AmpliTaq and AmpliTaq Gold DNA Polymerase - Applied Biosystems

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Neisseria miniature insertion sequences (nemis) are miniature DNA insertion sequences found inNeisseria species. Out of 57 elements closely flanking cellular genes analyzed by PCR, mostwere conserved in Neisseria meningitidis but not in N. lactamica strains. Since mRNAs spanningnemis are processed by RNase III at hairpins formed by element termini, gene sets couldselectively be regulated in meningococci at the posttranscriptional level.Sheehan, B. J., J. T. Bosse, et al. (2003). "Identification of Actinobacillus pleuropneumoniae GenesImportant for Survival during Infection in Its Natural Host." Infect. Immun. 71(7): 3960-3970.http://iai.asm.org/cgi/content/abstract/71/7/3960Actinobacillus pleuropneumoniae is a strict respiratory tract pathogen of swine and is thecausative agent of porcine pleuropneumonia. We have used signature-tagged mutagenesis(STM) to identify genes required for survival of the organism within the pig. A total of 2,064signature-tagged Tn10 transposon mutants were assembled into pools of 48 each, and used toinoculate pigs by the endotracheal route. Out of 105 mutants that were consistently attenuated invivo, only 11 mutants showed a >2-fold reduction in growth in vitro compared to the wild type,whereas 8 of 14 mutants tested showed significant levels of attenuation in pig as evidenced fromcompetitive index experiments. Inverse PCR was used to generate DNA sequence of thechromosomal domains flanking each transposon insertion. Only one sibling pair of mutants wasidentified, but three apparent transposon insertion hot spots were found--an anticipatedconsequence of the use of a Tn10-based system. Transposon insertions were found within 55different loci, and similarity (BLAST) searching identified possible analogues or homologues forall but four of these. Matches included proteins putatively involved in metabolism and transport ofvarious nutrients or unknown substances, in stress responses, in gene regulation, and in theproduction of cell surface components. Ten of the sequences have homology with genes involvedin lipopolysaccharide and capsule production. The results highlight the importance of genesinvolved in energy metabolism, nutrient uptake and stress responses for the survival of A.pleuropneumoniae in its natural host: the pig.Berberov, E. M., Y. Zhou, et al. (2004). "Relative Importance of Heat-Labile Enterotoxin in the Causationof Severe Diarrheal Disease in the Gnotobiotic Piglet Model by a Strain of EnterotoxigenicEscherichia coli That Produces Multiple Enterotoxins." Infect. Immun. 72(7): 3914-3924.http://iai.asm.org/cgi/content/abstract/72/7/3914Enterotoxigenic Escherichia coli (ETEC) strains that produce multiple enterotoxins are importantcauses of severe dehydrating diarrhea in human beings and animals, but the relative importanceof these enterotoxins in the pathogenesis is poorly understood. Gnotobiotic piglets were used tostudy the importance of heat-labile enterotoxin (LT) in infection with an ETEC strain that producesmultiple enterotoxins. LT- ({Delta}eltAB) and complemented mutants of an F4+ LT+ STb+EAST1+ ETEC strain were constructed, and the virulence of these strains was compared ingnotobiotic piglets expressing receptors for F4+ fimbria. Sixty percent of the piglets inoculatedwith the LT- mutant developed severe dehydrating diarrhea and septicemia compared to 100% ofthose inoculated with the nalidixic acid-resistant (Nalr) parent and 100% of those inoculated withthe complemented mutant strain. Compared to piglets inoculated with the Nalr parent, the meanrate of weight loss (percent per hour) of those inoculated with the LT- mutant was 67% lower (P
severe dehydrating diarrhea and postdiarrheal septicemia in ETEC infections of swine anddemonstrate that EAST1, LT, and STb may be concurrently expressed by porcine ETEC strains.Altet, L., O. Francino, et al. (2002). "Mapping and Sequencing of the Canine NRAMP1 Gene andIdentification of Mutations in Leishmaniasis-Susceptible Dogs." Infect. Immun. 70(6): 2763-2771.http://iai.asm.org/cgi/content/abstract/70/6/2763The NRAMP1 gene (Slc11a1) encodes an ion transporter protein involved in the control ofintraphagosomal replication of parasites and in macrophage activation. It has been described inmice as the determinant of natural resistance or susceptibility to infection with antigenicallyunrelated pathogens, including Leishmania. Our aims were to sequence and map the canineSlc11a1 gene and to identify mutations that may be associated with resistance or susceptibility toLeishmania infection. The canine Slc11a1 gene has been mapped to dog chromosome CFA37and covers 9 kb, including a 700-bp promoter region, 15 exons, and a polymorphic microsatellitein intron 1. It encodes a 547-amino-acid protein that has over 87% identity with the Slc11a1proteins of different mammalian species. A case-control study with 33 resistant and 84susceptible dogs showed an association between allele 145 of the microsatellite and susceptibledogs. Sequence variant analysis was performed by direct sequencing of the cDNA and thepromoter region of four unrelated beagles experimentally infected with Leishmania infantum tosearch for possible functional mutations. Two of the dogs were classified as susceptible and theother two were classified as resistant based on their immune responses. Two important mutationswere found in susceptible dogs: a G-rich region in the promoter that was common to both animalsand a complete deletion of exon 11, which encodes the consensus transport motif of the protein,in the unique susceptible dog that needed an additional and prolonged treatment to avoidcontinuous relapses. A study with a larger dog population would be required to prove theassociation of these sequence variants with disease susceptibility.Baranova, I., T. Vishnyakova, et al. (2002). "Lipopolysaccharide Down Regulates Both ScavengerReceptor B1 and ATP Binding Cassette Transporter A1 in RAW Cells." Infect. Immun. 70(6):2995-3003.http://iai.asm.org/cgi/content/abstract/70/6/2995Lipopolysaccharide (LPS) has recently been shown to facilitate macrophage foam cell formationand has been suggested to be a proatherogenic factor. The mechanism of LPS inducedcholesterol accumulation, however, is unclear. In this report, using the macrophage-like RAW264.7 cell line, we provide experimental evidence that LPS's proatherogenic effects may at leastin part reflect altered cholesterol metabolism. Data presented demonstrate that in a dosedependentmanner, LPS is able to down regulate the mRNA expression of the two primary highdensitylipoprotein (HDL) receptors, scavenger receptor B1 (SR-B1) and ATP binding cassette A1(ABCA1), with a 50% inhibitory concentration of less than 0.2 ng/ml, as well as to decrease SR-B1 protein expression by 80%. We also found that LPS treatment resulted in a significantdecrease (to 20% of the control level) of the specific 125I-HDL binding as well as in 50%inhibition of the HDL-mediated cholesterol efflux compared to untreated cells. In addition, wecompared the potencies of various modified LPS preparations and demonstrated that thephosphorylated lipid A portion of LPS, which is highly conserved among gram-negativemicroorganisms, including Chlamydia, is primarily responsible for the effects of LPS on SR-B1and ABCA1 expression. Inhibitors of NF-{kappa}B activation were observed to efficiently blockthe suppressive effect of LPS on SR-B1 and ABCA1, suggesting a mechanism involving NF-{kappa}B. These data indicate that the LPS effects on cholesterol metabolism may contribute tothe proatherogenic properties of LPS.
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Page 12 and 13: Post, D. M. B., N. J. Phillips, et
Page 14 and 15: 700.http://iai.asm.org/cgi/content/
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Page 20 and 21: Behrens, M., J. Sheikh, et al. (200
Page 22: The flagellum protein flagellin of
Page 25 and 26: histopathology. The mucosal immune
Page 27 and 28: Kohler, A. K., D. M. Stone, et al.
Page 29 and 30: Immun. 70(10): 5556-5561.http://iai

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