single isoform, GcDsx-C). Knockdown of all Dsx isoforms resulted in intersex phenotype both in male and female. Also, knockdown of all female-specific isoforms transformed females to intersex phenotype, while did not affect male phenotype. Our results clearly illustrate the important function of Gcdsx in determining sex-specific trait expression in both sexes. PMID:27404087

  5. Molecular Cloning, Characterization and mRNA Expression of a Chitin Synthase 2 Gene from the Oriental Fruit Fly, Bactrocera dorsalis (Diptera: Tephritidae)

    PubMed Central

    Chen, Li; Yang, Wen-Jia; Cong, Lin; Xu, Kang-Kang; Wang, Jin-Jun

    2013-01-01

    Chitin synthase (CHS), a potential target for eco-friendly insecticides, plays an essential role in chitin formation in insects. In this study, a full-length cDNA encoding chitin synthase 2 (BdCHS2) was cloned and characterized in the oriental fruit fly, Bactrocera dorsalis. The BdCHS2 cDNA had 4417 nucleotides, containing an open reading frame of 4122 nucleotides, which encoded 1373 amino acid residues with a predicted molecular weight of 158.5 kDa. Phylogenetic analysis with other insect CHSs suggested that BdCHS2 belongs to insect CHS2. The BdCHS2 transcript was predominately found in midgut but was detected at low levels in fat body, Malpighian tubules, integument, and trachea. Moreover, BdCHS2 was expressed in all developmental stages, and highly expressed in the feeding stages. There was a positive relationship between BdCHS2 expression and total chitin content during development. Furthermore, both the gene expression and chitin content in midgut decreased when the insect was fed for 24 h, then starved for 24 h, while they increased dramatically and rapidly under the condition of starvation for 24 h then feeding for 24 h. These results suggest that BdCHS2 may play an important role in regulating chitin content of the midgut, and subsequently affect the growth and development of B. dorsalis. PMID:23965972

  6. Molecular cloning and expression of the gene for a major leucine-rich protein from human hepatoblastoma cells (HepG2).

    PubMed

    Hou, J; Wang, F; McKeehan, W L

    1994-02-01

    The human hepatoblastoma cell line, HepG2, exhibits an array of stable properties in culture that have made it a popular cell culture model for studies on regulation of liver-specific gene expression and properties of hepatoma cells. In contrast to other hepatoma cell lines, HepG2 cells overexpress a characteristic detergent-extractable, wheat germ lectin-binding protein with apparent molecular mass of 130 kDa. Using an antibody to screen a phage expression library of HepG2 complementary DNA (cDNA), we identified and cloned a 4734 base pair cDNA which codes for a 130-kDa leucine-rich protein (lrp 130) when expressed in transfected cells. The deduced sequence of lrp130 exhibits sequences weakly homologous to the consensus sequence for the ATP binding site in ATP-dependent kinases and the protein kinase C phosphorylation site of the epidermal growth factor receptor. Consistent with the higher levels of expression of lrp130 antigen, Northern hybridization analysis indicated that HepG2 cells express high levels of the major 4.8 kilobase lrp130 mRNA relative to other hepatoma cells. Although currently of unknown function, lrp130 may be of utility as a marker for liver cell lineages represented by the HepG2 cell line. PMID:8012652

  7. Molecular cloning and characterization of a ToxA-like gene from the maize pathogen Cochliobolus heterostrophus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ToxA, the first discovered fungal proteinaceous host-selective toxin, was originally identified from the tan spot fungus Pyrenophora tritici-repentis (Ptr). Homologues of the PtrToxA gene have not been identified from any other ascomycetes except the leaf/glume blotch fungus Stagonospora nodorum, w...

  8. Molecular cloning and characterization of the mouse carboxyl ester lipase gene and evidence for expression in the lactating mammary gland

    SciTech Connect

    Lidmer, A.S.; Lundberg, L.; Kannius, M.; Bjursell, G.

    1995-09-01

    DNA hybridization was used to isolate a 2.04-kb cDNA encoding carboxyl ester lipase (CEL) from a mouse lactating mammary gland, {lambda}gt10 cDNA library. The cDNA sequence translated into a protein of 599 amino acids, including 20 amino acids of a putative signal peptide. Comparison of the deduced amino acid sequence of the mouse CEL with CEL from five other species revealed that there is a high degree of a homology between the different species. The mouse CEL gene was also isolated and found to span approximately 7.2 kb and to include 11 exons. This organization is similar to those of the recently reported human and rat CEL genes. We have also analyzed expression of the CEL gene in the mammary glands from other species by performing a Northern blot analysis with RNA from goat and cow. The results show that the gene is expressed in both species. 36 refs., 6 figs., 1 tab.

  9. Molecular cloning, sequencing, and identification of a metalloprotease gene from Listeria monocytogenes that is species specific and physically linked to the listeriolysin gene.

    PubMed Central

    Domann, E; Leimeister-Wächter, M; Goebel, W; Chakraborty, T

    1991-01-01

    The entire nucleotide sequence of an open reading frame located immediately downstream of the listeriolysin gene from a virulent Listeria monocytogenes serotype 1/2a strain was determined. The product of the open reading frame was 510 amino acids with a predicted molecular weight of 57,400. The deduced amino acid sequence of this open reading frame is highly similar to that of a family of secreted metalloproteases produced by various members of the genus Bacillus, of which thermolysin is the prototype. Immunoblots performed with specific antisera raised against thermolysin from Bacillus stearothermophilus allowed the detection of a 60-kDa polypeptide, corresponding to the pro-form of the protease, in culture supernatants of L. monocytogenes strains. In maxicell experiments, Escherichia coli recombinants harboring this open reading frame also specifically directed production of a 60-kDa protein. Protease activity was low to undetectable in both Listeria strains and E. coli recombinants. This is due to lack of processing of the inactive pro-form of the protease to its mature active form in both species. We have designated this gene mpl for metalloprotease of L. monocytogenes. The gene was present only in pathogenic L. monocytogenes strains, in which it was physically linked to the listeriolysin gene. Images PMID:1898903

  10. MOLECULAR CLONING, CHARACTERIZATION, AND REGULATION OF A PSEUDOMONAS PICKETTII PK01 GENE IN ENCODING PHENOL HYDROXYLASE AND EXPRESSION OF THE GENE IN PSEUDOMONAS AERUGINOSA PA01C

    EPA Science Inventory

    A 26 kilobase BamHI restriction endonuclease DNA fragment has been cloned from Pseudomonas pickettii PK01, a strain isolated from a soil microcosm that had been amended with benzene, toluene, and xylene. This DNA fragment, cloned into vector plasmid pR0l727 and designated pR0l957...

  11. Molecular cloning and expression analysis of tyrosinase gene in the skin of Jining Gray Goat (Capra hircus).

    PubMed

    Chen, Weiyun; Wang, Hui; Dong, Bin; Dong, Zhongdian; Zhou, Fenna; Fu, Yong; Zeng, Yongqing

    2012-07-01

    Tyrosinase is the key regulatory enzyme of melanogenesis and plays a major role in mammal coat color. For the first time, we have sequenced and characterized the tyrosinase (TYR) of Jining Gray Goat (Capra hircus), which is the world-famous fur-bearing animal with its special color and pattern. The full-length cDNA was cloned by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) method. As a result, one 2131-bp nucleotide sequence representing the full-length cDNA of TYR was obtained. The entire open reading frame (ORF) of the TYR is 1593 bp and encodes for 530 amino acids, which is well conserved compared with TYR of various species with higher degree of sequence similarity with other mammalian (74-99 %) than amphibian, aves, and fishes (56-73 %). The deduced amino acids contained one signal peptide, one transmembrane domain, five N-linked glycosylation sites, and two copper binding sites. The result of real-time quantitative PCR showed that the expression level of TYR was the highest in the dark-gray goats and the lowest in the light-gray ones, while the goats of dark-gray individuals have more than 50 % black fiber and light-gray ones less than 30 %. During the whole life of Jining gray goat, TYR expression level changes with certain regularity and their coat color will change correspondingly by investigating the expression level in ten development stages. After comparing the result and the coat phenotype, we presume that it seems to have a positive relationship between the color depth of coat and the expression level of TYR. PMID:22407568

  12. Cloning, expression, and molecular characterization of the gene encoding an extremely thermostable [4Fe-4S] ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus.

    PubMed Central

    Heltzel, A; Smith, E T; Zhou, Z H; Blamey, J M; Adams, M W

    1994-01-01

    The gene for ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus was cloned, sequenced, and expressed in Escherichia coli. The coding region confirmed the determined amino acid sequence. Putative archaeon-type transcriptional regulatory elements were identified. The fdxA gene appears to be an independent transcriptional unit. Recombinant ferredoxin was indistinguishable from the protein purified from P. furiosus in its thermal stability and in the potentiometric and spectroscopic properties of its [4Fe-4S] cluster. PMID:8045914

  13. Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea

    SciTech Connect

    Stein, J.C.; Howlett, B.; Boyes, D.C.; Nasrallah, M.E.; Nasrallah, J.B. )

    1991-10-01

    Self-recognition between pollen and stigma during pollination in Brassica oleracea is genetically controlled by the multiallelic self-incompatibility locus (S). The authors describe the S receptor kinase (SRK) gene, a previously uncharacterized gene that residues at the S locus. The nucleotide sequences of genomic DNA and of cDNAs corresponding to SRK predict a putative transmembrane receptor having serine/threonine-specific protein kinase activity. Its extracellular domain exhibits striking homology to the secreted product of the S-locus genotypes are highly polymorphic and have apparently evolved in unison with genetically linked alleles of SLG. SRK directs the synthesis of several alternative transcripts, which potentially encode different protein products, and these transcripts were detected exclusively in reproductive organs. The identification of SRK may provide new perspectives into the signal transduction mechanism underlying pollen recognition.

  14. Molecular cloning and characterization of GbMECT and GbMECP gene promoters from Ginkgo biloba.

    PubMed

    Cheng, S Y; Li, L L; Yuan, H H; Xu, F; Cheng, H

    2015-01-01

    Ginkgolides are key pharmaceutical components in Ginkgo biloba. Using the cDNA sequence of the MECP and MECT genes to design primers, we obtained the promoters of these genes from Ginkgo genomic DNA using the genome walking method. The two promoters were 744 and 982 bp in length, respectively. The cis-elements of the GbMECPs and GbMECT promoters were predicted and analyzed using the plant cis-acting regulatory element database. We found major cis-elements in the sequence of the GbMECT and GbMECPs promoters. The GbMECP promoter contains six TATA boxes and eight CAAT boxes. The GbMECT contains five TATA boxes and seven CAAT boxes. Furthermore, some cis-elements in the promoters of GbMECPs and GbMECT included hormone and light-regulated elements, UB-B-induced elements, and stress-related dehydration-responsive elements. Expression analysis results showed that the MECP gene is mainly involved in responses to CCC (cycocel) and UV-B, and that MECT is mainly involved in responses to wounding treatment. These results also showed that the expression model was consistent with the cis-elements present. During the annual growth cycle, the level of GbMECPs was significantly correlated with terpene lactones accumulation in leaves. A fitted quadratic curve showed the best model for correlating GbMECPs with terpene lactones in leaves. These results will help us to understand the transcriptional regulatory mechanisms involved in key gene expression and ginkgolide accumulation in G. biloba. PMID:26634474

  15. Molecular cloning of a novel NF2/ERM/4.1 superfamily gene, ehm2, that is expressed in high-metastatic K1735 murine melanoma cells.

    PubMed

    Shimizu, K; Nagamachi, Y; Tani, M; Kimura, K; Shiroishi, T; Wakana, S; Yokota, J

    2000-04-15

    We have cloned a novel gene, Ehm2, that is expressed in high-metastatic but not in low-metastatic K-1735 murine melanoma cells. The Ehm2 gene encodes a protein of 527 amino acid residues, showing up to 41% amino acid identity with the FERM domain of NF2/ERM/4.1 superfamily proteins, which have the function of connecting cell surface transmembrane proteins to cytoskeletal molecules. The Ehm2 gene was mapped to chromosome 4 and was expressed in the liver, lung, kidney, and testis and in 7- to 17-day embryos. The highest level of homology was observed with NBL4, which is a new subfamily protein of the NF2/ERM/4.1 superfamily. A human homologue of the mouse Ehm2 gene, showing significant homology (83% identity), was identified in the genomic DNA and EST databases. Furthermore, seven rat EST clones and one pig EST clone in the GenBank EST database were identified as having 83-92% sequence homology with the cDNA sequence of the mouse Ehm2 gene. Thus, Ehm2 is a highly conserved gene that encodes a novel member of the NF2/ERM/4.1 superfamily proteins. PMID:10783258

  16. Molecular Cloning and Functional Characterization of Two Brachypodium distachyon UBC13 Genes Whose Products Promote K63-Linked Polyubiquitination

    PubMed Central

    Guo, Huiping; Wen, Rui; Liu, Zhi; Datla, Raju; Xiao, Wei

    2016-01-01

    Living organisms are constantly subject to DNA damage from environmental sources. Due to the sessile nature of plants, UV irradiation is a major genotoxic agent and imposes a significant threat on plant survival, genome stability and crop yield. In addition, other environmental chemicals can also influence the stability of the plant genome. Eukaryotic organisms have evolved a mechanism to cope with replication-blocking lesions and stabilize the genome. This mechanism is known as error-free DNA damage tolerance, and is mediated by K63-linked PCNA polyubiquitination. Genes related to K63-linked polyubiquitination have been isolated recently from model plants like Arabidopsis and rice, but we are unaware of such reports on the crop model Brachypodium distachyon. Here, we report the identification and functional characterization of two B. distachyon UBC13 genes. Both Ubc13s form heterodimers with Uevs from other species, which are capable of catalyzing K63 polyubiquitination in vitro. Both genes can functionally rescue the yeast ubc13 null mutant from killing by DNA-damaging agents. These results suggest that Ubc13-Uev-promoted K63-linked polyubiquitination is highly conserved in eukaryotes including B. distachyon. Consistent with recent findings that K63-linked polyubiquitination is involved in several developmental and stress-responsive pathways, the expression of BdUbc13s appears to be constitutive and is regulated by abnormal temperatures. PMID:26779244

  17. Molecular cloning and expression analysis of the MTN gene during adventitious root development in IBA-induced tetraploid black locust.

    PubMed

    Quan, Jine; Zhang, Chunxia; Zhang, Sheng; Meng, Sen; Zhao, Zhong; Xu, Xuexuan

    2014-12-15

    5'-Methylthioadenosine (MTA) nucleosidase (MTN) plays a key role in the methionine (Met) recycling pathway of plants. Here, we report the isolation of the 1158 bp full-length, cDNA sequence encoding tetraploid black locust (Robinia pseudoacacia L.) MTN (TrbMTN), which contains an open reading frame of 810 bp that encodes a 269 amino acid protein. The amino acid sequence of TrbMTN has more than 88% sequence identity to the MTNs from other plants, with a closer phylogenetic relationship to MTNs from legumes than to MTNs from other plants. Subcellular localization analysis revealed that the TrbMTN gene localizes mainly to the cell membrane and cytoplasm of onion epidermal cells. Indole-3-butyric acid (IBA)-treated cuttings showed higher TrbMTN transcript levels than untreated control cuttings during root primordium and adventitious root formation. TrbMTN and key Met cycle genes showed differential expression in shoots, leaves, stems, and roots, with the highest expression observed in stems. IBA-treated cuttings also showed higher TrbMTN activity than control cuttings during root primordium and adventitious root formation. These results indicate that TrbMTN gene might play an important role in the regulation of IBA-induced adventitious root development in tetraploid black locust cuttings. PMID:25305345

  18. Molecular cloning and activity analysis of a seed-specific FAD2-1B gene promoter from Glycine max.

    PubMed

    Zhao, Y; Sha, W; Wang, Q Y; Zhai, Y; Zhao, Y; Shao, S L

    2015-01-01

    Microsomal omega-6 fatty acid desaturase (FAD2-1B) is an enzyme that regulates the polyunsaturated fatty acid content in soybeans (Glycine max). In this study, the FAD2-1B gene was determined to be highly expressed in soybean seeds using quantitative real-time PCR(qRT-PCR). To investigate the expression pattern and activity of the FAD2-1B promoter, a 1929 bp 5'-upstream genomic DNA fragment, named PF, was isolated according to the soybean genomic sequence. Sequence analysis revealed the presence of many motifs related to seed-specific promoters in the PF fragment, such as E-box, SEF4, Skn-1 motif, AACACA, AATAAA and so on. Tobacco transgenics carrying the gus reporter gene driven by the PF and/or 35S promoters were confirmed by PCR and RT-PCR. qRT-PCR and histochemical GUS assays showed that the PF promoter could regulate gus gene accumulation in seeds and the expression level was higher than in other organs. In the meantime, it exhibited similar activity to the 35S promoter in seeds, which could be associated with seed-related cis-elements found in the 1-248 bp, 451-932 bp, and 1627-1803 bp regions of the promoter. PMID:26386665

  19. Molecular cloning of class III chitinase gene from Avicennia marina and its expression analysis in response to cadmium and lead stress.

    PubMed

    Wang, Li-Ying; Wang, You-Shao; Zhang, Jing-Ping; Gu, Ji-Dong

    2015-10-01

    Mangrove species have high tolerance to heavy metal pollution. Chitinases have been widely reported as defense proteins in response to heavy metal stress in terrestrial plants. In this study, a full-length cDNA sequence encoding an acidic and basic class III chitinase (AmCHI III) was cloned by using RT-PCR and RACE methods in Avicennia marina. AmCHI III mRNA expression in leaf of A. marina were investigated under Cd, Pb stresses on using real-time quantitative PCR. The deduced AmCHI III protein consists of 302 amino acids, including a signal putative peptide region, and a catalytic domain. Homology modeling of the catalytic domain revealed a typical molecular structure of class III plant chitinases. Results further demonstrated that the regulation of AmCHI III mRNA expression in leaves was strongly dependent on Cd, Pb stresses. AmCHI III mRNA expressions were significantly increased in response to Cd, Pb, and peaked at 7 days Cd-exposure, 7 days Pb-exposure, respectively. AmCHI III mRNA expression exhibited more sensitive to Pb stress than Cd stress. This work was the first time cloing chitinase from A. marina, and it brought evidence on chitinase gene involving in heavy metals (Cd(2+) and Pb(2+)) resistance or detoxification in plants. Further studies including the promoter and upstream regulation, gene over-expression and the response of mangrove chitinases to other stresses will shed more light on the role of chitinase in mangrove plants. PMID:26044930

  20. Molecular cloning of the sex-related gene PSI in Bemisia tabaci and its alternative splicing properties.

    PubMed

    Liu, Yating; Xie, Wen; Yang, Xin; Guo, Litao; Wang, Shaoli; Wu, Qingjun; Yang, Zezhong; Zhou, Xuguo; Zhang, Youjun

    2016-04-15

    The P-element somatic inhibitor (PSI) is gene known to regulate the transcription of doublesex (dsx) when transformer (tra) is absent in Bombyx mori. In this study, we identified and characterized a PSI homolog in Bemisia tabaci (BtPSI). BtPSI cDNA had a total length of 5700 bp and contained a predicted open reading frame (ORF) of 2208 nucleotides encoding for 735 amino acids. Multiple sequence alignments of the common regions of PSI proteins from B. tabaci and five other insect species revealed a high degree of sequence conservation. BtPSI is expressed in all stages of B. tabaci development, and expression did not significantly differ between female and male adult. A total of 92 BtPSI isoforms (78 in female and 22 in male) were identified, and a marker indicating the female-specific form was found. These results increase the understanding of genes that may determine sex in B. tabaci and provide a foundation for research on the sex determination mechanism in this insect. PMID:26773355

  1. Molecular cloning, expression profiling and trans-activation property studies of a DREB2-like gene from chrysanthemum (Dendranthema vestitum).

    PubMed

    Liu, Liqing; Zhu, Kai; Yang, Yanfang; Wu, Jian; Chen, Fadi; Yu, Deyue

    2008-03-01

    Dehydration responsive element binding (DREB) proteins are important transcription factors in plant stress response and signal transduction. In this study, a DREB homolog gene, DvDREB2A, was isolated from chrysanthemum (Dendranthema vestitum) by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. It contained an open reading frame (ORF) of 1,471 bp encoding 366 amino acid residues and was classified as a DREB2 subfamily member based on multiple sequence alignment. The predicted protein sequence contained a typical AP2/EREBP DNA-binding domain near the N-terminal region. In yeast one-hybrid analysis DvDREB2A protein was specifically bound to DRE elements (core sequence, A/GCCGAC) and activated the expression of the reporter HIS3 and LacZ. Transient expression experiment suggested that DvDREB2A protein was localized to the nucleus of onion epidermis cells. Quantitative real-time PCR (QRT-PCR) experiments showed that expression level of DvDREB2A was significantly affected by heat, low temperature, drought, abscisic acid (ABA) and high salinity treatments. These results indicated that the DvDREB2A gene is a new member of the DREB transcription factors, which may play an important role in providing tolerance to environmental stresses. PMID:18224275

  2. Molecular cloning and expression of a heat-shock cognate 70 (hsc70) gene from swordtail fish ( Xiphophorus helleri)

    NASA Astrophysics Data System (ADS)

    Li, Ningqiu; Fu, Xiaozhe; Han, Jingang; Shi, Cunbin; Huang, Zhibin; Wu, Shuqin

    2013-07-01

    Heat shock proteins are a family of molecular chaperones that are involved in many aspects of protein homeostasis. In the present study, a full-length cDNA, encoding the constitutively expressed 70-kDa heat shock cognate protein (Hsc70), was isolated from swordtail fish ( Xiphophorus helleri) and designated as XheHsc70. The Xhehsc70 cDNA was 2 104 bp long with an open reading frame of 1 941 bp, and it encoded a protein of 646 amino acids with a theoretical molecular weight of 70.77 kDa and an isoelectric point of 5.04. The deduced amino acid sequence shared 94.1%-98.6% identities with the Hsc70s from a number of other fish species. Tissue distribution results show that the Xhehsc70 mRNA was expressed in brain, heart, head kidney, kidney, spleen, liver, muscle, gill, and peripheral blood. After immunization with formalin-killed Vibrio alginolyticus cells there was a significant increase in the Xhehsc70 mRNA transcriptional level in the head kidney of the vaccinated fish compared with in the control at 6, 12, 24, and 48 h as shown by quantitative real time RT-PCR. Based on an analysis of the amino acid sequence of XheHsc70, its phylogeny, and Xhehsc70 mRNA expression, XheHsc70 was identified as a member of the cytoplasmic Hsc70 (constitutive) subfamily of the Hsp70 family of heat shock proteins, suggesting that it may play a role in the immune response. The Xhehsc70 cDNA sequence reported in this study was submitted to GenBank under the accession number JF739182.

  3. Molecular cloning of the BLADE-ON-PETIOLE gene and expression analyses during nodule development in Lupinus luteus.

    PubMed

    Frankowski, Kamil; Wilmowicz, Emilia; Kućko, Agata; Zienkiewicz, Agnieszka; Zienkiewicz, Krzysztof; Kopcewicz, Jan

    2015-05-01

    The BLADE-ON-PETIOLE (BOP) genes have been recently shown to play an essential role in many physiological processes, including embryogenesis, meristem determinacy, leaf patterning and nodule development. In our research we used Lupinus luteus, a plant with great agronomic potential due to its high protein content and nitrogen fixation ability. In this work, LlBOP in L. luteus was identified for the first time and its expression during nodule development was analyzed. The high expression levels of LlBOP and LlLbI (LEGHEMOGLOBIN), essential to nitrogen-fixing symbiosis, were noted in the developing root nodules and were correlated with the occurrence of leghemoglobin. All of these data indicate that LlBOP is an important regulator of root nodule formation and functioning in L. luteus. PMID:25817415

  4. Molecular cloning of a malyl coenzyme A lyase gene from Pseudomonas sp. strain AM1, a facultative methylotroph.

    PubMed Central

    Fulton, G L; Nunn, D N; Lidstrom, M E

    1984-01-01

    A genomic library containing HindIII partial digests of Pseudomonas sp. strain AM1 DNA was constructed in the broad-host-range cosmid pVK100. PCT57, a Pseudomonas sp. strain AM1 methanol mutant deficient in malyl coenzyme A lyase activity, was complemented to a methanol-positive phenotype by mobilization of the pVK100 library into PCT57 recipients with the ColE1/RK2 mobilizing plasmid pRK2013. Six different complemented isolates all contained a recombinant plasmid carrying the same 19.6-kilobase-pair Pseudomonas sp. strain AM1 DNA insert. Subcloning and complementation analysis demonstrated that the gene deficient in PCT57 (mcl-1) was located in a 1.6-kilobase-pair region within a 7.4-kilobase-pair EcoRI-HindIII fragment. PMID:6094488

  5. Molecular cloning and sequence analysis of the gene encoding OmpL1, a transmembrane outer membrane protein of pathogenic Leptospira spp.

    PubMed Central

    Haake, D A; Champion, C I; Martinich, C; Shang, E S; Blanco, D R; Miller, J N; Lovett, M A

    1993-01-01

    Pathogenic Leptospira spp. are spirochetes that have a low transmembrane outer membrane protein content relative to that of enteric gram-negative bacteria. In a previous study we identified a 31-kDa surface protein that was present in strains of Leptospira alstoni in amounts which correlated with the outer membrane particle density observed by freeze fracture electron microscopy (D. A. Haake, E. M. Walker, D. R. Blanco, C. A. Bolin, J. N. Miller, and M. A. Lovett, Infect. Immun. 59:1131-1140, 1991). The N-terminal amino acid sequence was used to design a pair of oligonucleotides which were utilized to screen a lambda ZAP II library containing EcoRI fragments of L. alstoni DNA. A 2.5-kb DNA fragment which contained the entire structural ompL1 gene was identified. The structural gene deduced from the sequence of this DNA fragment would encode a 320-amino-acid polypeptide with a 24-amino-acid leader peptide and a leader peptidase I cleavage site. Processing of OmpL1 results in a mature protein with a predicted molecular mass of 31,113 Da. Secondary-structure prediction identified repeated stretches of amphipathic beta-sheets typical of outer membrane protein membrane-spanning sequences. A topological model of OmpL1 containing 10 transmembrane segments is suggested. A recombinant OmpL1 fusion protein was expressed in Escherichia coli in order to immunize rabbits with the purified protein. Upon Triton X-114 extraction of L. alstoni and phase separation, anti-OmpL1 antiserum recognized a single band on immunoblots of the hydrophobic detergent fraction which was not present in the hydrophilic aqueous fraction. Immunoelectron microscopy with anti-OmpL1 antiserum demonstrates binding to the surface of intact L. alstoni. DNA hybridization studies indicate that the ompL1 gene is present in a single copy in all pathogenic Leptospira species that have been tested and is absent in nonpathogenic Leptospira species. OmpL1 may be the first spirochetal transmembrane outer membrane

  6. Molecular Cloning, Promoter Analysis and Expression Profiles of the sox3 Gene in Japanese Flounder, Paralichthys olivaceus

    PubMed Central

    Gao, Jinning; Li, Peizhen; Zhang, Wei; Wang, Zhigang; Wang, Xubo; Zhang, Quanqi

    2015-01-01

    Sox3, which belongs to the SoxB1 subgroup, plays major roles in neural and gonadal development. In the present study, Japanese flounder Paralichthys olivaceus sox3 gene (Posox3) and its promoter sequence were isolated and characterized. The deduced PoSox3 protein contained 298 amino acids with a characteristic HMG-box domain. Alignment and phylogenetic analyses indicated that PoSox3 shares highly identical sequence with Sox3 homologues from different species. The promoter region of Posox3 has many potential transcription factor (TF) binding sites. The expression profiles of Posox3 in different developmental stages and diverse adult tissues were analyzed by quantitative real-time RT-PCR (qRT-PCR). Posox3 mRNA was maternally inherited, and maintained at a considerably high expression level between the blastula stage and the hatching stage during embryonic development. Posox3 was abundantly expressed in the adult brain and showed sexually dimorphic expression pattern. In situ hybridization (ISH) was carried out to investigate the cellular distribution of Posox3 in the ovary, and results showed the uniform distribution of Posox3 throughout the cytoplasm of oogonia and stage I–III oocytes. These results indicate that Posox3 has potentially vital roles in embryonic and neural development and may be involved in the oogenesis process. Our work provides a fundamental understanding of the structure and potential functions of Sox3 in Paralichthys olivaceus. PMID:26610486

  7. Screening and molecular identification of overproducing γ-linolenic acid fungi and cloning the delta 6-desaturase gene.

    PubMed

    Lu, He; Zhu, Yu

    2015-01-01

    This research aims at isolating and identifying γ-linolenic acid (GLA)-producing fungi in the traditional Chinese salt-fermented soybean food, Douchi, from Yongchuan, People's Republic of China. In this study, Rhizopus oryzae DR3 was identified as a novel fungal species that produces large amounts of GLA. A full-length cDNA, designated as RoD6 D, with high homology to fungal △6 fatty acid desaturase genes was isolated from R. oryzae by using the rapid amplification of cDNA ends method. It had an open reading frame of 1,176 bp encoding a deduced polypeptide of 391 amino acids. Bioinformatics analysis characterized the putative RoD6 D protein as a typical membrane-bound desaturase, including three conserved histidine-rich motifs, a hydropathy profile, and a cytochrome b5 -like domain in the N-terminus. When the coding sequence was expressed in the Saccharomyces cerevisiae strain INVScl, the encoded product of RoD6 D exhibited △6 fatty acid desaturase activity that led to the accumulation of GLA. The results show that Douchi contains a large natural diverse composition, and some strains could be selected as starters for functional fermented foods. This study has also laid a foundation for developing functional Douchi products for further research. PMID:25169017

  8. Molecular cloning, expression, and stress response of the estrogen-related receptor gene (AccERR) from Apis cerana cerana

    NASA Astrophysics Data System (ADS)

    Zhang, Weixing; Zhu, Ming; Zhang, Ge; Liu, Feng; Wang, Hongfang; Guo, Xingqi; Xu, Baohua

    2016-04-01

    Estrogen-related receptor (ERR), which belongs to the nuclear receptor superfamily, has been implicated in diverse physiological processes involving the estrogen signaling pathway. However, little information is available on ERR in Apis cerana cerana. In this report, we isolated the ERR gene and investigated its involvement in antioxidant defense. Quantitative real-time polymerase chain reaction (qPCR) revealed that the highest mRNA expression occurred in eggs during different developmental stages. The expression levels of AccERR were highest in the muscle, followed by the rectum. The predicted transcription factor binding sites in the promoter of AccERR suggested that AccERR potentially functions in early development and in environmental stress responses. The expression of AccERR was induced by cold (4 °C), heat (42 °C), ultraviolet light (UV), HgCl2, and various types of pesticides (phoxim, deltamethrin, triadimefon, and cyhalothrin). Western blot was used to measure the expression levels of AccERR protein. These data suggested that AccERR might play a vital role in abiotic stress responses.

  9. Molecular cloning, expression and antioxidant characterisation of a typical thioredoxin gene (AccTrx2) in Apis cerana cerana.

    PubMed

    Yao, Pengbo; Hao, Lili; Wang, Fang; Chen, Xiaobo; Yan, Yan; Guo, Xingqi; Xu, Baohua

    2013-09-15

    Thioredoxins (Trxs) are a family of small, highly conserved and ubiquitous proteins that are involved in protecting organisms against toxic reactive oxygen species (ROS). In this study, a typical thioredoxin 2 gene was isolated from Apis cerana cerana, AccTrx2. The full-length cDNA sequence of AccTrx2 was composed of 407 bp containing a 318 bp open reading frame (ORF) that encodes a predicted protein of 105 amino acids, 11.974 kDa and an isoelectric point of 4.45. Expression profile of AccTrx2 as determined by a quantitative real-time PCR (qRT-PCR) analysis was higher in brain than in other tissues, with its highest transcript occurring on the 15day post-emergence adult and upregulated by such abiotic stresses as 4 °C, 16 °C, 25 °C, H2O2, cyhalothrin, acaricide, paraquat, phoxime and mercury (HgCl2) treatments. However, AccTrx2 was slightly repressed when exposed to 42 °C treatment. Characterisation of the recombinant protein showed that the purified AccTrx2 had insulin disulfide reductase activity and could protect DNA from ROS damage. These results indicate that AccTrx2 functions as an antioxidant that plays an important role in response to oxidative stress. PMID:23747404

  10. Molecular cloning, expression, purification and characterization of a novel cellulase gene (Bh-EGaseI) in the beetle Batocera horsfieldi.

    PubMed

    Mei, Hui-Zhen; Xia, Ding-Guo; Zhao, Qiao-Ling; Zhang, Guo-Zheng; Qiu, Zhi-Yong; Qian, Ping; Lu, Cheng

    2016-01-15

    Wood-feeding insects depend heavily on the secretion of a combination of cellulases, mainly endoglucanases and other glucanases such as exoglucanases and xylanases, to achieve efficient digestion of the cellulose of cellulosic materials. In this paper, we report a novel cellulose Bh-EGaseI belonging to the glycoside hydrolase family 45(gh45-1) obtained from the beetle Batocera horsfieldi. The Bh-EGaseI gene spans 714 bp and consists of three exons coding 237 amino acid residues. The cDNA encoding Bh-EGaseI was expressed as 25 KDa in baculovirus-infected Bombyx mori larvae. The expression products of Bh-EGaseI from larval hemolymph showed a specific enzymatic activity of approximately 1030.87 IU per mg. The enzyme was active over a wide range of pH and temperatures; optimal activity was observed at 40 °C and pH 4.0. The effects of ions on Bh-EGaseI activity were also studied, and results indicated that activity decreased to different extents upon addition of ions. Investigations on Bh-EGaseI facilitate their potential application in the production of bioenergy and biomaterials from cellulosic biomass in the future. PMID:26410410

  11. Molecular cloning and characterization of a phospholipid hydroperoxide glutathione peroxidase gene from a blood fluke Schistosoma japonicum.

    PubMed

    Zhang, Ying; He, Yuan; He, Li; Zong, Hong-Ying; Cai, Guo-Bin

    2015-01-01

    Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a major antioxidant enzyme and plays critical roles in the protection of cells against oxidative stress by catalysing reduction of lipid hydroperoxides. A full-length cDNA sequence corresponding to GPx gene from Schistosoma japonicum (designated SjGPx) was isolated and characterized. SjGPx contained an in-frame TGA codon for selenocysteine (Sec) and a concurrent Sec insertion sequence in its 3'-untranslated region. Protein encoded by SjGPx demonstrated a primary structure characteristic to the PHGPx family, including preservation of catalytic domains and absence of the subunit interaction domains. Phylogenetic analysis revealed that the SjGPx was highly related to the other PHGPx-related members, and clustered into the trematode subclade II. Semi-quantitative reverse transcription PCR and western blotting showed that the SjGPx was mainly expressed in the female adults and eggs. RNA interference was employed to investigate the effects of knockdown of SjGPx. SjGPx expression level was significantly reduced on the 5th day post-RNAi. We observed a 53.86% reduction in total GPx activity and the eggs severely deformed. Oxidative stimulation of viable worms with H2O2 or paraquat resulted in 1.6- to 2.1-fold induction of the GPx activity. Our results revealed that the SjGPx protein is selenium-dependent PHGPx, which might actively participate in the detoxification of oxidative damage during egg production. PMID:26484892

  12. Molecular cloning, expression, and stress response of the estrogen-related receptor gene (AccERR) from Apis cerana cerana.

    PubMed

    Zhang, Weixing; Zhu, Ming; Zhang, Ge; Liu, Feng; Wang, Hongfang; Guo, Xingqi; Xu, Baohua

    2016-04-01

    Estrogen-related receptor (ERR), which belongs to the nuclear receptor superfamily, has been implicated in diverse physiological processes involving the estrogen signaling pathway. However, little information is available on ERR in Apis cerana cerana. In this report, we isolated the ERR gene and investigated its involvement in antioxidant defense. Quantitative real-time polymerase chain reaction (qPCR) revealed that the highest mRNA expression occurred in eggs during different developmental stages. The expression levels of AccERR were highest in the muscle, followed by the rectum. The predicted transcription factor binding sites in the promoter of AccERR suggested that AccERR potentially functions in early development and in environmental stress responses. The expression of AccERR was induced by cold (4 °C), heat (42 °C), ultraviolet light (UV), HgCl2, and various types of pesticides (phoxim, deltamethrin, triadimefon, and cyhalothrin). Western blot was used to measure the expression levels of AccERR protein. These data suggested that AccERR might play a vital role in abiotic stress responses. PMID:26922780

  13. Molecular cloning and characterization of a putative proline dehydrogenase gene in the Colorado potato beetle, Leptinotarsa decemlineata.

    PubMed

    Wan, Pin-Jun; Lü, Dong; Guo, Wen-Chao; Ahmat, Tursun; Yang, Lu; Mu, Li-Li; Li, Guo-Qing

    2014-04-01

    Leptinotarsa decemlineata adults exhibit a season-dependent activity. In spring, post-diapause beetles often fly a long distance from overwintering sites to potato fields. In summer and autumn, the flight ability is sharply reduced. Proline is the main energy substrate of L. decemlineata during flight and proline dehydrogenase (ProDH) catalyzes the first step in proline catabolism. Here we identified a putative LdProDH gene; it had three cDNA isoforms which shared the same 5'UTR and coding region, but differed in the lengths of 3'UTRs (515, 1 092 and 1 242 bp for isoforms-1, -2 and -3, respectively). LdProDH encoded a 616 amino acid protein that showed high sequence similarity to ProDH-like proteins from other insect species. LdProDH was expressed in the third and fourth instars larvae and adults, but not in pupae. Dietary ingestion of bacterially expressed LdProDH-dsRNA by adults significantly decreased its messenger RNA (mRNA) level, and caused an elevation of free proline content in the hemolymph. Further observation revealed that three canonical polyadenylation signals (AATAAA) were tandemly located in the 3'UTR of isoform-3. The first, second and third polyadenylation sites gave rise to isoforms-1, -2 and -3, respectively. Analysis of the genomic DNA uncovered that the three isoforms resulted from alternative polyadenylation. The mRNA level of isoform-1, which expressed at low levels in pre-diapause adults, became abundant in post-diapause beetles. It is indicated that the LdProDH expression is fine-tuned through 3'UTR to control proline catabolism for the season-dependent activity of L. decemlineata adults. PMID:23956209

  14. Molecular cloning and mRNA expression of a hepcidin gene from the spinyhead croaker, Collichthys lucidus.

    PubMed

    Sang, C; Lin, Y; Jiang, K; Zhang, F; Song, W

    2015-01-01

    Antimicrobial peptides are important components that participate in host innate immune activities and play crucial roles in host defense against microbial invasion. Hepcidin is an antimicrobial peptide and iron-regulatory molecule that primarily functions in the liver. In the present study, we first obtained a full-length cDNA sequence of hepcidin and its corresponding genomic DNA sequence from Collichthys lucidus using RT-PCR and rapid amplification of cDNA ends (RACE), and then analyzed these sequences using bioinformatics software. The results showed that C. lucidus hepcidin (CL-hepc) possesses two introns and three exons in the genomic DNA, with a length of 816 bp. The open reading frame was 264 bp, encoding an 87 amino acid peptide, and with high similarity (88.89%) to 83416593 Larimichthys crocea (ABC18307) and relatively low similarity (47.73%) to 158358729 L. crocea (ABY84845.1). The pre-peptide contained a signal peptide (28 amino acids), a prodomain (34 amino acids), and a mature peptide (25 amino acids). The predicted 25 amino acid hepcidin mature peptide included 8 conserved cysteine residues. Quantitative real-time reverse transcription-PCR analysis revealed specific expression patterns of CL-hepc, with the highest expression observed in the liver, relatively low expression observed in the gill and spleen, and almost no expression detected in other tissues analyzed. In conclusion, we identified a hepcidin from C. lucidus that has common expression patterns with other hepcidins. However, as this hepcidin is inconsistent with two other hepcidins from L. crocea in terms of the phylogenetic tree, the presence of another hepcidin gene warrants further investigation. PMID:26662398

  15. Molecular cloning and nucleotide sequence of the 1,2-alpha-D-mannosidase gene, msdS, from Aspergillus saitoi and expression of the gene in yeast cells.

    PubMed

    Inoue, T; Yoshida, T; Ichishima, E

    1995-12-01

    A full-length cDNA encoding 1,2-alpha-D-mannosidase (EC 3.2.1.113) from Aspergillus saitoi was cloned. Analysis of the 1718 bp nucleotide sequence of the cDNA revealed a single open reading frame with 1539 nucleotides of 1,2-alpha-D-mannosidase gene, msdS. The predicted amino-acid sequence of 1,2-alpha-D-mannosidase consists of 513 residues with a molecular mass of 55,767 and is 70%, 26% and 35% identity with those of Penicillium citrinum 1,2-alpha-D-mannosidase, yeast alpha-mannosidase, and mouse alpha-mannosidase. The cDNA of the msdS gene has been cloned and expressed in yeast cells. To identify the activity of expression product methyl-2-O-alpha-mannopyranosyl-alpha-mannopyranoside (Man alpha 1-->2Man-OMe) was used as a substrate at pH 5.0. PMID:8519794

  16. Molecular cloning, expression pattern, and 3D structural analysis of the MHC class IIB gene in the Chinese longsnout catfish (Leiocassis longirostris).

    PubMed

    Shen, Tong; Xu, Shixia; Yang, Mei; Pang, Shuying; Yang, Guang

    2011-05-15

    Major histocompatibility complex (MHC) class I and class II molecules encode glycoproteins which mediate the specificity of the vertebrate adaptive immune response. In this study, MHC class IIB gene from the Chinese longsnout catfish (Leiocassis longirostris) was cloned and sequenced, which encoded a predicted protein of 248 amino acids (28.06 kDa) containing a signal peptide, a beta 1 domain, a beta 2 domain, a connecting peptide, a transmembrane region, and a cytoplasmic tail. Using PCR with primers designed from known fish MHC class IIB sequences followed by elongation of the 5' and 3' ends using rapid amplification of cDNA ends (RACE), the full-length cDNA of longsnout catfish MHC class IIB was identified to be 1293 bp, consisting of a 26 bp 5'-terminal untranslated region (UTR), a 520 bp 3'-UTR, and a 747 bp open reading frame (ORF) bearing characteristics of the immunoglobulin C-type 1 (IGc1) family. The deduced amino acid sequences of the Chinese longsnout catfish MHC class IIB gene had 58-75% identity with those of other fishes. Six class IIB alleles were identified from five individuals. At most two different alleles observed in each individual may infer the existence of a single locus of class IIB gene in the Chinese longsnout catfish genome. An extensive study of polymorphism was examined in 60 individuals. A total of 11 haplotypes of exon 2 were detected in the sampled Chinese longsnout catfish. The rates of nonsynonymous substitutions (d(N)) occurred at a higher frequency than that of synonymous substitutions (d(S)), suggesting the polymorphism of exon 2 seemed to be maintained by the balancing selection. By using long PCR technique, the genomic sequence was further identified to be 2345 bp in length, which contained six exons and five introns. Interestingly, a 98 bp intron 5 cut the 3'-UTR into two parts. Real-time quantitative RT-PCR demonstrated high expression of MHC IIB in gills, spleen, head kidney, and intestine, moderate expression in liver and

  17. Molecular cloning, genomic organization, chromosome mapping, tissues expression pattern and identification of a novel splicing variant of porcine CIDEb gene.

    PubMed

    Li, YanHua; Li, AiHua; Yang, Z Q

    2016-09-01

    Cell death-inducing DNA fragmentation factor-α-like effector b (CIDEb) is a member of the CIDE family of apoptosis-inducing factors, CIDEa and CIDEc have been reported to be Lipid droplets (LDs)-associated proteins that promote atypical LD fusion in adipocytes, and responsible for liver steatosis under fasting and obese conditions, whereas CIDEb promotes lipid storage under normal diet conditions [1], and promotes the formation of triacylglyceride-enriched VLDL particles in hepatocytes [2]. Here, we report the gene cloning, chromosome mapping, tissue distribution, genetic expression analysis, and identification of a novel splicing variant of the porcine CIDEb gene. Sequence analysis shows that the open reading frame of the normal porcine CIDEb isoform covers 660bp and encodes a 219-amino acid polypeptide, whereas its alternative splicing variant encodes a 142-amino acid polypeptide truncated at the fourth exon and comprised of the CIDE-N domain and part of the CIDE-C domain. The deduced amino acid sequence of normal porcine CIDEb shows an 85.8% similarity to the human protein and 80.0% to the mouse protein. The CIDEb genomic sequence spans approximately 6KB comprised of five exons and four introns. Radiation hybrid mapping demonstrated that porcine CIDEb is located at chromosome 7q21 and at a distance of 57cR from the most significantly linked marker, S0334, regions that are syntenic with the corresponding region in the human genome. Tissue expression analysis indicated that normal CIDEb mRNA is ubiquitously expressed in many porcine tissues. It was highly expressed in white adipose tissue and was observed at relatively high levels in the liver, lung, small intestine, lymphatic tissue and brain. The normal version of CIDEb was the predominant form in all tested tissues, whereas the splicing variant was expressed at low levels in all examined tissues except the lymphatic tissue. Furthermore, genetic expression analysis indicated that CIDEb mRNA levels were

  18. Molecular cloning and expression of the male sterility-related CtYABBY1 gene in flowering Chinese cabbage (Brassica campestris L. ssp chinensis var. parachinensis).

    PubMed

    Zhang, X L; Zhang, L G

    2014-01-01

    Expression of the YABBY gene family in the abaxial surface of lateral plant organs determines abaxial destiny of cells, enhances growth and expansion of lateral organs, and plays an important role in polar establishment of lateral organs. However, the YABBY gene has not been studied in male sterility and fertility restoration. We homologously cloned the CtYABBY1 gene of male-sterile TC1 in Brassica campestris L. ssp chinensis var. parachinensis; its expression was analyzed by real-time PCR. A 937-bp sequence was cloned from TC1 and named CtYABBY1. The ORF of this gene has 702 bp, contains a "C2C2 zinc finger" motif at the N-terminal end, and a "YABBY" structural domain at the C-terminal end. This gene had the highest homology with DBC43-3-2 gene in B. campetris ssp pekinensis. Expression of CtYABBY1 gene has a wide range of functions. It is involved in growth and development of lateral organs, such as leaves and flowers, enhancing expansion of the area and volume of young organs. CtYABBY1 is a gene that promotes thermo-sensitive fertility restoration. At room temperature, expression level of this gene was found to be lower in the stamens of sterile flowers. After treating TC1 at a low temperature of 2°-6°C for 20 days, expression of this gene was upregulated in the stamen of fertile flowers. We conclude that male sterility in TC1 is negatively regulated by this gene, which facilitates transition from male sterility to fertility. PMID:25036178

  19. Molecular cloning of a cDNA for human {triangle}{sup 1}-pyrroline-5-carboxylate (P5C) dehydrogenase, the gene defective in type 2 hyperprolinemia

    SciTech Connect

    Hu, C.A.; Lin, W.; Valle, D.

    1994-09-01

    P5C dehydrogenase (EC 1.5.1.12) is a mitochondrial matrix NAD(P) dependent enzyme catalyzing the conversion of P5C, derived from either proline or ornithine, to glutamate. This reaction is an important component in the pathway interconnecting the urea cycle with the tricarboxylic acid cycle. Deficiency of P5C dehydrogenase causes type 2 hyperprolinemia (HPII), an autosomal recessive disorder characterized by seizures, hyperprolinemia and accumulation of P5C. To investigate the molecular basis of HPII and the pathophysiology of gyrate atrophy, a disorder of ornithine metabolism, we have cloned a cDNA for P5C dehydrogenase. Utilizing published sequences of peptides from purified human P5C dehydrogenase and the nucleotide sequence of yeast P5C dehydrogenase, we designed degenerate PCR primers to amplify cDNAs from a HepG2 cDNA library. We identified an amplified fragment of the correct size that encoded one of the many peptides and used it to clone near full length clones of the corresponding cDNA. The longest is 1.8 kb with a 1,485 bp ORF encoding a protein corresponding to the C terminal 495 residues of yeast P5C dehydrogenase. The predicted amino acid sequence of this clone has 100% identity to published sequence of human P5C dehydrogenase peptides and 42% identity with the corresponding sequence of the yeast enzyme. This cDNA detects a 2.3 kb transcript in Northern blots of fibroblast RNA. We conclude we have cloned a near full length cDNA for human P5C dehydrogenase. Studies investigating the molecular basis of HPII are in progress.

  20. Molecular and functional analysis of the TOL plasmid pWWO from Pseudomonas putida and cloning of genes for the entire regulated aromatic ring meta cleavage pathway.

    PubMed Central

    Franklin, F C; Bagdasarian, M; Bagdasarian, M M; Timmis, K N

    1981-01-01

    The genetic organization of the Pseudomonas putida plasmid pWWO-161, which encodes enzymes for the degradation of toluene and related aromatic hydrocarbons, has been investigated by transposition mutagenesis and gene cloning. Catabolic genes were localized to two clusters, one for upper pathway (hydrocarbon leads to carboxylic acid) enzymes and the other for lower pathway (carboxylic acid leads to tricarboxylic acid cycle) enzymes, that are separated by a 14-kilobase DNA segment. The physical organization of the catabolic genes thus reflects their functional organization into two regulatory blocks. The pWWO-161 DNA fragments Sst I fragment C and fragment D were cloned in a broad host range vector to produce plasmid pKT530. This hybrid encodes toluate oxygenase and all meta cleavage pathway enzymes, and it enables P. putida mt-2 and Escherichia coli K-12 cells to grow on m-toluate as sole carbon source. The pKT530 plasmid also carries xylS (a gene whose product has been postulated to regulate expression of the lower pathway genes) and the control sequences of the pathway that interact with this product, because catechol 2,3-oxygenase synthesis is specifically induced by m-toluate in both P. putida and E. coli. Evidence is presented that suggests the promoter operator of the meta pathway gene functions less effectively with the RNA polymerase or xylS product of E. coli than with the enzyme or product of P. putida. PMID:6950388

  1. Identification of two novel members of erbA superfamily by molecular cloning: the gene products of the two are highly related to each other.

    PubMed Central

    Miyajima, N; Kadowaki, Y; Fukushige, S; Shimizu, S; Semba, K; Yamanashi, Y; Matsubara, K; Toyoshima, K; Yamamoto, T

    1988-01-01

    Two v-erbA-related genes, named ear-2 and ear-3, have been identified in the human genome and characterized by cDNA cloning. These genes are predicted to encode proteins that are very similar in primary structure to receptors for steroid hormones or thyroid hormone (T3). In addition, amino acid sequences of the ear-2 and ear-3 gene products are very similar each other especially at the DNA binding domain (86% homology) and at the putative ligand binding domain (76% homology). Northern hybridization with ear DNA probes of RNAs from various tissues of a human fetus reveals that the expression of ear-2 is high in the liver whereas the expression of ear-3 is relatively ubiquitous. Hybridization analysis of DNAs from sorted chromosomes shows that the ear-2 gene is located on chromosome 19 and ear-3 on chromosome 5, indicating that the two genes are clearly different from each other. Images PMID:2905047

  2. [Mapping and cloning of low phosphorus tolerance genes in soybeans].

    PubMed

    Dan, Zhang; Haina, Song; Hao, Cheng; Deyue, Yu

    2015-04-01

    Soybean is a major source of edible oil and phytoprotein. Low phosphorus available in soil is an important factor limiting the current soybean production. Effective ways to solve the problem include identification of germplasms and genes tolerant to low-phosphorus stress, and cultivation of soybean varieties with high phosphorus efficiency. Recently many researches have been carrying out investigations to map and clone genes related to phosphorus efficiency in soybeans. However, due to the complexity of the soybean genome and little knowledge of functional genes, it has been difficult to understand the mechanism of soybean tolerance to low phosphorus. Although quantitative trait locus (QTL) mapping related to low phosphorus tolerance has made some progress, it remains elusive to obtain accurate candidate genes for molecular breeding applications, due to the limited accuracy of QTL. Even for the cloned soybean low phosphorus tolerance genes, the molecular mechanisms are largely unknown, further limiting the application to breeding. In this review, we summarize the progresses on mapping, cloning and functional characterization of soybean low phosphorus tolerance genes. PMID:25881699

  3. Molecular Cloning of HbPR-1 Gene from Rubber Tree, Expression of HbPR-1 Gene in Nicotiana benthamiana and Its Inhibition of Phytophthora palmivora

    PubMed Central

    Khunjan, Uraiwan; Ekchaweng, Kitiya; Panrat, Tanate; Tian, Miaoying; Churngchow, Nunta

    2016-01-01

    This is the first report to present a full-length cDNA (designated HbPR-1) encoding a putative basic HbPR-1 protein from rubber tree (Hevea brasiliensis) treated with salicylic acid. It was characterized and also expressed in Nicotiana benthamiana using Agrobacterium-mediated transient gene expression system in order to investigate the role of HbPR-1 gene in rubber tree against its oomycete pathogen Phytopthora palmivora and to produce recombinant HbPR-1 protein for microbial inhibition test. The HbPR-1 cDNA was 647 bp long and contained an open reading frame of 492 nucleotides encoding 163 amino acid residues with a predicted molecular mass of 17,681 Da and an isoelectric point (pI) of 8.56, demonstrating that HbPR-1 protein belongs to the basic PR-1 type. The predicted 3D structure of HbPR-1 was composed of four α-helices, three β-sheets, seven strands, and one junction loop. Expression and purification of recombinant HbPR-1 protein were successful using Agrobacterium-mediated transient expression and one-step of affinity chromatography. Heterologous expression of HbPR-1 in N. benthamiana reduced necrosis areas which were inoculated with P. palmivora zoospores, indicating that the expressed HbPR-1 protein played an important role in plant resistance to pathogens. The purified recombinant HbPR-1 protein was found to inhibit 64% of P. palmivora zoospore germination on a water agar plate compared with control, suggesting that it was an antimicrobial protein against P. palmivora. PMID:27337148

  4. Molecular Cloning of HbPR-1 Gene from Rubber Tree, Expression of HbPR-1 Gene in Nicotiana benthamiana and Its Inhibition of Phytophthora palmivora.

    PubMed

    Khunjan, Uraiwan; Ekchaweng, Kitiya; Panrat, Tanate; Tian, Miaoying; Churngchow, Nunta

    2016-01-01

    This is the first report to present a full-length cDNA (designated HbPR-1) encoding a putative basic HbPR-1 protein from rubber tree (Hevea brasiliensis) treated with salicylic acid. It was characterized and also expressed in Nicotiana benthamiana using Agrobacterium-mediated transient gene expression system in order to investigate the role of HbPR-1 gene in rubber tree against its oomycete pathogen Phytopthora palmivora and to produce recombinant HbPR-1 protein for microbial inhibition test. The HbPR-1 cDNA was 647 bp long and contained an open reading frame of 492 nucleotides encoding 163 amino acid residues with a predicted molecular mass of 17,681 Da and an isoelectric point (pI) of 8.56, demonstrating that HbPR-1 protein belongs to the basic PR-1 type. The predicted 3D structure of HbPR-1 was composed of four α-helices, three β-sheets, seven strands, and one junction loop. Expression and purification of recombinant HbPR-1 protein were successful using Agrobacterium-mediated transient expression and one-step of affinity chromatography. Heterologous expression of HbPR-1 in N. benthamiana reduced necrosis areas which were inoculated with P. palmivora zoospores, indicating that the expressed HbPR-1 protein played an important role in plant resistance to pathogens. The purified recombinant HbPR-1 protein was found to inhibit 64% of P. palmivora zoospore germination on a water agar plate compared with control, suggesting that it was an antimicrobial protein against P. palmivora. PMID:27337148

  5. Rapid cloning of any rearranged mouse immunoglobulin variable genes

    SciTech Connect

    Dattamajumdar, A.K.; Jacobson, D.P.; Hood, L.E.; Osman, G.E.

    1996-12-31

    Immunoglobulins (Ig) have been the focus of extensive study for several decades and have become an important research area for immunologists and molecular biologists. The use of polymerase chain reaction (PCR) technology has accelerated the cloning, sequencing, and characterization of genes of the immune system. However, cloning and sequencing the Ig variable (V) genes using the PCR technology has been a challenging task, primarily due to the very diverse nature of Ig V region genes. We have developed a simple, rapid, and reproducible PCR-based technique to clone any rearranged mouse Ig heavy or light chain genes. A close examination of all Ig heavy and light chain V gene families has resulted in the design of 5{prime} and 3{prime} universal primers from regions that are highly conserved across all heavy or light chain V gene families, and the joining or constant regions, respectively. We present our strategy for designing universal primers for Ig V gene families. These primers were able to rapidly amplify the rearranged Ig V genes, belonging to diverse Ig V gene families from very different cell lines, i.e., J558, MOPC-21, 36-60, and a chicken ovalbumin specific B-cell hybridoma. In addition, the present study provides the complete alignment of nucleotide sequences of all heavy and light chain variable gene families. This powerful method of cloning Ig V genes, therefore, allows rapid and precise analysis of B-cell hybridomas, B-cell repertoire, and B-cell ontogeny. 55 refs., 5 figs., 2 tabs.

  6. Molecular cloning of the cowpea leghemoglobin II gene and expression of its cDNA in Escherichia coli. Purification and characterization of the recombinant protein.

    PubMed Central

    Arredondo-Peter, R; Moran, J F; Sarath, G; Luan, P; Klucas, R V

    1997-01-01

    Cowpea (Vigna unguiculata) nodules contain three leghemoglobins (LbI, LbII, and LbIII) that are encoded by at least two genes. We have cloned and sequenced the gene that encodes for LbII (lbII), the most abundant Lb in cowpea nodules, using total DNA as the template for PCR. Primers were designed using the sequence of the soybean lbc gene. The lbII gene is 679 bp in length and codes for a predicted protein of 145 amino acids. Using sequences of the cowpea lbII gene for the synthesis of primers and total nodule RNA as the template, we cloned a cDNA for LbII into a constitutive expression vector (pEMBL19+) and then expressed it in Escherichia coli. Recombinant LbII (rLbII) and native LbII (nLbII) from cowpea nodules were purified to homogeneity using standard techniques. Properties of rLbII were compared with nLbII by partially sequencing the proteins and by sodium dodecyl sulfate- and isoelectric focusing polyacrylamide gel electrophoresis, western-blot analysis using anti-soybean Lba antibodies, tryptic and chymotryptic mapping, and spectrophotometric techniques. The data showed that the structural and spectral characteristics of rLbII and nLbII were similar. The rLbII was reversibly oxygenated/deoxygenated, showing that it is a functional hemoglobin. PMID:9193085

  7. Molecular characterization of the body site-specific human epidermal cytokeratin 9: cDNA cloning, amino acid sequence, and tissue specificity of gene expression.

    PubMed

    Langbein, L; Heid, H W; Moll, I; Franke, W W

    1993-12-01

    Differentiation of human plantar and palmar epidermis is characterized by the suprabasal synthesis of a major special intermediate-sized filament (IF) protein, the type I (acidic) cytokeratin 9 (CK 9). Using partial amino acid (aa) sequence information obtained by direct Edman sequencing of peptides resulting from proteolytic digestion of purified CK 9, we synthesized several redundant primers by 'back-translation'. Amplification by polymerase chain reaction (PCR) of cDNAs obtained by reverse transcription of mRNAs from human foot sole epidermis, including 5'-primer extension, resulted in multiple overlapping cDNA clones, from which the complete cDNA (2353 bp) could be constructed. This cDNA encoded the CK 9 polypeptide with a calculated molecular weight of 61,987 and an isoelectric point at about pH 5.0. The aa sequence deduced from cDNA was verified in several parts by comparison with the peptide sequences and showed the typical structure of type I CKs, with a head (153 aa), and alpha-helical coiled-coil-forming rod (306 aa), and a tail (163 aa) domain. The protein displayed the highest homology to human CK 10, not only in the highly conserved rod domain but also in large parts of the head and the tail domains. On the other hand, the aa sequence revealed some remarkable differences from CK 10 and other CKs, even in the most conserved segments of the rod domain. The nuclease digestion pattern seen on Southern blot analysis of human genomic DNA indicated the existence of a unique CK 9 gene. Using CK 9-specific riboprobes for hybridization on Northern blots of RNAs from various epithelia, a mRNA of about 2.4 kb in length could be identified only in foot sole epidermis, and a weaker cross-hybridization signal was seen in RNA from bovine heel pad epidermis at about 2.0 kb. A large number of tissues and cell cultures were examined by PCR of mRNA-derived cDNAs, using CK 9-specific primers. But even with this very sensitive signal amplification, only palmar

  8. Gene Cloning and Molecular Characterization of an Extracellular Poly(l-Lactic Acid) Depolymerase from Amycolatopsis sp. Strain K104-1

    PubMed Central

    Matsuda, Emiko; Abe, Naoki; Tamakawa, Hideyuki; Kaneko, Jun; Kamio, Yoshiyuki

    2005-01-01

    We have isolated a polylactide or poly(l-lactic acid) (PLA)-degrading bacterium, Amycolatopsis sp. strain K104-1, and purified PLA depolymerase (PLD) from the culture fluid of the bacterium. Here, we cloned and expressed the pld gene encoding PLD in Streptomyces lividans 1326 and characterized a recombinant PLD (rPLD) preparation. We also describe the processing mechanism from nascent PLD to mature PLD. The pld gene encodes PLD as a 24,225-Da polypeptide consisting of 238 amino acids. Biochemical and Western immunoblot analyses of PLD and its precursors revealed that PLD is synthesized as a precursor (prepro-type), requiring proteolytic cleavage of the N-terminal 35-amino-acid extension including the 26-amino-acid signal sequence and 9-residue prosequence to generate the mature enzyme of 20,904 Da. The cleavage of the prosequence was found to be autocatalytic. PLD showed about 45% similarity to many eukaryotic serine proteases. In addition, three amino acid residues, H57, D102, and S195 (chymotrypsin numbering), which are implicated in forming the catalytic triad necessary for cleavage of amide bond of substrates in eukaryotic serine proteases, were conserved in PLD as residues H74, D111, and S197. The G193 residue (chymotrypsin numbering), which is implicated in forming an oxyanion hole with residue S195 and forms an important hydrogen bond for interaction with the carbonyl group of the scissile peptide bond, was also conserved in PLD. The functional analysis of the PLD mutants H74A, D111A, and S197A revealed that residues H74, D111, and S197 are important for the depolymerase and caseinolytic activities of PLD and for cleavage of the prosequence from pro-type PLD to form the mature one. The PLD preparation had elastase activity which was not inhibited by 1 mM elastatinal, which is 10 times higher than needed for complete inhibition of porcine pancreatic elastase. The rPLD preparation degraded PLA with an average molecular mass of 220 kDa into lactic acid dimers

  9. Cloning, expression and sequencing of Helicobacter felis urease genes.

    PubMed

    Ferrero, R L; Labigne, A

    1993-07-01

    Urease genes from Helicobacter felis were cloned and expressed in Escherichia coli cells. A genomic bank of Sau3A-digested H. felis chromosomal DNA was created using a cosmid vector. Cosmid clones were screened for urease activity following subculture on a nitrogen-limiting medium. Subcloning of DNA from an urease-positive cosmid clone led to the construction of pILL205 (9.5 kb) which conferred a urease activity of 1.2 +/- 0.5 mumole urea min-1 mg-1 bacterial protein to E. coli HB101 bacteria grown on a nitrogen-limiting medium. Random mutagenesis using a MiniTn3-Km transposable element permitted the identification of three DNA regions on pILL205 which were necessary for the expression of an urease-positive phenotype in E. coli clones. To localize the putative structural genes of H. felis on pILL205, extracts of clones harbouring the mutated copies of the plasmid were analysed by Western blotting with anti-H. felis rabbit serum. One mutant clone did not synthesize the putative UreB subunit of H. felis urease and it was postulated that the transposable element had disrupted the corresponding structural gene. By sequencing the DNA region adjacent to the transposon insertion site two open reading frames, designated ureA and ureB, were identified. The polypeptides encoded by these genes had calculated molecular masses of 26,074 and 61,663 Da, respectively, and shared 73.5% and 88.2% identity with the corresponding gene products of Helicobacter pylori urease. PMID:8412683

  10. Molecular cloning, nucleotide sequence, and marker exchange mutagenesis of the exo-poly-alpha-D-galacturonosidase-encoding pehX gene of Erwinia chrysanthemi EC16.

    PubMed Central

    He, S Y; Collmer, A

    1990-01-01

    The pehX gene encoding extracellular exo-poly-alpha-D-galacturonosidase (exoPG; EC 3.2.1.82) was isolated from a genomic library of the pectate lyase-deficient Erwinia chrysanthemi mutant UM1005 (a Nalr Kanr delta pelABCE derivative of EC16) by immunoscreening 2,800 Escherichia coli HB101 transformants with an antibody against exoPG protein. The cloned pehX gene was expressed highly from its own promoter in E. coli, and most of the enzyme was localized in the periplasm. The nucleotide sequence of pehX revealed the presence of an amino-terminal signal peptide and an open reading frame encoding a preprotein of 64,608 daltons. The cloned pehX gene was insertionally inactivated with TnphoA and used to mutate the chromosomal pehX gene of E. chrysanthemi AC4150 (Nalr) and CUCPB5006 (Nalr Kans delta pelABCE) by marker exchange mutagenesis. Analysis of the resulting mutants, CUCPB5008 (Pel+ Peh-) and CUCPB5009 (Pel- Peh-), indicated that exoPG can contribute significantly to bacterial utilization of polygalacturonate and the induction of pectate lyase in the presence of extracellular pectic polymers. CUCPB5009 retained a slight ability to pit polygalacturonate semisolid agar and macerated chrysanthemum pith tissues when large numbers of bacteria were inoculated. Images PMID:2168372

  11. Molecular cloning and analysis of zebrafish voltage-gated sodium channel beta subunit genes: implications for the evolution of electrical signaling in vertebrates

    PubMed Central

    Chopra, Sameer S; Watanabe, Hiroshi; Zhong, Tao P; Roden, Dan M

    2007-01-01

    Background Action potential generation in excitable cells such as myocytes and neurons critically depends on voltage-gated sodium channels. In mammals, sodium channels exist as macromolecular complexes that include a pore-forming alpha subunit and 1 or more modulatory beta subunits. Although alpha subunit genes have been cloned from diverse metazoans including flies, jellyfish, and humans, beta subunits have not previously been identified in any non-mammalian species. To gain further insight into the evolution of electrical signaling in vertebrates, we investigated beta subunit genes in the teleost Danio rerio (zebrafish). Results We identified and cloned single zebrafish gene homologs for beta1-beta3 (zbeta1-zbeta3) and duplicate genes for beta4 (zbeta4.1, zbeta4.2). Sodium channel beta subunit loci are similarly organized in fish and mammalian genomes. Unlike their mammalian counterparts, zbeta1 and zbeta2 subunit genes display extensive alternative splicing. Zebrafish beta subunit genes and their splice variants are differentially-expressed in excitable tissues, indicating tissue-specific regulation of zbeta1-4 expression and splicing. Co-expression of the genes encoding zbeta1 and the zebrafish sodium channel alpha subunit Nav1.5 in Chinese Hamster Ovary cells increased sodium current and altered channel gating, demonstrating functional interactions between zebrafish alpha and beta subunits. Analysis of the synteny and phylogeny of mammalian, teleost, amphibian, and avian beta subunit and related genes indicated that all extant vertebrate beta subunits are orthologous, that beta2/beta4 and beta1/beta3 share common ancestry, and that beta subunits are closely related to other proteins sharing the V-type immunoglobulin domain structure. Vertebrate sodium channel beta subunit genes were not identified in the genomes of invertebrate chordates and are unrelated to known subunits of the para sodium channel in Drosophila. Conclusion The identification of conserved

  12. Molecular cloning, sequencing and tissue expression of vasotocin and isotocin precursor genes from Ostariophysian catfishes: phylogeny and evolutionary considerations in teleosts

    PubMed Central

    Banerjee, Putul; Chaube, Radha; Joy, Keerikkattil P.

    2015-01-01

    Basic and neutral neurohypophyseal (NH) nonapeptides have evolved from vasotocin (VT) by a gene duplication at the base of the gnathostome lineage. In teleosts, VT and IT are the basic and neutral peptides, respectively. In the present study, VT and IT precursor genes of Heteropneustes fossilis and Clarias batrachus (Siluriformes, Ostariophysi) were cloned and sequenced. The channel catfish Icatalurus punctatus NH precursor sequences were obtained from EST database. The catfish NH sequences were used along with the available Acanthopterygii and other vertebrate NH precursor sequences to draw phylogenetic inference on the evolutionary history of the teleost NH peptides. Synteny analysis of the NH gene loci in various teleost species was done to complement the phylogenetic analysis. In H. fossilis, the NH transcripts were also sequenced from the ovary. The cloned genes and the deduced precursor proteins showed conserved characteristics of the NH nonapeptide precursors. The genes are expressed in brain and ovary (follicular envelope) of H. fossilis with higher transcript abundance in the brain. The addition of the catfish sequences in the phylogenetic analysis revealed that the VT and IT precursors of the species-rich superorders of teleosts have a distinct phylogenetic history with the Acanthopterygii VT and IT precursors sharing a less evolutionary distance and the Ostariophysi VT and IT having a greater evolutionary distance. The genomic location of VT and IT precursors, and synteny analysis of the NH loci lend support to the phylogenetic inference and suggest a footprint of fish- specific whole genome duplication (3R) and subsequent diploidization in the NH loci. The VT and IT precursor genes are most likely lineage-specific paralogs resulting from differential losses of the 3R NH paralogs in the two superorders. The independent yet consistent retention of VT and IT in the two superorders might be directed by a stringent ligand-receptor selectivity. PMID:26029040

  13. Cloning, Expression Analysis, and Molecular Modeling of the Gamma-Aminobutyric Acid Receptor Alpha2 Subunit Gene from the Common Cutworm, Spodoptera litura

    PubMed Central

    Zuo, Hongliang; Gao, Lu; Hu, Zhen; Liu, Haiyuan; Zhong, Guohua

    2013-01-01

    Intensive research on the molecule structures of the gamma-nminobutyric acid (GABA) receptor in agricultural pests has great significance to the mechanism investigation, resistance prevention, and molecular design of novel pesticides. The GABA receptor a2 (SlGABARα2) subunit gene in Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) was cloned using the technologies of reverse transcription PCR and rapid amplification of cDNA ends. The gemonic DNA sequence of SlGABARα2 has 5164 bp with 8 exons and 7 introns that were in accordance with the GT-AG splicing formula. The complete mRNA sequence of SlGABARα2 was 1965 bp, with an open reading frame of 1500 bp encoding a protein of 499 amino acids. The GABA receptor is highly conserved among insects. The conserved regions include several N-glycosylation, Oglycosylation, and phosphorylation sites, as well as 4 transmembrane domains. The identities that SlGABARα2 shared with the GABA receptor a2 subunit of Spodoptera exigua, Heliothis virescens, Chilo suppressalis, Plutella xylostella, Bombyx mori ranged from 99.2% to 87.2% at the amino acid level. The comparative 3-dimensional model of SlGABARα2 showed that its tertiary structure was composed of 4 major α-helixes located at the 4 putative transmembrane domains on one side, with some β-sheets and 1 small α-helix on the other side. SlGABARα2 may be attached to the membrane by 4 α-helixes that bind ions in other conserved domains to transport them through the membrane. The results of quantitative real time PCR demonstrated that SlGABARα2 was expressed in all developmental stages of S. litura. The relative expression level of SlGABARα2 was the lowest in eggs and increased with larval growth, while it declined slightly in pupae and reached the peak in adults. The expressions of SlGABARα2 in larvae varied among different tissues; it was extremely high in the brain but was low in the midgut, epicuticle, Malpighian tube, and fat body. PMID:23909412

  14. Cloning, expression analysis, and molecular modeling of the gamma-aminobutyric acid receptor alpha2 subunit gene from the common cutworm, Spodoptera litura.

    PubMed

    Zuo, Hongliang; Gao, Lu; Hu, Zhen; Liu, Haiyuan; Zhong, Guohua

    2013-01-01

    Intensive research on the molecule structures of the gamma-nminobutyric acid (GABA) receptor in agricultural pests has great significance to the mechanism investigation, resistance prevention, and molecular design of novel pesticides. The GABA receptor a2 (SlGABARα2) subunit gene in Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) was cloned using the technologies of reverse transcription PCR and rapid amplification of cDNA ends. The gemonic DNA sequence of SlGABARα2 has 5164 bp with 8 exons and 7 introns that were in accordance with the GT-AG splicing formula. The complete mRNA sequence of SlGABARα2 was 1965 bp, with an open reading frame of 1500 bp encoding a protein of 499 amino acids. The GABA receptor is highly conserved among insects. The conserved regions include several N-glycosylation, Oglycosylation, and phosphorylation sites, as well as 4 transmembrane domains. The identities that SlGABARα2 shared with the GABA receptor a2 subunit of Spodoptera exigua, Heliothis virescens, Chilo suppressalis, Plutella xylostella, Bombyx mori ranged from 99.2% to 87.2% at the amino acid level. The comparative 3-dimensional model of SlGABARα2 showed that its tertiary structure was composed of 4 major α-helixes located at the 4 putative transmembrane domains on one side, with some β-sheets and 1 small α-helix on the other side. SlGABARα2 may be attached to the membrane by 4 α-helixes that bind ions in other conserved domains to transport them through the membrane. The results of quantitative real time PCR demonstrated that SlGABARα2 was expressed in all developmental stages of S. litura. The relative expression level of SlGABARα2 was the lowest in eggs and increased with larval growth, while it declined slightly in pupae and reached the peak in adults. The expressions of SlGABARα2 in larvae varied among different tissues; it was extremely high in the brain but was low in the midgut, epicuticle, Malpighian tube, and fat body. PMID:23909412

  15. Molecular cloning of cellular genes encoding retinoblastoma-associated proteins: identification of a gene with properties of the transcription factor E2F.

    PubMed Central

    Shan, B; Zhu, X; Chen, P L; Durfee, T; Yang, Y; Sharp, D; Lee, W H

    1992-01-01

    The retinoblastoma protein interacts with a number of cellular proteins to form complexes which are probably crucial for its normal physiological function. To identify these proteins, we isolated nine distinct clones by direct screening of cDNA expression libraries using purified RB protein as a probe. One of these clones, Ap12, is expressed predominantly at the G1-S boundary and in the S phase of the cell cycle. The nucleotide sequence of Ap12 has features characteristic of transcription factors. The C-terminal region binds to unphosphorylated RB in regions similar to those to which T antigen binds and contains a transactivation domain. A region containing a potential leucine zipper flanked by basic residues is able to bind an E2F recognition sequence specifically. Expression of Ap12 in mammalian cells significantly enhances E2F-dependent transcriptional activity. These results suggest that Ap12 encodes a protein with properties known to be characteristic of transcription factor E2F. Images PMID:1448092

  16. Molecular cloning and expression analysis of prostaglandin E receptor 2 gene in cashmere goat (Capra hircus) skin during hair follicle development.

    PubMed

    Geng, Rong-Qing; Yuan, Chao; Chen, Yu-Lin

    2014-04-01

    As a member of the four subtypes of receptors for prostaglandin E2 (PGE2), prostaglandin E receptor 2 (PTGER2) is in the family of G-protein coupled receptors and has been characterized to be involved in the development and growth of hair follicles. In this study, we cloned and characterized the full-length coding sequence (CDS) of PTGER2 gene from cashmere goat skin. The entire open reading frame (ORF) of PTGER2 gene was 1047 bp and encoded 348 amino acid residues. The deduced protein contained one G-protein coupled receptors family 1 signature, seven transmembrane domains, and other potential sites. Tissue expression analysis showed that PTGER2 gene was expressed strongly in the skin. The general expression tendency of PTGER2 gene at different hair follicle developmental stages in the skin was gradually decreased from anagen to catagen to telogen. After comparing with the expression of BMP4 gene and related reports, we further presume that it seems to have a relationship between the hair follicle cycle and the expression level of PTGER2 gene in cashmere goat skin. PMID:24555795

  17. The human osmoregulatory Na{sup +}/myo-inositol cotransporter gene (SLC5A3): Molecular cloning and localization to chromosome 21

    SciTech Connect

    Berry, G.T.; Mallee, J.J.; Muenke, M.

    1995-01-20

    A human Na{sup +}/myo-inositol cotransporter (SLC5A3) gene was cloned; sequencing revealed a single intron-free open reading frame of 2157 nucleotides. Containing 718 amino acid residues, the predicted protein is highly homologous to the product of the canine osmoregulatory SLC5A3 gene. The SLC5A3 protein is number 3 of the solute carrier family 5 and was previously designated SMIT. Using fluorescence in situ hybridization, the human SLC5A3 gene was localized to band q22 on chromosome 21. Many tissues including brain demonstrate gene expression. The inability of a trisomic 21 cell to downregulate expression of three copies of this osmoregulatory gene could result in increased flux of both myo-inositol and Na{sup +} across the plasma membrane. The potential consequences include perturbations in the cell membrane potential and tissue osmolyte levels. The SLC5A3 gene may play a role in the pathogenesis of Down syndrome. 54 refs., 4 figs.

  18. Molecular cloning of tissue-specific transcripts of a transketolase-related gene: Implications for the evolution of new vertebrate genes

    SciTech Connect

    Coy, J.F.; Duebel, S.; Kioschis, P.; Delius, H.; Poustka, A.

    1996-03-05

    As part of a systematic search for differentially expressed genes, we have isolated a novel transketolase-related gene (TKR) (HGMW-approved symbol TKT), located between the green color vision pigment gene (GCP) and the ABP-280 filamin gene (FLN1) in Xq28. Transcripts encoding tissue-specific protein isoforms could be isolated. Comparison with known transketolases (TK) demonstrated a TKR-specific deletion mutating one thiamine binding site. Genomic sequencing of the TKR gene revealed the presence of a pseudoexon as well as the acquisition of a tissue-specific spliced exon compared to TK. Since it has been postulated that the vertebrate genome arose by two cycles of tetraploidization from a cephalochordate genome, this could represent an example of the modulation of the function of a preexisting transketolase gene by gene duplication. Thiamine defiency is closely involved with two neurological disorders, Beriberi and Wernicke-Korsakoff syndromes, and in both of these conditions TK with altered activity are found. We discuss the possible involvement of TKR in explaining the observed variant transketolase forms. 34 refs., 4 figs., 1 tab.

  19. Positional cloning of disease genes on chromosome 16

    SciTech Connect

    Doggett, N.; Bruening, M.; Callen, D.; Gardiner, M.; Lerner, T.

    1996-04-01

    The project seeks to elucidate the molecular basis of an important genetic disease (Batten`s disease) by molecular cloning of the affected gene by utilizing an overlapping clone map of chromosome 16. Batten disease (also known as juvenile neuronal ceroid lipofuscinosis) is a recessively inherited neurodegenerative disorder of childhood characterized by progressive loss of vision, seizures, and psychomoter disturbances. The Batten disease gene was genetically mapped to the chromosome region 16p 12.1 in close linkage with the genetic markers D16S299 and D16S298. Exon amplification of a cosmid containing D16S298 yielded a candidate gene that was disrupted by a 1 kb genomic deletion in all patients containing the most common haplotype for the disease. Two separate deletions and a point mutation altering a splice site in three unrelated families have confirmed the gene as the Batten disease gene. The disease gene encodes a novel 438 amino acid membrane binding protein of unknown function.

  20. Molecular cloning and characterization of the oprQ gene coding for outer membrane protein OprE3 of Pseudomonas aeruginosa.

    PubMed

    Okamoto, K; Gotoh, N; Tsujimoto, H; Yamada, H; Yoshihara, E; Nakae, T; Nishino, T

    1999-01-01

    We cloned and characterized the oprQ gene coding for outer membrane protein OprE3 of Pseudomonas aeruginosa PAO1. The oprQ gene was composed of 1,275 base pairs including a sequence encoding for the signal sequence and a mature protein with a Mr of 44,602. Computer-aided alignment and hydropathy analyses of the predicted amino acid sequences suggested that OprE3 is a transmembrane protein homologous to outer membrane proteins of P. aeruginosa such as OprD2 (OprD) porin and OprE1 (OprE) porin. Susceptibility to several antibiotics of the strains lacking or overproducing OprE3 was indistinguishable from that of the wild-type strain, suggesting that OprE3 is unlikely involved in the diffusion of carbapenems and other beta-lactam antibiotics. PMID:10338201

  1. Cloning and molecular characterization of the potato RING finger protein gene StRFP1 and its function in potato broad-spectrum resistance against Phytophthora infestans.

    PubMed

    Ni, Xuemei; Tian, Zhendong; Liu, Jun; Song, Botao; Xie, Conghua

    2010-04-15

    Really interesting new gene (RING) finger proteins function as ubiquitin ligase and play key roles in biotic and abiotic stresses. A new RING-H2 finger protein gene, StRFP1, was cloned from Phytophthora infestans-inoculated leaves of potato (Solanum tuberosum) clone 386209.10, which is free of R1-R11 genes. The deduced amino acid sequence was characterized by an N-terminal transmembrane domain, a GLD region and a RING-H2 finger signature. StRFP1 is homologous to the tobacco NtACRE132 protein and belongs to the ATL family. The DNA gel blot analysis and mapping revealed that StRFP1, an intron-free gene, had one to two copies in the potato genome and was located on chromosome 3. RT-PCR assays showed that StRFP1 was constitutively expressed in potato plants and significantly induced in detached potato leaves by P. infestans and plant defense-related signal molecules, abscisic acid, salicylic acid and methyl jasmonate. Transient expression studies revealed that StRFP1 fused with GFP localized to the plasma membrane or out of that in onion epidermal cells. The function of StRFP1 in potato resistance against late blight was further investigated by constructing overexpression and RNA interference (RNAi) vectors, which were introduced into potato cv. E-potato 3, respectively. By challenging the detached leaves with mixture races of P. infestans, all of the StRFP1-overexpressing plants displayed slower disease development than non-transformed controls in terms of the lesion growth rate (LGR). In contrast, StRFP1-silencing plants through RNAi were more susceptible to pathogen infection. The present results demonstrate that StRFP1 contributes to broad-spectrum resistance against P. infestans in potato. PMID:20042252

  2. Molecular cloning and characterization of the gene encoding the DNA methyltransferase, M.CviBIII, from Chlorella virus NC-1A.

    PubMed Central

    Narva, K E; Wendell, D L; Skrdla, M P; Van Etten, J L

    1987-01-01

    The gene encoding the DNA methyltransferase, M.CviBIII, from Chlorella virus NC-1A was cloned and expressed in E. coli plasmid pUC8. Plasmid (pNC-1A.14.8) encoded M.CviBIII methylates adenine in TCGA sequences both in vivo in E. coli and in vitro. Transposon Tn5 mutagenesis localized the M.CviBIII functional domain to a 1.5 kbp region of pNC-1A.14.8 and also indicated that a virus promoter directs transcription of the gene in E. coli. The 2.1 kbp insert containing the M.CviBIII gene was sequenced and a single open reading frame of 1131 bp was identified within the domain determined by Tn5 mutagenesis. When the M.CviBIII gene was fused in-frame with the 19 amino-terminal codons of lacZ a 45 kD polypeptide was identified in maxicells as predicted by the DNA sequence. The M.CviBIII gene was not essential for virus replication since a virus M.CviBIII deletion mutant also replicated in Chlorella. Images PMID:3320956

  3. Molecular cloning, genomic structure, and genetic mapping of two Rdl-orthologous genes of GABA receptors in the diamondback moth, Plutella xylostella.

    PubMed

    Yuan, Guorui; Gao, Weiyue; Yang, Yihua; Wu, Yidong

    2010-06-01

    The Resistance to dieldrin (Rdl) gene encodes a subunit of the insect gamma-aminobutyric acid (GABA) receptor. Cyclodiene resistance in many insects is associated with replacement of a single amino acid (alanine at position 302) with either a serine or a glycine in the Rdl gene. Two Rdl-orthologous genes of GABA receptors (PxGABARalpha1 and PxGABARalpha2) were cloned and sequenced from a susceptible strain (Roth) of Plutella xylostella. PxGABARalpha1 and PxGABARalpha2 showed 84% and 77% identity with the Rdl gene of Drosophila melanogaster at an amino acid level, respectively. The coding regions of PxGABARalpha1 and PxGABARalpha2 both comprise ten exons, with two alternative RNA-splicing forms in exon 3 of both genes. At the orthologous position of alanine-302 in D. melanogaster Rdl, PxGABARalpha1 has a conserved alanine at position 282. PxGABARalpha2 has a serine instead of an alanine at the equivalent position. With two informative DNA markers, both PxGABARalpha1 and PxGABARalpha2 were mapped onto the Z chromosome of P. xylostella. PMID:20513056

  4. Molecular cloning and characterization of drought stress responsive abscisic acid-stress-ripening (Asr 1) gene from wild jujube, Ziziphus nummularia (Burm.f.) Wight & Arn.

    PubMed

    Padaria, Jasdeep Chatrath; Yadav, Radha; Tarafdar, Avijit; Lone, Showkat Ahmad; Kumar, Kanika; Sivalingam, Palaiyur Nanjappan

    2016-08-01

    Drought is a calamitous abiotic stress hampering agricultural productivity all over the world and its severity is likely to increase further. Abscisic acid-stress-ripening proteins (ASR), are a group of small hydrophilic proteins which are induced by abscisic acid, stress and ripening in many plants. In the present study, ZnAsr 1 gene was fully characterized for the first time from Ziziphus nummularia, which is one of the most low water forbearing plant. Full length ZnAsr 1 gene was characterised and in silico analysis of ZnASR1 protein was done for predicting its phylogeny and physiochemical properties. To validate transcriptional pattern of ZnAsr 1 in response to drought stress, expression profiling in polyethylene glycol (PEG) induced Z. nummularia seedlings was studied by RT-qPCR analysis and heterologous expression of the recombinant ZnAsr1 in Escherichia coli. The nucleotide sequence analysis revealed that the complete open reading frame of ZnAsr 1 is 819 bp long encoding a protein of 273 amino acid residues, consisting of a histidine rich N terminus with an abscisic acid/water deficit stress domain and a nuclear targeting signal at the C terminus. In expression studies, ZnAsr 1 gene was found to be highly upregulated under drought stress and recombinant clones of E. coli cells expressing ZnASR1 protein showed better survival in PEG containing media. ZnAsr1 was proven to enhance drought stress tolerance in the recombinant E.coli cells expressing ZnASR1. The cloned ZnAsr1 after proper validation in a plant system, can be used to develop drought tolerant transgenic crops. PMID:27209581

  5. Phenylalanine ammonia-lyase in tobacco. Molecular cloning and gene expression during the hypersensitive reaction to tobacco mosaic virus and the response to a fungal elicitor.

    PubMed Central

    Pellegrini, L; Rohfritsch, O; Fritig, B; Legrand, M

    1994-01-01

    A tobacco (Nicotiana tabacum L. cv Samsun NN) cDNA clone coding the enzyme phenylalanine ammonia-lyase (PAL) was isolated from a cDNA library made from polyadenylated RNA purified from tobacco mosaic virus (TMV)-infected leaves. Southern analysis indicated that, in tobacco, PAL is encoded by a small family of two to four unclustered genes. Northern analysis showed that PAL genes are weakly expressed under normal physiological conditions, they are moderately and transiently expressed after wounding, but they are strongly induced during the hypersensitive reaction to TMV or to a fungal elicitor. Ribonuclease protection experiments confirmed this evidence and showed the occurrence of two highly homologous PAL messengers originating from a single gene or from two tightly co-regulated genes. By in situ RNA-RNA hybridization PAL transcripts were shown to accumulate in a narrow zone of leaf tissue surrounding necrotic lesions caused by TMV infection or treatment with the fungal elicitor. In this zone, no cell specificity was observed and there was a decreasing gradient of labeling from the edge of necrosis. Some labeling was also found in various cell types of young, healthy stems and was shown to accumulate in large amounts in the same cell types after the deposition of an elicitor solution at the top of the decapitated plant. PMID:7824656

  6. Molecular cloning of the b subunit of mouse coagulation factor XIII and assignment of the gene to chromosome 1: Close evolutionary relationship to complement factor H

    SciTech Connect

    Nonaka, Mayumi; Nonaka, Masaru; Natsuume-Sakai, Shunnosuke ); Matsuda, Yoichi ); Shiroishi, Toshihiko; Moriwaki, Kazuo )

    1993-03-01

    The b subunit of human coagulation factor XIII (FXIII-b) is composed of 10 short consensus repeats (SCRs) characteristic of the regulatory proteins of complement activation system. A full-length cDNA clone of mouse FXIII-b was isolated and the entire sequence was determined. The predicted amino acid sequence showed 77.5% homology with human FXIII-b, although mouse FXIII-b contained seven extra amino acid residues at the carboxyl terminal. The strong reactivity of the translation product of this clone with rabbit anti-human FXIII-b antiserum confirmed that it encodes a mouse counterpart of the human FXIII-b. By in situ hybridization and mapping studies using 66 interspecific backcross mice, the mouse FXIII-b gene (designated F13b) was shown to be located on distal chromosome 1 closely linked to Cfh, extending a conserved linkage group between human and mouse chromosome 1. In addition, a significant structural similarity between FXIII-b and complement factor H is described. 29 refs., 6 figs., 1 tab.

  7. Escherichia coli invasion of brain microvascular endothelial cells in vitro and in vivo: molecular cloning and characterization of invasion gene ibe10.

    PubMed Central

    Huang, S H; Wass, C; Fu, Q; Prasadarao, N V; Stins, M; Kim, K S

    1995-01-01

    Most cases of neonatal Escherichia coli meningitis develop as a result of hematogenous spread, but it is not clear how circulating E. coli crosses the blood-brain barrier. In an attempt to identify E. coli structures contributing to invasion into the central nervous system (CNS), TnphoA mutagenesis was performed with an invasive CSF isolate of E. coli K1 strain RS218 (O18:K1:H7), and TnphoA mutants were examined for their noninvasive capability in brain microvascular endothelial cells (BMEC). The noninvasive mutants exhibited the invasive ability of < 1% of the parent strain. One of the noninvasive mutants (10A-23) with a single TnphoA insertion and no changes in phenotypic characteristics was found to be significantly less invasive into the CNS in the newborn rat model of hematogenous E. coli meningitis. The TnphoA inserts with flanking sequences were cloned and sequenced. A novel open reading frame (8.2 kDa) was identified. Open reading frame analysis indicated that the 8.2-kDa protein (Ibe10) contained multiple transmembrane domains. ibe10 was cloned into an expression vector, pQE30, and the purified Ibe10 was shown to inhibit invasion of BMEC by strain RS218. These findings indicate that ibe10 is one of the E. coli genes involved in the invasion of BMEC in vitro and in vivo. PMID:7591087

  8. Molecular cloning, expression pattern analysis of porcine Rb1 gene and its regulatory roles during primary dedifferentiated fat cells adipogenic differentiation.

    PubMed

    Hu, Xiaoming; Luo, Pei; Peng, Xuewu; Song, Tongxing; Zhou, Yuanfei; Wei, Hongkui; Peng, Jian; Jiang, Siwen

    2015-04-01

    Adipocytes are the main constituent of adipose tissue and are considered to be a corner stone in the homeostatic control of whole body metabolism. Recent reports evidenced that retinoblastoma 1 (Rb1) gene plays an important role in fat development and adipogenesis in mice. Here, we cloned the partial cDNA sequences of the porcine Rb1 gene which contains the complete coding sequences (CDS) of 2820bp encoding a protein of 939 amino acids. Bioinformatic analysis revealed that the CDS of porcine Rb1 was highly identical with those of cattle, human and mice. The porcine Rb1 has three typical conserved structural domains, including Rb-A pocket domain, CYCLIN domain and C-terminus domain, and the phylogenetic tree indicates a closer genetic relationship with cattle and human. Tissue distribution analysis showed that Rb1 expression appeared to be ubiquitously in various tissues, being higher in heart, liver, muscle, and stomach. Furthermore, significant downregulation of Rb1 was found at the initial stage of dedifferentiated fat (DFAT) cells adipogenic differentiation. With the knockdown of the Rb1 expression by siRNA, the number of DFAT cells recruited to white rather than brown adipogenesis was promoted, and mRNA levels of adipogenic markers, such as PPARγ, aP2, LPL and adiponectin and protein expression of PPARγ and adiponectin were increased after hormone stimulation. The underlying mechanisms may be that knockdown of Rb1 promotes the mitotic clonal expansion and PPARγ expression by derepressing the transcriptional activity of E2F so as to facilitate the first steps of adipogenesis. In summary, we cloned and characterized an important negative regulator in adipogenic commitment of porcine DFAT cells. PMID:25626122

  9. Molecular cloning and tissue expression of the fatty acid-binding protein (Es-FABP) gene in female Chinese mitten crab (Eriocheir sinensis)

    PubMed Central

    2010-01-01

    Background Fatty acid-binding proteins (FABPs), small cytosolic proteins that function in the uptake and utilization of fatty acids, have been extensively studied in higher vertebrates while invertebrates have received little attention despite similar nutritional requirements during periods of reproductive activity. Results Therefore, a cDNA encoding Eriocheir sinensis FABP (Es-FABP) was cloned based upon EST analysis of a hepatopancreas cDNA library. The full length cDNA was 750 bp and encoded a 131 aa polypeptide that was highly homologous to related genes reported in shrimp. The 9108 bp Es-FABP gene contained four exons that were interrupted by three introns, a genomic organization common among FABP multigene family members in vertebrates. Gene expression analysis, as determined by RT-PCR, revealed the presence of Es-FABP transcripts in hepatopancreas, hemocytes, ovary, gills, muscle, thoracic ganglia, heart, and intestine, but not stomach or eyestalk. Real-time quantitative RT-PCR analysis revealed that Es-FABP expression in ovary, hemocytes, and hepatopancreas was dependent on the status of ovarian development, with peak expression observed in January. Conclusions Evidence provided in the present report supports a role of Es-FABP in lipid transport during the period of rapid ovarian growth in E. sinensis, and indirectly confirms the participation of the hepatopancreas, ovary, and hemocytes in lipid nutrient absorption and utilization processes. PMID:20846381

  10. Cloning and molecular characterization of a mitogen-activated protein kinase gene from Poncirus trifoliata whose ectopic expression confers dehydration/drought tolerance in transgenic tobacco

    PubMed Central

    Huang, Xiao-San; Luo, Tao; Fu, Xing-Zheng; Fan, Qi-Jun; Liu, Ji-Hong

    2011-01-01

    The mitogen-activated protein kinase (MAPK) cascade plays pivotal roles in diverse signalling pathways related to plant development and stress responses. In this study, the cloning and functional characterization of a group-I MAPK gene, PtrMAPK, in Poncirus trifoliata (L.) Raf are reported. PtrMAPK contains 11 highly conserved kinase domains and a phosphorylation motif (TEY), and is localized in the nucleus of transformed onion epidermal cells. The PtrMAPK transcript level was increased by dehydration and cold, but was unaffected by salt. Transgenic overexpression of PtrMAPK in tobacco confers dehydration and drought tolerance. The transgenic plants exhibited better water status, less reactive oxygen species (ROS) generation, and higher levels of antioxidant enzyme activity and metabolites than the wild type. Interestingly, the stress tolerance capacity of the transgenic plants was compromised by inhibitors of antioxidant enzymes. In addition, overexpression of PtrMAPK enhanced the expression of ROS-related and stress-responsive genes under normal or drought conditions. Taken together, these data demonstrate that PtrMAPK acts as a positive regulator in dehydration/drought stress responses by either regulating ROS homeostasis through activation of the cellular antioxidant systems or modulating transcriptional levels of a variety of stress-associated genes. PMID:21778184

  11. Molecular cloning and characterization of an up-regulated UDP-glucosyltransferase gene induced by DON from Triticum aestivum L. cv. Wangshuibai.

    PubMed

    Lulin, Ma; Yi, Shang; Aizhong, Cao; Zengjun, Qi; Liping, Xing; Peidu, Chen; Dajun, Liu; Xiu-E, Wang

    2010-02-01

    Fusarium head blight, also called scab, is a serious disease of small grain cereals and maize. Scab can not only cause yield loss, more seriously is that it can also deteriorate seed quality by contaminating the infected grains with trichothecenes toxins harmful to human and animal health. Deoxynivalenol (DON) is one of the most important toxin members. It was proposed that DON acted first as a virulence factor during fungal pathogenesis and then accumulated in grain to levels posing a threat to human and animal health. In the present research, by expression analysis of DON-induced samples using GeneChip Wheat Genome Array ( http://www.affymetrix.com/products/arrays/specific/wheat.affx ), a DON-resistance related gene TaUGT3 (GenBank accession FJ236328) was cloned and characterized from a scab resistant wheat (Triticum aestivum L.) variety Wangshuibai. The full-length cDNA of TaUGT3 was 1,755 bp and contained a putative open reading frame (ORF) with 496 amino acids encoding a UDP-glucosyltransferase (UGT). TaUGT3 showed high similarity in amino acid level with DOGT1 gene in Arabidopsis, which is able to detoxify DON. TaUGT3 was located on the group 3 chromosomes of wheat using nulli-tetrasomic lines and deletion lines of Chinese Spring. Co-transformed of TaUGT3 with GFP genes to onion epidermis cells using transient transformation technique by microprojectile bombardment indicated the subcellular location of the protein encoded by TaUGT3 was in the plasma membrane and nuclear. Transformation and overexpression of the TaUGT3 gene in Arabidopsis could enhance tolerance against DON. PMID:19585272

  12. Molecular Cloning and Heterologous Expression of a Biosynthetic Gene Cluster for the Antitubercular Agent d-Cycloserine Produced by Streptomyces lavendulae▿

    PubMed Central

    Kumagai, Takanori; Koyama, Yusuke; Oda, Kosuke; Noda, Masafumi; Matoba, Yasuyuki; Sugiyama, Masanori

    2010-01-01

    In the present study, we successfully cloned a 21-kb DNA fragment containing a d-cycloserine (DCS) biosynthetic gene cluster from a DCS-producing Streptomyces lavendulae strain, ATCC 11924. The putative gene cluster consists of 10 open reading frames (ORFs), designated dcsA to dcsJ. This cluster includes two ORFs encoding d-alanyl-d-alanine ligase (dcsI) and a putative membrane protein (dcsJ) as the self-resistance determinants of the producer organism, indicated by our previous work. When the 10 ORFs were introduced into DCS-nonproducing Streptomyces lividans 66 as a heterologous host cell, the transformant acquired DCS productivity. This reveals that the introduced genes are responsible for the biosynthesis of DCS. As anticipated, the disruption of dcsG, seen in the DCS biosynthetic gene cluster, made it possible for the strain ATCC 11924 to lose its DCS production. We here propose the DCS biosynthetic pathway. First, l-serine is O acetylated by a dcsE-encoded enzyme homologous to homoserine O-acetyltransferase. Second, O-acetyl-l-serine accepts hydroxyurea via an O-acetylserine sulfhydrylase homolog (dcsD product) and forms O-ureido-l-serine. The hydroxyurea must be supplied by the catalysis of a dcsB-encoded arginase homolog using the l-arginine derivative, NG-hydroxy-l-arginine. The resulting O-ureido-l-serine is then racemized to O-ureido-d-serine by a homolog of diaminopimelate epimerase. Finally, O-ureido-d-serine is cyclized to form DCS with the release of ammonia and carbon dioxide. The cyclization must be done by the dcsG or dcsH product, which belongs to the ATP-grasp fold family of protein. PMID:20086163

  13. Molecular cloning of two distinct precursor genes of NdWFamide, a d-tryptophan-containing neuropeptide of the sea hare, Aplysia kurodai.

    PubMed

    Morishita, Fumihiro; Furukawa, Yasuo; Matsushima, Osamu

    2012-12-01

    NdWFamide (NdWFa) is a D-tryptophan-containing cardioexcitatory neuropeptide in gastropod mollusks, such as Aplysia kurodai and Lymanea stagnalis. In this study, we have cloned two cDNA encoding distinct precursors for NdWFa from the abdominal ganglion of A. kurodai. One of the predicted precursor proteins consisted of 90 amino acids (NWF90), and the other consisted of 87 amino acids (NWF87). Both of the predicted precursor proteins have one NWFGKR sequence preceded by the N-terminal signal peptide. Sequential double staining by in situ hybridization (ISH) and immunostaining with anti-NdWFa antibody suggested that NdWFa-precursor and NdWFa peptide co-exist in neurons located in the right-upper quadrant region of the abdominal ganglion. In ISH, NWF90-specific signal and NWF87-specific one were found in different subsets of neurons in the abdominal ganglia of Aplysia. The expression level of NWF90 gene estimated by RT-PCR is much higher than that of NWF87 gene. These results suggest that NWF90 precursor is the major source of NdWFa in Aplysia ganglia. PMID:23000476

  14. Molecular cloning, characterization and expression analysis of the protein arginine N-methyltransferase 1 gene (As-PRMT1) from Artemia sinica.

    PubMed

    Jiang, Xue; Yao, Feng; Li, Xuejie; Jia, Baolin; Zhong, Guangying; Zhang, Jianfeng; Zou, Xiangyang; Hou, Lin

    2015-07-01

    Protein arginine N-methyltransferase 1 (PRMT1) is an important epigenetic regulation factor in eukaryotic genomes. PRMT1 is involved in histone arginine loci methylation modification, changes in eukaryotic genomes' chromatin structure, and gene expression regulation. In the present paper, the full-length 1201-bp cDNA sequence of the PRMT1 homolog of Artemia sinica (As-PRMT1) was cloned for the first time. The putative As-PRMT1 protein comprises 346 amino acids with a SAM domain and a PRMT5 domain. Multiple sequence alignments revealed that the putative sequence of As-PRMT1 protein was relatively conserved across species, especially in the SAM domain. As-PRMT1 is widely expressed during embryo development of A. sinica. This is followed by a dramatic upregulation after diapause termination and then downregulation from the nauplius stage. Furthermore, As-PRMT1 transcripts are highly upregulated under conditions of high salinity and low temperature stress. These findings suggested that As-PRMT1 is a stress-related factor that might promote or inhibit the expression of certain genes, play a critical role in embryonic development and in resistance to low temperature and high salinity stress. PMID:25843627

  15. Molecular cloning of LIM homeodomain transcription factor Lhx2 as a transcription factor of porcine follicle-stimulating hormone beta subunit (FSHβ) gene.

    PubMed

    Kato, Takako; Ishikawa, Akio; Yoshida, Saishu; Sano, Yoshiya; Kitahara, Kousuke; Nakayama, Michie; Susa, Takao; Kato, Yukio

    2012-01-01

    We cloned the LIM-homeodomain protein LHX2 as a transcription factor for the porcine follicle-stimulating hormone β subunit gene (Fshβ) by the Yeast One-Hybrid Cloning System using the upstream region of -852/-746 bases (b) from the transcription start site, called Fd2, as a bait sequence. The reporter assay in LβT2 and CHO cells revealed the presence of an LHX2-responsive region other than Fd2. A potential LHX2 binding sequence was confirmed as AATTAAT containing a consensus homeodomain binding core sequence AATT by Systematic Evolution of Ligands by Exponential Enrichment analysis. DNase I footprinting demonstrated three AATTAAT sequences located at regions -835/-829, -818/-812 and -806/-800 b in the Fd2 region and 12 binding sites in the distal and proximal regions mostly containing an AATT-core sequence. RT-PCR analysis of Lhx2 expression during porcine fetal and postnatal pituitary development showed a gradual increase from fetal day (f) 40 to postnatal day (p) 8 followed by a slight decrease to p230, suggesting that LHX2 may play its role largely in the late fetal and postnatal periods. The analyses of Lhx2 expression in pituitary tumor-derived cell lines showed their expressions in cell lines including αT31, LβT2 and others. Since LHX2 was previously identified as a transcription factor for Cga and the in vitro experiments in the present study suggested that LHX2 regulated the expression of Fshβ, it is possible that LHX2 controls the synthesis of FSH at the transcription level. PMID:22134063

  16. Cloning of the human DNA methyltransferase gene

    SciTech Connect

    Ramchanani, S.K.; Rouleau, J.; Szyf, M.

    1994-09-01

    During the process of carcinogenesis it has been observed that DNA methylation is deregulated. At least two levels of regulation of the mouse DNA MeTase have been shown: at the transcriptional level, via its promoter, and at the post transcriptional level in a cell cycle dependent fashion. The sequence of the complete DNA MeTase gene and identification of the promoter has not yet been reported. Using a probe generated by PCR of the human DNA MeTase cDNA, a human genomic library was screened and a clone of approximately 22 kilobases (kb) was isolated. It was found that this clone contains the complete coding sequence of the DNA MeTase enzyme. Sequence analysis along with restriction enzyme digests have allowed us to construct a partial map of the physical structure of the human DNA MeTase gene. This partial structure has already revealed some interesting aspects related to the genetic evolution of the human DNA MeTase. First, the proposed catalytic domain of the human DNA MeTase is extremely homologous to all other cytosine DNA MeTases, even to those that are found in bacteria, and this catalytic domain is conserved within one complete exon in the human gene. This is very different from the structure of the 5{prime} region of the gene, which is fragmented into numerous little introns and exons. Within one of the small introns that have been identified, a trinucleotide repeat of ATG occurs (9 times in a row), and this repeat is upstream of the proposed start site of translation. Trinucleotide repeat expansion has been shown to be a genetic hot spot for mutation, but even more interesting is the nature of the repeat, ATG, which is the translation start codon; this repeat appears to be in frame with the {open_quotes}normal{close_quotes} coding sequence, the implications being that possible alternative methyltransferases may be translated under certain conditions such as cancer.

  17. Branched-chain-amino-acid biosynthesis in plants: molecular cloning and characterization of the gene encoding acetohydroxy acid isomeroreductase (ketol-acid reductoisomerase) from Arabidopsis thaliana (thale cress).

    PubMed Central

    Dumas, R; Curien, G; DeRose, R T; Douce, R

    1993-01-01

    Towards the goal of gaining a better understanding of the molecular mechanisms controlling branched-chain-amino-acid biosynthesis in plants, we have isolated, sequenced and characterized a gene encoding acetohydroxy acid isomero-reductase (ketol-acid reductoisomerase) from Arabidopsis thaliana (thale cress). Comparison between the acetohydroxy acid isomeroreductase cDNA and the genomic sequence has allowed us to determine the exon structure of the coding region. The isolated acetohydroxy acid isomeroreductase gene is distributed over approx. 4.5 kbp and contains nine introns (79-347 bp). The transcriptional start site was found to be 52 bp upstream of the translational initiation site. Southern-blot analysis of A. thaliana genomic DNA shows that the acetohydroxy acid isomeroreductase is encoded by a single-copy gene. Images Figure 3 Figure 5 PMID:8379936

  18. Molecular cloning, characterization and expression of heat shock protein 70 gene from the oyster Crassostrea hongkongensis responding to thermal stress and exposure of Cu(2+) and malachite green.

    PubMed

    Zhang, Zhanhui; Zhang, Qizhong

    2012-04-15

    Heat shock protein 70 (HSP70) acts mostly as a molecular chaperone and plays a key role in the process of protecting cells by facilitating the folding of nascent peptides and the cellular stress response. The cDNA of the oyster Crassostrea hongkongensis hsp70 (designated chhsp70) was cloned with the techniques of homological cloning and rapid amplification of cDNA ends (RACE). The full-length chhsp70 cDNA was 2251bp, consisting of a 130bp 5'-UTR, 216bp 3'-UTR with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1905bp, which encoded a polypeptide of 634 amino acids. Three classical HSP signature motifs were detected in ChHSP70, i.e., DLGTT-S-V, IFDLGGGTFDVSIL and VVLVGGSTRIPKIQK. BLAST analysis revealed that the ChHSP70 shared high identity with other bivalve HSP70. The phylogenetic analysis indicated that the ChHSP70 was a member of the HSP70 family. The chhsp70 mRNA transcripts were quantified by fluorescent real time RT-PCR under both unstressed and stressed conditions, i. e., heat shock and exposure to Cu(2+) and malachite green. Basal expression level was similar in mantle, gill, digestive gland, and heart, but higher in muscle than that in the others. A similar trend showed that the chhsp70 mRNA expression significantly increased at 3-6h, then dropped and returned to control level at 24h in the five tissues and organs mentioned above after heat shock. A clearly time-dependent expression pattern of chhsp70 mRNA in digestive gland and gill of the oyster was observed after exposure of Cu(2+) and malachite green. In the two tissues, the chhsp70 mRNA level reached the maximum at 6h after malachite green exposure and on day 4 after Cu(2+) exposure, and then decreased progressively to the control level. The results indicated that ChHSP70 of the oyster is an inducible protein, and plays an important role in response to the Cu(2+) and malachite green polluted stress, so chhsp70 might be used as a potential molecular

  19. Molecular cloning and characterization of a membrane associated NAC family gene, SiNAC from foxtail millet [Setaria italica (L.) P. Beauv].

    PubMed

    Puranik, Swati; Bahadur, Ranjit Prasad; Srivastava, Prem S; Prasad, Manoj

    2011-10-01

    The plant-specific NAC (NAM, ATAF, and CUC) transcription factors have diverse role in development and stress regulation. A transcript encoding NAC protein, termed SiNAC was identified from a salt stress subtractive cDNA library of S. italica seedling (Puranik et al., J Plant Physiol 168:280-287, 2011). This single/low copy gene containing four exons and four introns within the genomic-sequence encoded a protein of 462 amino acids. Structural analysis revealed that highly divergent C terminus contains a transmembrane domain. The NAC domain consisted of a twisted antiparallel beta-sheet packing against N terminal alpha helix on one side and a shorter helix on the other side. The domain was predicted to homodimerize and control DNA-binding specificity. The physicochemical features of the SiNAC homodimer interface justified the dimeric form of the predicted model. A 1539 bp fragment upstream to the start codon of SiNAC gene was cloned and in silico analysis revealed several putative cis-acting regulatory elements within the promoter sequence. Transactivation analysis indicated that SiNAC activated expression of reporter gene and the activation domain lied at the C terminal. The SiNAC:GFP was detected in the nucleus and cytoplasm while SiNAC ΔC(1-158):GFP was nuclear localized in onion epidermal cells. SiNAC transcripts mostly accumulated in young spikes and were strongly induced by dehydration, salinity, ethephon, and methyl jasmonate. These results suggest that SiNAC encodes a membrane associated NAC-domain protein that may function as a transcriptional activator in response to stress and developmental regulation in plants. PMID:21312005

  20. Molecular cloning and identification of mouse epididymis-specific gene mHong1, the homologue of rat HongrES1.

    PubMed

    Hu, Shuang-Gang; Du, Han; Yao, Guang-Xin; Zhang, Yong-Lian

    2012-07-01

    Previous studies have shown that rat epididymis-specific gene HongrES1 plays important roles in sperm capacitation and fertility. In this study, we cloned the mouse homologue gene by sequence alignment and RT-PCR methods and designated it as mHong1. The mHong1 gene is located on chromosome 12p14, spanning five exons. The cDNA sequence consists of 1257 nucleotides and encodes a 419 amino-acid protein with a predicted N-terminal signal peptide of 20 amino acids. The mHong1 mRNA shows similarity with HongrES1 in the expression patterns: (i) specific expression in epididymal tissue, especially in the cauda region; and (ii) androgen-dependence but testicular fluid factor independence. Its protein product shows 71% similarity with HongrES1 and contains a classical serpin domain as does HongrES1. A polyclonal antibody against mHong1 with high specificity and sensitivity was raised. Like HongrES1, the mHong1 protein shows a checker-board expression pattern in the epididymal epithelium and is secreted into the epididymal lumen. The mHong1 protein shows higher glycosylation than HongrES1. Although both of them are deposited onto the sperm head surface, mHong1 is localized to the equatorial segment, which is different from that of HongrES1. The mHong1 protein can be removed from the sperm membrane by high ionic strength and therefore can be classed as an extrinsic membrane protein. Collectively, we conclude that mHong1 is the homologue of HongrES1 and the present work paves the way for establishing animal models to elucidate the precise functions of HongrES1 and mHong1. PMID:22426594

  1. Molecular cloning of the human proto-oncogene Wnt-5A and mapping of the gene (WNT5A) to chromosome 3p14-p21

    SciTech Connect

    Clark, C.C.; Cohen, I.; Eichstetter, I.; Cannizzaro, L.A.; Iozzo, R.V. ); McPherson, J.D.; Wasmuth, J.J. )

    1993-11-01

    The highly conserved Wnt genes belong to a widely distributed family of presumptive signaling molecules that have been implicated not only in the regulation of normal pattern formation during embryogenesis and differentiation of cell lineages, but also in oncogenic events. All of the known vertebrate Wnt genes encode for 38- to 43-kDa cysteine-rich putative glycoproteins, which have features typical of secreted growth factors: A hydrophobic signal sequence, a conserved asparagine-linked oligosaccharide consensus sequence, and 22 conserved cysteine residues whose relative spacing is maintained. In this study, the authors report the cloning and sequencing of several overlapping cDNAs encoding [approximately]4.1 kb of the human homologue of Wnt-5A. The mature protein contained 343 residues (M[sub r] [approximately] 38,000 excluding any post-translational modifications) with a >93% homology to the reported sequences of other Wnt-5A proteins (>99% homologous to mouse Wnt-5A). This protein maintained certain features - a hydrophobic signal sequence, the Wnt-1 family [open quotes]signature sequence[close quotes] (CKCHGvSGSC), and a number of other conserved amino acid residues - 24 cysteine residues, 4 asparagine-linked oligosaccharide consensus sequences, and a tyrosine sulfation site - that have been found in all other Wnt-5A proteins. Reverse transcriptase PCR analysis of RNA from a variety of human embryonic, neonatal, and adult cells and/or tissues showed that human Wnt-5A expression was detected only in neonatal heart and lung. It may be relevant, however, that the 3[prime]-untranslated region contained numerous AT-rich motifs that could be involved in the rapid degradation of mRNA. Finally, using a combination of Southern blotting, PCR amplification, and in situ hybridization, the human Wnt-5A (WNT5A) gene was mapped to chromosome 3p14-p21. 36 refs., 7 figs., 2 tabs.

  2. Cloning and characterization of porcine resistin gene.

    PubMed

    Dai, M H; Xia, T; Chen, X D; Gan, L; Feng, S Q; Qiu, H; Peng, Y; Yang, Z Q

    2006-02-01

    Resistin is a member of resistin-like molecules (RELMs) and a hormone secreted from mature adipocytes in rodents and leukocytes in human. We now report the cloning and characterization of the full-length porcine resistin cDNA and gene. Sequence analysis indicated that the pig resistin cDNA sequence had an open reading frame of 330 bp encoding a 12 kDa protein of 109 amino acids. The deduced amino acid sequence showed 75.2% identity to the human resistin. The porcine resistin gene was composed of four exons and had exactly the same exon structure as the human resistin gene. The tissue distribution of porcine resistin mRNA was assessed by semi-quantitative RT-PCR. Resistin gene expression was the highest in porcine leukocytes and low in adipose tissue. Resistin protein could be detected in porcine serum by western blotting and it circulated in serum as dimers and trimers. We provided the first evidence that resistin was abundantly expressed in porcine leukocytes and had an expression pattern similar to that in human resistin mRNA and protein. This suggests that the pig may be a suitable animal model for studying the function of resistin in human insulin resistance. PMID:16023825

  3. Molecular cloning and expression analysis on LPL of Coilia nasus.

    PubMed

    Wang, Meiyao; Xu, Dongpo; Liu, Kai; Yang, Jian; Xu, Pao

    2016-06-01

    Coilia nasus is one important commercial anadromous species which mainly distributed in the Yangtze River in China. At present, it has been on the "National Key Protective Species List" because of its severe resource damage. Lipid metabolism is very important during its long-distance migration. To make further research on lipid metabolism of C. nasus, we cloned lipoprotein lipase gene with homologous cloning method. A full-length cDNA of LPL of C. nasus was cloned from liver which covered 3537 bp with a 1519 bp open reading frame encoding 505 deduced amino acids whose molecular mass was 57.5 kDa and theoretical isoelectric point was 7.58. The deduced amino acids had high similarity with the reported LPL sequence of other species. It had typical conserved domain of LPL protein containing catalytic triad, N-linked glycosylation sites and conserved heparin-binding site, etc. We adopted quantitative real-time RT-PCR method to detect the mRNA expression of LPL of C. nasus in ten tissues including mesenteric adipose, liver, muscle, stomach, spleen, heart, head kidney, trunk kidney, gill and brain with β-actin as internal reference. LPL expressed in all the detected tissues. The highest expression was in mesenteric adipose, and followed by liver, muscle, stomach. Lipid expressed lowly in spleen, heart, head kidney, trunk kidney, gill and brain. The research on the cloning and differential expression of LPL of C. nasus will lay foundation for further research on lipid metabolism of C. nasus. PMID:26877109

  4. Molecular cloning and chromosomal mapping of bone marrow stromal cell surface gene, BST2, that may be involved in pre-B-cell growth

    SciTech Connect

    Ishikawa, Jun; Kaisho, Tsuneyasu; Tomizawa, Hitoshi

    1995-04-10

    Bone marrow stromal cells regulate B-cell growth and development through their surface molecules and cytokines. In this study, we generated a mAb, RS38, that recognized a novel human membrane protein, BST-2, expressed on bone marrow stromal cell lines and synovial cell lines. We cloned a cDNA encoding BST-2 from a rheumatoid arthritis-derived synovial cell line. BST-2 is a 30- to 36-kDa type II transmembrane protein, consisting of 180 amino acids. The BST-2 gene (HGMW-approved symbol BST2) is located on chromosome 19p13.2. BST-2 is expressed not only on certain bone marrow stromal cell lines but also on various normal tissues, although its expression pattern is different from that of another bone marrow stromal cell surface molecule, BST-1. BST-2 surface expression on fibroblast cell lines facilitated the stromal cell-dependent growth of a murine bone marrow-derived pre-B-cell line, DW34. The results suggest that BST-2 may be involved in pre-B-cell growth. 45 refs., 7 figs., 2 tabs.

  5. Molecular cloning and functional analysis of a H(+)-dependent phosphate transporter gene from the ectomycorrhizal fungus Boletus edulis in southwest China.

    PubMed

    Wang, Junling; Li, Tao; Wu, Xiaogang; Zhao, Zhiwei

    2014-01-01

    Phosphate transporters (PTs), as entry points for phosphorus (P) in organisms, are involved in a number of P nutrition processes such as phosphate uptake, transport, and transfer. In the study, a PT gene 1632 bp long (named BePT) was cloned, identified, and functionally characterized from Boletus edulis. BePT was expected to encode a polypeptide with 543 amino acid residues. The BePT polypeptide belonged to the major facilitator superfamily and showed a high degree of sequence identity to the Pht1 family. A topology model revealed that BePT exhibited 12 transmembrane helices, divided into two halves, and connected by a large hydrophilic loop in the middle. A yeast mutant complementation analysis suggested that BePT was a functional PT which mediated orthophosphate uptake of yeast at micromolar concentrations. Green fluorescent protein-BePT fusion proteins expressed were extensively restricted to the plasma membrane in BePT transformed yeast, and its activity was dependent on electrochemical membrane potential. In vitro, quantitative PCR confirmed that the expression of BePT was significantly upregulated at lower phosphorus availability, which may enhance phosphate uptake and transport under phosphate starvation. Our results suggest that BePT plays a key role in phosphate acquisition in the ectomycorrhizal fungus B. edulis. PMID:24863474

  6. Isolation and properties of Drosophila melanogaster ferritin--molecular cloning of a cDNA that encodes one subunit, and localization of the gene on the third chromosome.

    PubMed

    Charlesworth, A; Georgieva, T; Gospodov, I; Law, J H; Dunkov, B C; Ralcheva, N; Barillas-Mury, C; Ralchev, K; Kafatos, F C

    1997-07-15

    Ferritin was purified from iron-fed Drosophila melanogaster extracts by centrifugation in a gradient of potassium bromide. On polyacrylamide gel electrophoresis, the product showed two protein bands corresponding to the ferritin monomer and dimer. Electrophoresis following dissociation with SDS and 2-mercaptoethanol revealed three strong bands of approximately 25, 26, and 28 kDa. N-terminal amino acid sequences were identical for the 25-kDa and 26-kDa subunits, but different for the 28-kDa subunit. Conserved ferritin PCR primers were used to amplify a 360-bp cDNA product, which was used to isolate a clone from a D. melanogaster cDNA library that contained the complete coding sequence for a ferritin subunit. Additional 5' sequence obtained by the RACE method revealed the presence of a putative iron regulatory element. The PCR product was also used to locate the position of the ferritin subunit gene at region 99F on the right arm of the third chromosome. The deduced amino acid sequence of the D. melanogaster ferritin subunit contained a signal sequence and resembled most closely ferritin of the mosquito Aedes aegypti. The evolution of ferritin sequences is discussed. PMID:9266686

  7. Molecular cloning, functional expression, and characterization of isolectin genes of hemolytic lectin CEL-III from the marine invertebrate Cucumaria echinata.

    PubMed

    Shimizu, Yoshiki; Yamazaki, Hiroshi; Yoshida, Shigeto; Yonekura, Masami; Kouzuma, Yoshiaki

    2012-01-01

    CEL-III is a hemolytic lectin purified from the marine invertebrate Cucumaria echinata. Previous research has indictated that CEL-III is composed of several isoforms. Here we identified five CEL-III isolectin genes, designated CEL-III-L1, CEL-III-L2, CEL-III-S1, CEL-III-S2, and CEL-III-LS1, by cDNA cloning. The deduced amino acid sequences suggested they shared 94.0-99.8% identical residues. Among the amino acid residues involved in carbohydrate binding, the His residue, which contributes to stacking with sugar, in subdomain 1α was replaced by Tyr in CEL-III-L2. The recombinant proteins were expressed in Escherichia coli or insect cells. rCEL-III-L2 showed higher hemolytic activity than those of the other isolectins. Furthermore, an apparent oligomer band of rCEL-III-L2 was detected on erythrocyte membranes, although the other isolectins showed smear bands. These results suggest that Tyr36 of CEL-III-L2 is important for the expression of hemolytic activity and oligomerization. PMID:22313748

  8. Polyhydroxyalkanoate production by a novel bacterium Massilia sp. UMI-21 isolated from seaweed, and molecular cloning of its polyhydroxyalkanoate synthase gene.

    PubMed

    Han, Xuerong; Satoh, Yasuharu; Kuriki, Yumi; Seino, Teruyuki; Fujita, Shinji; Suda, Takanori; Kobayashi, Takanori; Tajima, Kenji

    2014-11-01

    We successfully isolated one microorganism (UMI-21) from Ulva, a green algae that contains starch. The strain UMI-21 can produce polyhydroxyalkanoate (PHA) from starch, maltotriose, or maltose as a sole carbon source. Taxonomic studies and 16S rDNA sequence analysis revealed that strain UMI-21 was phylogenetically related to species of the genus Massilia. The PHA content under the cultivation condition using a 10-L jar fermentor was 45.5% (w/w). This value was higher than that obtained after cultivation in a flask, suggesting the possibility of large-scale PHA production by UMI-21 from starch. A major issue for the industrial production of microbial PHAs is the very high production cost. Starch is a relatively inexpensive substrate that is also found in abundant seaweeds such as Ulva. Therefore, the strain isolated in this study may be very useful for producing PHA from seaweeds containing polysaccharides such as starch. In addition, a 3.7-kbp DNA fragment containing the whole PHA synthase gene (phaC) was obtained from the strain UMI-21. The results of open reading frame (ORF) analysis suggested that the DNA fragment contained two ORFs, which were composed of 1740 (phaC) and 564 bp (phaR). The deduced amino acid sequence of PhaC from strain UMI-21 shared high similarity with PhaC from Ralstonia eutropha, which is a representative PHA-producing bacterium with a class I PHA synthase. This is the first report for the cloning of the PHA synthase gene from Massilia species. PMID:24932969

  9. CYTOLOGICAL AND MOLECULAR ANALYSIS OF NORMAL AND CLONED BULLS’ MEIOSIS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cytological and molecular analysis of meiotic cells from two bull clones and three non-clones was performed in order to detect effects of somatic cell nuclear transfer (SNCT) on the meiotic process. Pachytene cells were analyzed by immunohistology using antibodies against the synaptonemal complex pr...

  10. Molecular and biological characterization of a neurovirulent molecular clone of simian immunodeficiency virus.

    PubMed Central

    Flaherty, M T; Hauer, D A; Mankowski, J L; Zink, M C; Clements, J E

    1997-01-01

    To identify the molecular determinants of neurovirulence, we constructed an infectious simian immunodeficiency virus (SIV) molecular clone, SIV/17E-Fr, that contained the 3' end of a neurovirulent strain of SIV, SIV/17E-Br, derived by in vivo virus passage. SIV/17E-Fr is macrophage tropic in vitro and neurovirulent in macaques. In contrast, a molecular clone, SIV/17E-Cl, that contains the SU and a portion of the TM sequences of SIV/17E-Br is macrophage tropic but not neurovirulent. To identify the amino acids that accounted for the replication differences between SIV/17E-Fr and SIV/17E-Cl in primary macaque cells in vitro, additional infectious molecular clones were constructed. Analysis of these recombinant viruses revealed that changes in the TM portion of the envelope protein were required for the highest level of replication in primary macaque macrophages and brain cells derived from the microvessel endothelium. In addition, a full-length Nef protein is necessary for optimum virus replication in both of these cell types. Finally, viruses expressing a full-length Nef protein in conjunction with the changes in the TM had the highest specific infectivity in a sMAGI assay. Thus, changes in the TM and nef genes between SIV/17E-Cl and SIV/17E-Fr account for replication differences in vitro and correlate with replication in the central nervous system in vivo. PMID:9223467

  11. Gene cloned for enzyme used to make cheese

    SciTech Connect

    Not Available

    1982-02-15

    Scientists at Collaborative Research in Waltham, Mass., working under a contract with Dow Chemical, Midland, Mich. are reported to have cloned the gene rennin, an enzyme used in the production of cheese. The gene was cloned in both yeast and the bacterium Escherichia coli using standard recombinant DNA techniques. Rennin is the first enzyme of industrial importance to be cloned and it is hoped that rennin will be commercially available by the mid-1980's.

  12. Molecular Cloning, Characterization, and Expression of a Catalase Gene in the Japanese Scallop Mizuhopecten yessoensis Induced in the Presence of Cadmium

    NASA Astrophysics Data System (ADS)

    Gao, Jialong; Ishizaki, Shoichiro; Nagashima, Yuji

    2016-03-01

    Cadmium (Cd) is known to influence the oxidative status of marine organisms and can induce the formation of reactive oxygen species (ROS). Catalase (CAT) is one of the important enzymes involved in scavenging high levels of ROS. In present study, we cloned CAT cDNA and investigated the response of this enzyme at the transcriptional level in the Japanese scallop Mizuhopecten yessoensis exposed to Cd. The full-length CAT cDNA (MyCAT) of 1,870 nucleotides including a 57 bp 5'-UTR, a coding sequence of 1,500 bp and a 313 bp 3'-UTR were identified from the scallop. The deduced amino acid sequence of MyCAT corresponds to 499 amino acids with predicted molecular weight of 56.48 kDa and contains highly conserved motifs of the proximal heme-binding site RLFSYSTH, proximal active signature FNRERIPERVVHAKGGG and three catalytic amino acid residues His72, Asn145, and Tyr355. Its significant homology to CATs from multiple alignments revealed that MyCAT had a high identity with CATs from other mollusks. CAT mRNA expression analysis revealed that expression level was highest in the digestive gland ( p < 0.01) but weak in muscle. Following exposure to 200 and 400 µg/l of Cd, a high amount of Cd was found to have accumulated in the digestive gland and CAT mRNA expression had significantly increased in this organ among 7-day exposed scallops ( p < 0.001). The result demonstrated that antioxidant enzymes such as CAT play important roles in counteracting Cd stress in M. yessoensis.

  13. Molecular cloning and characterization of the gonadotropin subunits GPα, FSHβ, and LHβ genes in the stinging catfish Heteropneustes fossilis: phylogeny, seasonal expression and pituitary localization.

    PubMed

    Acharjee, Arup; Chaube, Radha; Joy, Keerikkattil Paily

    2015-10-01

    Gonadotropins are heterodimeric glycoproteins secreted by the pituitary, and consist of a common glycoprotein hormone alpha (GPα) and the function-specific follicle-stimulating hormone beta subunit (FSHβ) or luteinizing hormone beta subunit (LHβ). In the present study, the subunit protein genes were cloned and characterized from the pituitary of the catfish Heteropneustes fossilis. Full-length cDNAs of GPα, FSHβ, and LHβ are 511 base pairs (bp), 659 bp and 660 bp long, and encode 92, 108, and 112 aminoacids long mature proteins, respectively. GPα has 10 cysteines with 2 N-linked glycosylation sites while LHβ contains 12 cysteines with a single N-linked glycosylation site. In contrast, FSHβ has 13 cysteines, 1 additional over the conserved 12 cysteines of other vertebrates, and a single glycosylation site between Cys 3 and Cys 4. Phylogenetic analyses of the deduced proteins confirm their homology and relationships with the respective gonadotropin subunit proteins of gnathostome vertebrates. Tissue expression analysis by semi-quantitative RT-PCR shows that GPα mRNA is expressed only in the pituitary while both FSHβ and LHβ mRNA are expressed in extra-pituitary sites. The subunit mRNAs show both seasonal and sex dimorphic variations especially in the expression of FSHβ and LHβ transcripts. In the sexually quiescent phase, the transcript expression is low while in the recrudescent phase, the expressions are differential, high, and varied with regard to sex and reproductive phase. In situ hybridization of the mRNAs gave positive signals in gonadotropes in the pars distalis of the pituitary, which exhibited seasonal variation in staining intensity and numbers. PMID:26205349

  14. Molecular cloning of the mouse grb2 gene: differential interaction of the Grb2 adaptor protein with epidermal growth factor and nerve growth factor receptors.

    PubMed Central

    Suen, K L; Bustelo, X R; Pawson, T; Barbacid, M

    1993-01-01

    We report the isolation and molecular characterization of the mouse grb2 gene. The product of this gene, the Grb2 protein, is highly related to the Caenorhabditis elegans sem-5 gene product and the human GRB2 protein and displays the same SH3-SH2-SH3 structural motifs. In situ hybridization studies revealed that the mouse grb2 gene is widely expressed throughout embryonic development (E9.5 to P0). However, grb2 transcripts are not uniformly distributed, and in certain tissues (e.g., thymus) they appear to be regulated during development. Recent genetic and biochemical evidence has implicated the Grb2 protein in the signaling pathways that link cell surface tyrosine kinase receptors with Ras. We have investigated the association of the Grb2 protein with epidermal growth factor (EGF) and nerve growth factor (NGF) receptors in PC12 pheochromocytoma cells. EGF treatment of PC12 cells results in the rapid association of Grb2 with the activated EGF receptors, an interaction mediated by the Grb2 SH2 domain. However, Grb2 does not bind to NGF-activated Trk receptors. Mitogenic signaling of NGF in NIH 3T3 cells ectopically expressing Trk receptors also takes place without detectable association between Grb2 and Trk. These results suggest that whereas EGF and NGF can activate the Ras signaling pathway in PC12 cells, only the EGF receptor is likely to do so through a direct interaction with Grb2. Finally, binding studies with glutathione S-transferase fusion proteins indicate that Grb2 binds two distinct subsets of proteins which are individually recognized by its SH2 and SH3 domains. These observations add further support to the concept that Grb2 is a modular adaptor protein. Images PMID:7689150

  15. Molecular cloning and sequence analysis of the gene encoding LipL41, a surface-exposed lipoprotein of pathogenic Leptospira species.

    PubMed Central

    Shang, E S; Summers, T A; Haake, D A

    1996-01-01

    We report the cloning of the gene encoding a surface-exposed leptospiral lipoprotein, designated LipL41. In a previous study, a 41-kDa protein antigen was identified on the surface of Leptospira kirschneri (D. A. Haake, E. M. Walker, D. R. Blanco, C. A. Bolin, J. N. Miller, and M. A. Lovett, Infect. Immun. 59:1131-1140, 1991). We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment in order to design an oligonucleotide probe.A Lambda ZAP II library containing EcoRI fragments of L. kirschneri DNA was screened, and a 2.3-kb DNA fragment which contained the entire structural lipL41 gene was identified. The deduced amino acid sequence of LipL41 would encode a 355-amino-acid polypeptide with a 19-amino-acid signal peptide, followed by an L-X-Y-C lipoprotein signal peptidase cleavage site. A recombinant His6-LipL41 fusion protein was expressed in Escherichia coli in order to generate specific rabbit antiserum. LipL41 is solubilized by Triton X-114 extraction of L. kirschneri; phase separation results in partitioning of LipL41 exclusively into the detergent phase. At least eight proteins, including LipL41 and the other major Triton X-114 detergent phase proteins, are intrinsically labeled during incubation of L. kirschneri in media containing [3H] palmitate. Processing of LipL41 is inhibited by globomycin, a selective inhibitor of lipoprotein signal peptidase. Triton X-100 extracts of L. kirschneri contain immunoprecipitable OmpL1 (porin), LipL41, and another lipoprotein, LipL36. However, in contrast to LipL36, only LipL41 and OmpL1 were exposed on the surface of intact organisms. Immunoblot analysis of a panel of Leptospira species reveals that LipL41 expression is highly conserved among leptospiral pathogens. PMID:8675344

  16. Molecular cloning of chicken aggrecan. Structural analyses.

    PubMed Central

    Chandrasekaran, L; Tanzer, M L

    1992-01-01

    The large, aggregating chondroitin sulphate proteoglycan of cartilage, aggrecan, has served as a generic model of proteoglycan structure. Molecular cloning of aggrecans has further defined their amino acid sequences and domain structures. In this study, we have obtained the complete coding sequence of chicken sternal cartilage aggrecan by a combination of cDNA and genomic DNA sequencing. The composite sequence is 6117 bp in length, encoding 1951 amino acids. Comparison of chicken aggrecan protein primary structure with rat, human and bovine aggrecans has disclosed both similarities and differences. The domains which are most highly conserved at 70-80% identity are the N-terminal domains G1 and G2 and the C-terminal domain G3. The chondroitin sulphate domain of chicken aggrecan is smaller than that of rat and human aggrecans and has very distinctive repeat sequences. It has two separate sections, one comprising 12 consecutive Ser-Gly-Glu repeats of 20 amino acids each, adjacent to the other which has 23 discontinuous Ser-Gly-Glu repeats of 10 amino acids each; this latter region, N-terminal to the former one, appears to be unique to chicken aggrecan. The two regions contain a total of 94 potential chondroitin sulphate attachment sites. Genomic comparison shows that, although chicken exons 11-14 are identical in size to the rat and human exons, chicken exon 10 is the smallest of the three species. This is also reflected in the size of its chondroitin sulphate coding region and in the total number of Ser-Gly pairs. The putative keratan sulphate domain shows 31-45% identity with the other species and lacks the repetitive sequences seen in the others. In summary, while the linear arrangement of specific domains of chicken aggrecan is identical to that in the aggrecans of other species, and while there is considerable identity of three separate domains, chicken aggrecan demonstrates unique features, notably in its chondroitin sulphate domain and its keratan sulphate

  17. Cloning, expression and characterisation of a promising mosquitocidal gene.

    PubMed

    Zhang, Lingling; Huang, Enjiong; Gelbic, Ivan; Guan, Chunyu; Guan, Yi; Guan, Xiong

    2009-10-01

    A new mosquitocidal gene, cyt1Aa6, was isolated and cloned from the novel Bacillus thuringiensis strain LLP29, previously isolated from the phylloplane of Magnolia denudata. Nucleotide sequence analysis of cyt1Aa6 indicated that the open reading frame consisted of 750 base pairs, encoding 249 amino acid sequences with a calculated molecular weight of 27.3681 kDa and a PI value of 4.77. An homological comparison revealed that the cyt1Aa6 amino acid sequence was 99% identical with those of known Cyt1Aa proteins. In addition, the cyt1Aa6 gene was successfully expressed in Escherichia coli BL21. Bioassays on Aedes albopictus showed that Bt LLP29 and the expressed BL21 were both toxic to 3rd-instar mosquito larvae. The isolation of cyt1Aa6 provides new opportunities for selecting new strains and to obtain novel B. thuringiensis products based on its toxins. PMID:20112806

  18. Expression cloning of genes encoding human peroxisomal proteins

    SciTech Connect

    Spathaky, J.M.; Tate, A.W.; Cox, T.M.

    1994-09-01

    Numerous metabolic disorders associated with diverse peroxisomal defects have been identified but their molecular characterization has been hampered by difficulties associated with the purification of proteins from this fragile organelle. We have utilized antibodies directed against the C-terminal tripeptide peroxisomal targeting signal to detect hitherto unknown peroxisomal proteins in tissue fractions and to isolate genes encoding peroxisonal proteins from human expression libraries. We immunized rabbits with a peptide conjugate encompassing the C-terminal nine amino acids of rat peroxisomal acyl CoA oxidase. Immunoprecipitation assays using radio-labelled peptide showed that the antibody specifically recognizes the terminal SKL motif as well as C-terminal SHL and SRL but not SHL at an internal position. Affinity-purified antibody was used to probe Western blots of crude and peroxisome-enriched monkey liver preparations and detected 8-10 proteins specifically in the peroxisome fractions. 100 positive clones were identified on screening a human liver cDNA expression library in {lambda}-gt11. Sequence analysis has confirmed the identity of cDNA clones for human acyl CoA oxidase and epoxide hydrolase. Four clones show no sequence identity and their putative role in the human peroxisome is being explored.

  19. Molecular cloning of the extracellular endodextranase of Streptococcus salivarius.

    PubMed Central

    Lawman, P; Bleiweis, A S

    1991-01-01

    We report the cloning in Escherichia coli of the gene encoding an extracellular endodextranase (alpha-1,6-glucanhydrolase, EC 3.2.1.11) from Streptococcus salivarius PC-1. Recombinants from a S. salivarius PC-1-Lambda ZAP II genomic library specifying dextranase activity were identified as plaques surrounded by zones of clearing on blue dextran agar. One such clone, PD1, had a 6.3-kb EcoRI fragment insert which encoded a 190-kDa protein with dextranase activity. The recombinant strain also produced two lower-molecular-mass polypeptides (90 and 70 kDa) that had dextranase activity. Native dextranase was recovered from concentrated culture fluids of S. salivarius as a single 110-kDa polypeptide. PD1 phage lysate and PC-1 culture supernatant fluid extract were used to measure substrate specificity of the recombinant and native forms of dextranase, respectively. Analysis of these reaction products by thin-layer chromatography revealed the expected isomaltosaccharide products yielded by the recombinant-specified enzyme but was unable to resolve the larger polysaccharide products of the native enzyme. Furthermore, S. salivarius utilized neither the substrates nor the products of dextran hydrolysis for growth. Images FIG. 1 FIG. 2 FIG. 3 FIG. 4 PMID:1938938

  20. [A brief introduction to the methods for novel gene cloning].

    PubMed

    Sun, C X; Yu, A C

    2000-01-01

    There are a lot of methods for novel gene cloning, but how to clone candidate gene(s) quickly and correctly? This is a brief introduction to methods of novel gene cloning, these methods includes: differential display reverse transcriptase polymerase chain reaction(DD RT-PCR), suppression subtractive hybridization(SSH), RNA arbitrarily primed PCR(RAP-PCR), representational difference analysis(RDA), yeast two-hybrid system, cDNA capturation, et al. We not only introduced these methods, but also discussed the advantages and disadvantages of them. However, no single method is omnipotent, one should pick up the method most suitable for a special purpose. PMID:12532765

  1. Molecular characterization of the MLL-SEPT6 fusion gene in acute myeloid leukemia: identification of novel fusion transcripts and cloning of genomic breakpoint junctions.

    PubMed

    Cerveira, Nuno; Micci, Francesca; Santos, Joana; Pinheiro, Manuela; Correia, Cecília; Lisboa, Susana; Bizarro, Susana; Norton, Lucília; Glomstein, Anders; Asberg, Ann E; Heim, Sverre; Teixeira, Manuel R

    2008-07-01

    One of the MLL fusion partners in leukemia is the SEPT6 gene, which belongs to the evolutionarily conserved family of genes of septins. In this work we aimed to characterize at both the RNA and DNA levels three acute myeloid leukemias with cytogenetic evidence of a rearrangement between 11q23 and Xq24. Molecular analysis led to the identification of several MLL-SEPT6 fusion transcripts in all cases, including a novel MLL-SEPT6 rearrangement (MLL exon 6 fused with SEPT6 exon 2). Genomic DNA breakpoints were found inside or near Alu or LINE repeats in the MLL breakpoint cluster region, whereas the breakpoint junctions in the SEPT6 intron 1 mapped to the vicinity of GC-rich low-complexity repeats, Alu repeats, and a topoisomerase II consensus cleavage site. These data suggest that a non-homologous end-joining repair mechanism may be involved in the generation of MLL-SEPT6 rearrangements in acute myeloid leukemia. PMID:18492691

  2. Cloning and characterization of nanos gene in silkworm Bombyx mori.

    PubMed

    Zhao, Guoli; Chen, Keping; Yao, Qin; Wang, Weihua

    2008-02-01

    Gene nanos is a maternal posterior group gene required for normal development of abdominal segments and the germ line in Drosophila. Expression of nanos-related genes is associated with the germ line in a broad variety of other taxa. In this study, the 5'-RACE method and the in silico cloning method are used to isolate the new nanos-like gene of Bombyx mori and the gene obtained is analyzed with bioinformatics tools. The putative protein is expressed in Escherichia coli and the antiserum has been produced in New Zealand white rabbits. The result shows that the nanos cDNA is 1,913 bp in full length and contains a 954 bp open reading frame. The deduced protein has 317 amino acid residues, with a predicted molecular weight of 35 kDa, isoelectric point of 5. 38, and contains a conserved nanos RNA binding domain. The conserved region of the deduced protein shares 73% homology with the nanos protein conserved region of Honeybee (Apis mellifera). This gene has been registered in the GenBank under the accession number EF647589. One encoding sequence of the nanos fragment has been successfully expressed in E. coli. Western blotting analysis indicates that homemade antiserum can specifically detect nanos protein expressed in prokaryotic cells. PMID:18407054

  3. Short Communication Molecular cloning and expression pattern of the porcine 5-aminolevulinate synthase 1 (ALAS1) gene and its association with reproductive traits.

    PubMed

    Liu, L Q; Li, F E; Deng, C Y

    2016-01-01

    5-Aminolevulinate synthase 1 (ALAS1) is the first enzyme in the heme biosynthetic pathway and is upregulated in follicular tissue during the early stages of ovulation. In this study, we isolated the complete coding sequence of the porcine ALAS1 gene and its 2-9 intron sequence, identified a single nucleotide polymorphism (SNP; T/C) in intron 9, and developed a PCR-MspI-restriction fragment length polymorphism genotyping assay. Association of the SNP with litter size was assessed in two populations [purebred Large White and the experimental synthetic (DIV) line]. Statistical analysis demonstrated that for total number of piglets born (TNB) in all parities, pigs with the CC genotype had an additional 0.68 and 0.74 piglets compared to the TC and TT animals (P < 0.05) in the DIV line, respectively. Purebred Large White sows inheriting the CC and TC genotypes gave birth to an additional 0.96 and 0.70 piglets compared to the TT animals (P < 0.05) in all parities, respectively. In addition, for TNB in all parities, a significant additive effect of 0.48 ± 0.23 and 0.37 ± 0.17 piglets/ litter was detected in sows of both populations (P < 0.05), respectively. The highest levels of ALAS1 gene expression were observed in isolated ovarian granulosa cells 2 and 12 h after stimulation with pregnant mare serum gonadotropin human chorionic gonadotropin, which represents the time of follicular development and ovulation, respectively. Therefore, the ALAS1 gene was significantly associated with litter size in two populations and could be a useful molecular marker for the selection of increasing litter size in pigs. PMID:26910002

  4. Molecular Cloning, Heterologous Expression, and Functional Characterization of an NADPH-Cytochrome P450 Reductase Gene from Camptotheca acuminata, a Camptothecin-Producing Plant

    PubMed Central

    Chen, Fei; Yang, Yun; Yang, Lixia; Zhang, Guolin; Luo, Yinggang

    2015-01-01

    Camptothecin (CAM), a complex pentacyclic pyrroloqinoline alkaloid, is the starting material for CAM-type drugs that are well-known antitumor plant drugs. Although many chemical and biological research efforts have been performed to produce CAM, a few attempts have been made to uncover the enzymatic mechanism involved in the biosynthesis of CAM. Enzyme-catalyzed oxidoreduction reactions are ubiquitously presented in living organisms, especially in the biosynthetic pathway of most secondary metabolites such as CAM. Due to a lack of its reduction partner, most catalytic oxidation steps involved in the biosynthesis of CAM have not been established. In the present study, an NADPH-cytochrome P450 reductase (CPR) encoding gene CamCPR was cloned from Camptotheca acuminata, a CAM-producing plant. The full length of CamCPR cDNA contained an open reading frame of 2127-bp nucleotides, corresponding to 708-amino acid residues. CamCPR showed 70 ~ 85% identities to other characterized plant CPRs and it was categorized to the group II of CPRs on the basis of the results of multiple sequence alignment of the N-terminal hydrophobic regions. The intact and truncate CamCPRs with N- or C-terminal His6-tag were heterologously overexpressed in Escherichia coli. The recombinant enzymes showed NADPH-dependent reductase activity toward a chemical substrate ferricyanide and a protein substrate cytochrome c. The N-terminal His6-tagged CamCPR showed 18- ~ 30-fold reduction activity higher than the C-terminal His6-tagged CamCPR, which supported a reported conclusion, i.e., the last C-terminal tryptophan of CPRs plays an important role in the discrimination between NADPH and NADH. Co-expression of CamCPR and a P450 monooxygenase, CYP73A25, a cinnamate 4-hydroxylase from cotton, and the following catalytic formation of p-coumaric acid suggested that CamCPR transforms electrons from NADPH to the heme center of P450 to support its oxidation reaction. Quantitative real-time PCR analysis showed that

  5. Molecular Cloning and Characterization of Novel Morus alba Germin-Like Protein Gene Which Encodes for a Silkworm Gut Digestion-Resistant Antimicrobial Protein

    PubMed Central

    Patnaik, Bharat Bhusan; Kim, Dong Hyun; Oh, Seung Han; Song, Yong-Su; Chanh, Nguyen Dang Minh; Kim, Jong Sun; Jung, Woo-jin; Saha, Atul Kumar; Bindroo, Bharat Bhushan; Han, Yeon Soo

    2012-01-01

    Background Silkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances) and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens. Methodology/Principal Findings Silkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4), at a temperature of 60°C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC). SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC). The activity of the purified protein was tested against selected Gram +/− bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS) and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp). In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5′- and 3′-rapid amplification of cDNA ends (RACE-PCR). The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps) involved in plant development and defense. Conclusions/Significance The study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was found

  6. Molecular cloning and identification of a flavanone 3-hydroxylase gene from Lycium chinense, and its overexpression enhances drought stress in tobacco.

    PubMed

    Song, Xinyu; Diao, Jinjin; Ji, Jing; Wang, Gang; Guan, Chunfeng; Jin, Chao; Wang, Yurong

    2016-01-01

    Flavonoids, as plant secondary metabolites, are widespread throughout the plant kingdom and involved in many physiological and biochemical processes. Drought resistance is attributed to flavonoids with respect to protective functions in the cell wall and membranes. The flavanone 3-hydroxylase (F3H) gene which encodes flavanone 3-hydroxylase, is essential in flavonoids biosynthetic pathway. Lycium chinense (L. chinense) is a deciduous woody perennial halophyte that grows under a large variety of environmental conditions and survives under extreme drought stress. A novel cDNA sequence coding a F3H gene in Lycium chinense (LcF3H, GenBank: KJ636468.1) was isolated. The open reading frame of LcF3H comprised 1101 bp encoding a polypeptide of 366 amino acids with a molecular weight of about 42 kDa and an isoelectric point of 5.32. The deduced LcF3H protein showed high identities with other plant F3Hs, and the conserved motifs were found in LcF3H at similar positions like other F3Hs. The recombinant protein converted naringen into dihydrokaempferol in vitro. Since studies have shown that amongst flavonoids, flavan-3-ols (catechin and epicatechin) have direct free radical scavenging activity to maintain the normal physiological function of cells in vivo, these data support the possible relationship between the oxidative damage and the regulation of LcF3H gene expression in L. chinense under drought stress. In order to better understand the biotechnological potential of LcF3H, gene overexpression was conducted in tobacco. The content of flavan-3-ols and the tolerance to drought stress were increased in LcF3H overexpressing tobacco. Analysis of transgenic tobacco lines also showed that antioxidant enzyme activities were increased meanwhile the malondialdehyde (MDA) content and the content of H2O2 were reduced comparing to nontransformed tobacco plants. Furthermore, the photosynthesis rate was less decreased in the transgenetic plants. These results suggest that LcF3H

  7. Cas9-Assisted Targeting of CHromosome segments CATCH enables one-step targeted cloning of large gene clusters

    PubMed Central

    Jiang, Wenjun; Zhao, Xuejin; Gabrieli, Tslil; Lou, Chunbo; Ebenstein, Yuval; Zhu, Ting F.

    2015-01-01

    The cloning of long DNA segments, especially those containing large gene clusters, is of particular importance to synthetic and chemical biology efforts for engineering organisms. While cloning has been a defining tool in molecular biology, the cloning of long genome segments has been challenging. Here we describe a technique that allows the targeted cloning of near-arbitrary, long bacterial genomic sequences of up to 100 kb to be accomplished in a single step. The target genome segment is excised from bacterial chromosomes in vitro by the RNA-guided Cas9 nuclease at two designated loci, and ligated to the cloning vector by Gibson assembly. This technique can be an effective molecular tool for the targeted cloning of large gene clusters that are often expensive to synthesize by gene synthesis or difficult to obtain directly by traditional PCR and restriction-enzyme-based methods. PMID:26323354

  8. Human retinoblastoma susceptibility gene: cloning, identification, and sequence

    SciTech Connect

    Lee, W.; Bookstein, R.; Hong, F.; Young, L.; Shew, J.; Lee, E.Y.P.

    1987-03-13

    Recent evidence indicates the existence of a genetic locus in chromosome region 13q14 that confers susceptibility to retinoblastoma, a cancer of the eye in children. A gene encoding a messenger RNA of 4.6 kilobases (kb), located in the proximity of esterase D, was identified as the retinoblastoma susceptibility (RB) gene on the basis of chromosomal location, homozygous deletion, and tumor-specific alterations in expression. Transcription of this gene was abnormal in six of six retinoblastomas examined: in two tumors, RB mRNA was not detectable, while four others expressed variable quantities of RB mRNA with decreased molecular size of about 4.0 kb. In contrast, full-length RB mRNA was present in human fetal retina and placenta, and in other tumors such as neuroblastoma and medulloblastoma. DNA from retinoblastoma cells had a homozygous gene deletion in one case and hemizygous deletion in another case, while the remainder were not grossly different from normal human control DNA. The gene contains at least 12 exons distributed in a region of over 100 kb. Sequence analysis of complementary DNA clones yielded a single long open reading frame that could encode a hypothetical protein of 816 amino acids.

  9. Molecular cloning and expression analysis of the retinoid X receptor (RXR) gene in golden pompano Trachinotus ovatus fed Artemia nauplii with different enrichments.

    PubMed

    Yang, Qibin; Zheng, Panlong; Ma, Zhenhua; Li, Tao; Jiang, Shigui; Qin, Jian G

    2015-12-01

    The retinoid X receptors (RXRs) are involved in the skeletal development and other biological process such as blood vessel formation and metabolism. Partial sequences of RXRα and β genes were obtained, and their expressions were quantified on golden pompano Trachinotus ovatus at 28 days post hatching (DPH) to explore the molecular response to nutritional manipulation in fish larvae. As live food, Artemia nauplii were separately enriched with Nannochloropsis and Algamac 3080 and non-enriched Artemia nauplii (control) for fish feeding. The expressions of RXRs were detected in the embryos and fish larvae at early stages, suggesting that the skeletal development in golden pompano initiated before yolk re-sorption completion. Fish fed non-enriched Artemia nauplii ended up with higher jaw malformation. The highest specific growth rate was obtained when fish were fed with the Artemia nauplii enriched with Algamac 3080, and the lowest growth rate was observed when fish were fed with unenriched Artemia nauplii. The highest survival was obtained when fish were fed with non-enriched or Nannochloropsis-enriched Artemia nauplii. This study indicates that the use of enriched formula for Artemia nauplii can significantly affect the expression levels of RXRs and jaw malformation of golden pompano larvae, but there is no clear correlation between RXRs expressions and malformation rates when fish are subjected to nutrient challenge. PMID:26159320

  10. Molecular cloning and characterization of l-methionine γ-lyase from Streptomyces avermitilis.

    PubMed

    Kudou, Daizou; Yasuda, Eri; Hirai, Yoshiyuki; Tamura, Takashi; Inagaki, Kenji

    2015-10-01

    A pyridoxal 5'-phosphate-dependent methionine γ-lyase (MGL) was cloned from Streptomyces avermitilis catalyzed the degradation of methionine to α-ketobutyrate, methanethiol, and ammonia. The sav7062 gene (1,242 bp) was corresponded to 413 amino acid residues with a molecular mass of 42,994 Da. The deduced amino acid sequence showed a high degree of similarity to those of other MGL enzymes. The sav7062 gene was overexpressed in Escherichia coli. The enzyme was purified to homogeneity and exhibited the MGL catalytic activities. We cloned the enzyme that has the MGL activity in Streptomyces for the first time. PMID:25817696

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

    PubMed

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

    1998-12-11

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

  12. Cloning and expression of a gene segment encoding the enzymatic moiety of Pseudomonas aeruginosa exotoxin A.

    PubMed Central

    Mozola, M A; Wilson, R B; Jordan, E M; Draper, R K; Clowes, R C

    1984-01-01

    Using the broad-host-range plasmid vector pRO1614, we cloned a segment of the gene from Pseudomonas aeruginosa PA103 encoding the enzymatically active part of the exotoxin A protein. Expression of the cloned gene segment has been achieved both in Escherichia coli and in a nontoxigenic P. aeruginosa host, as assayed by the production of exotoxin A-related antigen and by the ability of the gene product to ADP-ribosylate elongation factor 2. Western blot hybridization analysis revealed a series of polypeptides antigenically related to exotoxin A, the largest of which had a molecular weight of ca. 50,000. Images PMID:6086583

  13. Molecular cloning, sequencing, and expression of omp-40, the gene coding for the major outer membrane protein from the acidophilic bacterium Thiobacillus ferrooxidans.

    PubMed

    Guiliani, N; Jerez, C A

    2000-06-01

    Thiobacillus ferrooxidans is one of the chemolithoautotrophic bacteria important in industrial biomining operations. Some of the surface components of this microorganism are probably involved in adaptation to their acidic environment and in bacterium-mineral interactions. We have isolated and characterized omp40, the gene coding for the major outer membrane protein from T. ferrooxidans. The deduced amino acid sequence of the Omp40 protein has 382 amino acids and a calculated molecular weight of 40,095.7. Omp40 forms an oligomeric structure of about 120 kDa that dissociates into the monomer (40 kDa) by heating in the presence of sodium dodecyl sulfate. The degree of identity of Omp40 amino acid sequence to porins from enterobacteria was only 22%. Nevertheless, multiple alignments of this sequence with those from several OmpC porins showed several important features conserved in the T. ferrooxidans surface protein, such as the approximate locations of 16 transmembrane beta strands, eight loops, including a large external L3 loop, and eight turns which allowed us to propose a putative 16-stranded beta-barrel porin structure for the protein. These results together with the previously known capacity of Omp40 to form ion channels in planar lipid bilayers strongly support its role as a porin in this chemolithoautotrophic acidophilic microorganism. Some characteristics of the Omp40 protein, such as the presence of a putative L3 loop with an estimated isoelectric point of 7.21 allow us to speculate that this can be the result of an adaptation of the acidophilic T. ferrooxidans to prevent free movement of protons across its outer membrane. PMID:10831405

  14. The toxin-coregulated pilus (TCP) of Vibrio cholerae: molecular cloning of genes involved in pilus biosynthesis and evaluation of TCP as a protective antigen in the infant mouse model.

    PubMed

    Sharma, D P; Stroeher, U H; Thomas, C J; Manning, P A; Attridge, S R

    1989-12-01

    A serum containing antibodies to non-lipopolysaccharide (non-LPS) protective antigens of Vibrio cholerae has been used, after extensive absorption, to facilitate the cloning of genes involved in the synthesis of toxin-coregulated pili (TCP). A gene bank was constructed from V. cholerae Z17561 DNA using a mobilizable cosmid vector in Escherichia coli, and subsequently transferred by conjugation into V. cholerae O17. This strain does not produce TCP in vitro and lacks non-LPS protective antigens. Eight positive clones were isolated, and of these, four produced TCP as determined by electron microscopic and immunoblotting analyses. TCP-positive O17 clones were 70-fold more virulent than TCP-negative clones or O17 in the infant mouse cholera model. Only the former could remove protective antibodies from the clone-probing serum by absorption. As a corollary, serum containing antibodies to TCP protected mice from challenge with TCP-positive clones, but not with TCP-negative clones or O17. Our data indicate that TCP can function as both a virulence determinant and a protective antigen in the infant mouse model. PMID:2576091

  15. Molecular Cloning of Endo-β-d-1,4-Glucanase Genes, rce1, rce2, and rce3, from Rhizopus oryzae

    PubMed Central

    Moriya, Tatsuki; Murashima, Koichiro; Nakane, Akitaka; Yanai, Koji; Sumida, Naomi; Koga, Jinichiro; Murakami, Takeshi; Kono, Toshiaki

    2003-01-01

    Three endoglucanase genes, designated the rce1, rce2, and rce3 genes, were isolated from Rhizopus oryzae as the first cellulase genes from the subdivision Zygomycota. All the amino acid sequences deduced from the rce1, rce2, and rce3 genes consisted of three distinct domains: cellulose binding domains, linker domains, and catalytic domains belonging to glycosyl hydrolase family 45. The rce3 gene had two tandem repeated sequences of cellulose binding domains, while rce1 and rce2 had only one. rce1, rce2, and rce3 had various lengths of linker sequences. PMID:12591897

  16. Molecular cloning and characterization of canine ICOS.

    PubMed

    Lee, Je-Hwan; Joo, Young-Don; Yim, Daesong; Lee, Richard; Ostrander, Elaine A; Loretz, Carol; Little, Marie-Térèse; Storb, Rainer; Kuhr, Christian S

    2004-10-01

    Inducible costimulatory receptor (ICOS) is one recently identified member of the CD28 family of costimulatory molecules. Evidence suggests ICOS functions as a critical immune regulator and, to evaluate these effects, we employed the canine model system that has been used to develop strategies currently in clinical use for hematopoietic stem cell transplantation. To investigate the effects of blocking the ICOS pathway in the canine hematopoietic cell transplantation model, we tested existing murine and human reagents and cloned the full length of the open reading frame of canine ICOS cDNA to allow the development of reagents specific for the canine ICOS. Canine ICOS contains a major open reading frame of 624 nucleotides, encoding a protein of 208 amino acids, and localizes to chromosome 37. Canine ICOS shares 79% sequence identity with human ICOS, 70% with mouse, and 69% with rat. Canine ICOS expression is limited to stimulated PBMC. PMID:15475250

  17. Fluorescence in situ hybridization of 16S rRNA gene clones (Clone-FISH) for probe validation and screening of clone libraries.

    PubMed

    Schramm, Andreas; Fuchs, Bernhard M; Nielsen, Jeppe L; Tonolla, Mauro; Stahl, David A

    2002-11-01

    A method is presented for fluorescence in situ hybridization (FISH) of 16S rRNA gene clones targeting in vivo transcribed plasmid inserts (Clone-FISH). Several different cloning approaches and treatments to generate target-rRNA in the clones were compared. Highest signal intensities of Clone-FISH were obtained using plasmids with a T7 RNA polymerase promoter and host cells with an IPTG-inducible T7 RNA polymerase. Combined IPTG-induction and chloramphenicol treatment of those clones resulted in FISH signals up to 2.8-fold higher than signals of FISH with probe EUB338 to cells of Escherichia coli. Probe dissociation curves for three oligonucleotide probes were compared for reference cells containing native (FISH) or cloned (Clone-FISH) target sequences. Melting behaviour and calculated T(d) values were virtually identical for clones and cells, providing a format to use 16S rRNA gene clones instead of pure cultures for probe validation and optimization of hybridization conditions. The optimized Clone-FISH protocol was also used to screen an environmental clone library for insert sequences of interest. In this application format, 13 out of 82 clones examined were identified to contain sulphate-reducing bacterial rRNA genes. In summary, Clone-FISH is a simple and fast technique, compatible with a wide variety of cloning vectors and hosts, that should have general utility for probe validation and screening of clone libraries. PMID:12460279

  18. Cloning and phylogenetic analysis of polyphenol oxidase genes in common wheat and related species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cloning and phylogenetic analysis of polyphenol oxidase (PPO) genes in common wheat and its relatives would greatly advance the understanding of molecular mechanisms of grain PPO activity. In the present study, six wheat relative species, including T. urartu, T. boeoticum, T. monococcum, T. dicoccoi...

  19. Molecular cloning of three pyranose dehydrogenase-encoding genes from Agaricus meleagris and analysis of their expression by real-time RT-PCR.

    PubMed

    Kittl, Roman; Sygmund, Christoph; Halada, Petr; Volc, Jindrich; Divne, Christina; Haltrich, Dietmar; Peterbauer, Clemens K

    2008-02-01

    Sugar oxidoreductases such as cellobiose dehydrogenase or pyranose oxidase are widespread enzymes among fungi, whose biological function is largely speculative. We investigated a similar gene family in the mushroom Agaricus meleagris and its expression under various conditions. Three genes (named pdh1, pdh2 and pdh3) putatively encoding pyranose dehydrogenases were isolated. All three genes displayed a conserved structure and organization, and the respective cDNAs contained ORFs translating into polypeptides of 602 or 600 amino acids. The N-terminal sections of all three genes encode putative signal peptides consistent with the enzymes extracellular secretion. We cultivated the fungus on different carbon sources and analyzed the mRNA levels of all three genes over a period of several weeks using real-time RT-PCR. The glyceraldehyde-3-phosphate dehydrogenase gene from A. meleagris was also isolated and served as reference gene. pdh2 and pdh3 are essentially transcribed constitutively, whereas pdh1 expression is upregulated upon exhaustion of the carbon source; pdh1 appears to be additionally regulated under conditions of oxygen limitation. These data are consistent with an assumed role in lignocellulose degradation. PMID:18097667

  20. Rapid Molecular Characterization of Acinetobacter baumannii Clones with rep-PCR and Evaluation of Carbapenemase Genes by New Multiplex PCR in Hospital District of Helsinki and Uusimaa

    PubMed Central

    Pasanen, Tanja; Koskela, Suvi; Mero, Sointu; Tarkka, Eveliina; Tissari, Päivi; Vaara, Martti; Kirveskari, Juha

    2014-01-01

    Multidrug-resistant Acinetobacter baumannii (MDRAB) is an increasing problem worldwide. Prevalence of carbapenem resistance in Acinetobacter spp. due to acquired carbapenemase genes is not known in Finland. The purpose of this study was to examine prevalence and clonal spread of multiresistant A. baumannii group species, and their carbapenemase genes. A total of 55 Acinetobacter isolates were evaluated with repetitive PCR (DiversiLab) to analyse clonality of isolates, in conjunction with antimicrobial susceptibility profile for ampicillin/sulbactam, colistin, imipenem, meropenem, rifampicin and tigecycline. In addition, a new real-time PCR assay, detecting most clinically important carbapenemase genes just in two multiplex reactions, was developed. The assay detects genes for KPC, VIM, IMP, GES-1/-10, OXA-48, NDM, GIM-1, SPM-1, IMI/NMC-A, SME, CMY-10, SFC-1, SIM-1, OXA-23-like, OXA-24/40-like, OXA-58 and ISAbaI-OXA-51-like junction, and allows confident detection of isolates harbouring acquired carbapenemase genes. There was a time-dependent, clonal spread of multiresistant A. baumannii strongly correlating with carbapenamase gene profile, at least in this geographically restricted study material. The new carbapenemase screening assay was able to detect all the genes correctly suggesting it might be suitable for epidemiologic screening purposes in clinical laboratories. PMID:24465749

  1. Molecular biological enhancement of coal desulfurization: Cloning and expression of the sulfoxide/sulfone/sulfonate/sulfate genes in Pseudomonads and Thiobacillae

    SciTech Connect

    Krawiec, S.

    1991-08-30

    The DbtS{sup +} phenotype is defined as the selective ability to oxidize the sulfur in dibenzothiophene (DBT) successively to dibenzothiophene-5-oxide, dibenzosulfone, and, finally, either o, o'-biphenol or monohydroxybiphenyl. By using a fluorescent assay, many Pseudomonas putida isolates having a DbtS{sup +} phenotype have been obtained. The ability of the isolates to generate o, o'-biphenol was confirmed with HPLC shortly after the time of isolation. The broad-host-range plasmid, R68.45, was introduced from P. putida PRS 2003 into many soil isolates. The plasmid was able to mobilize the determinants for the DbtS{sup +} phenotype. Accordingly, R68.45 and the determinants of the phenotype could be transferred simultaneously form soil isolates to P. aeruginosa 27853. The DbtS{sup +} phenotype in the isolates and in P. aeruginosa 27853 has proven to be unstable. Whether the instability is genetic, physiological, some combination of these two, or is founded on some other phenomenon is not known. Fresh Gram-positive isolates with the DbtS{sup +} phenotype have been isolated using the sulfur bioavailability assay. The DbtS{sup +} phenotype in these isolates appears to be stable. The product of desulfurization of DBT of dibenzosulfone is monohydroxybiphenyl. The nature of the endproduct has been confirmed by HPLC, colorimetry, GC/mass spectroscopy, and UV absorption. The kinetics of monohydroxybiphenyl production are being studied in batch and continuous culture. Study of the basis of cloning with R68.45 has continued. Data regarding in vivo cloning with R68.45 will be important when the genetic determinants for the DbtS{sup +} phenotype must be moved from one species to another by natural processes'' rather than through methods of genetic engineering. 8 refs., 3 figs.

  2. Molecular cloning, sequence identification and tissue expression profile of three novel genes Sfxn1, Snai2 and Cno from Black-boned sheep (Ovis aries).

    PubMed

    Xi, Dongmei; He, Yiduo; Sun, Yongke; Gou, Xiao; Yang, Shuli; Mao, Huaming; Deng, Weidong

    2011-03-01

    The complete coding sequences of three of Black-boned sheep (Ovis aries) genes Sfxn1, Snai2 and Cno were amplified using the reverse transcriptase polymerase chain reaction (RT-PCR) according to the conserved sequence information of the cattle or other mammals and known highly homologous sheep ESTs. Black-boned sheep Sfxn1 gene encodes a protein of 322 amino acids which has high homology with the Sfxn1 proteins of five species--cattle 98%, pig 95%, human 95%, rat 93%, and mouse 93%. Black-boned sheep Snai2 gene encodes a protein of 268 amino acids that has high identity with the Snai2 proteins of six species--cattle 99%, pig 94%, human 93%, dog 93%, rat 91%, and mouse 90%. Black-boned sheep Cno gene encodes a protein of 214 amino acids that has high homology with the Cno proteins of four species--cattle 97%, human 75%, mouse 67%, and rat 65%. The phylogenetic tree analysis demonstrated that Black-boned sheep Sfxn1, Snai2 and Cno proteins have close relationship with cattle Sfxn1, Snai2 and Cno proteins. The tissue expression analysis indicated that Black-boned sheep Sfxn1, Snai2 and Cno genes were expressed in a range of tissues including leg muscle, kidney, skin, longissimus dorsi muscle, spleen, heart and liver. Our experiment is the first to provide the primary foundation for further insight into these three sheep genes. PMID:20853147

  3. Evolution of the thioester-containing proteins (TEPs) of the arthropoda, revealed by molecular cloning of TEP genes from a spider, Hasarius adansoni.

    PubMed

    Sekiguchi, Reo; Fujito, Naoko T; Nonaka, Masaru

    2012-02-01

    The thioester-containing protein (TEP) family of genes, found in most Eumetazoan genomes, is classified into two subfamilies: the alpha-2-macroglobulin (A2M) subfamily and the C3 subfamily. Many A2M subfamily members, including insect TEP (iTEP), have been reported from the Arthropoda, whereas the C3 subfamily members have been reported only from two horseshoe crab species thus far. To elucidate the evolution of these genes among the Arthropoda, TEP genes were isolated from a spider, Hasarius adansoni (Chelicerata), by reverse transcription polymerase chain reaction (RT-PCR) amplification using universal degenerate primers specific for the thioester region. Four different TEP genes were identified. Phylogenetic analysis using the entire amino acid sequences of these and various other TEP sequences from the Eumetazoa indicated that two of the spider genes are type C3 (HaadC3-1 and HaadC3-2), one is type A2M (HaadA2M) and the other is closely related to iTEP (HaadiTEP). These results suggest that the common ancestor of the Arthropoda possessed at least three TEP genes, C3, A2M and iTEP and that they were lost differentially in the Crustacean and Hexapodan lineages. PMID:21663759

  4. Express primer tool for high-throughput gene cloning and expression.

    SciTech Connect

    Yoon, J. R.; Laible, P. D.; Gu, M.; Scott, H. N.; Collart, F. R.; Biosciences Division

    2002-12-01

    High-throughput approaches for gene cloning and expression require the development of new nonstandard tools for molecular biologists and biochemists. We introduce a Web-based tool to design primers specifically for the generation of expression clones for both laboratory-scale and high-throughput projects. The application is designed not only to allow the user complete flexibility to specify primer design parameters but also to minimize the amount of manual intervention needed to generate a large number of primers for the simultaneous amplification of multiple target genes.

  5. Molecular cloning and functional characterization of avian interleukin-19

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The present study describes the cloning and functional characterization of avian interleukin (IL)-19, a cytokine that, in mammals, alters the balance of Th1 and Th2 cells in favor of the Th2 phenotype. The full-length avian IL-19 gene, located on chromosome 26, was amplified from LPS-stimulated chi...

  6. Elevating crop disease resistance with cloned genes

    PubMed Central

    Jones, Jonathan D. G.; Witek, Kamil; Verweij, Walter; Jupe, Florian; Cooke, David; Dorling, Stephen; Tomlinson, Laurence; Smoker, Matthew; Perkins, Sara; Foster, Simon

    2014-01-01

    Essentially all plant species exhibit heritable genetic variation for resistance to a variety of plant diseases caused by fungi, bacteria, oomycetes or viruses. Disease losses in crop monocultures are already significant, and would be greater but for applications of disease-controlling agrichemicals. For sustainable intensification of crop production, we argue that disease control should as far as possible be achieved using genetics rather than using costly recurrent chemical sprays. The latter imply CO2 emissions from diesel fuel and potential soil compaction from tractor journeys. Great progress has been made in the past 25 years in our understanding of the molecular basis of plant disease resistance mechanisms, and of how pathogens circumvent them. These insights can inform more sophisticated approaches to elevating disease resistance in crops that help us tip the evolutionary balance in favour of the crop and away from the pathogen. We illustrate this theme with an account of a genetically modified (GM) blight-resistant potato trial in Norwich, using the Rpi-vnt1.1 gene isolated from a wild relative of potato, Solanum venturii, and introduced by GM methods into the potato variety Desiree. PMID:24535396

  7. Elevating crop disease resistance with cloned genes.

    PubMed

    Jones, Jonathan D G; Witek, Kamil; Verweij, Walter; Jupe, Florian; Cooke, David; Dorling, Stephen; Tomlinson, Laurence; Smoker, Matthew; Perkins, Sara; Foster, Simon

    2014-04-01

    Essentially all plant species exhibit heritable genetic variation for resistance to a variety of plant diseases caused by fungi, bacteria, oomycetes or viruses. Disease losses in crop monocultures are already significant, and would be greater but for applications of disease-controlling agrichemicals. For sustainable intensification of crop production, we argue that disease control should as far as possible be achieved using genetics rather than using costly recurrent chemical sprays. The latter imply CO₂ emissions from diesel fuel and potential soil compaction from tractor journeys. Great progress has been made in the past 25 years in our understanding of the molecular basis of plant disease resistance mechanisms, and of how pathogens circumvent them. These insights can inform more sophisticated approaches to elevating disease resistance in crops that help us tip the evolutionary balance in favour of the crop and away from the pathogen. We illustrate this theme with an account of a genetically modified (GM) blight-resistant potato trial in Norwich, using the Rpi-vnt1.1 gene isolated from a wild relative of potato, Solanum venturii, and introduced by GM methods into the potato variety Desiree. PMID:24535396

  8. Molecular cloning of Renibacterium salmoninarum DNA fragments.

    PubMed

    Etchegaray, J P; Martínez, M A; Krauskopf, M; León, G

    1991-03-15

    A Renibacterium salmoninarum enriched recombinant DNA library was constructed to isolate DNA fragments which could be used as probes to detect gene sequences specific for the causative agent of bacterial kidney disease in salmonid fish. One fragment of 149 base pairs was isolated and its specificity and sequence determined. This probe may prove useful in the design of diagnostic tests for the disease in asymptomatic fish and ova. PMID:2044941

  9. Map-Based Cloning of Genes Important for Maize Anther Development

    NASA Astrophysics Data System (ADS)

    Anaya, Y.; Walbot, V.; Nan, G.

    2012-12-01

    Map-Based cloning for maize mutant MS13 . Scientists still do not understand what decides the fate of a cell in plants. Many maize genes are important for anther development and when they are disrupted, the anthers do not shed pollen, i.e. male sterile. Since the maize genome has been fully sequenced, we conduct map-based cloning using a bulk segregant analysis strategy. Using PCR (polymerase chain reaction), we look for biomarkers that are linked to our gene of interest, Male Sterile 13 (MS13). Recombinations occur more often if the biomarkers are further away from the gene, therefore we can estimate where the gene is and design more PCR primers to get closer to our gene. Genetic and molecular analysis will help distinguish the role of key genes in setting cell fates before meiosis and for being in charge of the switch from mitosis to meiosis.

  10. Cloning, characterization, and sequence of the yeast DNA topoisomerase I gene.

    PubMed Central

    Thrash, C; Bankier, A T; Barrell, B G; Sternglanz, R

    1985-01-01

    The structural gene for yeast DNA topoisomerase I (TOP1) has been cloned from two yeast genomic plasmid banks. Integration of a plasmid carrying the gene into the chromosome and subsequent genetic mapping shows that TOP1 is identical to the gene previously called MAK1. Seven top1 (mak1) mutants including gene disruptions are viable, demonstrating that DNA topoisomerase I is not essential for viability in yeast. A 3787-base-pair DNA fragment including the gene has been sequenced. The protein predicted from the DNA sequence has 769 amino acids and a molecular weight of 90,020. Images PMID:2989818

  11. Molecular cloning and characterization of amh and dax1 genes and their expression during sex inversion in rice-field eel Monopterus albus

    PubMed Central

    Hu, Qing; Guo, Wei; Gao, Yu; Tang, Rong; Li, Dapeng

    2015-01-01

    The full-length cDNAs of amh and dax1 in the hermaphrodite, rice-field eel (Monopterus albus), were cloned and characterized in this study. Multiple sequence alignment revealed Dax1 was well conserved among vertebrates, whereas Amh had a low degree of similarity between different vertebrates. Their expression profiles in gonads during the course of sex inversion and tissues were investigated. The tissue distribution indicated amh was expressed mostly in gonads and was scarcely detectable in other tissues, whereas the expression of dax1 was widespread among the different tissues, especially liver and gonads. amh was scarcely detectable in ovaries whereas it was abundantly expressed in both ovotestis and testis. By contrast, dax1 was highly expressed in ovaries, especially in ♀IV (ovaries in IV stage), but it was decreased significantly in ♀/♂I (ovotestis in I stage). Its expression was increased again in ♀/♂III (ovotestis in III stage), and then decreased to a low level in testis. These significant different expression patterns of amh and dax1 suggest the increase of amh expression and the decline of dax1 expression are important for the activation of testis development, and the high level of amh and a low level of dax1 expression are necessary for maintenance of testis function. PMID:26578091

  12. Molecular cloning and characterization of amh and dax1 genes and their expression during sex inversion in rice-field eel Monopterus albus.

    PubMed

    Hu, Qing; Guo, Wei; Gao, Yu; Tang, Rong; Li, Dapeng

    2015-01-01

    The full-length cDNAs of amh and dax1 in the hermaphrodite, rice-field eel (Monopterus albus), were cloned and characterized in this study. Multiple sequence alignment revealed Dax1 was well conserved among vertebrates, whereas Amh had a low degree of similarity between different vertebrates. Their expression profiles in gonads during the course of sex inversion and tissues were investigated. The tissue distribution indicated amh was expressed mostly in gonads and was scarcely detectable in other tissues, whereas the expression of dax1 was widespread among the different tissues, especially liver and gonads. amh was scarcely detectable in ovaries whereas it was abundantly expressed in both ovotestis and testis. By contrast, dax1 was highly expressed in ovaries, especially in ♀IV (ovaries in IV stage), but it was decreased significantly in ♀/♂I (ovotestis in I stage). Its expression was increased again in ♀/♂III (ovotestis in III stage), and then decreased to a low level in testis. These significant different expression patterns of amh and dax1 suggest the increase of amh expression and the decline of dax1 expression are important for the activation of testis development, and the high level of amh and a low level of dax1 expression are necessary for maintenance of testis function. PMID:26578091

  13. Positional Cloning and Characterization of AltSB, a Major Aluminum Tolerance Gene in Sorghum: Toward the Identification of the Molecular and Physiological basis of Allelic effects

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aluminum toxicity is a major constraint for agriculture on acid soils, which comprise over half of the world’s potentially arable lands. However, the molecular basis underlying the most accepted tolerance mechanism based on Al-induced organic acid release by root apices, is only now being elucidate...

  14. Molecular cloning, characterization and expression analysis of TLR9, MyD88 and TRAF6 genes in common carp (Cyprinus carpio)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Induction of innate immune pathways is critical for early host defense but there is limited understanding of how teleost fish recognize pathogen molecules and activate these pathways. In mammals, cells of the innate immune system detect pathogenic molecular structures using pattern recognition rece...

  15. Molecular cloning and sequence analysis of the Sta58 major antigen gene of Rickettsia tsutsugamushi: sequence homology and antigenic comparison of Sta58 to the 60-kilodalton family of stress proteins.

    PubMed Central

    Stover, C K; Marana, D P; Dasch, G A; Oaks, E V

    1990-01-01

    The scrub typhus 58-kilodalton (kDa) antigen (Sta58) of Rickettsia tsutsugamushi is a major protein antigen often recognized by humans infected with scrub typhus rickettsiae. A 2.9-kilobase HindIII fragment containing a complete sta58 gene was cloned in Escherichia coli and found to express the entire Sta58 antigen and a smaller protein with an apparent molecular mass of 11 kDa (Stp11). DNA sequence analysis of the 2.9-kilobase HindIII fragment revealed two adjacent open reading frames encoding proteins of 11 (Stp11) and 60 (Sta58) kDa. Comparisons of deduced amino acid sequences disclosed a high degree of homology between the R. tsutsugamushi proteins Stp11 and Sta58 and the E. coli proteins GroES and GroEL, respectively, and the family of primordial heat shock proteins designated Hsp10 Hsp60. Although the sequence homology between the Sta58 antigen and the Hsp60 protein family is striking, the Sta58 protein appeared to be antigenically distinct among a sample of other bacterial Hsp60 homologs, including the typhus group of rickettsiae. The antigenic uniqueness of the Sta58 antigen indicates that this protein may be a potentially protective antigen and a useful diagnostic reagent for scrub typhus fever. Images PMID:2108930

  16. Molecular Cloning and Functional Analysis of Gene Clusters for the Biosynthesis of Indole-Diterpenes in Penicillium crustosum and P. janthinellum

    PubMed Central

    Nicholson, Matthew J.; Eaton, Carla J.; Stärkel, Cornelia; Tapper, Brian A.; Cox, Murray P.; Scott, Barry

    2015-01-01

    The penitremane and janthitremane families of indole-diterpenes are abundant natural products synthesized by Penicillium crustosum and P. janthinellum. Using a combination of PCR, cosmid library screening, and Illumina sequencing we have identified gene clusters encoding enzymes for the synthesis of these compounds. Targeted deletion of penP in P. crustosum abolished the synthesis of penitrems A, B, D, E, and F, and led to accumulation of paspaline, a key intermediate for paxilline biosynthesis in P. paxilli. Similarly, deletion of janP and janD in P. janthinellum abolished the synthesis of prenyl-elaborated indole-diterpenes, and led to accumulation in the latter of 13-desoxypaxilline, a key intermediate for the synthesis of the structurally related aflatremanes synthesized by Aspergillus flavus. This study helps resolve the genetic basis for the complexity of indole-diterpene natural products found within the Penicillium and Aspergillus species. All indole-diterpene gene clusters identified to date have a core set of genes for the synthesis of paspaline and a suite of genes encoding multi-functional cytochrome P450 monooxygenases, FAD dependent monooxygenases, and prenyl transferases that catalyse various regio- and stereo- specific oxidations that give rise to the diversity of indole-diterpene products synthesized by this group of fungi. PMID:26213965

  17. Molecular cloning of genomic DNA and chromosomal assignment of the gene for human aromatic L-amino acid decarboxylase, the enzyme for catecholamine and serotonin biosynthesis

    SciTech Connect

    Sumi-Ichinose, Chiho ); Ichinose, Hiroshi; Nagatsu, Toshiharu ); Takahashi, Eiichi; Hori, Tadaaki )

    1992-03-03

    Aromatic L-amino acid decarboxylase (AADC) catalyzes the decarboxylation of both L-3,4-dihydroxyphenylalanine and L-5-hydroxytryptophan to dopamine and serotonin, respectively, which are major mammalian neurotransmitters and hormones belonging to catecholamines and indoleamines. This report describes the organization of the human AADC gene. The authors proved that the gene of human AADC consists of 15 exons spanning more than 85 kilobases and exists as a single copy in the haploid genome. The boundaries between exon and intron followed the AG/GT rule. The sizes of exons and introns ranged from 20 to 400 bp and from 1.0 to 17.7 kb, respectively, while the sizes of four introns were not determined. Untranslated regions located in the 5{prime} region of mRNA were encoded by two exons, exons 1 and 2. The transcriptional starting point was determined around G at position {minus}111 by primer extension and S1 mapping. There were no typical TATA box' and CAAT box' within 540 bp from the transcriptional starting point. The human AADC gene was mapped to chromosome band 7p12.1-p12.3 by fluorescence in situ hybridization. This is the first report on the genomic structure and chromosomal localization of the AADC gene in mammals.

  18. Molecular cloning and expression of PoIR2, a novel gene involved in immune response in Japanese flounder ( Paralichthys olivaceus)

    NASA Astrophysics Data System (ADS)

    Li, Shuo; Li, Chunmei; Wang, Xubo; Wang, Yanan; Liu, Zhipeng; Zhai, Teng; Zhang, Quanqi

    2010-03-01

    A novel immune-related gene was expressed in Japanese flounder ( Paralichthys olivaceus) injected with Vibrio anguillarum. The complete cDNA contained a 169 bp 5’UTR, a 336 bp open reading frame (ORF) encoding 111 amino acids and a 556bp 3’UTR. Six exons and five introns were identified in the PoIR2 gene. Blastp similarity comparison showed its encoding protein had 50% similarity to Danio rerio neuromedin S (NMS), but further alignment indicated they did not have NMS C-terminal conservational signature domain. So it was not defined as an NMS homologue. Protein structure analysis indicated it had a 26aa signal peptide and was a secretory pathway protein. RT-PCR demonstrated that the expression of PoIR2 was quickly induced and drastically increased in liver, kidney, spleen, gills, intestine, heart, and skeletal muscle after infected with V. anguillarum. These results indicated that the PoIR2 might play some important role in Japanese flounder immune response system. This gene was named PoIR2 ( P.olivaceus immune-related gene 2, GenBank accession number: EU224372). The mature PoIR2 peptide was expressed in BL21(DE3) pLysS using pET-32a(+) vector and a great part of the recombinant mature peptide existed as soluble type.

  19. Molecular cloning of NCCRP-1 gene from orange-spotted grouper (Epinephelus coioides) and characterization of NCCRP-1(+) cells post Cryptocaryon irritans infection.

    PubMed

    Huang, Xia-Zi; Li, Yan-Wei; Mai, Yong-Zhan; Luo, Xiao-Chun; Dan, Xue-Ming; Li, An-Xing

    2014-10-01

    Nonspecific cytotoxic cells (NCCs) are an important cytotoxic cell population in the innate teleost immune system. The receptor designated "NCC receptor protein 1" (NCCRP-1) has been reported to be involved in the recognition and activation of NCCs. In this study, the full-length cDNA of Epinephelus coioides NCCRP-1 (ecnccrp-1) was cloned. The open reading frame (ORF) of ecnccrp-1 is 699 bp, encoding a 232 amino acid protein that includes proline-rich motifs at the N-terminus and is related to the F-box associated family. Although a bioinformatics analysis showed that EcNCCRP-1 had no signal peptide or transmembrane helices, a polyclonal antibody directed against recombinant EcNCCRP-1 efficiently labeled a membrane protein in the head kidney, detected with Western blot analysis, which indicated that the protein localized to the cell surface. RT-PCR showed that the constitutive expression of ecnccrp-1 was higher in the lymphoid organs, such as the trunk kidney, spleen, head kidney, and thymus, and lower in brain, heart, fat, liver, muscle, and skin. After infection with Cryptocaryon irritans, the transcription of ecnccrp-1 was analyzed at the infected sites (skin and gills) and in the systemic immune organs (head kidney and spleen). At the infected sites, especially the skin, ecnccrp-1 expression was upregulated at 6h post infection, reaching peak expression on day 3 post the primary infection. However, the expression patterns differed in the systemic immune organs. In the spleen, ecnccrp-1 was gradually increased in the early infection period and decreased sharply on day 3 post the primary infection, whereas in the head kidney, the transcription of ecnccrp-1 was depressed during almost the whole course of infection. An immunohistochemical analysis showed that EcNCCRP-1(+) cells accumulated at the sites of infection with C. irritans. These results suggested that NCCs were involved in the process of C. irritans infection in E. coioides. PMID:24844613

  20. Molecular cloning, characterization and expression analysis of Toll-like receptor 5M gene in Japanese sea perch (Lateolabrax japonicas) after bacterial infection.

    PubMed

    Wang, Chengyang; Zhao, Chao; Fu, Mingjun; Bao, Weiyang; Qiu, Lihua

    2016-09-01

    Toll-like receptor 5M belongs to Toll-like receptors (TLRs) family, which plays a crucial role in innate immunity due to its important role in the recognition of bacteria invasion and in the activation of immune related pathways downstream. In the present study, we firstly cloned the full-length cDNAs of TLR 5M (LjTLR 5M) from Japanese sea perch (Lateolabrax japonicas). The full-length cDNAs of LjTLR 5M include an open reading frame (ORF) of 2676 bp encoding a polypeptide of 891 amino acid residues. The deduced amino acid sequence analysis showed that LiTLR 5M contains LRRs (extracellular leucine rich repeats), transmembrane and TIR (Toll/interleukin-1 receptor) domain. Transcriptional expression analysis indicated that LiTLR 5M mRNAs were ubiquitously expressed in wide array of tissues and the peak level was observed in the head-kidney. The expression patterns of LjTLR 5M after Vibro harveyi and Streptococus agalactiae infection were detected by qRT-PCR, and the results showed that LjTLR 5M was significant up-regulated in spleen, liver and head-kidney. Additionally, the expression patterns of LjTLR 5M in infected spleen and head-kidney were further validated by in situ hybridization (ISH). In summary, these findings indicate that LjTLR 5M is significant induced after different bacterial infection and is involved in immune response. Furthermore, this study will provide foundational information for other TLRs research of L. japonicas against different bacterial pathogens invasion. PMID:27417233

  1. Cloning and characterisation of JAZ gene family in Hevea brasiliensis.

    PubMed

    Hong, H; Xiao, H; Yuan, H; Zhai, J; Huang, X

    2015-05-01

    Mechanical wounding or treatment with exogenous jasmonates (JA) induces differentiation of the laticifer in Hevea brasiliensis. JA is a key signal for latex biosynthesis and wounding response in the rubber tree. Identification of JAZ (jasmonate ZIM-domain) family of proteins that repress JA responses has facilitated rapid progress in understanding how this lipid-derived hormone controls gene expression and related physiological processes in plants. In this work, the full-length cDNAs of six JAZ genes were cloned from H. brasiliensis (termed HbJAZ). These HbJAZ have different lengths and sequence diversity, but all of them contain Jas and ZIM domains, and two of them contain an ERF-associated amphiphilic repression (EAR) motif in the N-terminal. Real-time RT-PCR analyses revealed that HbJAZ have different expression patterns and tissue specificity. Four HbJAZ were up-regulated, one was down-regulated, while two were less effected by rubber tapping treatment, suggesting that they might play distinct roles in the wounding response. A yeast two-hybrid assay revealed that HbJAZ proteins interact with each other to form homologous or heterogeneous dimer complexes, indicating that the HbJAZ proteins may expand their function through diverse JAZ-JAZ interactions. This work lays a foundation for identification of the JA signalling pathway and molecular mechanisms of latex biosynthesis in rubber trees. PMID:25399518

  2. Utilization of a novel deuterostome model for the study of regeneration genetics: molecular cloning of genes that are differentially expressed during early stages of larval sea star regeneration.

    PubMed

    Vickery, M C; Vickery, M S; McClintock, J B; Amsler, C D

    2001-01-10

    Sea stars share many characteristics with vertebrates, including deuterostome type development. We previously reported that sea star larvae are capable of complete regeneration (with organogenesis) of missing body parts. Here we report the first application of whole-body cDNA subtractive hybridization for the identification of regeneration-specific gene expression in a deuterostome. We identified nine novel cDNAs from genes differentially expressed during early larval sea star regeneration, including a serine protease which may have a function similar to that of trypsin/plasmin-like proteases during vertebrate wound repair and regeneration. This study demonstrates that sea star larvae can provide a valuable new deuterostome model for the study of regeneration genetics, with potential applications in vertebrate regeneration. PMID:11179669

  3. Phagmids and genetic engineering: analysis of cloned gene libraries

    SciTech Connect

    Mel'nikov, A.A.; Fodor, I.

    1985-07-01

    Phagmids are bi-replicon DNA molecules which, depending on the conditions, can form phage particles and lyse E. coli cells or be maintained in the cell in the plasmid state on account of a plasmid replicator. The authors suggest a new method for the selection of genes from cloned gene libraries, created on the basis of lambda phage. Phages from individual transparent plaques were reproduced, the DNA isolated, and the structure of the DNA was analyzed using restriction endonucleases and the method of hybridization. The authors used a fragment of the interferon A gene with which recombination was performed, as well as DNA fragments used for hybridization. The main evidence that clones containing interferon genes were selected by this method consists of the fact that recombination in vivo was performed with the 3'-end of the DNA of the interferon A gene, while DNA-DNA hybridization in the clones revealed the 5'-terminal sequences of the DNA of the gene. Hybridization of the EcoRI-BglII fragment of (/sup 32/P)-DNA of interferon A, both isolated from polyacrylamide gel and cloned in the vector M13mp8, showed that in five (lambda I2, I7, I8, I9, I11), of the ten selected phages, there are 5'-terminal fragments of the DNA of the interferon gene. The clones lambda I7, lambda I9, and lambda I11, have the same structure according to the data of restriction and hybridization analyses. The clones lambda I4, lambda I5, and lambda I6, are also identical and hybridize only with the 3'-terminal sequence of interferon A DNA.

  4. An adenine nucleotide translocase (ANT) gene from Apostichopus japonicus; molecular cloning and expression analysis in response to lipopolysaccharide (LPS) challenge and thermal stress.

    PubMed

    Liu, Qiu-Ning; Chai, Xin-Yue; Tu, Jie; Xin, Zhao-Zhe; Li, Chao-Feng; Jiang, Sen-Hao; Zhou, Chun-Lin; Tang, Bo-Ping

    2016-02-01

    The adenine nucleotide translocases (ANTs) play a vital role in energy metabolism via ADP/ATP exchange in eukaryotic cells. Apostichopus japonicus (Echinodermata: Holothuroidea) is an important economic species in China. Here, a cDNA representing an ANT gene of A. japonicus was isolated and characterized from respiratory tree and named AjANT. The full-length AjANT cDNA is 1924 bp, including a 5'-untranslated region (UTR) of 38 bp, 3'-UTR of 980 bp and an open reading frame (ORF) of 906 bp encoding a polypeptide of 301 amino acids. The protein contains three homologous repeat Mito_carr domains (Pfam00153). The deduced AjANT protein sequence has 49-81% in comparison to ANT proteins from other individuals. The predicted tertiary structure of AjANT protein is highly similar to animal ANT proteins. Phylogenetic analysis showed that the AjANT is closely related to Holothuroidea ANT genes. Real-time quantitative reverse transcription-PCR (qPCR) analysis showed that AjANT expression is higher in the respiratory tree than in other examined tissues. After thermal stress or LPS challenge, expression of AjANT was significantly fluctuant compared to the control. These results suggested that changes in the expression of ANT gene might be involved in immune defense and in protecting A. japonicus against thermal stress. PMID:26706223

  5. Molecular cloning and characterization of the protein 4.1O gene, a novel member of the protein 4.1 family with focal expression in ovary.

    PubMed

    Ni, Xiaohua; Ji, Chaoneng; Cao, Gentao; Cheng, Haipeng; Guo, Lingchen; Gu, Shaohua; Ying, Kang; Zhao, Robert C; Mao, Yumin

    2003-01-01

    Protein 4.1 is an important structural protein that is expressed in erythroid and in a variety of nonerythroid tissues. In mammalian erythrocytes, it plays a key role in regulating the physical properties of mechanical stability and deformability in membranes by stabilizing the spectrin-actin interaction. The protein 4.1 family mainly comprises 4.1R, 4.1G (general type), 4.1B (brain type), and 4.1N (neuron type). We identified a novel human 4.1 ( 4.1O) gene that is 2312 bp in length and encodes a protein of 553 amino acid residues. The protein shared homology with mouse protein 4.1B (identity 38%, similarity 55%) with a FERM domain. The expression pattern of the human 4.1O gene in 16 tissues showed that there was a transcript only in ovary, whereas in the remaining 15 tissues, specific bands of the transcript could not be detected. In eight human fetal tissues, the specific bands of the transcript could be detected in skeletal muscle, with lower levels detected in thymus and brain. The 4.1O gene consists of 14 exons and 13 introns and was mapped to Chromosome 9q21-9q22 by bioinformatics analysis. PMID:12601556

  6. Molecular Cloning and Characterization of a Newly Isolated Pyrethroid-Degrading Esterase Gene from a Genomic Library of Ochrobactrum anthropi YZ-1

    PubMed Central

    Song, Jinlong; Shi, Yanhua; Li, Kang; Zhao, Bin; Yan, Yanchun

    2013-01-01

    A novel pyrethroid-degrading esterase gene pytY was isolated from the genomic library of Ochrobactrum anthropi YZ-1. It possesses an open reading frame (ORF) of 897 bp. Blast search showed that its deduced amino acid sequence shares moderate identities (30% to 46%) with most homologous esterases. Phylogenetic analysis revealed that PytY is a member of the esterase VI family. pytY showed very low sequence similarity compared with reported pyrethroid-degrading genes. PytY was expressed, purified, and characterized. Enzyme assay revealed that PytY is a broad-spectrum degrading enzyme that can degrade various pyrethroids. It is a new pyrethroid-degrading gene and enriches genetic resource. Kinetic constants of Km and Vmax were 2.34 mmol·L−1 and 56.33 nmol min−1, respectively, with lambda-cyhalothrin as substrate. PytY displayed good degrading ability and stability over a broad range of temperature and pH. The optimal temperature and pH were of 35°C and 7.5. No cofactors were required for enzyme activity. The results highlighted the potential use of PytY in the elimination of pyrethroid residuals from contaminated environments. PMID:24155944

  7. A simple and rapid strategy for the molecular cloning and monitoring of mouse HtrA2 serine protease.

    PubMed

    Kim, Goo-Young; Nam, Min-Kyung; Kim, Sang-Soo; Kim, Ho-Young; Lee, Sang-Kyu; Rhim, Hyangshuk

    2008-03-01

    A simple and rapid strategy for molecular cloning using a gel-free and antibiotic selection method is described which allows for the complete elimination of DNA extraction by gel electrophoresis, and thus has several advantages over gel-based cloning methods, including: (i) a cloning efficiency that is approximately 10-times higher due to the prevention of ethidium bromide ultraviolet-induced DNA damage and contamination with ligase inhibitors; (ii) the amount of plasmid DNA required is approximately five times less; and (iii) the cloning time is several hours less. Once the target gene, such as mouse HtrA2 serine protease, was cloned into the pEGFP-N3 plasmid, the integrity of the kanamycin-resistant molecular clone encoding the GFP fusion protein was verified by immunoblot and immunofluorescence assays. In addition, the integrity of the ampicillin-resistant molecular clone was directly evaluated by analyzing the expression and affinity purification of the GST fusion protein and by measuring its enzymatic activity. Therefore, this method is suitable for the routine construction of a plasmid expressing the gene of interest, and the usefulness of this strategy can be demonstrated by monitoring the expression of the target gene in E. coli and mammalian cells. PMID:17939055

  8. Molecular cloning and characterization of UDP-glucose: furaneol glucosyltransferase gene from grapevine cultivar Muscat Bailey A (Vitis labrusca × V. vinifera).

    PubMed

    Sasaki, Kanako; Takase, Hideki; Kobayashi, Hironori; Matsuo, Hironori; Takata, Ryoji

    2015-10-01

    2,5-Dimethyl-4-hydroxy-3(2H)-furanone (furaneol) is an important aroma compound in fruits, such as pineapple and strawberry, and is reported to contribute to the strawberry-like note in some wines. Several grapevine species are used in winemaking, and furaneol is one of the characteristic aroma compounds in wines made from American grape (Vitis labrusca) and its hybrid grape. Furaneol glucoside was recently isolated as an important furaneol derivative from the hybrid grapevine cultivar, Muscat Bailey A (V. labrusca × V. vinifera), and this was followed by its isolation from some fruits such as strawberry and tomato. Furaneol glucoside is a significant 'aroma precursor of wine' because furaneol is liberated from it during alcoholic fermentation. In this study, a glucosyltransferase gene from Muscat Bailey A (UGT85K14), which is responsible for the glucosylation of furaneol was identified. UGT85K14 was expressed in the representative grape cultivars regardless of species, indicating that furaneol glucoside content is regulated by the biosynthesis of furaneol. On the other hand, furaneol glucoside content in Muscat Bailey A berry during maturation might be controlled by the expression of UGT85K14 along with the biosynthesis of furaneol. Recombinant UGT85K14 expressed in Escherichia coli is able to transfer a glucose moiety from UDP-glucose to the hydroxy group of furaneol, indicating that this gene might be UDP-glucose: furaneol glucosyltransferase in Muscat Bailey A. PMID:26160581

  9. Molecular cloning and sequence analysis of the gene coding for the 57-kDa major soluble antigen of the salmonid fish pathogen Renibacterium salmoninarum.

    PubMed

    Chien, M S; Gilbert, T L; Huang, C; Landolt, M L; O'Hara, P J; Winton, J R

    1992-09-15

    The complete sequence coding for the 57-kDa major soluble antigen of the salmonid fish pathogen, Renibacterium salmoninarum, was determined. The gene contained an opening reading frame of 1671 nucleotides coding for a protein of 557 amino acids with a calculated M(r) value of 57,190. The first 26 amino acids constituted a signal peptide. The deduced sequence for amino acid residues 27-61 was in agreement with the 35 N-terminal amino acid residues determined by microsequencing, suggesting the protein is synthesized as a 557-amino acid precursor and processed to produce a mature protein of M(r) 54,505. Two regions of the protein contained imperfect direct repeats. The first region contained two copies of an 81-residue repeat, the second contained five copies of an unrelated 25-residue repeat. Also, a perfect inverted repeat (including three in-frame UAA stop codons) was observed at the carboxyl-terminus of the gene. PMID:1383085

  10. Cloning and characterization of the Bacillus licheniformis gene coding for alkaline phosphatase.

    PubMed Central

    Hulett, F M

    1984-01-01

    The structural gene for alkaline phosphatase (orthophosphoric monoester phosphohydrolase; EC 3.1.3.1) of Bacillus licheniformis MC14 was cloned into the Pst1 site of pMK2004 from chromosomal DNA. The gene was cloned on an 8.5-kilobase DNA fragment. A restriction map was developed, and the gene was subcloned on a 4.2-kilobase DNA fragment. The minimum coding region of the gene was localized to a 1.3-kilobase region. Western blot analysis was used to show that the gene coded for a 60,000-molecular-weight protein which cross-reacts with anti-alkaline phosphatase prepared against the salt-extractable membrane alkaline phosphatase of B. licheniformis MC14 . Images PMID:6327655

  11. Cloning and characterization of the Bacillus licheniformis gene coding for alkaline phosphatase.

    PubMed

    Hulett, F M

    1984-06-01

    The structural gene for alkaline phosphatase (orthophosphoric monoester phosphohydrolase; EC 3.1.3.1) of Bacillus licheniformis MC14 was cloned into the Pst1 site of pMK2004 from chromosomal DNA. The gene was cloned on an 8.5-kilobase DNA fragment. A restriction map was developed, and the gene was subcloned on a 4.2-kilobase DNA fragment. The minimum coding region of the gene was localized to a 1.3-kilobase region. Western blot analysis was used to show that the gene coded for a 60,000-molecular-weight protein which cross-reacts with anti-alkaline phosphatase prepared against the salt-extractable membrane alkaline phosphatase of B. licheniformis MC14 . PMID:6327655

  12. Molecular cloning and functional analysis of the fatty acid-binding protein (Sp-FABP) gene in the mud crab (Scylla paramamosain)

    PubMed Central

    Zeng, Xianglan; Ye, Haihui; Yang, Ya’nan; Wang, Guizhong; Huang, Huiyang

    2013-01-01

    Intracellular fatty acid-binding proteins (FABPs) are multifunctional cytosolic lipid-binding proteins found in vertebrates and invertebrates. In this work, we used RACE to obtain a full-length cDNA of Sp-FABP from the mud crab Scylla paramamosain. The open reading frame of the full length cDNA (886 bp) encoded a 136 amino acid polypeptide that showed high homology with related genes from other species. Real-time quantitative PCR identified variable levels of Sp-FABP transcripts in epidermis, eyestalk, gill, heart, hemocytes, hepatopancreas, muscle, ovary, stomach and thoracic ganglia. In ovaries, Sp-FABP expression increased gradually from stage I to stage IV of development and decreased in stage V. Sp-FABP transcripts in the hepatopancreas and hemocytes were up-regulated after a bacterial challenge with Vibrio alginnolyficus. These results suggest that Sp-FABP may be involved in the growth, reproduction and immunity of the mud crab. PMID:23569421

  13. Molecular Clone and Expression of a NAD+-Dependent Glycerol-3-Phosphate Dehydrogenase Isozyme Gene from the Halotolerant alga Dunaliella salina

    PubMed Central

    Cai, Ma; He, Li-Hong; Yu, Tu-Yuan

    2013-01-01

    Glycerol is an important osmotically compatible solute in Dunaliella. Glycerol-3-phosphate dehydrogenase (G3PDH) is a key enzyme in the pathway of glycerol synthesis, which converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate. Generally, the glycerol-DHAP cycle pathway, which is driven by G3PDH, is considered as the rate-limiting enzyme to regulate the glycerol level under osmotic shocks. Considering the peculiarity in osmoregulation, the cDNA of a NAD+-dependent G3PDH was isolated from D. salina using RACE and RT-PCR approaches in this study. Results indicated that the length of the cDNA sequence of G3PDH was 2,100 bp encoding a 699 amino acid deduced polypeptide whose computational molecular weight was 76.6 kDa. Conserved domain analysis revealed that the G3PDH protein has two independent functional domains, SerB and G3PDH domains. It was predicted that the G3PDH was a nonsecretory protein and may be located in the chloroplast of D. salina. Phylogenetic analysis demonstrated that the D. salina G3PDH had a closer relationship with the G3PDHs from the Dunaliella genus than with those from other species. In addition, the cDNA was subsequently subcloned in the pET-32a(+) vector and was transformed into E. coli strain BL21 (DE3), a expression protein with 100 kDa was identified, which was consistent with the theoretical value. PMID:23626797

  14. Molecular cloning of peroxinectin gene and its expression in response to peptidoglycan and Vibrio harveyi in Indian white shrimp Fenneropenaeus indicus.

    PubMed

    Shanthi, Sathappan; Manju, Sivalingam; Rajakumaran, Perumal; Vaseeharan, Baskaralingam

    2014-12-01

    The cDNA sequence of peroxinectin was obtained from the haemocytes of Indian white shrimp Fenneropenaeus indicus using RT-PCR and RACE. Fenneropenaeus indicus peroxinectin (Fi-Pxn) sequence has an open reading frame (ORF) of 2415 bp encoding a protein of 804 amino acids with 21 residues signal sequence. The mature protein has molecular mass of 89.8 kDa with an estimated pI of 8.6. Two putative integrin-binding motifs, RGD and KGD, were observed at the basic N-terminal and C-terminal part of the mature aminoacid sequence. Fi-Pxn nucleotide sequence comparison showed high homology to mud crab Scylla serrata (89%) and to various vertebrate and invertebrate species. qRT-PCR showed peroxinectin mRNA transcript in haemocytes of F. indicus increased at 6 h post injection of peptidoglycan and Vibrio harveyi. The Fi-Pxn was mainly expressed in the tissues of haemocytes and the heart. The moulting stage responses showed Fi-Pxn expression in premoult stages D0/1 and D0/2. PMID:25072536

  15. Molecular cloning, characterization, and stress-responsive expression of genes encoding glycine-rich RNA-binding proteins in Camelina sativa L.

    PubMed

    Kwak, Kyung Jin; Kang, Hunseung; Han, Kyung-Hwan; Ahn, Sung-Ju

    2013-07-01

    Camelina sativa L. is an oil-seed crop that has potential for biofuel applications. Although the importance of C. sativa as a biofuel crop has increased in recent years, reports demonstrating the stress responsiveness of C. sativa and characterizing the genes involved in stress response of C. sativa have never been published. Here, we isolated and characterized three genes encoding glycine-rich RNA-binding proteins (GRPs) from camelina: CsGRP2a, CsGRP2b, and CsGRP2c. The three CsGRP2 proteins were very similar in amino acid sequence and contained a well-conserved RNA-recognition motif at the N-terminal region and glycine-rich domain at the C-terminal region. To understand the functional roles of CsGRP2s under stress conditions, we investigated the expression patterns of CsGRP2s under various environmental stress conditions. The expressions of the three CsGRP2s were highly up-regulated under cold stress. The expression of CsGRP2a was up-regulated under salt or dehydration stress, whereas the transcript levels of CsGRP2b and CsGRP2c were decreased under salt or dehydration stress conditions. The three CsGRP2s had the ability to complement cold-sensitive Escherichia coli mutants at low temperatures and harbored transcription anti-termination and nucleic acid-melting activities, indicating that the CsGRP2s possess RNA chaperone activity. The CsGRP2a protein was localized to both the nucleus and the cytoplasm. Expression of CsGRP2a in cold-sensitive Arabidopsis grp7 mutant plants resulted in decreased electrolyte leakage at freezing temperatures. Collectively, these results suggest that the stress-responsive CsGRP2s play a role as an RNA chaperone during the stress adaptation process in camelina. PMID:23628924

  16. Molecular cloning and functional analysis of a UV-B photoreceptor gene, MdUVR8 (UV Resistance Locus 8), from apple.

    PubMed

    Zhao, Cheng; Mao, Ke; You, Chun-Xiang; Zhao, Xian-Yan; Wang, Shu-Hui; Li, Yuan-Yuan; Hao, Yu-Jin

    2016-06-01

    UVR8 (UV Resistance Locus 8) is an ultraviolet-B (UV-B; 280-315nm) light receptor that is involved in regulating many aspects of plant growth and development. UV-B irradiation can increase the development of flower and fruit coloration in many fruit trees, such as grape, pear and apple. Previous investigations of the structure and functions of UVR8 in plants have largely focused on Arabidopsis. Here, we isolated the UVR8 gene from apple (Malus domestica) and analyzed its function in transgenic Arabidopsis. Genomic and protein sequence analysis showed that MdUVR8 shares high similarity with the AtUVR8 protein from Arabidopsis, including the conserved seven-bladed β-propeller, the C27 region, the 3 "GWRHT" motifs and crucial amino-acid residues (14 Trps, 2 Args). A point mutation prediction and three-dimensional structural analysis of MdUVR8 indicated that it has a similar structure to AtUVR8 and that the crucial residues are also important in MdUVR8. In terms of transcript levels, MdUVR8 expression was up-regulated by UV-B light, which suggests that its expression follows a 24-h circadian rhythm. Using heterologous expression of MdUVR8 in both uvr8-1 mutant and wild-type (WT) Arabidopsis, we found that MdUVR8 regulates hypocotyl elongation and gene expression under UV-B light. These data provide functional evidence for a role of MdUVR8 in controlling photomorphogenesis under UV-B light and indicate that the function of UVR8 is conserved between Arabidopsis and apple. Furthermore, we examined the interaction between MdUVR8 and MdCOP1 (constitutive photomorphogenic1) using a yeast two-hybrid assay and a co-immunoprecipitation assay. This interaction provides a direction for investigating the regulatory mechanisms of the UV-B-light pathway in apple. PMID:27095405

  17. Cloning and expression of a Salmonella enteritidis fimbrin gene in Escherichia coli.

    PubMed Central

    Feutrier, J; Kay, W W; Trust, T J

    1988-01-01

    A gene bank of DNA from a human isolate of Salmonella enteritidis was constructed in the cosmid pHC79 in Escherichia coli HB101. Five clones containing 35- to 45-kilobase inserts of S. enteritidis DNA reacted in colony immunoblot assays with a polyclonal antiserum prepared against purified S. enteritidis fimbriae. Electron microscopy showed that none of the five fimbrin-producing clones produced fimbriae, yet radioimmunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis located the 14,400-molecular-weight S. enteritidis in the outer membrane fraction of three of the clones and in the periplasmic fraction of all five clones. By using an oligonucleotide probe homologous to the 5' region of the fimbrin structural gene, the fimbrin gene was located on a 5.3-kilobase HindIII fragment. In vitro transcription-translation analysis verified that this HindIII fragment subcloned into plasmid pTZ18R produced unprocessed S. enteritidis fimbrin of molecular weight 16,400. Dot blot hybridization against a selection of strains of the family Enterobacteriaceae indicated a limited distribution of the S. enteritidis fimbrin gene. Images PMID:2900832

  18. Molecular cloning, pathologically-correlated expression and functional characterization of the colonystimulating factor 1 receptor (CSF-1R) gene from a teleost, Plecoglossus altivelis

    PubMed Central

    CHEN, Qiang; LU, Xin-Jiang; LI, Ming-Yun; CHEN, Jiong

    2016-01-01

    Colony-stimulating factor 1 receptor (CSF-1R) is an important regulator of monocytes/macrophages (MO/MΦ). Although several CSF-1R genes have been identified in teleosts, the precise role of CSF- 1R in ayu (Plecoglossus altivelis) remains unclear. In this study, we characterized the CSF-1R homologue from P. altivelis, and named it PaCSF-1R. Multiple sequence alignment and phylogenetic tree analysis showed that PaCSF-1R was most closely related to that of Japanese ricefish (Oryzias latipes). Tissue distribution and expression analysis showed that the PaCSF-1R transcript was mainly expressed in the head kidney-derived MO/MΦ, spleen, and head kidney, and its expression was significantly altered in various tissues upon Vibrio anguillarum infection. After PaCSF-1R neutralization for 48 h, the phagocytic activity of MO/MΦ was significantly decreased, suggesting that PaCSF-1R plays a role in regulating the phagocytic function of ayu MO/MΦ. PMID:27029867

  19. Molecular cloning, organization, expression and 3D structural analysis of the MHC class Ia gene in the whitespotted bamboo shark (Chiloscyllium plagiosum).

    PubMed

    Shen, Tong; Lei, Meiling; Wang, Jingru; He, Xiaoshun; Li, Xiuming; Li, Jianming

    2014-01-15

    Cartilaginous fishes are the oldest jawed vertebrates, from which the major histocompatibility complex (MHC) derived approximately 500 MYA; however, full-length genomic sequences for MHC genes in these species remain undescribed. This lack of basic information about MHC organization in cartilaginous fish is hindering investigations into the relationship between MHC polymorphism and disease, and leaves a large gap in our understanding of shark MHC evolution. Here, we obtained a complete 4887 bp genomic DNA of chplUAA (designated as chplUAA) from the whitespotted bamboo shark (Chiloscyllium plagiosum) using long PCR. The full-length cDNA sequence was 1385 bp, with a 1029 bp open reading frame (ORF) encoding 343 amino acids. Six unique sequences (chplUAA*01-06) were detected from 51 sequences from three samples. No more than two sequences were found in each individual, suggesting that only one UAA locus was amplified in each sample. Phylogenetic analysis supports monophyly of all available shark classical class Ia sequences. PMID:24315118

  20. Molecular Cloning, Characterization and Expression Analysis of the SAMS Gene during Adventitious Root Development in IBA-Induced Tetraploid Black Locust

    PubMed Central

    Quan, Jine; Zhang, Sheng; Zhang, Chunxia; Meng, Sen; Zhao, Zhong; Xu, Xuexuan

    2014-01-01

    S-Adenosylmethionine synthetase (SAMS) catalyzes the synthesis of S-adenosylmethionine (SAM), a precursor for ethylene and polyamine biosynthesis. Here, we report the isolation of the 1498 bp full-length cDNA sequence encoding tetraploid black locust (Robinia pseudoacacia L.) SAMS (TrbSAMS), which contains an open reading frame of 1179 bp encoding 392 amino acids. The amino acid sequence of TrbSAMS has more than 94% sequence identity to SAMSs from other plants, with a closer phylogenetic relationship to SAMSs from legumes than to SAMS from other plants. The TrbSAMS monomer consists of N-terminal, central, and C-terminal domains. Subcellular localization analysis revealed that the TrbSAMS protein localizes mainly to in the cell membrane and cytoplasm of onion epidermal cells and Arabidopsis mesophyll cell protoplasts. Indole-3-butyric acid (IBA)-treated cuttings showed higher levels of TrbSAMS transcript than untreated control cuttings during root primordium and adventitious root formation. TrbSAMS and its downstream genes showed differential expression in shoots, leaves, bark, and roots, with the highest expression observed in bark. IBA-treated cuttings also showed higher SAMS activity than control cuttings during root primordium and adventitious root formation. These results indicate that TrbSAMS might play an important role in the regulation of IBA-induced adventitious root development in tetraploid black locust cuttings. PMID:25285660

  1. Molecular cloning and characterization of autophagy-related gene TmATG8 in Listeria-invaded hemocytes of Tenebrio molitor.

    PubMed

    Tindwa, Hamisi; Jo, Yong Hun; Patnaik, Bharat Bhusan; Lee, Yong Seok; Kang, Sang Sun; Han, Yeon Soo

    2015-07-01

    Macroautophagy (hereinafter called autophagy) is a highly regulated process used by eukaryotic cells to digest portions of the cytoplasm that remodels and recycles nutrients and disposes of unwanted cytoplasmic constituents. Currently 36 autophagy-related genes (ATG) and their homologs have been characterized in yeast and higher eukaryotes, including insects. In the present study, we identified and functionally characterized the immune function of an ATG8 homolog in a coleopteran insect, Tenebrio molitor (TmATG8). The cDNA of TmATG8 comprises of an ORF of 363 bp that encodes a protein of 120 amino acid residues. TmATG8 transcripts are detected in all the developmental stages analyzed. TmAtg8 protein contains a highly conserved C-terminal glycine residue (Gly116) and shows high amino acid sequence identity (98%) to its Tribolium castaneum homolog, TcAtg8. Loss of function of TmATG8 by RNAi led to a significant increase in the mortality rates of T. molitor larvae against Listeria monocytogenes. Unlike dsEGFP-treated control larvae, TmATG8-silenced larvae failed to turn-on autophagy in hemocytes after injection with L. monocytogenes. These data suggest that TmATG8 play a role in mediating autophagy-based clearance of Listeria in T. molitor. PMID:25727880

  2. Molecular cloning and characterization of cgt, the Brucella abortus cyclic beta-1,2-glucan transporter gene, and its role in virulence.

    PubMed

    Roset, Mara S; Ciocchini, Andrés E; Ugalde, Rodolfo A; Iñón de Iannino, Nora

    2004-04-01

    The animal pathogen Brucella abortus contains a gene cgt, which complemented Sinorhizobium meliloti nodule development (ndvA) and Agrobacterium tumefaciens chromosomal virulence (chvA) mutants. Complemented strains recovered the presence of anionic cyclic beta-1,2-glucan, motility, tumor induction in A. tumefaciens, and nodule occupancy in S. meliloti, all traits strictly associated with the presence of cyclic beta-1,2-glucan in the periplasm. Nucleotide sequencing revealed that B. abortus cgt contains a 1,797-bp open reading frame coding for a predicted membrane protein of 599 amino acids (65.9 kDa) that is 58.5 and 59.9% identical to S. meliloti NdvA and A. tumefaciens ChvA, respectively. Additionally, B. abortus cgt, like S. meliloti ndvA and A. tumefaciens chvA possesses ATP-binding motifs and the ABC signature domain features of a typical ABC transporter. Characterization of Cgt was carried out by the construction of null mutants in B. abortus 2308 and S19 backgrounds. Both mutants do not transport cyclic beta-1,2-glucan to the periplasm, as shown by the absence of anionic cyclic glucan, and they display reduced virulence in mice and defective intracellular multiplication in HeLa cells. These results suggest that cyclic beta-1,2-glucan must be transported into the periplasmatic space to exert its action as a virulence factor. PMID:15039351

  3. Molecular cloning, characterization and expression analysis of the SAMS gene during adventitious root development in IBA-induced tetraploid black locust.

    PubMed

    Quan, Jine; Zhang, Sheng; Zhang, Chunxia; Meng, Sen; Zhao, Zhong; Xu, Xuexuan

    2014-01-01

    S-Adenosylmethionine synthetase (SAMS) catalyzes the synthesis of S-adenosylmethionine (SAM), a precursor for ethylene and polyamine biosynthesis. Here, we report the isolation of the 1498 bp full-length cDNA sequence encoding tetraploid black locust (Robinia pseudoacacia L.) SAMS (TrbSAMS), which contains an open reading frame of 1179 bp encoding 392 amino acids. The amino acid sequence of TrbSAMS has more than 94% sequence identity to SAMSs from other plants, with a closer phylogenetic relationship to SAMSs from legumes than to SAMS from other plants. The TrbSAMS monomer consists of N-terminal, central, and C-terminal domains. Subcellular localization analysis revealed that the TrbSAMS protein localizes mainly to in the cell membrane and cytoplasm of onion epidermal cells and Arabidopsis mesophyll cell protoplasts. Indole-3-butyric acid (IBA)-treated cuttings showed higher levels of TrbSAMS transcript than untreated control cuttings during root primordium and adventitious root formation. TrbSAMS and its downstream genes showed differential expression in shoots, leaves, bark, and roots, with the highest expression observed in bark. IBA-treated cuttings also showed higher SAMS activity than control cuttings during root primordium and adventitious root formation. These results indicate that TrbSAMS might play an important role in the regulation of IBA-induced adventitious root development in tetraploid black locust cuttings. PMID:25285660

  4. Molecular cloning and expression analysis of a Cu/Zn SOD gene (BcCSD1) from Brassica campestris ssp. chinensis.

    PubMed

    Cui, Lijie; Huang, Qiang; Yan, Bin; Wang, Yao; Qian, Zhongyin; Pan, Jingxian; Kai, Guoyin

    2015-11-01

    Superoxide dismutases (SODs) are a family of metalloproteins extensively exists in eukaryote, which plays an essential role in stress-tolerance of higher plants. A full-length cDNA encoding Cu/Zn SOD (BcCSD1) was isolated from young seedlings of non-heading Chinese cabbage (Brassica campestris ssp. chinensis) by rapid amplification of cDNA ends (RACE). Bioinformatics analysis revealed that BcCSD1 belonged to the plant SOD super family and had the closest relationship with SOD from Brassica napus. Tissue expression pattern analysis revealed that the BcCSD1 was constitutively expressed in all the tested tissues, and strongest in leaf, moderate in stem, lowest in root. The expression profiles under different stress treatments such as drought, NaCl, high temperature and ABA were also investigated, and the results revealed that BcCSD1 was a stress-responsive gene, especially to ABA. These results provide useful information for further understanding the role of BcCSD1 resistant to abiotic stress in Brassica campestris in the future. PMID:25976826

  5. Molecular cloning, characterization, and expression pattern of the ultraspiracle gene homolog (RXR/USP) from the hemimetabolous insect Periplaneta americana (Dictyoptera, Blattidae) during vitellogenesis.

    PubMed

    Elgendy, Azza M; Elmogy, Mohamed; Takeda, Makio

    2014-02-01

    Ecdysteroid and sequiterpenoids juvenile hormones play a gonadotrophic role in the insect adult female vitellogenesis. The molecular basis of hormone action has been analyzed in great detail in flies and moths, but rarely in primitive insect orders. The primitive hemimetabolous insect Periplaneta americana was used, as a model, to isolate and characterize, for the first time, two cDNAs of RXR/USP, a component of the heterodimeric ecdysone receptor. These two cDNAs correspond to two isoforms, named PamRXR-S (short form) and PamRXR-L (long form). Both are identical except for 25 amino acids deletion/insertion located in the loop between helices H1 and H3 of the ligand-binding domain. The two isoforms are differentially expressed in different tissues as revealed by RT-PCR and northern blot analysis. In fat body, brain, ovary, and muscle tissues, the predominant form was PamRXR-S, whereas PamRXR-L was abundant in ovaries. The PamRXR transcript was detected during all stages of vitellogenesis in the fat body with different levels. It was little low during the early vitellogenic period (days 2, 3), then a peak of increase was detected during days 4-6 (day 5) which was followed by another peak of increase at the end of vitellogenesis, day 9. We assumed that PamRXR might play a dual role of induction of vitellogenin through JH at early vitellogenesis and suppression through 20E during late vitellogenesis. The present work will pave the way for several other investigations to understand both the ecdysteroid-dependent genetic hierarchy and JH mechanism controlling vitellogenesis in the American cockroach, P. americana. PMID:23873076

  6. Molecular cloning, sequence characteristics, and polymorphism analyses of the tyrosinase-related protein 2 / DOPAchrome tautomerase gene of black-boned sheep (Ovis aries).

    PubMed

    Deng, Weidong; Tan, Yuwen; Wang, Xinyu; Xi, Dongmei; He, Yiduo; Yang, Shuli; Mao, Huaming; Gao, Shizheng

    2009-12-01

    Tyrosinase-related protein 2 (TYRP2) plays a pivotal role in the biosynthesis of eumelanin. Black-boned sheep have excessive melanin and eumelanin, resulting in dark (black) muscles and organs. This study was designed to investigate the effects of variants of the TYRP2 gene on black traits and coat colour of black-boned sheep. Melanin traits were measured in three populations of sheep (Nanping black-boned, Nanping normal, and Romney Marsh) and compared in this study. From the TYRP2 cDNA, all 8 exons and their flanking regions were amplified and characterized. Fifteen single nucleotide polymorphisms (SNPs) were identified in the exons and their flanking regions. Five exonic polymorphic sites, including two synonymous (c.93T>G and c.1140C>T) and three non-synonymous mutations (c.163C>T (p.R55W), c.605G>A (p.R202H), and c.1141A>G (p.T381A)), were retrieved. PCR-RFLP analysis of c.605G>A showed that the frequencies of allele G in the Nanping black-boned, Nanping normal, and Romney Marsh sheep were 0.632, 0.603, and 0.886, respectively. Sheep with the GG genotype had significantly (P < 0.05) lower tyrosinase activity, alkali-soluble melanin content, and ratio of eumelanin : total melanin than sheep with GA and AA genotypes when measured across all investigated samples but not when samples within each population of sheep were compared. However, there was no association of TYRP2 genotype at a single SNP position with coat colour across populations. Nonetheless, the two breeds with higher overall tyrosinase activity did produce darker and more varied coat colours than the breed with lower tyrosinase activity. PMID:19953128

  7. Molecular cloning and stress-dependent expression of a gene encoding Delta(12)-fatty acid desaturase in the Antarctic microalga Chlorella vulgaris NJ-7.

    PubMed

    Lu, Yandu; Chi, Xiaoyuan; Yang, Qingli; Li, Zhaoxin; Liu, Shaofang; Gan, Qinhua; Qin, Song

    2009-11-01

    The psychrotrophic Antarctic alga, Chlorella vulgaris NJ-7, grows under an extreme environment of low temperature and high salinity. In an effort to better understand the correlation between fatty acid metabolism and acclimation to Antarctic environment, we analyzed its fatty acid compositions. An extremely high amount of Delta(12) unsaturated fatty acids was identified which prompted us to speculate about the involvement of Delta(12) fatty acid desaturase in the process of acclimation. A full-length cDNA sequence, designated CvFAD2, was isolated from C. vulgaris NJ-7 via reverse transcription polymerase chain reaction (RT-PCR) and RACE methods. Sequence alignment and phylogenetic analysis showed that the gene was homologous to known microsomal Delta(12)-FADs with the conserved histidine motifs. Heterologous expression in yeast was used to confirm the regioselectivity and the function of CvFAD2. Linoleic acid (18:2), normally not present in wild-type yeast cells, was detected in transformants of CvFAD2. The induction of CvFAD2 at an mRNA level under cold stress and high salinity is detected by real-time PCR. The results showed that both temperature and salinity motivated the upregulation of CvFAD2 expression. The accumulation of CvFAD2 increased 2.2-fold at 15 degrees C and 3.9-fold at 4 degrees C compared to the alga at 25 degrees C. Meanwhile a 1.7- and 8.5-fold increase at 3 and 6% NaCl was detected. These data suggest that CvFAD2 is the enzyme responsible for the Delta(12) fatty acids desaturation involved in the adaption to cold and high salinity for Antarctic C. vugaris NJ-7. PMID:19728010

  8. Cloning and characterization of the Bacteroides fragilis metalloprotease toxin gene.

    PubMed Central

    Franco, A A; Mundy, L M; Trucksis, M; Wu, S; Kaper, J B; Sears, C L

    1997-01-01

    Strains of Bacteroides fragilis that produce a ca. 20-kDa heat-labile protein toxin (termed B. fragilis toxin [BFT]) have been associated with diarrheal disease of animals and humans. BFT alters the morphology of intestinal epithelial cells both in vitro and in vivo and stimulates secretion in ligated intestinal segments of rats, rabbits, and lambs. Previous genetic and biochemical data indicated that BFT was a metalloprotease which hydrolyzed G (monomeric) actin, gelatin, and azocoll in vitro. In this paper, the cloning and sequencing of the entire B. fragilis toxin gene (bft) from enterotoxigenic B. fragilis (ETBF) 86-5443-2-2 is reported. The bft gene from this ETBF strain consists of one open reading frame of 1,191 nucleotides encoding a predicted 397-residue holotoxin with a calculated molecular weight of 44,493. Comparison of the predicted BFT protein sequence with the N-terminal amino acid sequence of purified BFT indicates that BFT is most probably synthesized by ETBF strains as a preproprotein. These data predict that BFT is processed to yield a biologically active toxin of 186 residues with a molecular mass of 20.7 kDa which is secreted into the culture supernatant. Analysis of the holotoxin sequence predicts a 20-residue amphipathic region at the carboxy terminus of BFT. Thus, in addition to the metalloprotease activity of BFT, the prediction of an amphipathic domain suggests that oligomerization of BFT may permit membrane insertion of the toxin with creation of a transmembrane pore. Comparison of the sequences available for the bft genes from ETBF 86-5443-2-2 and VPI 13784 revealed two regions of reduced homology. Hybridization of oligonucleotide probes specific for each bft to toxigenic B.fragilis strains revealed that 51 and 49% of toxigenic strains contained the 86-5433-2-2 and VPI 13784 bft genes, respectively. No toxigenic strain hybridized with both probes. We propose that these two subtypes of bft be termed bft-1 (VPI 13784) and bft-2 (86

  9. Molecular Cloning of a cDNA Encoding for Taenia solium TATA-Box Binding Protein 1 (TsTBP1) and Study of Its Interactions with the TATA-Box of Actin 5 and Typical 2-Cys Peroxiredoxin Genes

    PubMed Central

    Rodríguez-Lima, Oscar; García-Gutierrez, Ponciano; Jiménez, Lucía; Zarain-Herzberg, Ángel; Lazzarini, Roberto; Landa, Abraham

    2015-01-01

    TATA-box binding protein (TBP) is an essential regulatory transcription factor for the TATA-box and TATA-box-less gene promoters. We report the cloning and characterization of a full-length cDNA that encodes a Taenia solium TATA-box binding protein 1 (TsTBP1). Deduced amino acid composition from its nucleotide sequence revealed that encodes a protein of 238 residues with a predicted molecular weight of 26.7 kDa, and a theoretical pI of 10.6. The NH2-terminal domain shows no conservation when compared with to pig and human TBP1s. However, it shows high conservation in size and amino acid identity with taeniids TBP1s. In contrast, the TsTBP1 COOH-terminal domain is highly conserved among organisms, and contains the amino acids involved in interactions with the TATA-box, as well as with TFIIA and TFIIB. In silico TsTBP1 modeling reveals that the COOH-terminal domain forms the classical saddle structure of the TBP family, with one α-helix at the end, not present in pig and human. Native TsTBP1 was detected in T. solium cysticerci´s nuclear extract by western blot using rabbit antibodies generated against two synthetic peptides located in the NH2 and COOH-terminal domains of TsTBP1. These antibodies, through immunofluorescence technique, identified the TBP1 in the nucleus of cells that form the bladder wall of cysticerci of Taenia crassiceps, an organism close related to T. solium. Electrophoretic mobility shift assays using nuclear extracts from T. solium cysticerci and antibodies against the NH2-terminal domain of TsTBP1 showed the interaction of native TsTBP1 with the TATA-box present in T. solium actin 5 (pAT5) and 2-Cys peroxiredoxin (Ts2-CysPrx) gene promoters; in contrast, when antibodies against the anti-COOH-terminal domain of TsTBP1 were used, they inhibited the binding of TsTBP1 to the TATA-box of the pAT5 promoter gene. PMID:26529408

  10. Molecular Cloning of a cDNA Encoding for Taenia solium TATA-Box Binding Protein 1 (TsTBP1) and Study of Its Interactions with the TATA-Box of Actin 5 and Typical 2-Cys Peroxiredoxin Genes.

    PubMed

    Rodríguez-Lima, Oscar; García-Gutierrez, Ponciano; Jiménez, Lucía; Zarain-Herzberg, Ángel; Lazzarini, Roberto; Landa, Abraham

    2015-01-01

    TATA-box binding protein (TBP) is an essential regulatory transcription factor for the TATA-box and TATA-box-less gene promoters. We report the cloning and characterization of a full-length cDNA that encodes a Taenia solium TATA-box binding protein 1 (TsTBP1). Deduced amino acid composition from its nucleotide sequence revealed that encodes a protein of 238 residues with a predicted molecular weight of 26.7 kDa, and a theoretical pI of 10.6. The NH2-terminal domain shows no conservation when compared with to pig and human TBP1s. However, it shows high conservation in size and amino acid identity with taeniids TBP1s. In contrast, the TsTBP1 COOH-terminal domain is highly conserved among organisms, and contains the amino acids involved in interactions with the TATA-box, as well as with TFIIA and TFIIB. In silico TsTBP1 modeling reveals that the COOH-terminal domain forms the classical saddle structure of the TBP family, with one α-helix at the end, not present in pig and human. Native TsTBP1 was detected in T. solium cysticerci´s nuclear extract by western blot using rabbit antibodies generated against two synthetic peptides located in the NH2 and COOH-terminal domains of TsTBP1. These antibodies, through immunofluorescence technique, identified the TBP1 in the nucleus of cells that form the bladder wall of cysticerci of Taenia crassiceps, an organism close related to T. solium. Electrophoretic mobility shift assays using nuclear extracts from T. solium cysticerci and antibodies against the NH2-terminal domain of TsTBP1 showed the interaction of native TsTBP1 with the TATA-box present in T. solium actin 5 (pAT5) and 2-Cys peroxiredoxin (Ts2-CysPrx) gene promoters; in contrast, when antibodies against the anti-COOH-terminal domain of TsTBP1 were used, they inhibited the binding of TsTBP1 to the TATA-box of the pAT5 promoter gene. PMID:26529408

  11. Molecular cloning and amino acid sequence of human 5-lipoxygenase

    SciTech Connect

    Matsumoto, T.; Funk, C.D.; Radmark, O.; Hoeoeg, J.O.; Joernvall, H.; Samuelsson, B.

    1988-01-01

    5-Lipoxygenase (EC 1.13.11.34), a Ca/sup 2 +/- and ATP-requiring enzyme, catalyzes the first two steps in the biosynthesis of the peptidoleukotrienes and the chemotactic factor leukotriene B/sub 4/. A cDNA clone corresponding to 5-lipoxygenase was isolated from a human lung lambda gt11 expression library by immunoscreening with a polyclonal antibody. Additional clones from a human placenta lambda gt11 cDNA library were obtained by plaque hybridization with the /sup 32/P-labeled lung cDNA clone. Sequence data obtained from several overlapping clones indicate that the composite DNAs contain the complete coding region for the enzyme. From the deduced primary structure, 5-lipoxygenase encodes a 673 amino acid protein with a calculated molecular weight of 77,839. Direct analysis of the native protein and its proteolytic fragments confirmed the deduced composition, the amino-terminal amino acid sequence, and the structure of many internal segments. 5-Lipoxygenase has no apparent sequence homology with leukotriene A/sub 4/ hydrolase or Ca/sup 2 +/-binding proteins. RNA blot analysis indicated substantial amounts of an mRNA species of approx. = 2700 nucleotides in leukocytes, lung, and placenta.

  12. Cloning and expression of the potato alternative oxidase gene

    SciTech Connect

    Hiser, C.; McIntosh, L. Michigan State Univ., East Lansing )

    1990-05-01

    Mitochondria from 24-hour-aged potato slices possess an alternative path capacity and a 36kD protein not present in fresh potato mitochondria. This 36kD protein was identified by a monoclonal antibody against the Sauromatum guttatum alternative oxidase. These results suggest de novo synthesis of the 36kD protein during the aging process. To investigate this phenomenon, a clone containing a potato alternative oxidase gene was isolated from a cDNA library using the S. guttatum gene as a probe. This clone shows areas of high homology to the S. guttatum gene. Norther blots of RNA from fresh and 24-hour-aged potato slices are being probed with the potato gene to examine its expression in relation to the appearance of the 36kD protein.

  13. Molecular cloning and physical mapping of murine cytomegalovirus DNA.

    PubMed Central

    Ebeling, A; Keil, G M; Knust, E; Koszinowski, U H

    1983-01-01

    Murine cytomegalovirus (MCMV) Smith strain DNA is cleaved by restriction endonuclease HindIII into 16 fragments, ranging in size from 0.64 to 22.25 megadaltons. Of the 16 HindIII fragments, 15 were cloned in plasmid pACYC177 in Escherichia coli HB101 (recA). The recombinant plasmid clones were characterized by cleavage with the enzymes XbaI and EcoRI. In addition, fragments generated by double digestion of cloned fragments with HindIII and XbaI were inserted into the plasmid vector pACYC184. The results obtained after hybridization of 32P-labeled cloned fragments to Southern blots of MCMV DNA cleaved with HindIII, XbaI, EcoRI, BamHI, ApaI, ClaI, EcoRV, or KpnI allowed us to construct complete physical maps of the viral DNA for the restriction endonucleases HindIII, XbaI, and EcoRI. On the basis of the cloning and mapping experiments, it was calculated that the MCMV genome spans about 235 kilobase pairs, corresponding to a molecular weight of 155,000,000. All fragments were found to be present in equimolar concentrations, and no cross-hybridization between any of the fragments was seen. We conclude that the MCMV DNA molecule consists of a long unique sequence without large terminal or internal repeat regions. Thus, the structural organization of the MCMV genome is fundamentally different from that of the human cytomegalovirus or herpes simplex virus genome. Images PMID:6312075

  14. Development and characterization of an in vivo pathogenic molecular clone of equine infectious anemia virus.

    PubMed

    Cook, R F; Leroux, C; Cook, S J; Berger, S L; Lichtenstein, D L; Ghabrial, N N; Montelaro, R C; Issel, C J

    1998-02-01

    An infectious nonpathogenic molecular clone (19-2-6A) of equine infectious anemia virus (EIAV) was modified by substitution of a 3.3-kbp fragment amplified by PCR techniques from a pathogenic variant (EIAV(PV)) of the cell culture-adapted strain of EIAV (EIAV(PR)). This substitution consisted of coding sequences for 77 amino acids at the carboxyl terminus of the integrase, the S1 (encoding the second exon of tat), S2, and S3 (encoding the second exon of rev) open reading frames, the complete env gene (including the first exon of rev), and the 3' long terminal repeat (LTR). Modified 19-2-6A molecular clones were designated EIAV(PV3.3), and infection of a single pony (678) with viruses derived from a mixture of five of these molecular clones induced clinical signs of acute equine infectious anemia (EIA) at 23 days postinfection (dpi). As a consequence of this initial study, a single molecular clone, EIAV(PV3.3#3) (redesignated EIAV(UK)), was selected for further study and inoculated into two ponies (613 and 614) and two horses (700 and 764). Pony 614 and the two horses developed febrile responses by 12 dpi, which was accompanied by a 48 to 64% reduction in platelet number, whereas pony 613 did not develop fever (40.6 degrees C) until 76 dpi. EIAV could be isolated from the plasma of these animals by 5 to 7 dpi, and all became seropositive for antibodies to this virus by 21 dpi. Analysis of the complete nucleotide sequence demonstrated that the 3.3-kbp 3' fragment of EIAV(UK) differed from the consensus sequence of EIAV(PV) by just a single amino acid residue in the second exon of the rev gene. Complete homology with the EIAV(PV) consensus sequence was observed in the hypervariable region of the LTR. However, EIAV(UK) was found to contain an unusual 68-bp nucleotide insertion/duplication in a normally conserved region of the LTR sequence. These results demonstrate that substitution of a 3.3-kbp fragment from the EIAV(PV) strain into the infectious nonpathogenic

  15. Cloning and expression of calmodulin gene in Scoparia dulcis.

    PubMed

    Saitoh, Daisuke; Asakura, Yuki; Nkembo, Marguerite Kasidimoko; Shite, Masato; Sugiyama, Ryuji; Lee, Jung-Bum; Hayashi, Toshimitsu; Kurosaki, Fumiya

    2007-06-01

    A homology-based cloning strategy yielded a cDNA clone, designated Sd-cam, encoding calmodulin protein from Scoparia dulcis. The restriction digests of genomic DNA of S. dulcis showed a single hybridized signal when probed with the fragment of this gene in Southern blot analyses, suggesting that Sd-cam occurs as a sole gene encoding calmodulin in the plant. The reverse-transcription polymerase chain reaction analysis revealed that Sd-cam was appreciably expressed in leaf, root and stem tissues. It appeared that transcription of this gene increased transiently when the leaf cultures of S. dulcis were treated with methyl jasmonate and calcium ionophore A23187. These results suggest that transcriptional activation of Sd-cam is one of the early cellular events of the methyl jasmonate-induced responses of S. dulcis. PMID:17541174

  16. Molecular cloning and expression vector construction of bovine TRIM28.

    PubMed

    Ma, X; Zhai, Z C; Zhang, M L; Song, B H; Zhu, Y R; Yang, S B; Dong, X Q; Su, L Y; Wang, C F; Ma, H X; Luan, W M

    2016-01-01

    The bovine TRIM28 gene was amplified from ovary tissue by using RT-PCR. The TRIM28 gene was inserted into the eukaryotic expression vector pIRES2-EGFP and transfected into bovine fetal fibroblasts by using Lipofectamine 3000. TRIM28 mRNA and protein were detected by fluorescence microscope and western blotting. The results showed that the full length of TRIM28 was cloned and pIRES2-EGFP-TRIM28 was constructed successfully. EGFP expression was observed, and the pIRES2-EGFP-TRIM28 transfected group expressed more TRIM28 protein than that by the pIRES2-EGFP group. The TIMR28 gene has been successfully transferred into bovine fetal fibroblasts. PMID:27420979

  17. Expression cloning of a candidate gene for Mucolipidosis type IV

    SciTech Connect

    Gama Sosa, M.A.; De Gasperi, R.; Battistini, S.

    1994-09-01

    Mucolipidosis IV is an autosomal recessive lysosomal storage disease characterized by progressive psychomotor retardation and opthalmological abnormalities, namely corneal opacity and retinal degeneration. Biochemically, it is characterized by the lysosomal accumulation of diverse compounds such as gangliosides, phospholipids and acidic mucopolysaccharides. To date, the basic biochemical defect causing this storage disease is still unknown and the relevant gene has also not been identified. An expression cloning strategy was used to identify human kidney cDNA clones capable of reverting in transient gene expression assays the PAS+ phenotype typical of Mucolipidosis IV cells to the normal PAS- phenotype. By this method, a candidate cDNA clone (Mu cDNA) capable of clearing Mucolipidosis IV fibroblasts of their PAS+ positive storage material was isolated. Nucleotide sequence analysis indicated the presence of 2 open reading frames. In vitro translation of T7 transcribed Mu RNA showed protein products of 7,000 and 6,000 mw. Altered expression of the Mu gene may result in the onset of Mucolipidosis type IV.

  18. Cloning and analysis of phycoerythrin genes in Gracilaria Lemaneiformis (Rhodophyceae)

    NASA Astrophysics Data System (ADS)

    Sui, Zheng-Hong; Zhang, Xue-Cheng

    2000-03-01

    Cloning and sequencing of the genes coding for the α- and β- subunit of phycoerythrin (PE) of a red alga— Gracilaria lemaneiformis (GL) are reported. Alignment of 1084 nucleotides sequenced with three known red algal PE genes, Rhodella violacea (RV), Polysiphonia boldii (PB) and Aglaothamnion neglectum (AN), showed high level of conservation, and similarities of 77,6% (between GL and RV), 77.9% (GL and AN) and 79.0% (GL and PB). The similarities of amino acids were 84.8% (between GL and RV), 85.7% (GL and PB), and 80.6% (AN and GL), higher than those among nucleotides.

  19. Functional Identification and Characterization of Genes Cloned from Halophyte Seashore Paspalum Conferring Salinity and Cadmium Tolerance

    PubMed Central

    Chen, Yu; Chen, Chuanming; Tan, Zhiqun; Liu, Jun; Zhuang, Lili; Yang, Zhimin; Huang, Bingru

    2016-01-01

    Salinity-affected and heavy metal-contaminated soils limit the growth of glycophytic plants. Identifying genes responsible for superior tolerance to salinity and heavy metals in halophytes has great potential for use in developing salinity- and Cd-tolerant glycophytes. The objective of this study was to identify salinity- and Cd-tolerance related genes in seashore paspalum (Paspalum vaginatum), a halophytic perennial grass species, using yeast cDNA expression library screening method. Based on the Gateway-compatible vector system, a high-quality entry library was constructed, which contained 9.9 × 106 clones with an average inserted fragment length of 1.48 kb representing a 100% full-length rate. The yeast expression libraries were screened in a salinity-sensitive and a Cd-sensitive yeast mutant. The screening yielded 32 salinity-tolerant clones harboring 18 salinity-tolerance genes and 20 Cd-tolerant clones, including five Cd-tolerance genes. qPCR analysis confirmed that most of the 18 salinity-tolerance and five Cd-tolerance genes were up-regulated at the transcript level in response to salinity or Cd stress in seashore paspalum. Functional analysis indicated that salinity-tolerance genes from seashore paspalum could be involved mainly in photosynthetic metabolism, antioxidant systems, protein modification, iron transport, vesicle traffic, and phospholipid biosynthesis. Cd-tolerance genes could be associated with regulating pathways that are involved in phytochelatin synthesis, HSFA4-related stress protection, CYP450 complex, and sugar metabolism. The 18 salinity-tolerance genes and five Cd-tolerance genes could be potentially used as candidate genes for genetic modification of glycophytic grass species to improve salinity and Cd tolerance and for further analysis of molecular mechanisms regulating salinity and Cd tolerance. PMID:26904068

  20. Functional Identification and Characterization of Genes Cloned from Halophyte Seashore Paspalum Conferring Salinity and Cadmium Tolerance.

    PubMed

    Chen, Yu; Chen, Chuanming; Tan, Zhiqun; Liu, Jun; Zhuang, Lili; Yang, Zhimin; Huang, Bingru

    2016-01-01

    Salinity-affected and heavy metal-contaminated soils limit the growth of glycophytic plants. Identifying genes responsible for superior tolerance to salinity and heavy metals in halophytes has great potential for use in developing salinity- and Cd-tolerant glycophytes. The objective of this study was to identify salinity- and Cd-tolerance related genes in seashore paspalum (Paspalum vaginatum), a halophytic perennial grass species, using yeast cDNA expression library screening method. Based on the Gateway-compatible vector system, a high-quality entry library was constructed, which contained 9.9 × 10(6) clones with an average inserted fragment length of 1.48 kb representing a 100% full-length rate. The yeast expression libraries were screened in a salinity-sensitive and a Cd-sensitive yeast mutant. The screening yielded 32 salinity-tolerant clones harboring 18 salinity-tolerance genes and 20 Cd-tolerant clones, including five Cd-tolerance genes. qPCR analysis confirmed that most of the 18 salinity-tolerance and five Cd-tolerance genes were up-regulated at the transcript level in response to salinity or Cd stress in seashore paspalum. Functional analysis indicated that salinity-tolerance genes from seashore paspalum could be involved mainly in photosynthetic metabolism, antioxidant systems, protein modification, iron transport, vesicle traffic, and phospholipid biosynthesis. Cd-tolerance genes could be associated with regulating pathways that are involved in phytochelatin synthesis, HSFA4-related stress protection, CYP450 complex, and sugar metabolism. The 18 salinity-tolerance genes and five Cd-tolerance genes could be potentially used as candidate genes for genetic modification of glycophytic grass species to improve salinity and Cd tolerance and for further analysis of molecular mechanisms regulating salinity and Cd tolerance. PMID:26904068

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

    SciTech Connect

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

    1994-09-01

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

  2. Porin protein of Neisseria gonorrhoeae: cloning and gene structure.

    PubMed Central

    Gotschlich, E C; Seiff, M E; Blake, M S; Koomey, M

    1987-01-01

    The outer membrane porin molecule of Neisseria gonorrhoeae is known as protein I (PI). Among different strains of gonococci there is variability of PI, and two main classes, PIA and PIB, have been recognized. A lambda gt11 bank of gonococcal DNA was screened using monoclonal antibodies directed to a PIB-type porin molecule of N. gonorrhoeae, and three immunoreactive clones were isolated. DNA sequence analysis indicated that each contained only portions of the PI structural gene, but that together they contained the complete gene, and its structure was determined. The DNA sequence predicts a protein of 348 amino acids with a typical 19 amino acid signal peptide. The PI protein resembles Escherichia coli porins in size, lack of long hydrophobic sequences, and absence of cysteine residues. Sequence homologies between PI and the E. coli porins were found, particularly in the 100 N-terminal and the 110 C-terminal amino acids. In addition to the coding sequence of PI, the complementary strand contains a large open reading frame. At the 3' end of the PI gene, immediately following an inverted repeat (probably the transcription terminator), the clone contains an unusual sequence consisting of 31 perfect repeats of the heptamer CTGTTTT. Hybridization analysis suggests that there is a single structural gene for PI and that it is homologous to the gene found in a PIA-bearing strain of gonococcus. Images PMID:2825179

  3. Characterization of cDNA clones for the human c-yes gene.

    PubMed Central

    Sukegawa, J; Semba, K; Yamanashi, Y; Nishizawa, M; Miyajima, N; Yamamoto, T; Toyoshima, K

    1987-01-01

    Three c-yes cDNA clones were obtained from poly(A)+ RNA of human embryo fibroblasts. Sequence analysis of the clones showed that they contained inserts corresponding to nearly full-length human c-yes mRNA, which could encode a polypeptide of 543 amino acids with a relative molecular weight (Mr) of 60,801. The predicted amino acid sequence of the protein has no apparent membrane-spanning region or suspected ligand binding domain and closely resembles pp60c-src. Comparison of the sequences of c-yes and v-yes revealed that the v-yes gene contains most of the c-yes coding sequence except the region encoding its extreme carboxyl terminus. The region missing from the v-yes protein is the part that is highly conserved in cellular gene products of the protein-tyrosine kinase family. PMID:2436037

  4. Cloning and characterization of a murine SIL gene

    SciTech Connect

    Collazo-Garcia, N.; Scherer, P.; Aplan, P.D.

    1995-12-10

    The human SIL gene is disrupted by a site-specific interstitial deletion in 25% of children with T-cell acute lymphoblastic leukemia. Since transcriptionally active genes are prone to recombination events, the recurrent nature of this lesion suggests that the SIL gene product is transcriptionally active in the cell type that undergoes this interstitial deletion and that the SIL gene product may play a role in normal lymphoid development. To facilitate studies of SIL gene function, we have cloned and characterized a murine SIL gene. The predicted murine SIL protein is 75% identical to the human gene, with good homology throughout the open reading frame. An in vitro translated SIL cDNA generated a protein slightly larger than the predicted 139-kDa protein. Although a prior report detected SIL mRNA expression exclusively in hematopoietic tissues, a sensitive RT-PCR assay demonstrated SIL expression to be ubiquitous, detectable in all tissues examined. Since the RT-PCR assay suggested that SIL mRNA expression was higher in rapidly proliferating tissues, we assayed SIL mRNA expression using a murine erythroleukemia model of terminal differentiation and found it to be dramatically decreased in conjunction with terminal differentiation. These studies demonstrate that the human SIL gene product is quite well conserved in rodents and suggest that the SIL gene product may play a role in cell proliferation. 26 refs., 6 figs.

  5. Molecular and cytogenetic characterization of expanded B-cell clones from multiclonal versus monoclonal B-cell chronic lymphoproliferative disorders

    PubMed Central

    Henriques, Ana; Rodríguez-Caballero, Arancha; Criado, Ignacio; Langerak, Anton W.; Nieto, Wendy G.; Lécrevisse, Quentin; González, Marcos; Cortesão, Emília; Paiva, Artur; Almeida, Julia; Orfao, Alberto

    2014-01-01

    Chronic antigen-stimulation has been recurrently involved in the earlier stages of monoclonal B-cell lymphocytosis, chronic lymphocytic leukemia and other B-cell chronic lymphoproliferative disorders. The expansion of two or more B-cell clones has frequently been reported in individuals with these conditions; potentially, such coexisting clones have a greater probability of interaction with common immunological determinants. Here, we analyzed the B-cell receptor repertoire and molecular profile, as well as the phenotypic, cytogenetic and hematologic features, of 228 chronic lymphocytic leukemia-like and non-chronic lymphocytic leukemia-like clones comparing multiclonal (n=85 clones from 41 cases) versus monoclonal (n=143 clones) monoclonal B-cell lymphocytosis, chronic lymphocytic leukemia and other B-cell chronic lymphoproliferative disorders. The B-cell receptor of B-cell clones from multiclonal cases showed a slightly higher degree of HCDR3 homology than B-cell clones from mono clonal cases, in association with unique hematologic (e.g. lower B-lymphocyte counts) and cytogenetic (e.g. lower frequency of cytogenetically altered clones) features usually related to earlier stages of the disease. Moreover, a subgroup of coexisting B-cell clones from individual multiclonal cases which were found to be phylogenetically related showed unique molecular and cytogenetic features: they more frequently shared IGHV3 gene usage, shorter HCDR3 sequences with a greater proportion of IGHV mutations and del(13q14.3), than other unrelated B-cell clones. These results would support the antigen-driven nature of such multiclonal B-cell expansions, with potential involvement of multiple antigens/epitopes. PMID:24488564

  6. Molecular cloning of cecropin B responsive endonucleases in Yersinia ruckeri

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have previously demonstrated that Yersinia ruckeri resists cecropin B in an inducible manner. In this study, we sought to identify the molecular changes responsible for the inducible cecropin B resistance of Y. ruckeri. Differences in gene expression associated with the inducible resistance were ...

  7. Molecular cloning and functional analysis of a 10-epi-junenol synthase from Inula hupehensis.

    PubMed

    Gou, Jun-Bo; Li, Zhen-Qiu; Li, Chang-Fu; Chen, Fang-Fang; Lv, Shi-You; Zhang, Yan-Sheng

    2016-09-01

    Junenol based-eudesmanolides have been detected in many compositae plant species and were reported to exhibit various pharmacological activities. So far, the gene encoding junenol synthase has never been isolated. Here we report the molecular cloning and functional analysis of a 10-epi-junenol synthase from Inula hupehensis (designated IhsTPS1). IhsTPS1 converts the substrate farnesyl diphosphate into multiple sesquiterpenes with the product 10-epi-junenol being predominant. The transcript levels of IhsTPS1 correlate well with the accumulation pattern of 10-epi-junenol in I. hupehensis organs, supporting its biochemical roles in vivo. PMID:27231873

  8. Molecularly tagged genes and quantitative trait loci in cucumber

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Since the release of the cucumber draft genome, significant progress has been made in molecular mapping, tagging or cloning of horticulturally important genes and quantitative trait loci (QTLs) in cucumber, which provides the foundation for practicing marker-assisted selection in cucumber breeding. ...

  9. Ab initio multiple cloning algorithm for quantum nonadiabatic molecular dynamics

    SciTech Connect

    Makhov, Dmitry V.; Shalashilin, Dmitrii V.; Glover, William J.; Martinez, Todd J.

    2014-08-07

    We present a new algorithm for ab initio quantum nonadiabatic molecular dynamics that combines the best features of ab initio Multiple Spawning (AIMS) and Multiconfigurational Ehrenfest (MCE) methods. In this new method, ab initio multiple cloning (AIMC), the individual trajectory basis functions (TBFs) follow Ehrenfest equations of motion (as in MCE). However, the basis set is expanded (as in AIMS) when these TBFs become sufficiently mixed, preventing prolonged evolution on an averaged potential energy surface. We refer to the expansion of the basis set as “cloning,” in analogy to the “spawning” procedure in AIMS. This synthesis of AIMS and MCE allows us to leverage the benefits of mean-field evolution during periods of strong nonadiabatic coupling while simultaneously avoiding mean-field artifacts in Ehrenfest dynamics. We explore the use of time-displaced basis sets, “trains,” as a means of expanding the basis set for little cost. We also introduce a new bra-ket averaged Taylor expansion (BAT) to approximate the necessary potential energy and nonadiabatic coupling matrix elements. The BAT approximation avoids the necessity of computing electronic structure information at intermediate points between TBFs, as is usually done in saddle-point approximations used in AIMS. The efficiency of AIMC is demonstrated on the nonradiative decay of the first excited state of ethylene. The AIMC method has been implemented within the AIMS-MOLPRO package, which was extended to include Ehrenfest basis functions.

  10. Ab initio multiple cloning algorithm for quantum nonadiabatic molecular dynamics

    NASA Astrophysics Data System (ADS)

    Makhov, Dmitry V.; Glover, William J.; Martinez, Todd J.; Shalashilin, Dmitrii V.

    2014-08-01

    We present a new algorithm for ab initio quantum nonadiabatic molecular dynamics that combines the best features of ab initio Multiple Spawning (AIMS) and Multiconfigurational Ehrenfest (MCE) methods. In this new method, ab initio multiple cloning (AIMC), the individual trajectory basis functions (TBFs) follow Ehrenfest equations of motion (as in MCE). However, the basis set is expanded (as in AIMS) when these TBFs become sufficiently mixed, preventing prolonged evolution on an averaged potential energy surface. We refer to the expansion of the basis set as "cloning," in analogy to the "spawning" procedure in AIMS. This synthesis of AIMS and MCE allows us to leverage the benefits of mean-field evolution during periods of strong nonadiabatic coupling while simultaneously avoiding mean-field artifacts in Ehrenfest dynamics. We explore the use of time-displaced basis sets, "trains," as a means of expanding the basis set for little cost. We also introduce a new bra-ket averaged Taylor expansion (BAT) to approximate the necessary potential energy and nonadiabatic coupling matrix elements. The BAT approximation avoids the necessity of computing electronic structure information at intermediate points between TBFs, as is usually done in saddle-point approximations used in AIMS. The efficiency of AIMC is demonstrated on the nonradiative decay of the first excited state of ethylene. The AIMC method has been implemented within the AIMS-MOLPRO package, which was extended to include Ehrenfest basis functions.

  11. Ab initio multiple cloning algorithm for quantum nonadiabatic molecular dynamics.

    PubMed

    Makhov, Dmitry V; Glover, William J; Martinez, Todd J; Shalashilin, Dmitrii V

    2014-08-01

    We present a new algorithm for ab initio quantum nonadiabatic molecular dynamics that combines the best features of ab initio Multiple Spawning (AIMS) and Multiconfigurational Ehrenfest (MCE) methods. In this new method, ab initio multiple cloning (AIMC), the individual trajectory basis functions (TBFs) follow Ehrenfest equations of motion (as in MCE). However, the basis set is expanded (as in AIMS) when these TBFs become sufficiently mixed, preventing prolonged evolution on an averaged potential energy surface. We refer to the expansion of the basis set as "cloning," in analogy to the "spawning" procedure in AIMS. This synthesis of AIMS and MCE allows us to leverage the benefits of mean-field evolution during periods of strong nonadiabatic coupling while simultaneously avoiding mean-field artifacts in Ehrenfest dynamics. We explore the use of time-displaced basis sets, "trains," as a means of expanding the basis set for little cost. We also introduce a new bra-ket averaged Taylor expansion (BAT) to approximate the necessary potential energy and nonadiabatic coupling matrix elements. The BAT approximation avoids the necessity of computing electronic structure information at intermediate points between TBFs, as is usually done in saddle-point approximations used in AIMS. The efficiency of AIMC is demonstrated on the nonradiative decay of the first excited state of ethylene. The AIMC method has been implemented within the AIMS-MOLPRO package, which was extended to include Ehrenfest basis functions. PMID:25106573

  12. Analysis of nuclear reprogramming in cloned miniature pig embryos by expression of Oct-4 and Oct-4 related genes

    SciTech Connect

    Lee, Eugine; Lee, So Hyun; Kim, Sue

    2006-10-06

    Xenotransplantation is a rapidly expanding field of research and cloned miniature pigs have been considered as a model animal for it. However, the efficiency of somatic cell nuclear transfer (SCNT) is extremely low, with most clones resulting in early lethality and several kinds of aberrant development. A possible explanation for the developmental failure of SCNT embryos is insufficient reprogramming of the somatic cell nucleus by the oocyte. In order to test this, we analyzed the reprogramming capacity of differentiated fibroblast cell nuclei and embryonic germ cell nuclei with Oct-4 and Oct-4 related genes (Ndp5211, Dppa2, Dppa3, and Dppa5), which are important for embryonic development, Hand1 and GATA-4, which are important for placental development, as molecular markers using RT-PCR. The Oct-4 expression level was significantly lower (P < 0.05) in cloned hatched blastocysts derived from fibroblasts and many of fibroblast-derived clones failed to reactivate at least one of the tested genes, while most of the germ cell clones and control embryos correctly expressed these genes. In conclusion, our results suggest that the reprogramming of fibroblast-derived cloned embryos is highly aberrant and this improper reprogramming could be one reason of the early lethality and post-implantation anomalies of somatic cell-derived clones.

  13. Biological, molecular, and structural analysis of a cytopathic variant from a molecularly cloned simian immunodeficiency virus.

    PubMed Central

    LaBranche, C C; Sauter, M M; Haggarty, B S; Vance, P J; Romano, J; Hart, T K; Bugelski, P J; Hoxie, J A

    1994-01-01

    Some isolates of simian immunodeficiency virus (SIV) have been shown to infect Sup-T1 cells with slow kinetics and in the absence of cytopathic effects, including cell fusion or CD4 down-modulation (J. A. Hoxie, B. S. Haggarty, S. Bonser, J. Rackowski, H. Shan, and P. Kanki, J. Virol. 62:2557-2568, 1988). In the present study, we describe the isolation and characterization of a SIVmac variant, derived from the BK28 infectious molecular clone, that became highly cytopathic for Sup-T1 cells. This variant, termed CP-MAC, exhibited a number of differences from BK28, including (i) an altered tropism which largely restricted its host range to Sup-T1 cells, (ii) the ability to induce cell fusion and CD4 down-modulation, and (iii) a highly stable interaction of its external (SU) and transmembrane (TM) envelope glycoproteins. In addition, a marked increase in the level of surface envelope glycoproteins was observed both on CP-MAC-infected cells and on virions. The CP-MAC env gene was PCR amplified from infected cells, and sequence analysis identified five amino acid changes in SU and six in TM compared with BK28. The introduction of these changes into BK28 was shown to fully reconstitute the biological and morphological properties of CP-MAC. The limited number of mutations in CP-MAC should enable the molecular determinants to be more precisely defined and help to identify the underlying mechanisms responsible for the striking biological and structural alterations exhibited by this virus. Images PMID:8057433

  14. Molecular cloning and characterization of a novel repeat-containing Leishmania major gene, ppg1, that encodes a membrane-associated form of proteophosphoglycan with a putative glycosylphosphatidylinositol anchor.

    PubMed

    Ilg, T; Montgomery, J; Stierhof, Y D; Handman, E

    1999-10-29

    Leishmania parasites secrete a variety of proteins that are modified by phosphoglycan chains structurally similar to those of the cell surface glycolipid lipophosphoglycan. These proteins are collectively called proteophosphoglycans. We report here the cloning and sequencing of a novel Leishmania major proteophosphoglycan gene, ppg1. It encodes a large polypeptide of approximately 2300 amino acids. The N-terminal domain of approximately 70 kDa exhibits 11 imperfect amino acid repeats that show some homology to promastigote surface glycoproteins of the psa2/gp46 complex. The large central domain apparently consists exclusively of approximately 100 repetitive peptides of the sequence APSASSSSA(P/S)SSSSS(+/-S). Gene fusion experiments demonstrate that these peptide repeats are the targets of phosphoglycosylation in Leishmania and that they form extended filamentous structures reminiscent of mammalian mucins. The C-terminal domain contains a functional glycosylphosphatidylinositol anchor addition signal sequence, which confers cell surface localization to a normally secreted Leishmania acid phosphatase, when fused to its C terminus. Antibody binding studies show that the ppg1 gene product is phosphoglycosylated by phosphoglycan repeats and cap oligosaccharides. In contrast to previously characterized proteophosphoglycans, the ppg1 gene product is predominantly membrane-associated and it is expressed on the promastigote cell surface. Therefore this membrane-bound proteophosphoglycan may be important for direct host-parasite interactions. PMID:10531342

  15. Cloning and expression pattern of akirin2 gene in broiler.

    PubMed

    Man, Chaolai; Chang, Yang; Mu, Weitao; Zhao, Dongxue

    2014-12-01

    Akirin2 is an important nuclear factor which plays functions in innate immune response, myogenesis, muscle development, and carcinogenesis. In this study, akirin2 genes were cloned from 4-day-old Sanhuang and AA(+) broiler, and its expression patterns were analyzed by RT-PCR. The results showed that there were four SNPs in the 5'-terminal region of akirin2 coding sequences. Expression profile analysis showed that the akirin2 transcripts were constitutively expressed in 15 tissues tested, and similar expression patterns were found between the two breeds of broilers. In addition, one of the interesting findings was that the akirin2 gene is highly expressed in blood and lowly expressed in heart, respectively. These data can serve as a foundation for further studying functions of akirin2 gene. PMID:25098451

  16. Yeast vectors and assays for expression of cloned genes.

    PubMed

    Reynolds, A; Lundblad, V; Dorris, D; Keaveney, M

    2001-05-01

    This unit describes some of the most commonly used yeast vectors, as well as the cloned yeast genes that form the basis for these plasmids. Yeast vectors can be grouped into five general classes, based on their mode of replication in yeast: YIp, YRp, YCp, YEp, and YLp plasmids. With the exception of the YLp plasmids (yeast linear plasmids), all of these plasmids can be maintained in E. coli as well as in S. cerevisiae and thus are referred to as shuttle vectors. The nomenclature of different classes of yeast vectors, as well as details about their mode of replication in yeast are discussed. PMID:18265101

  17. Cloning of the glutamine synthetase gene from group B streptococci.

    PubMed

    Suvorov, A N; Flores, A E; Ferrieri, P

    1997-01-01

    The glnA gene from the human pathogen Streptococcus agalactiae was cloned from a genomic library prepared with the lambda phage vector lambdaDASHII. A 4.6-kb DNA fragment of one of the recombinant phages was subcloned in pUC18. This Escherichia coli clone expressed a 52-kDa protein encoded by a 1,341-bp open reading frame. The nucleotide sequence of the open reading frame and the deduced amino acid sequence shared a significant degree of homology with the sequences of other glutamine synthetases (GS). The highest homology was between our deduced protein and GS of gram-positive bacteria such as Bacillus subtilis, Bacillus cereus, and Staphylococcus aureus. Plasmids with the cloned streptococcal glnA were able to complement E. coli glnA mutants grown on minimal media. Rabbit antisera to streptococcal GS recombinant protein recognized not only the recombinant protein but also a similar-sized band in mutanolysin extracts of all group B streptococcal strains tested, regardless of polysaccharide type or surface protein profile. The amino acid sequence of the deduced protein had similarities to other streptococcal cell-surface-bound proteins. The possible functional role of the immunological features of streptococcal GS is discussed. PMID:8975911

  18. Cloning

    MedlinePlus

    ... DNA Reproductive cloning, which creates copies of whole animals Therapeutic cloning, which creates embryonic stem cells. Researchers hope to use these cells to grow healthy tissue to replace injured or diseased tissues in the human body. NIH: National Human Genome Research Institute

  19. In vitro expression of human p53 cDNA clones and characterization of the cloned human p53 gene.

    PubMed

    Wolf, D; Laver-Rudich, Z; Rotter, V

    1985-08-01

    The human p53 gene was cloned and characterized by using a battery of p53 DNA clones. A series of human cDNA clones of various sizes and relative localizations to the mRNA molecule were isolated by using the human p53-H14 (2.35-kilobase) cDNA probe which we previously cloned. One such isolate, clone p53-H7 (2.65 kilobases), spans the entire human mature p53 mRNA molecule. Construction of the human cDNA clones in the pSP65 RNA transcription vector facilitated the generation of p53 transcripts by the SP6 bacteriophage RNA polymerase. The p53-specific RNA transcripts obtained without further processing were translated into p53 proteins in a cell-free system. By using this rapid in vitro transcription-translation assay, we found that whereas clone p53-H7 (2.65 kilobases) coded for a mature-sized p53 protein, a shorter cDNA clone, p53-H13 (1.8 kilobases), dictated the synthesis of a smaller-sized p53 protein (45 kilodaltons). The p53 proteins synthesized in vitro immunoprecipitated efficiently with human-specific anti-p53 antibodies. Genomic analysis of human DNA revealed the presence of a single p53 gene residing within two EcoRI fragments. Heteroduplex analysis between the full-length cDNA clone p53-H7 and the cloned p53 gene indicated the presence of seven major exons. PMID:3018534

  20. Cloning of the Thermomonospora fusca Endoglucanase E2 gene in Streptomyces lividans: Affinity purification and functional domains of the cloned gene product

    SciTech Connect

    Ghangas, G.S.; Wilson, D.B. )

    1988-10-01

    Thermomonospora fusca YX grown in the presence of cellulose produces a number of {beta}-1-4-endoglucanases, some of which bind to microcrystalline cellulose. By using a multicopy plasmid, pIJ702, a gene coding for one of these enzymes (E2) was cloned into Streptomyces lividans and then mobilized into both Escherichia coli and Streptomyces albus. The gene was localized to a 1.6-kilobase PvuII-ClaI segment of the originally cloned 3.0-kilobase SstI fragment of Thermomonospora DNA. The culture supernatants of Streptomyces transformants contain a major endoglucanase that cross-reacts with antibody against Thermomonospora cellulase E2 and has the same molecular weight (43,000) as T. fusca E2. This protein binds quickly and tightly to Avicel. It also binds to filter paper but at a slower rate than to Avicel. Several large proteolytic degradation products of this enzyme generated in vivo lose the ability to bind to Avicel and have higher activity on carboxymethyl cellulose than the native enzyme. Other smaller products bind to Avicel but lack activity. A weak cellobiose-binding site not observed in the native enzyme was present in one of the degradation products. In E. coli, the cloned gene produced a cellulase that also binds tightly to Avicel but appeared to be slightly larger than T. fusca E2. The activity of intact E2 from all organisms can be inactivated by Hg{sup 2+} ions. Dithiothreitol protected against Hg{sup 2+} inactivation and reactivated both unbound and Avicel-bound Hg{sub 2+}-inhibited E2, but at different rates.