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Comprehensive Studies of Drug Resistance Mediated by Overexpression of Response Regulators of Two-Component Signal Transduction Systems in Escherichia coli. Hidetada Hirakawa, 2003.In Escherichia coli, there are 32 open reading frames (ORFs) that are assumed to be response regulator genes of two-component signal transduction systems on the basis of sequence similarities . We cloned all of these 32 ORFs into a multicopy expression vector and investigated whether or not they confer drug resistance via control of drug resistance determinants . Fifteen of these ORFs, i.e., baeR, citB, cpxR, evgA, fimZ, kdpE, narL, narP, ompR, rcsB, rstA, torR, yedW, yehT, and dcuR, conferred increased single- or multidrug resistance . Two-thirds of them conferred deoxycholate resistance . Five of them, i.e., evgA, baeR, ompR, cpxR, and rcsB, modulated the expression of several drug exporter genes . The drug resistance mediated by evgA, baeR, and cpxR could be assigned to drug exporters by using drug exporter gene knockout strains . Optimization Strategies for DNA Microarray-Based Detection of Bacteria with 16S rRNA-Targeting Oligonucleotide Probes. Jörg Peplies, 2003.The usability of the DNA microarray format for the specific detection of bacteria based on their 16S rRNA genes was systematically evaluated with a model system composed of six environmental strains and 20 oligonucleotide probes . Parameters such as secondary structures of the target molecules and steric hindrance were investigated to better understand the mechanisms underlying a microarray hybridization reaction, with focus on their influence on the specificity of hybridization . With adequate hybridization conditions, false-positive signals could be almost completely prevented, resulting in clear data interpretation . Among 199 potential nonspecific hybridization events, only 1 false-positive signal was observed, whereas false-negative results were more common (17 of 41) . Subsequent parameter analysis revealed that this was mainly an effect of reduced accessibility of probe binding sites caused by the secondary structures of the target molecules . False-negative results could be prevented and the overall signal intensities could be adjusted by introducing a new optimization strategy called directed application of capture oligonucleotides . The small number of false-positive signals in our data set is discussed, and a general optimization approach is suggested . Our results show that, compared to standard hybridization formats such as fluorescence in situ hybridization, a large number of oligonucleotide probes with different characteristics can be applied in parallel in a highly specific way without extensive experimental effort .
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