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Real-Time PCR for Dihydrofolate Reductase Gene Single-Nucleotide Polymorphisms in Plasmodium vivax Isolates. Sara Brega, 2004.Mutations in the dhfr gene of Plasmodium vivax (pvdhfr) are associated with resistance to the antifolate antimalarial drugs . Polymorphisms in the pvdhfr gene were assessed by hybridization probe technology on the LightCycler instrument with 134 P . vivax-infected blood samples from Turkey (n = 24), Azerbaijan (n = 39), Thailand (n = 16), Indonesia (n = 53), and travelers (n = 19) . Double mutations (S58R and S117N) or quadruple mutations (F57L/I, S58R, T61M, and S117N) in the pvdhfr genes were found in all Thai samples (100%) . pvdhfr mutant-type alleles were significantly more common in samples from travelers (42%) than in those from patients from Indonesia (5%) . Surprisingly, the pvdhfr single-mutation allele (S117N) was identified at a high frequency in parasites from Turkey and Azerbaijan (71 and 36%, respectively), where sulfadoxine-pyrimethamine is not recommended for the treatment of P . vivax malaria by the World Health Organization and the Malaria National Programs . Global Analysis of Genes Regulated by EvgA of the Two-Component Regulatory System in Escherichia coli. Kunihiko Nishino, 2003.The response regulator EvgA controls expression of multiple genes conferring antibiotic resistance in Escherichia coli (K . Nishino and A . Yamaguchi, J . Bacteriol . 184:2319-2323, 2002) . To understand the whole picture of EvgA regulation, DNA macroarray analysis of the effect of EvgA overproduction was performed . EvgA activated genes related to acid resistance, osmotic adaptation, and drug resistance . Spatial and Temporal Analysis of the Microbial Community in Slow Sand Filters Used for Treating Horticultural Irrigation Water. Leo A. Calvo-Bado, 2003.An experimental slow sand filter (SSF) was constructed to study the spatial and temporal structure of a bacterial community suppressive to an oomycete plant pathogen, Phytophthora cryptogea . Passage of water through the mature sand column resulted in complete removal of zoospores of the plant pathogen . To monitor global changes in the microbial community, bacterial and fungal numbers were estimated on selective media, direct viable counts of fungal spores were made, and the ATP content was measured . PCR amplification of 16S rRNA genes and denaturing gradient gel electrophoresis (DGGE) were used to study the dynamics of the bacterial community in detail . The top layer (1 cm) of the SSF column was dominated by a variable and active microbial population, whereas the middle (50 cm) and bottom (80 cm) layers were dominated by less active and diverse bacterial populations . The major changes in the microbial populations occurred during the first week of filter operation, and these populations then remained to the end of the study . Spatial and temporal nonlinear mapping of the DGGE bands provided a useful visual representation of the similarities between SSF samples . According to the DGGE profile, less than 2% of the dominating bands present in the SSF column were represented in the culturable population . Sequence analysis of DGGE bands from all depths of the SSF column indicated that a range of bacteria were present, with 16S rRNA gene sequences similar to groups such as Bacillus megaterium, Cytophaga, Desulfovibrio, Legionella, Rhodococcus rhodochrous, Sphingomonas, and an uncharacterized environmental clone . This study describes the characterization of the performance, and microbial composition, of SSFs used for the treatment of water for use in the horticultural industry . Utilization of naturally suppressive population of microorganisms either directly or by manipulation of the environment in an SSF may provide a more reproducible control method for the future . Is the In Situ Accessibility of the 16S rRNA of Escherichia coli for Cy3-Labeled Oligonucleotide Probes Predicted by a Three-Dimensional Structure Model of the 30S Ribosomal Subunit?. Sebastian Behrens, 2003.Systematic studies on the hybridization of fluorescently labeled, rRNA-targeted oligonucleotides have shown strong variations in in situ accessibility . Reliable predictions of target site accessibility would contribute to more-rational design of probes for the identification of individual microbial cells in their natural environments . During the past 3 years, numerous studies of the higher-order structure of the ribosome have advanced our understanding of its spatial conformation . These studies range from the identification of rRNA-rRNA interactions based on covariation analyses to physical imaging of the ribosome for the identification of protein-rRNA interactions . Here we reevaluate our Escherichia coli 16S rRNA in situ accessibility data with regard to a tertiary-structure model of the small subunit of the ribosome . We localized target sequences of 176 oligonucleotides on a 3.0-Å-resolution three-dimensional (3D) model of the 30S ribosomal subunit . Little correlation was found between probe hybridization efficiency and the proximity of the probe target region to the surface of the 30S ribosomal subunit model . We attribute this to the fact that fluorescence in situ hybridization is performed on fixed cells containing denatured ribosomes, whereas 3D models of the ribosome are based on its native conformation . The effects of different fixation and hybridization protocols on the fluorescence signals conferred by a set of 10 representative probes were tested . The presence or absence of the strongly denaturing detergent sodium dodecyl sulfate had a much more pronounced effect than a change of fixative from paraformaldehyde to ethanol .
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