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Plasmids of Azotobacter vinelandii

Description: Nineteen laboratory strains of Azotobacter vinelandii and two organisms of the same species isolated from water samples were screened for the presence of plasmid deoxyribonucleic acid (DNA). Three laboratory strains and both organisms isolated from water samples contained one plasmid each. The migration distances of the plasmids in agarose gel electrophoresis were different molecular weights. The plasmids were cured by SDS or ethidium bromide treatment of the cultures.
Date: May 1986
Creator: Maia, Mauricio Silva
Partner: UNT Libraries

Physical characterization and restriction mapping of the SAL plasmid from Pseudomonas putida

Description: Physical and restriction mapping of the salicylate catabolic plasmid SAL from Pseudomonas putida strain PpG 2119 was carried out by standard multiple restriction analysis and by cross hybridization studies using radioactively labeled restriction fragment probes. The total numbers of fragments produced, their respective sizes, the arrangement of the restriction fragments on the plasmid and the map locations of the enzyme recognition sites for Hpal, Xhol, Dral and Smal are given.
Date: December 1987
Creator: Asghari, Abdolkarim
Partner: UNT Libraries

Structural Analysis of the TOL pDK1 xylGFJQK Region and Partial Characterization of the xylF and xylG Gene Products

Description: TOL plasmids encode enzymes responsible for utilization of toluene and related aromatic compounds by Pseudomonas putida, ultimately converting them to central metabolic intermediates. The nucleotide sequence for the 5.6 kb xylGFJQK region of the pDK1 TOL meta operon was determined. DNA sequence analysis revealed the presence of five open reading frames corresponding to xylG (1458 bp), xylF (846 bp), xylJ (783 bp), xylQ (936 bp) and xylK (1047 bp), encoding predicted protein products of 51.6, 31.3, 27.8, 32.8, and 36.6 kDa in size, respectively. The average G+C content of the xylLTEGFJQK region was 65.7%, somewhat higher than the 58.9% seen in the immediately upstream xylXYZ region and substantially more than the 50% G+C content reported for the upper TOL operon of this plasmid. Homology comparisons were made with genes and proteins of related catabolic plasmids. The dmpCDEFG and pWWO xylGFJQK regions exhibit consistently high levels of nucleotide and amino acid homology to pDK1 xylGFJQK throughout the entire region. In contrast, although the nucleotide sequence homology of the Acinetobacter atdCDE region to xylGFJ is high, the homology of atdFG to xylQK is markedly less. Such radical changes in homology between corresponding regions of different operons, combined with variable base and codon usage patterns within and between operons, provides additional support for the idea that the upper and lower operons encoding enzymes of aromatic pathways have evolved independently of one another and that these operons have continued to exchange genetic material with homologous expression units through a series of recombination events. Recombinant plasmids were constructed for individual expression of each of the xylGFJQK genes. HMSD (XylG) and HMSH (XylF) were partially purified and characterized with respect to substrate specificity and kinetic mechanism. Evidence was obtained suggesting that the HMSD reaction occurs via a steady state ordered mechanism or a random mechanism where ...
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Date: December 1999
Creator: Poulter, Melinda D.
Partner: UNT Libraries

Physical and Functional Characterization of the xy1XYZ Region From TOL Plasmid pDK1 and its Associated Downstream Regulatory Elements

Description: The nucleotide sequence for the pDKl TOL plasmid region encoding toluate-1,2-dioxygenase (Xy1XYZ, TO) was determined. TO is the first enzyme in the meta-cleavage operon, responsible for the conversion of toluates and benzoates to their carboxy-substituted diols. DNA sequence analysis revealed the presence of three open reading frames (ORF). The three ORFs correspond to xylX (1353 bp), xylY (486 bp) and xylZ (1008 bp), encoding predicted protein products of 51370 Da, 19368 Da and 36256 Da, respectively.
Date: August 1998
Creator: Hares, Douglas R. (Douglas Ryan)
Partner: UNT Libraries

The integrated microbial genomes (IMG) system in 2007: datacontent and analysis tool extensions

Description: The Integrated Microbial Genomes (IMG) system is a data management, analysis and annotation platform for all publicly available genomes. IMG contains both draft and complete JGI microbial genomes integrated with all other publicly available genomes from all three domains of life, together with a large number of plasmids and viruses. IMG provides tools and viewers for analyzing and annotating genomes, genes and functions, individually or in a comparative context. Since its first release in 2005, IMG's data content and analytical capabilities have been constantly expanded through quarterly releases. IMG is provided by the DOE-Joint Genome Institute (JGI) and is available from http://img.jgi.doe.gov.
Date: August 1, 2007
Creator: Markowitz, Victor M.; Szeto, Ernest; Palaniappan, Krishna; Grechkin, Yuri; Chu, Ken; Chen, I-Min A. et al.
Partner: UNT Libraries Government Documents Department

Isolation of the xylE-xylL region of Pseudomonas putida plasmid pDKR1 and determination of the complete nucleotide sequence of the xylE gene encoding catechol 2,3-dioxygenase

Description: A 5.1 kbEcoR I fragment from Pseudomonas putida TOL plsmid pDKR1, carrying the xylE and xylL genes, was inserted into pBR 325 and transformed into E. coli. The xylE region, coding for catechol 2,3-dioxygenase, was subjected to Maxam-Gilbert sequencing reactions.
Date: May 1989
Creator: Voss, John A. (John Andrew)
Partner: UNT Libraries

Nucleotide Sequence Determination, Subcloning, Expression and Characterization of the xy1LT Region of the Pseudomonas putida TOL Plasmid pDK1

Description: The complete nucleotide sequence of the region encoding the DHCDH function of the pDK1 lower operon was determined. DNA analysis has shown the presence of two open reading frames, one gene consisting of 777 nucleotides encoding a polypeptide of 27.85 kDa and another gene of 303 nucleotides encoding a polypeptide of 11.13 kDa. The results of enzymatic expression studies suggest that DHCDH activity is associated only with xy1L. However although the addition of xy1T cell-free extracts to xy1L cell-free extracts does not produce an increase in DHCDH activity, subclones carrying both xy1L and xy1T exhibit 300- 400% more DHCDH activity than subclones carrying only xy1L.
Date: December 1992
Creator: Baker, Ronald F. (Ronald Fredrick)
Partner: UNT Libraries

Characterization of the OCT Plasmid-Encoded Mercury Resistance Genetic Locus in Pseudomonas putida

Description: A 17.1 Kb genetic element encoding for mercury resistance (OCT-Hg^r) was shown to translocate from its original location on the OCT plasmid to the resistance plasmid, RPl, in Pseudomonas putida. Analysis of RPl-Hg^r recombinant plasmids revealed that insertion of mercury resistance genes into RPl could occur at a variety of sites, with all recombinants having common EcoRI restriction fragments of 9.4, 3.8, 2.3, and 1.6 Kb, derived from the insertion. Hybridization analysis suggested the existence of extensive homology between this insertion and the prototypic mercury resistance transposon, Tn501, as well as the location of a similar merA sequence. Although the overall size was shown to be quite different from Tn501, striking physical similarities are shared between these two elements.
Date: May 1989
Creator: Armbruster, Steven C. (Steven Christopher)
Partner: UNT Libraries

Structural Analysis of the Genes Encoding the Oxalocrotonate Branch of the Pseudomonas putida TOL Plasmid pDKI meta-cleavage Pathway and the Expression of the xy1G Gene Product in Escherichia coli

Description: Three overlapping DNA fragments from the lower operon of Pseudomonas putida TOL plasmid pDK1, covering the xy1IH genes and downstream flanking region, were cloned into pUC19. They include a 2.8 kbp XhoI fragment, a 2.7 kbp PstI fragment and a 2.0 kbp EcoRI-HindIII fragment. They were subjected to DNA sequence analysis. The xy1I (4-oxalocrotonate decarboxylase) and xy1H (4-oxalocrotonate tautomerase) genes were found to possess coding regions of 792 and 189 nucleotides, respectively. A possible transcriptional terminator resembling E. coli rho-independent terminators was identified downstream of the translational stop of xy1H. An additional stem and loop structure was found in the intergenic region between xy1I and xy1H. The individual ORF's of the oxalocrotonate branch (xy1G, xy1I and xy1H) have been cloned into pUC18/19. The expression of the xy1G gene in E. coli was successfully assayed spectrophotometrically.
Date: December 1992
Creator: Luo, Xuebin
Partner: UNT Libraries

A Novel Mechanism for Site-Directed Mutagenesis of Large Catabolic Plasmids Using Natural Transformation

Description: Natural transformation is the process by which cells take up DNA from the surrounding medium under physiological conditions, altering the genotype in a heritable fashion. This occurs without chemical or physical treatment of the cells. Certain Acinetobacter strains exhibit a strong tendency to incorporate homologous DNA into their chromosomes by natural transformation. Transformation in Acinetobacter exhibits several unique properties that indicate this system's superiority as a model for transformation studies or studies which benefit from the use of transformation as an experimental method of gene manipulation. Pseudomonas putida is the natural host of TOL plasmids, ranging between 50 kbp and 300 kbp in size and encoding genes for the catabolism of toluene, meta-toluate, and xylene. These very large, single-copy plasmids are difficult to isolate, manipulate, or modify in vitro. In this study, the TOL plasmid pDKR1 was introduced into Acinetobacter calcoaceticus strains and genetically engineered utilizing natural transformation as part of the process. Following engineering by transformation, the recombinant DNA molecule was returned to the native genetic background of the original host P. putida strain. Specific parameters for the successful manipulation of large plasmids by natural transformation in Acinetobacter were identified and are outlined. The effects of growth phase, total transforming DNA concentration, transforming DNA conformation, and gene dosage on transformation efficiency are presented. Addition of Acinetobacter plasmid DNA sequences to the manipulated constructs did not have an effect on transformation rates. Results suggest that a broadly applicable and efficient method to carry out site-directed genetic manipulations of large plasmids has been identified. The ability to easily reintroduce the recombinant DNA molecules back into the original host organism was maintained.
Date: August 2001
Creator: Williamson, Phillip C.
Partner: UNT Libraries

Discovery and Characterization of Two Tn5 Generated pyrA Mutants in Pseudomonas putida and the Generation of Hfr Strains

Description: A pyrA mutation in Pseudomonas putida was isolated using transposon mutagenesis for the first time. Transposon Tn5 was used to inactivate the pyrA gene for carbamoylphosphate synthetase in these mutants. Accordingly, these mutants were defective in pyrimidine and arginine biosynthesis. The suicide vector, pM075, from Pseudomonas aeruginosa, was used to introduce the transposon into the cells. Tn5 was subsequently used to supply homology so that the plasmid pM075 could be introduced in its entirety into the Pseudomonas putida chromosome at the locus of the Tn5 insertion in the pyrA gene. Consequently, these strains exhibited high frequency of recombination and were capable of chromosome mobilization.
Date: August 1994
Creator: Liljestrand, Laura Gail
Partner: UNT Libraries

The Methanosarcina barkeri genome: comparative analysis withMethanosarcina acetivorans and Methanosarcina mazei reveals extensiverearrangement within methanosarcinal genomes

Description: We report here a comparative analysis of the genome sequence of Methanosarcina barkeri with those of Methanosarcina acetivorans and Methanosarcina mazei. All three genomes share a conserved double origin of replication and many gene clusters. M. barkeri is distinguished by having an organization that is well conserved with respect to the other Methanosarcinae in the region proximal to the origin of replication with interspecies gene similarities as high as 95%. However it is disordered and marked by increased transposase frequency and decreased gene synteny and gene density in the proximal semi-genome. Of the 3680 open reading frames in M. barkeri, 678 had paralogs with better than 80% similarity to both M. acetivorans and M. mazei while 128 nonhypothetical orfs were unique (non-paralogous) amongst these species including a complete formate dehydrogenase operon, two genes required for N-acetylmuramic acid synthesis, a 14 gene gas vesicle cluster and a bacterial P450-specific ferredoxin reductase cluster not previously observed or characterized in this genus. A cryptic 36 kbp plasmid sequence was detected in M. barkeri that contains an orc1 gene flanked by a presumptive origin of replication consisting of 38 tandem repeats of a 143 nt motif. Three-way comparison of these genomes reveals differing mechanisms for the accrual of changes. Elongation of the large M. acetivorans is the result of multiple gene-scale insertions and duplications uniformly distributed in that genome, while M. barkeri is characterized by localized inversions associated with the loss of gene content. In contrast, the relatively short M. mazei most closely approximates the ancestral organizational state.
Date: May 19, 2006
Creator: Maeder, Dennis L.; Anderson, Iain; Brettin, Thomas S.; Bruce,David C.; Gilna, Paul; Han, Cliff S. et al.
Partner: UNT Libraries Government Documents Department

Single molecule study of a processivity clamp sliding on DNA

Description: Using solution based single molecule spectroscopy, we study the motion of the polIII {beta}-subunit DNA sliding clamp ('{beta}-clamp') on DNA. Present in all cellular (and some viral) forms of life, DNA sliding clamps attach to polymerases and allow rapid, processive replication of DNA. In the absence of other proteins, the DNA sliding clamps are thought to 'freely slide' along the DNA; however, the abundance of positively charged residues along the inner surface may create favorable electrostatic contact with the highly negatively charged DNA. We have performed single-molecule measurements on a fluorescently labeled {beta}-clamp loaded onto freely diffusing plasmids annealed with fluorescently labeled primers of up to 90 bases. We find that the diffusion constant for 1D diffusion of the {beta}-clamp on DNA satisfies D {le} 10{sup -14} cm{sup 2}/s, much slower than the frictionless limit of D = 10{sup -10} cm{sup 2}/s. We find that the {beta} clamp remains at the 3-foot end in the presence of E. coli single-stranded binding protein (SSB), which would allow for a sliding clamp to wait for binding of the DNA polymerase. Replacement of SSB with Human RP-A eliminates this interaction; free movement of sliding clamp and poor binding of clamp loader to the junction allows sliding clamp to accumulate on DNA. This result implies that the clamp not only acts as a tether, but also a placeholder.
Date: July 5, 2007
Creator: Laurence, T A; Kwon, Y; Johnson, A; Hollars, C; O?Donnell, M; Camarero, J A et al.
Partner: UNT Libraries Government Documents Department

High-Throughput Plasmid cDNA Library Screening

Description: Libraries of cDNA clones are valuable resources foranalysing the expression, structure, and regulation of genes, as well asfor studying protein functions and interactions. Full-length cDNA clonesprovide information about intron and exon structures, splice junctionsand 5'- and 3'-untranslated regions (UTRs). Open reading frames (ORFs)derived from cDNA clones can be used to generate constructs allowingexpression of native proteins and N- or C-terminally tagged proteins.Thus, obtaining full-length cDNA clones and sequences for most or allgenes in an organism is critical for understanding genome functions.Expressed sequence tag (EST) sequencing samples cDNA libraries at random,which is most useful at the beginning of large-scale screening projects.However, as projects progress towards completion, the probability ofidentifying unique cDNAs via EST sequencing diminishes, resulting in poorrecovery of rare transcripts. We describe an adapted, high-throughputprotocol intended for recovery of specific, full-length clones fromplasmid cDNA libraries in five days.
Date: May 24, 2006
Creator: Wan, Kenneth H.; Yu, Charles; George, Reed A.; Carlson, JosephW.; Hoskins, Roger A.; Svirskas, Robert et al.
Partner: UNT Libraries Government Documents Department

Molecular Cloning and Functional Analysis of Transposable Mercury Resistance Genes Encoded by the OCT Plasmid

Description: Translocation of a 17.1 kilobase region of the OCT plasmid encoding mercury resistance (mer) in Pseudomonas putida was shown to occur in a recombination-deficient host with plasmid PP1 serving as a recipient replicon. The frequency of transposition in Pseudomonas was estimated at 10^3 -10 -^2, but undetectable in Escherichia soli. ' DNA comprising all of mr as well as subregions there of were cloned and subjected to DNA sequence analysis. Like other transposons, mer was found to contain inverted repeat sequences at its termini. These were similar to, but not identical to the inverted repeat structures found in the prototypical mercury resistance transposon Tn501 from E. aeruginosa.
Date: August 1991
Creator: Wang, Chien-Sao
Partner: UNT Libraries

Horizontal gene transfer as adaptive response to heavy metal stress in subsurface microbial communities. Final report for period October 15, 1997 - October 15, 2000

Description: Horizontal gene transfer as adaptive response to heavy metal stress in the presence of heavy metal stress was evaluated in oligotrophic subsurface soil laboratory scale microcosms. Increasing levels of cadmium (10, 100 and 1000 mM) were applied and an E. coli donor was used to deliver the target plasmids, pMOL187 and pMOL222, which contained the czc and ncc operons, and the helper plasmid RP4. Plasmid transfer was evaluated through monitoring of the heavy metal resistance and presence of the genes. The interactive, clearly revealed, effect of biological and chemical external factors on the extent of plasmid-DNA propagation in microbial communities in contaminated soil environments was observed in this study. Additionally, P.putida LBJ 415 carrying a suicide construct was used to evaluate selective elimination of a plasmid donor.
Date: December 21, 2001
Creator: Smets, B. F.
Partner: UNT Libraries Government Documents Department

Controlled production of cellulases in plants for biomass conversion. Annual report, March 11, 1997--March 14, 1998

Description: The goal of this project is to facilitate conversion of plant biomass to usable energy by developing transgenic plants that express genes for microbial cellulases, which can be activated after harvest of the plants. In particular, the feasibility of targeting an endoglucanase and a cellobiohydrolase to the plant apoplast (cell wall milieu) is to be determined. To avoid detrimental effects of cellulose expression in plants, enzymes with high temperature optima were chosen; the genes for these enzymes are from thermophilic organisms that can use cellulose as a sole energy source. During the past year (year 2 of the grant), efforts have been focused on testing expression of endoglucanase E{sub 1}, from Acidothermus cellulolyticus, in the apoplast of both tobacco suspension cells and Arabidopsis thaliana plants. Using the plasmids constructed during the first year, transgenic cells and plants that contain the gene for the E{sub 1} catalytic domain fused to a signal peptide sequence were obtained. This gene was constructed so that the fusion protein will be secreted into the apoplast. The enzyme is made in large quantities and is secreted into the apoplast. More importantly, it is enzymatically active when placed under optimal reaction conditions (high temperature). Moreover, the plant cells and intact plants exhibit no obvious problems with growth and development under laboratory conditions. Work has also continued to improve binary vectors for Agrobacterium-mediated transformation, to determine activity of E{sub 1} at various temperatures, and to investigate the activity of the 35S Cauliflower Mosaic Virus promoter in E. coli. 9 figs.
Date: June 1, 1998
Creator: Danna, K.J.
Partner: UNT Libraries Government Documents Department

[Genome plasticity and catabolic potential of pseudomonas cepacia]. Progress report

Description: This progress report describes efforts directed at understanding the genomic structure of Pseudomonas cepacia. Variously reported are descriptions of the replicons in the genome, organization of macrorestriction fragments comprising the genome, use of a Tn-5- 751S to insertionally inactivate and map selected genes, construction of IS407 derivatives containing a trimethoprim resistance marker and SwaI site, and analysis of nucleotide sequences of IS401 and IS408.
Date: December 31, 1993
Partner: UNT Libraries Government Documents Department

Biochemistry and genetics of autotrophy in Methanococcus. Progress report

Description: In the last two years of this research, the most exciting results have come from the work on the genetics of methanococci. First, the author demonstrated that the cryptic plasmid from Methanococcus maripaludis C5, pURB500, could be transformed into Methanococcus maripaludis JJ. Strain JJ is the type strain of M. maripaludis and has only about 65% DNA:DNA hybridization to strain C5. Because of the low relatedness of these strains, it was not obvious that pURB500 could be transferred between them. This goal was achieved by first transforming strain C5 with a series of suicide plasmids containing the pac cassette, which possessed the selectable puromycin resistance marker, and different cloned fragments of pURB500. From the puromycin-resistant transformants, a plasmid was isolated that transformed strain JJ. However, when this plasmid was electroporated into E. coli, only rearrangement products were obtained that contained small portions of the original pURB500. These plasmids no longer transformed Methanococcus. While these experiments did not yield a shuttle vector, they demonstrated that pURB500 could replicate in strain JJ.
Date: September 1, 1998
Creator: Whitman, W.B.
Partner: UNT Libraries Government Documents Department

Exploration of new perspectives and limitations in Agrobacterium mediated gene transfer technology. Progress report, [June 1, 1992-- May 31, 1994]

Description: This report describes progress aimed at constructing gene-transfer technology for Nicotiana plumbaginifolia. Most actual effort as described herein has so far been directed at exploring new perspectives and limitations in Agrobacterium mediated gene transfer. Accomplishments are described using a core homologous gene targeting vector.
Date: December 31, 1994
Creator: Marton, L.
Partner: UNT Libraries Government Documents Department

Sequential cloning of chromosomes

Description: A method for sequential cloning of chromosomal DNA and chromosomal DNA cloned by this method are disclosed. The method includes the selection of a target organism having a segment of chromosomal DNA to be sequentially cloned. A first DNA segment, having a first restriction enzyme site on either side. homologous to the chromosomal DNA to be sequentially cloned is isolated. A first vector product is formed by ligating the homologous segment into a suitably designed vector. The first vector product is circularly integrated into the target organism`s chromosomal DNA. The resulting integrated chromosomal DNA segment includes the homologous DNA segment at either end of the integrated vector segment. The integrated chromosomal DNA is cleaved with a second restriction enzyme and ligated to form a vector-containing plasmid, which is replicated in a host organism. The replicated plasmid is then cleaved with the first restriction enzyme. Next, a DNA segment containing the vector and a segment of DNA homologous to a distal portion of the previously isolated DNA segment is isolated. This segment is then ligated to form a plasmid which is replicated within a suitable host. This plasmid is then circularly integrated into the target chromosomal DNA. The chromosomal DNA containing the circularly integrated vector is treated with a third, retrorestriction enzyme. The cleaved DNA is ligated to give a plasmid that is used to transform a host permissive for replication of its vector. The sequential cloning process continues by repeated cycles of circular integration and excision. The excision is carried out alternately with the second and third enzymes.
Date: December 31, 1991
Creator: Lacks, S.A.
Partner: UNT Libraries Government Documents Department

Subcloning and Nucleotide Sequence of the xylO/PUWCMA Region from the Pseudomonas putida TOL Plasmid pDK1

Description: The TOL plasmids of Pseudomonas putida encode enzymes required for the oxidation of toluene and other related aromatic compounds. These genes are organized into two operons, the xylUWCMABN operon (upper), and the xylXYZLTEGFJQKIH operon (lower). Here we report the nucleotide sequence of a 7107 bp segment of the TOL pDK1 plasmid encoding the region just upstream of the "upper" operon through the genes encoding xylUWCMA. Sequence analysis, comparison of base-usage patterns, codon-usage patterns, and intergenic distances between genes help support the idea that the "upper" and "lower" operons have evolved independently in different genetic backgrounds and have only more recently been brought together in TOL and related catabolic plasmids.
Date: December 1997
Creator: Guigneaux, Michelle M. (Michelle Marie)
Partner: UNT Libraries

Subcloning and Nucleotide Sequence of Two Positive Acting Regulatory Genes, xy1R and xy1S, from the Pseudomonas putida HS1 TOL Plasmid PDK1

Description: TOL plasmids of Pseudomonas putida encode enzymes for the degradation of toluene and related aromatics. These genes are organized into two operons regulated by the Xy1R and Xy1S transcriptional activators. Previous analysis of the TOL pDK1 catechol-2,3-dioxygenase gene (xy1E) and a comparison of this gene to xy1E from the related TOL plasmid pWW0, revealed the existance of a substantial level of sequence homology (82%).
Date: May 1992
Creator: Chang, Teh-Tsai
Partner: UNT Libraries