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DNA Library (dna + library)
Kinds of DNA Library Selected AbstractsProkaryotic diversity and metabolically active microbial populations in sediments from an active mud volcano in the Gulf of MexicoENVIRONMENTAL MICROBIOLOGY, Issue 10 2006Robert J. Martinez Summary In this study, ribosomes and genomic DNA were extracted from three sediment depths (0,2, 6,8 and 10,12 cm) to determine the vertical changes in the microbial community composition and identify metabolically active microbial populations in sediments obtained from an active seafloor mud volcano site in the northern Gulf of Mexico. Domain-specific Bacteria and Archaea 16S polymerase chain reaction primers were used to amplify 16S rDNA gene sequences from extracted DNA. Complementary 16S ribosomal DNA (crDNA) was obtained from rRNA extracted from each sediment depth that had been subjected to reverse transcription polymerase chain reaction amplification. Twelve different 16S clone libraries, representing the three sediment depths, were constructed and a total of 154 rDNA (DNA-derived) and 142 crDNA (RNA-derived) Bacteria clones and 134 rDNA and 146 crDNA Archaea clones obtained. Analyses of the 576 clones revealed distinct differences in the composition and patterns of metabolically active microbial phylotypes relative to sediment depth. For example, ,- Proteobacteria rDNA clones dominated the 0,2 cm clone library whereas ,-Proteobacteria dominated the 0,2 cm crDNA library suggesting , to be among the most active in situ populations detected at 0,2 cm. Some microbial lineages, although detected at a frequency as high as 9% or greater in the total DNA library (i.e. Actinobacteria, ,- Proteobacteria), were markedly absent from the RNA-derived libraries suggesting a lack of in situ activity at any depth in the mud volcano sediments. This study is one of the first to report the composition of the microbial assemblages and physiologically active members of archaeal and bacterial populations extant in a Gulf of Mexico submarine mud volcano. [source] Cloning, 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] DNA aptamers developed against a soman derivative cross-react with the methylphosphonic acid core but not with flanking hydrophobic groupsJOURNAL OF MOLECULAR RECOGNITION, Issue 3 2009John G. Bruno Abstract Twelve rounds of systematic evolution of ligands by exponential enrichment (SELEX) were conducted against a magnetic bead conjugate of the para -aminophenylpinacolylmethylphosphonate (PAPMP) derivative of the organophosphorus (OP) nerve agent soman (GD). The goal was to develop DNA aptamers that could scavenge GD in vivo, thereby reducing or eliminating the toxic effects of this dangerous compound. Aptamers were sequenced and screened in peroxidase-based colorimetric plate assays after rounds 8 and 12 of SELEX. The aptamer candidate sequences exhibiting the highest affinity for the GD derivative from round 8 also reappeared in several clones from round 12. Each of the highest affinity PAPMP-binding aptamers also bound methylphosphonic acid (MPA). In addition, the aptamer with the highest overall affinity for PAPMP carried a sequence motif (TTTAGT) thought to bind MPA based on previously published data (J. Fluoresc 18: 867,876, 2008). This sequence motif was found in several other relatively high affinity PAPMP aptamer candidates as well. In studies with the nerve agent GD, pre-incubation of a large molar excess of aptamer candidates failed to protect human butyrylcholinesterase (BuChE) from inhibition. With the aid of three-dimensional molecular modeling of the GD derivative it appears that a hydrophilic cleft sandwiched between the pinacolyl group and the p -aminophenyl ring might channel nucleotide interactions to the phosphonate portion of the immobilized GD derivative. However, bona fide GD free in solution may be repulsed by the negative phosphate backbone of aptamers and rotate its phosphonate and fluorine moieties away from the aptamer to avoid being bound. Future attempts to develop aptamers to GD might benefit from immobilizing the pinacolyl group of bona fide GD to enhance exposure of the phosphonate and fluorine to the random DNA library. Copyright © 2008 John Wiley & Sons, Ltd. [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] Isolation and characterization of 102 new microsatellite loci in Murray cod, Maccullochella peelii peelii (Percichthyidae), and assessment of cross-amplification in 13 Australian native and six introduced freshwater speciesMOLECULAR ECOLOGY RESOURCES, Issue 6 2007MEAGHAN ROURKE Abstract We have isolated 102 polymorphic microsatellite loci from an enriched Murray cod DNA library and also assessed their amplification success in 13 native and six introduced freshwater fish species. The loci will serve the dual purpose of assessing wild population genetic structure for future conservation efforts, and for identifying markers for key quantitative trait loci important for aquaculture. [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] Optimization of novel polymorphic microsatellites in muskox (Ovibos Moschatus) leads to an increased estimate of muskox microsatellite diversityMOLECULAR ECOLOGY RESOURCES, Issue 4 2004PETER J. VAN COEVERDEN Abstract Initial microsatellite studies suggested that muskoxen display the lowest microsatellite polymorphism in a large ungulate. We optimized 17 di-nucleotide microsatellites with longer repeats from a muskox DNA library and surveyed 18 animals from across their natural range. Loci with perfect repeats were more variable than imperfect loci: Hperfect = 0.504 ± 0.045 (± SE) vs. Himperfect = 0.067 ± 0.055. Our Hperfect estimate is higher than previous low estimates of HE = 0.018 and HE = 0.059. [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] Characterization of microsatellite loci in a seed chalcid, Megastigmus spermotrophus (Hymenoptera: Torymidae)MOLECULAR ECOLOGY RESOURCES, Issue 3 2003S. Boivin Abstract Highly polymorphic microsatellite markers can supply demographic information on founder events and range expansion following initial introduction of invasive insect species. Six microsatellite loci were isolated from a partial DNA library in order to study the invasion patterns of a seed chalcid, Megastigmus spermotrophus, introduced to Europe and New Zealand. Allelic diversity at all described loci was high, ranging from 17 to 30 alleles per locus. All six loci were successfully amplified in 15 congeneric species. [source] Isolation and characterization of microsatellite loci from Asparagus acutifolius (Liliaceae)MOLECULAR ECOLOGY RESOURCES, Issue 2 2003S. Aceto Abstract The isolation of molecular markers in Asparagus acutifolius, a wild edible plant species, is important to characterize local ecotypes that could be cultivated and preserved. We isolated and characterized polymorphic microsatellite loci from A. acutifolius by constructing and screening an enriched DNA library. Primer pairs were designed for 12 loci. Seven primer pairs worked well during amplification reactions and were tested on a wild population from Pontecagnano (SA), Italy. These loci showed a high level of genetic variability, with the numbers of alleles identified ranging from two to five and observed heterozygosity ranging from 0.20 to 0.73. [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] Identification and expression analysis of a MYB family transcription factor in the parasitic plant Orobanche ramosaANNALS OF APPLIED BIOLOGY, Issue 2 2007C.I. González-Verdejo Abstract MYB proteins are transcription factors (TFs) involved in the regulation of developmental processes in eukaryotes. A number of MYB genes have been identified from plants, but they have not been studied in parasitic plants. In this work, a member of the R2R3 MYB family of TFs was isolated from a complementary DNA library representing different developmental stages of the parasitic plant Orobanche ramosa. The pattern of expression of the gene was studied by in situ hybridisation. Alignment of the deduced Or-MYB1 protein with members of the MYB family showed the highest overall identity with MYB.Ph3 from petunia (Petunia hybrida), NtMYBAS1/S2 from tobacco (Nicotiana tabacum) and AtMYB101 from Arabidopsis thaliana. Amino acid sequence comparisons of DNA-binding domains showed that Or-MYB1 protein forms a closely related group with these proteins. Transcripts of Or-MYB1 were detected during all the developmental stages analysed, and in situ hybridisation showed that the expression was restricted to the parenchymatic cells proximal to the vascular vessels. These findings are consistent with a role of Or-MYB1 during early stages of development of O. ramosa, probably through the phenylpropanoid pathway. [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] |