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Genomic DNA Library (genomic + dna_library)
Selected AbstractsCloning, expression and characterization of a gene encoding nitroalkane-oxidizing enzyme from Streptomyces ansochromogenesFEBS JOURNAL, Issue 24 2002Jihui Zhang A nitroalkane-oxidizing enzyme gene (naoA) was cloned from a genomic DNA library of Streptomyces ansochromogenes 7100. The deduced protein (NaoA) of this gene contains 363 amino acids and has high similarity to several nitroalkane-oxidizing enzymes from various micro-organisms. The naoA gene was subcloned into an expression vector pET23b and overexpressed in Escherichia coli BL21(DE3). The protein was then purified, and its characteristics were studied. Experimental results showed that NaoA can convert 1-nitropropane, 2-nitropropane and nitroethane into the corresponding carbonyl compounds. The optimal pH and temperature for NaoA was found to be pH 7,8 and 48,56 °C, respectively. The Km of NaoA for nitroethane is ,,26.8 mm. NADH and nitro blue tetrazolium are strong inhibitors of NaoA, and thiol compounds and superoxide dismutase partially inhibit the enzyme activity. Therefore, superoxide may be an essential intermediate in the oxidation of nitroalkane by NaoA. [source] Sequencing and characterization of a novel serine metalloprotease from Burkholderia pseudomalleiFEMS MICROBIOLOGY LETTERS, Issue 1 2000May-Ann Lee Abstract Burkholderia pseudomallei, a Gram-negative bacterium is found in the soil and water, mainly in Southeast Asia and Northern Australia. It is responsible for melioidosis in human and animals. The bacteria produce several potential virulent factors such as extracellular protease, hemolysin, lipase and lecithinase. The isolation of virulence genes and the study of their functions will contribute to our understanding of bacterial pathogenesis. Previous studies have implicated protease as a contributing virulence factor in the pathogenesis of some bacteria. Three out of 5000 clones screened from a genomic DNA library of B. pseudomallei were found to express protease activity. The clones were found to have the same sequence. The nucleotide sequence revealed an open reading frame (designated as metalloprotease A, mprA) encoding a 500-amino acid protein, MprA, with an estimated molecular mass of 50,241 Da. The predicted amino acid sequence shares homology with the subtilisin family of serine proteases. [source] Large-scale screening of intracellular protein localization in living fission yeast cells by the use of a GFP-fusion genomic DNA libraryGENES TO CELLS, Issue 3 2000Da-Qiao Ding Background Intracellular localization is an important part of the characterization of a gene product. In an attempt to search for genes based on the intracellular localization of their products, we constructed a green fluorescent protein (GFP)-fusion genomic DNA library of S. pombe. Results We constructed the S. pombe GFP-fusion genomic DNA library by fusing, in all three reading frames, random fragments of genomic DNA to the 5, end of the GFP gene in such a way that expression of potential GFP-fusion proteins would be under the control of the own promoters contained in the genomic DNA fragments. Fission yeast cells were transformed with this plasmid library, and microscopic screening of 49 845 transformants yielded 6954 transformants which exhibited GFP fluorescence, of which 728 transformants showed fluorescence localized to distinct intracellular structures such as the nucleus, the nuclear membrane, and cytoskeletal structures. Plasmids were isolated from 516 of these transformants, and a determination of their DNA sequences identified 250 independent genes. The intracellular localizations of the 250 GFP-fusion constructs was categorized as an image database; using this database, DNA sequences can be searched for based on the localizations of their products. Conclusions A number of new intracellular structural components were found in this library. The library of GFP-fusion constructs also provides useful fluorescent markers for various intracellular structures and cellular activities, which can be readily used for microscopic observation in living cells. [source] Isolation and characterization of microsatellite loci in the deep-sea marine fish, the roundnose grenadier (Coryphaenoides rupestris)MOLECULAR ECOLOGY RESOURCES, Issue 5 2008HALVOR KNUTSEN Abstract We developed polymerase chain reaction primers for eight dinucleotide microsatellite loci in the marine deep sea fish, roundnose grenadier (Coryphaenoides rupestris). All markers were obtained from a partial genomic DNA library, and characterized in 90 unrelated individuals from one putative population sampled on the Mid-Atlantic Ridge. The number of alleles ranged from two to 61 with an average of 21 per locus. The observed heterozygosity levels ranged from 0.301 to 0.987 with an average of 0.672. Several of the markers amplified multiple alleles from either the Atlantic cod (Gadus morhua) or the deep-sea fish roughhead grenadier (Macrourus berglax). [source] PERMANENT GENETIC RESOURCES: Characterization of eight microsatellite loci in the woolly mouse opossum, Micoureus paraguayanus, isolated from Micoureus demeraraeMOLECULAR ECOLOGY RESOURCES, Issue 2 2008I. M. G. DIAS Abstract Eight novel microsatellite markers were isolated from the woolly mouse opossum from the Amazon Forest in Peru, Micoureus demerarae, using a partial genomic DNA library and an enrichment protocol. These loci were polymorphic in M. demerarae and Micoureus paraguayanus populations from the Atlantic Forest in Brazil with the number of alleles ranging from two to 23. Those eight loci plus another five already described for M. paraguayanus will allow for the evaluation of genetic diversity of populations from the ,Rio Doce' Park, one of the last Atlantic Forest fragments in Minas Gerais state, Brazil. [source] Isolation and characterization of 17 polymorphic microsatellites in grass carpMOLECULAR ECOLOGY RESOURCES, Issue 6 2007JIA LE LI Abstract Here we report the isolation and characterization of 17 polymorphic loci isolated from a partial genomic DNA library of grass carp (Ctenopharyngodon idellus) enriched for CA repeats. We tested variability of these microsatellites on 24 unrelated individuals collected in China. All microsatellites were polymorphic. The average allele number was 7.9 per locus, ranging from four to 13. The observed heterozygosity was from 0.46 to 0.88 with an average of 0.71, whereas the average expected heterozygosity was 0.78. Sixteen of the 17 microsatellites conformed to Hardy,Weinberg equilibrium, and inherited independently. These microsatellites can be used to study genetic diversity and population structure of wild populations, and facilitate selective breeding of cultured broodstocks. [source] PCR primers for trinucleotide and tetranucleotide microsatellites in greater amberjack, Seriola dumeriliMOLECULAR ECOLOGY RESOURCES, Issue 4 2006MARK A. RENSHAW Abstract Eighteen nuclear-encoded microsatellites from a genomic DNA library of greater amberjack, Seriola dumerili, were isolated and characterized. The microsatellites include 13 perfect (five tetranucleotide and eight trinucleotide) and five imperfect (three tetranucleotide, one trinucleotide and one combination dinucleotide/trinucleotide) repeat motifs. The number of alleles at the 18 microsatellites among a sample of 29 fish ranged from two to 20; gene diversity (expected heterozygosity) ranged from 0.068 to 0.950, whereas observed heterozygosity ranged from 0.069 to 0.966. Following Bonferroni correction, genotypes at all 18 microsatellites fit expectations of Hardy,Weinberg equilibrium, and all pairwise comparisons of microsatellites did not deviate significantly from genotypic equilibrium. Greater amberjack support commercial and recreational fisheries along both the Atlantic and the Gulf coasts of the USA and represent a species with potential for worldwide aquaculture. The microsatellites developed will be useful for population genetic studies of ,wild' populations and breeding studies of domesticated populations. [source] Isolation and characterization of microsatellite loci from Larix kaempferiMOLECULAR ECOLOGY RESOURCES, Issue 3 2006KEIYA ISODA Abstract Microsatellites were isolated and characterized for Japanese larch, Larix kaempferi, a conifer species distributed in Japan. A larch genomic DNA library enriched for (AG)n repeats was screened using the colony polymerase chain reaction method and 145 unique microsatellite containing sequences were obtained. Seventy-two primer pairs were designed and 30 produced single-locus products, and 19 of them were polymorphic. The expected heterozygosity ranged from 0.566 to 0.951. These 19 polymorphic microsatellite loci should be valuable markers for genetic studies on Japanese larch. [source] Characterization of seven polymorphic microsatellite loci in the Baja California endemic black-tailed brush lizard Urosaurus nigricaudusMOLECULAR ECOLOGY RESOURCES, Issue 2 2006R. RODRIGUEZ-ESTRELLA Abstract Seven microsatellite loci were developed for the Baja California endemic black-tailed brush lizard Urosaurus nigricaudus, using an enriched genomic DNA library. All loci were polymorphic and overall presented high levels of variation. Number of alleles ranged from five to 16 (average 12.14), and observed heterozygosities from 0.535 to 0.923 (average 0.752). Cross-species amplification was successful and polymorphism was detected for all the loci using the congeners Urosaurus lahtelai and Urosaurus ornatus. These markers will be useful to study fragmented populations of U. nigricaudus on agricultural landscape of the Baja California Peninsula. [source] Development and characterization of novel microsatellite markers from the olive ridley sea turtle (Lepidochelys olivacea)MOLECULAR ECOLOGY RESOURCES, Issue 1 2004Ramesh K. Aggarwal Abstract Olive ridley turtles, although widely distributed globally and in Indian coastal waters, have undergone declines in recent years due to anthropogenic factors, particularly fishery-related mortality. Assessment of genetic variability in existing populations is critical to the development of effective conservation strategies. Here we describe the development of six highly polymorphic microsatellite loci from a simple sequence repeat-enriched genomic DNA library of olive ridley turtle. Characterization of five of these loci using 83 individual olive ridley turtles revealed eight to 24 alleles per locus, high observed and expected heterozygosity values and broad cross-species amplifications. The sixth microsatellite was found to be monomorphic in the olive ridley samples but was polymorphic in two related marine turtle species. These microsatellites thus provide efficient genetic markers to understand the population structure, phylogeography and species relationships of olive ridley and other marine turtle species. [source] Development and variability analysis of microsatellite markers in peachPLANT BREEDING, Issue 1 2002M. J. Aranzana Abstract A genomic DNA library enriched with AG/CT repeats has been developed from the peach cultivar ,Merrill O'Henry'. The enrichment method was efficient, with 61% of the clones obtained carrying a microsatellite sequence and a yield of one polymorphic microsatellite every 2.17 sequenced clones. From 35 microsatellites detected, 24 were polymorphic in a set of 25 cultivars including 14 peaches and 11 nectarines. A total of 82 alleles were found with the polymorphic microsatellites, with an average of a 37% of observed heterozygosity. Microsatellites with a high number of repeats were generally those having the largest number of alleles. All cultivars except two (,Spring Lady' and ,Queencrest') could be individually distinguished with the markers used. Just three selected microsatellites were enough for the discrimination of 24 out of the 25 possible genotypes. Cluster analysis grouped all nectarines in a single cluster. Peaches, with 75 of the 82 alleles found, were more variable than nectarines, with only 64. Microsatellites appear to be powerful and suitable markers for application in peach genetics and breeding. [source] Mapping of rabbit chromosome 1 markers generated from a microsatellite-enriched chromosome-specific libraryANIMAL GENETICS, Issue 5 2001R. Korstanje A genomic DNA library was produced from flow-sorted rabbit chromosome 1 and enriched for fragments containing CA-repeats. Clones containing CA-repeats were identified and primers for amplification of the microsatellite were developed after sequencing the clone. The degree of polymorphism was tested in rabbits from different breeds. This approach identified 12 microsatellite markers which could be used for studying linkage relationships in the progeny of an F2 -intercross: (AX/JUxIIIVO/JU) F2, and two backcrosses: (OS/JxX/J)X/J and (WH/JxX/J)X/J. Seven of these markers were mapped on chromosome 1. [source] Characterisation of distant Alstrogmeria hybrids: application of highly repetitive DNA sequences from A. ligtu ssp. ligtuANNALS OF APPLIED BIOLOGY, Issue 3 2003SHUJUN ZHOU Summary Clones from a Sau 3A family of eight highly repetitive sequences previously isolated from a genomic DNA library of Alstroemeria ligtu ssp. ligtu were sequenced and found to be highly conserved. A trinucleotide microsatellite repeat [GCA]3,4 was present. A second, unrelated, Sau 3A repeat was also characterised. Southern analysis proved that the isolated repeats were specific for the A. ligtu subspecies and could not be detected in other Chilean or Brazilean Alstroemeria species. As shown by in situ hybridisation, the Sau 3A family and the unrelated Sau 3A repeat co-localised at distinct sites along most chromosomes of Alstroemeria ligtu ssp. ligtu and Alstroemeria ligtu ssp. simsii. The present set of species-specific repetitive sequences enables the identification of A. ligtu chromosomes, and thus the tracking of chromosome transmission to interspecific hybrids and their progeny. [source] | ||||||||||