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Generation and molecular characterization of new temperature-sensitive plasmids intended for genetic engineering of Pasteurellaceae.

ABSTRACT: Temperature-sensitive (TS) plasmids were generated through chemical mutagenesis of a derivative of the streptomycin resistance parent plasmid pD70, isolated from Mannheimia hemolytica serotype 1. Three TS plasmids which failed to replicate at or above 42 degrees C in M. hemolytica but which were fully functional below 31 degrees C were selected for further analysis. Two of the TS plasmids were shown by sequencing to possess unique single-base-pair mutations. The third TS plasmid contained a unique base pair substitution and a second mutation that had been previously identified. These mutations were clustered within a 200-bp region of the presumed plasmid origin of replication. Site-directed single-nucleotide substitutions were introduced into the wild-type pD70 origin of replication to confirm that mutations identified by sequencing had conferred thermoregulated replication. Deletion analysis on the wild-type pD70 plasmid replicon revealed that approximately 720 bp are necessary for plasmid maintenance. Replication of the TS plasmids was thermoregulated in Pasteurella multocida and Haemophilus somnus as well. To consistently transform H. somnus with TS plasmid, in vitro DNA methylation with commercially available HhaI methyltransferase was necessary to protect against the organism's restriction enzyme HsoI (recognition sequence 5'-GCGC-3') characterized herein.

SUBMITTER: Briggs RE

PROVIDER: S-EPMC1287723 | biostudies-literature | 2005 Nov

REPOSITORIES: biostudies-literature

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Generation and molecular characterization of new temperature-sensitive plasmids intended for genetic engineering of Pasteurellaceae.

Briggs Robert E RE Tatum Fred M FM

Applied and environmental microbiology 20051101 11

Temperature-sensitive (TS) plasmids were generated through chemical mutagenesis of a derivative of the streptomycin resistance parent plasmid pD70, isolated from Mannheimia hemolytica serotype 1. Three TS plasmids which failed to replicate at or above 42 degrees C in M. hemolytica but which were fully functional below 31 degrees C were selected for further analysis. Two of the TS plasmids were shown by sequencing to possess unique single-base-pair mutations. The third TS plasmid contained a uniq ...[more]

PMID: 16269758

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Project description:BackgroundMovement regularities are inherently present in automated goal-directed motions of the primate's arm system. They can provide important signatures of intentional behaviours driven by sensory-motor strategies, but it remains unknown if during motor learning new regularities can be uncovered despite high variability in the temporal dynamics of the hand motions.MethodsWe investigated the conservation and violation of new movement regularity obtained from the hand motions traced by two untrained monkeys as they learned to reach outwardly towards spatial targets while avoiding obstacles in the dark. The regularity pertains to the transformation from postural to hand paths that aim at visual goals.ResultsIn length-minimizing curves the area enclosed between the Euclidean straight line and the curve up to its point of maximum curvature is 1/2 of the total area. Similar trend is found if one examines the perimeter. This new movement regularity remained robust to striking changes in arm dynamics that gave rise to changes in the speed of the reach, to changes in the hand path curvature, and to changes in the arm's postural paths. The area and perimeter ratios characterizing the regularity co-varied across repeats of randomly presented targets whenever the transformation from posture to hand paths was compliant with the intended goals. To interpret this conservation and the cases in which the regularity was violated and recovered, we provide a geometric model that characterizes arm-to-hand and hand-to-arm motion paths as length minimizing curves (geodesics) in a non-Euclidean space. Whenever the transformation from one space to the other is distance-metric preserving (isometric) the two symmetric ratios co-vary. Otherwise, the symmetric ratios and their co-variation are violated. As predicted by the model we found empirical evidence for the violation of this movement regularity whenever the intended goals mismatched the actions. This was manifested in unintended curved "after-effect" trajectories executed in the absence of obstacles. In this case, the system was "perturbed" away from the symmetry but after several repeats it recovered its default state.ConclusionsWe propose this movement regularity as a sensory-motor transformation invariant of intentional acts.

Project description:Domestic pigs are considered as one of the main intermediate hosts in the zoonotic transmission of Toxoplasma gondii in many countries. Serological and molecular studies are warranted to better understand the epidemiology and transmission patterns of this parasite worldwide. To date, seroepidemiological information on T. gondii in domestic pigs in Cuba is very scarce and there are no reports of T. gondii genotypes circulating in this country. Here, we aimed to estimate the seroprevalence of T. gondii and provide genetic characterization of the strains circulating in slaughtered pigs intended for human consumption in Central Cuba. Seroprevalence was determined in 450 serum samples from slaughtered pigs in Villa Clara province using ELISA. Anti-Toxoplasma gondii IgG antibodies were detected in 100 animals (22.2%, 95% CI: 18.5-26.2). Conventional PCR of the 529-bp marker of T. gondii was performed in hearts and diaphragm tissues of all ELISA-seropositive pigs. Toxoplasma gondii DNA was detected in four animals. Further genetic characterization of the positive DNA samples was performed by multilocus PCR-RFLP and PCR-sequencing typing tools. Molecular analysis revealed four different genetic profiles that were combinations of type I, II, III and u-1 alleles, suggesting the circulation of non-clonal genotypes of T. gondii in domestic pigs in Cuba. Our results indicate that T. gondii is widely distributed in slaughtered pigs in this country, which might have important implications for public health. To the best of our knowledge, this is the first report on genetic characterization of T. gondii in Cuba. Although preliminary, the results suggest a high genetic diversity of T. gondii in the study region. Further studies based on parasite isolation are needed to definitively identify the genotypes circulating and characterize the virulence of strains detected in pigs in Cuba, and to assess the risk of zoonotic transmission from pork products in this country.

Dataset Information

Generation and molecular characterization of new temperature-sensitive plasmids intended for genetic engineering of Pasteurellaceae.

Publications

Generation and molecular characterization of new temperature-sensitive plasmids intended for genetic engineering of Pasteurellaceae.

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