DNA hybridization

To explore how microbial interactions within the rhizosphere influence the diversity and functional roles of bacterial communities, we isolated 21 bacterial strains from soil samples collected near Rocky Branch Creek on the University of South Carolina campus. Our findings revealed that a significant proportion of the isolated bacterial strains are lysogenic. Contrary to predictions of a narrow host range, most of the bacteriophages derived from these lysogenic bacteria demonstrated the ability to infect a broad range of bacterial strains. These results suggest that the bacterial community shares a complex phage community, creating an intricate web of interactions. This study enhances our understanding of the relationships between phages and their bacterial hosts in soil ecosystems, with implications for ecological balance and agricultural practices aimed at improving plant health through microbial management strategies.

We demonstrate, using transmission electron microscopy and immunopurification with an antibody specific for RNA/DNA hybrid, that intact mtDNA replication intermediates (mtRIs) are essentially duplex throughout their length, but contain extensive RNA tracts on one strand. However, the extent of preservation of RNA in such molecules is highly dependent on the preparative method used. These findings strongly support the strand-coupled model of mtDNA replication involving RNA incorporation throughout the lagging strand (RITOLS).

DNA:DNA colony hybridization techniques were used to select isolates from freshwater sediment samples that contain genes homologous to plasmid pSS50, coding for 4-chlorobiphenyl biodegradation. A high degree of resolution was achieved in which target organisms representing 0.3°7o of the total population were discerned. Initially, eight positive cultures were obtained, these were lbund to exist as consortia populations. Pure cultures, from the consortia, were then isolated and screened for 4-chlorobiphenyl degradative genes "by DNA:DNA colony hybridization. Each strain demonstrating positive hybridization was subsequently shown to biodegrade 4-chlorobiphenyl to 4-chlorobenzoate. Following phenotypic characterization of the pure cultures it was found that three different organisms were repeatedly isolated from the various consortia populations. Field sampling to isolation of positive strains was accomplished within one week and completely avoided primary enrichment cultivation.

This study was conducted to understand the seasonal distribution of Vibrio vulnificus in oysters from two estuaries and the effect of environmental factors on the abundance of V. vulnificus in tropical waters. V. vulnificus was detected in 56.6% of the samples tested by colony hybridization with an alkaline phosphatase-labeled oligonucleotide probe (VV-AP), and the counts ranged from <10/g during the summer months to 10 3 /g in the monsoon season at both sites. The density of V. vulnificus appeared to be controlled more by salinity than by temperature. A nested PCR used in this study detected V. vulnificus in 85% of the samples following 18 h of enrichment in alkaline peptone water.

Lactobacilli play an important role in maintaining vaginal health. However, during bacterial vaginosis lactobacilli decrease for unknown reasons. Our preliminary study showed that phages could infect vaginal lactobacilli. Therefore, the aim of this study was to analyze the distribution, virulence, and types of vaginal Lactobacillus phages isolated from women of two countries: the United States and Turkey. A total of 209 vaginal lactobacilli were isolated from reproductive-aged women in the United States ( n = 107) and Turkey ( n = 102). By analysis of 16S rRNA gene sequence and by comparison of protein profiles, most lactobacilli were identified as L. crispatus , L. gasseri , and L. jensenii . After mitomycin C induction, 28% of American lactobacilli and 36% of Turkish lactobacilli released phages. A total of 67 phages were isolated and further characterized by their host range, electron microscopy, and DNA homology. All 67 phages were infective against lactobacilli from both collec...

It is frequently claimed that equal rates of DNA evolution are observed in birds, but specific tests necessary to demonstrate this are rarely performed. I demonstrated statistically significant differences in rates of DNA evolution for a few passefine birds that vitiate the role of DNA hybridization as the direct indicator of kinship. The differences in evolutionary rates observed may be substantial enough to introduce ambiguity into the clustering of taxa. Researchers frequently fail to perform the specific experiments needed to distinguish between real differences in relative rates of DNA evolution vs. differences that can be attributed to experimental error. They also fail to draw attention to and account for either erroneous or problematical data for some birds, or fail to perform the experiments necessary to determine the cause of unexpectedly problematical results. The DNA molecular clock is shown to be calibrated using speculative and questionable data. It involves diverse organisms that cannot be shown to evolve at the same rate, and that probably did not. Yet, DNA hybridization is a valuable tool that probably cannot lead one to make major systematic errors providing the data are not incorrect because of technical or computational errors and are taken from a sufficient diversity of relevant taxa.

Opinions on the systematic relationships of birds in the avian order Gruiformes have been as diverse as the families included within it. Despite ongoing debate over monophyly of the order and relationships among its various members, recent opinion has converged on the monophyly of a ''core'' group of five families classified as the suborder Grues: the rails (Rallidae), the cranes (Gruidae), the Limpkin (Aramidae), the trumpeters (Psophiidae), and the finfoots (Heliornithidae). We present DNA sequence data from four mitochondrial (cytochrome b, 12S rRNA, Valine tRNA, and 16S rRNA) and three nuclear loci (intron 7 of b-fibrinogen, intron 5 of alcohol dehydrogenase-I, and introns 3 through 5 of glyceraldehyde-3-phosphate dehydrogenase) to test previous hypotheses of interfamilial relationships within Grues, with particular attention to the enigmatic family Heliornithidae. Separate and combined analyses of these gene sequences confirm the monophyly of Grues as a whole, and of the five families individually, including all three species of Heliornithidae. The preferred topology unambiguously supports relationships among four of the five families, with only the position of Psophiidae remaining equivocal. Bayesian ''relaxed-clock'' dating methods suggest that the divergences of the three heliornithid species occurred in the mid-Tertiary, suggesting that their present disjunct pantropical distribution is a result of early-to mid-Tertiary dispersal.

Opinions on the systematic relationships of birds in the avian order Gruiformes have been as diverse as the families included within it. Despite ongoing debate over monophyly of the order and relationships among its various members, recent opinion has converged on the monophyly of a ''core'' group of five families classified as the suborder Grues: the rails (Rallidae), the cranes (Gruidae), the Limpkin (Aramidae), the trumpeters (Psophiidae), and the finfoots (Heliornithidae). We present DNA sequence data from four mitochondrial (cytochrome b, 12S rRNA, Valine tRNA, and 16S rRNA) and three nuclear loci (intron 7 of b-fibrinogen, intron 5 of alcohol dehydrogenase-I, and introns 3 through 5 of glyceraldehyde-3-phosphate dehydrogenase) to test previous hypotheses of interfamilial relationships within Grues, with particular attention to the enigmatic family Heliornithidae. Separate and combined analyses of these gene sequences confirm the monophyly of Grues as a whole, and of the five families individually, including all three species of Heliornithidae. The preferred topology unambiguously supports relationships among four of the five families, with only the position of Psophiidae remaining equivocal. Bayesian ''relaxed-clock'' dating methods suggest that the divergences of the three heliornithid species occurred in the mid-Tertiary, suggesting that their present disjunct pantropical distribution is a result of early-to mid-Tertiary dispersal.

Forty-six Mesorhizobium strains associated with the leguminous plants Leucaena leucocephala and Sesbania herbacea in an uncultivated Mexican field were characterized using a polyphasic approach. The strains were identified as Mesorhizobium plurifarium based upon the close relationships with the reference strains for this species in PCR-based restriction fragment length polymorphism analyses, sequencing of 16S rRNA genes, multilocus enzyme electrophoresis, and DNA-DNA hybridization. Although the strains isolated from both plants formed the same group in multilocus enzyme electrophoresis and cross-nodulations were observed in the laboratory, different electrophoretic types were obtained from the two plants grown in natural soils, indicating the existence of a preferable association between the plants and the rhizobia. The M. plurifarium strains from Mexico and the reference strains from Africa and Brazil formed different phenotypic clusters in a numerical taxonomy. The Mexican strains did not grow at 37 °C and were sensitive to salty-alkaline conditions, while the reference strains from Africa and Brazil grew at 42 °C and were more resistant to salty-alkaline conditions. These results demonstrate that both the plants and environmental factors affected the evolution of rhizo-bia and that the Mexican strains had adapted to the neutral soils and the cool climate where they were isolated.

The formation of G-quadruplexes in G-rich regions of DNA is believed to affect DNA transcription and replication. However, it is currently unclear how this formation occurs in the presence of a complementary strand. We have used atomic force microscopy (AFM) to image stable RNA/DNA hybrid loops generated by transcription of the plasmid pPH600, which contains a 604-bp fragment of the murine immunoglobulin Sc3 switch region. We show that the non-RNA-containing portion folds into G-quadruplexes, consistent with computational predictions. We also show that hybrid formation prevents further transcription from occurring, implying a regulatory role. After in vitro transcription, almost all (93%) of the plasmids had an asymmetric loop, a large asymmetric blob or a spur-like projection at the appropriate position on the DNA contour. The loops disappeared following treatment of the transcribed plasmid with RNase H, which removes mRNA hybridized with the template strand. Replacement of K + in the transcription buffer with either Na + or Li + caused a reduction in the percentage of plasmids containing loops, blobs or spurs, consistent with the known effects of monovalent cations on G-quadruplex stability. The minimal sample preparation required for AFM imaging has permitted direct observation of the structural changes resulting from G-quadruplex formation.

Eighteen strains isolated from human specimens or from food products were characterized as atypical variants of mannitol-negative Streptococcus bovis. They were tested for extended biochemical criteria, ribotyping and DNA-DNA hybridization in order to define their taxonomic status. These strains were demonstrated to constitute a DNA relatedness group that includes strains of DNA group 4 of Farrow et al. (1984). Comparative analysis of 16S rRNA sequences demonstrated that these strains represent a new species which belongs to the Streptococcus bovis/Streptococcus equinus complex and which has been provisionally named S. infantarius by Bouvet et al. (1997). Biotyping and ribotyping allowed differentiation of these strains from the aesculinpositive strains of S. bovis belonging to the previously described biotypes I, II.1 and II.2. The results of the ribotyping and hybridization assays demonstrated the presence of two different DNA subgroups within the 18 strains. On the basis of these data, the names S. infantarius subsp. infantarius (aesculin-negative for five strains out of seven, including the type strain HDP 90056 T l NCDO 599 T ) and S. infantarius subsp. coli (aesculin-positive, reference strain HDP 90248 l NCDO 2620) are proposed as the names for these two subspecies within the S. infantarius species.

A cDNA clone encoding a nonspecific lipid transfer protein from spinach (Spinacia oleracea) was isolated by probing a library with synthetic oligonucleotides based on the amino acid sequence of the protein. Determination of the DNA sequence indicated a 354-nucleotide open reading frame which encodes a 118-amino acid residue polypeptide. The first 26 amino acids of the open reading frame, which are not present in the mature protein, have all the characteristics of a signal sequence which is normally associated with the synthesis of membrane proteins or secreted proteins. In vitro transcription of the cDNA and translation in the presence of canine pancreatic microsomes or microsomes from cultured maize endosperm cells indicated that proteolytic processing of the preprotein to the mature form was associated with cotranslational insertion into the microsomal membranes. Because there is no known mechanism by which the polypeptide could be transferred from the microsomal membranes to the ...

We documented the first record of the non-native species Piaractus brachypomus (Characiformes: Serrasalmidae) in Lago Petén-Itzá, Petén, Guatemala. One brined specimen was donated by a local fisherman in San Benito, El Petén. The captured specimen was reportedly collected in the southern basin of the lake. We used the mitochondrial gene cytochrome oxidase I (COI) to validate identity of the specimen. We hypothesize that the local aquarium trade is the most likely source of introduction of the non-native Pacu in Lago Petén-Itzá. Documentamos el primer registro de la especie no nativa Piaractus brachypomus (Characiformes: Serrasalmidae) en el lago Petén Itzá, Petén, Guatemala. Un espécimen colectado en la cuenca sur del lago por pescadores locales de San Benito, Petén nos fue donado. La identificación a nivel de especie fue realizada utilizando el gen mitocondrial citocromo oxidasa I (COI). Hipotetizamos que individuos provenientes de acuarios ornamentales son la fuente más plausible ...

Although widely found in the cytoplasm of eucaryotes, the copper-zinc form of superoxide dismutase (CuZnSOD) has been identified in only a small number of bacterial species. One species is the freshwater bacterium Caulobacter crescentus, which also contains an SOD with iron as the metal cofactor (FeSOD). To investigate the function of this CuZnSOD and its structural relationship to the eucaryotic CuZnSODs, the gene encoding CuZnSOD (sodC) of C. crescentus CB15 was cloned and sequenced. By hybridization to pulsed-field electrophoresis gels, sodC was mapped near cysE in the C. crescentus chromosome. Through analysis of spheroplasts, the two SODs of C. crescentus were shown to be differently localized, CuZnSOD in the periplasm and FeSOD in the cytoplasm. In its natural habitat, C. crescentus is frequently associated with blue-green algae (cyanobacteria). The oxygen evolved by these photosynthetic algae may create an extraceliular oxidative stress against which the periplasmic CuZnSOD may defend more effectively than the cytoplasmic FeSOD. Amino acid sequence alignments of C. crescentus CuZnSOD with eucaryotic CuZnSODs and with CuZnSOD of Photobacterium leiognathi (the only other bacterium from which CuZnSOD has been isolated and sequenced) suggest

Although widely found in the cytoplasm of eucaryotes, the copper-zinc form of superoxide dismutase (CuZnSOD) has been identified in only a small number of bacterial species. One species is the freshwater bacterium Caulobacter crescentus, which also contains an SOD with iron as the metal cofactor (FeSOD). To investigate the function of this CuZnSOD and its structural relationship to the eucaryotic CuZnSODs, the gene encoding CuZnSOD (sodC) of C. crescentus CB15 was cloned and sequenced. By hybridization to pulsed-field electrophoresis gels, sodC was mapped near cysE in the C. crescentus chromosome. Through analysis of spheroplasts, the two SODs of C. crescentus were shown to be differently localized, CuZnSOD in the periplasm and FeSOD in the cytoplasm. In its natural habitat, C. crescentus is frequently associated with blue-green algae (cyanobacteria). The oxygen evolved by these photosynthetic algae may create an extraceliular oxidative stress against which the periplasmic CuZnSOD may defend more effectively than the cytoplasmic FeSOD. Amino acid sequence alignments of C. crescentus CuZnSOD with eucaryotic CuZnSODs and with CuZnSOD of Photobacterium leiognathi (the only other bacterium from which CuZnSOD has been isolated and sequenced) suggest

Although widely found in the cytoplasm of eucaryotes, the copper-zinc form of superoxide dismutase (CuZnSOD) has been identified in only a small number of bacterial species. One species is the freshwater bacterium Caulobacter crescentus, which also contains an SOD with iron as the metal cofactor (FeSOD). To investigate the function of this CuZnSOD and its structural relationship to the eucaryotic CuZnSODs, the gene encoding CuZnSOD (sodC) of C. crescentus CB15 was cloned and sequenced. By hybridization to pulsed-field electrophoresis gels, sodC was mapped near cysE in the C. crescentus chromosome. Through analysis of spheroplasts, the two SODs of C. crescentus were shown to be differently localized, CuZnSOD in the periplasm and FeSOD in the cytoplasm. In its natural habitat, C. crescentus is frequently associated with blue-green algae (cyanobacteria). The oxygen evolved by these photosynthetic algae may create an extracellular oxidative stress against which the periplasmic CuZnSOD m...

Aim-To know the treatment outcome of all TB patients registered under Tuberculosis Unit, Karad. Objectives-To study the prevalence of different types of TB cases under Karad Tuberculosis Unit and to study the outcome of all registered TB cases at Karad Tuberculosis Unit. This is a record based, cross sectional study which included all types of tuberculosis cases registered for treatment at Karad Tuberculosis Unit for the year 2014. Data was collected by referring quarterly reports of RNTCP. Statistical Analysis-Data was entered in Excel, and proportions and various performance indicators were calculated for the year 2014. The sputum conversion rate at the end of IP among Cat I Cases was 91.1%. Among Cat II cases, the sputum conversion rate at the end of IP was found to be 50%. Among the pulmonary tuberculosis cases, 61% was the cure rate with 14% of treatment completion. The success rate was 74.5%. The death rate at Karad TU was very high at 13.8% with high failure rate 6.5% and default rate 4.5%. The transferred out rate was 0.8%. Among extrapulmonary tuberculosis cases, the success rate was 90.3% with a high death rate of 5.5%. The default rate of 2.8%, transferred out rate of 1.4% and zero percent failure rate were reported in year 2014. The suspicion rate of >220/lakh/year was achieved, sputum positive rate of 5%-15% was achieved although on lower side, new sputum positive target of 50% was also achieved.

On the basis of phenotypic and genotypic characteristics and analysis of 16S rRNA sequences, a novel species belonging to the genus Pseudoalteromonas is described. Two pale-orange-pigmented strains, KMM 300 T and KMM 290, isolated respectively from a mussel, Crenomytilus grayanus, and a scallop, Patinopecten yessoensis, are marine, Gram-negative, aerobic, rod-shaped bacteria that produce a number of antimicrobial compounds. The strains are able to degrade gelatin, elastin, starch, DNA and Tween 80. Chitin and agar are not degraded. The isolates from marine invertebrates grew at NaCl concentrations of 1-9 % and a temperature range of 10-35 SC and did not utilize most of the wide range of carbohydrates tested, with the exception of D- glucose, cellobiose and sucrose. The DNA GMC content was 484-489 mol %. The level of DNA homology of the two strains was 98 %. DNA from the strains isolated from marine invertebrates showed 5-15 % genetic relatedness to the DNA of other type strains of the genus Pseudoalteromonas. 16S rRNA analysis indicated a clear affiliation of the novel bacteria to other species of the genus. The strains are assigned to a novel species, Pseudomonas ruthenica sp. nov., with the type strain KMM 300 T (l LMG 19699 T l CIP 106857 T ).

Background DiGeorge syndrome (DGS; OMIM #188400), also known as 22q11.2 deletion syndrome, is characterized by cardiac defects, abnormal facial features, thymic hypoplasia, cleft palate, and hypocalcemia. The syndrome is ranked as the second most common chromosomal change after Down's syndrome, accounting for 1 in 2000 newborns. DGS syndrome is typically diagnosed through CGH and/or FISH analysis in developed countries. However, in lowresource healthcare settings, diagnosis often relies primarily on clinical manifestations due to limited access to genetic testing. The present study, the first of its kind, seeks to deepen the understanding of both the clinical and genetic aspects of DGS among Sudanese patients by employing FISH analysis as a confirmatory test.

We demonstrate effective mixing of microliter droplets using electrically tunable superhydrophobic nanostructured surfaces. By applying electrical voltage and current, droplets can be reversibly switched from a wetting to a non-wetting state, which induces fluid motion within the droplet. This mixing concept was verified using a DNA hybridization assay, in which a single droplet reversibility accelerated the hybridization reaction by an order of magnitude as compared to mixing by passive diffusion. This work offers a new method to effectively mix droplets for a variety of microfluidics applications.

In previous reports, we described a PCR cycle control approach in which the hybridization state of optically labeled L-DNA enantiomers of the D-DNA primers and targets determined when the thermal cycle was switched from cooling to heating and heating to cooling. A consequence of this approach is that it also "adapts" the cycling conditions to compensate for factors that affect the hybridization kinetics of primers and targets. It assumes, however, that the hybridization state of the labeled L-DNA analogs accurately reflects the hybridization state of the D-DNA primers and targets. In this report, the Van't Hoff equation is applied to determine the L-DNA concentration and ratio of L-DNA strands required by this assumption. Simultaneous fluorescence and temperature measurements were taken during L-DNA controlled cycling, and the optical and thermal switch points compared as a function of both total L-DNA concentration and ratio of strands. Based on the Van't Hoff relationship and these experimental results, L-DNA best mirrors the hybridization of PCR primers and targets when total L-DNA concentration is set equal to the initial concentration of the D-DNA primer of interest. In terms of strand ratios, L-DNA hybridization behavior most closely matches the behavior of their D-DNA counterparts throughout the reaction when one of the L-DNA strands is far in excess of the other. The L-DNA control algorithm was then applied to the practical case of the SARS-CoV-2 N2 reaction, which has been shown to fail or have a delayed Cq when PCR was performed without nucleic acid extraction. PCR Cq values for simulated "unextracted" PCR samples in a nasopharyngeal background and in an NaCl concentration similar to that of viral transport media were determined using either the L-DNA control algorithm (N = 6) or preset cycling conditions (N = 3) and compared to water background controls run in parallel. For preset cycling conditions, the presence of nasopharyngeal background or a high salt background concentration significantly increased Cq, but the L-DNA control algorithm had no significant delay. This suggests that a carefully designed L-DNA-based control algorithm "adapts" the cycling conditions to compensate for hybridization errors of the PCR D-DNA reactants that produce false negatives.

The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein a subunits (Ga) including Gsa, G.1la, Gi.2a, Gi.3a, and Gza (or Gxa), where G, and G1 are proteins that stimulate or inhibit adenylyl cyclase, respectively, and Gz is a protein that may mediate pertussis toxin-insensitive events. Other Ga-related mRNA transcripts were detected in fetal RPE cells by low-stringency hybridization to Gi.2a and Gsa protein-coding cDNA probes. The diversity of G proteins in RPE cells was further studied by cDNA amplification with reverse transcriptase and the polymerase chain reaction. This approach revealed that, besides the above mentioned members of the Ga gene family, at least two other Ga subunits are expressed in RPE cells. Human retinal cDNA clones that encode one of the additional Gca subunits were isolated and characterized. The results indicate that this Ga subunit belongs to a separate subfamily of G proteins that may be insensitive to inhibition by pertussis toxin.

The dissemination of cephamycin resistance in Enterobacteriaceae and its correlation with a transposon-like DNA element consisting of a specific tnpA-bla CMY-2 -blc-sugE structure were investigated. Methods: A total of 140 enterobacterial isolates belonging to 17 species (10 genera) of Enterobacteriaceae phenotypically characterized as putative AmpC-producers were evaluated. The isolates were examined by PCR analysis, DNA-DNA hybridization and nucleotide sequencing. The bla CMY-2 -carrying element was detected in 34 isolates from 10 species (9 genera), including all 14 Salmonella and 4 Shigella isolates as well as 7 of the 10 Escherichia coli isolates tested. The remaining 9 isolates were from 112 isolates of the other 14 species tested. The genetic structure of the bla CMY-2 -carrying element was identical in 29 isolates, while in 3 E. coli and 2 Citrobacter isolates an additional insertion sequence IS1 was found inserted at various nucleotide positions close to the 3 0 end, either within or downstream, of tnpA. In 12 of the 14 representative isolates examined, the bla CMY-2 -carrying element was found inserted in the finQ gene of various-sized plasmids with highly conserved 8 bp direct repeats flanking the junction regions. Among the other 106 non-CMY-2-producing isolates, plasmid-mediated ampC genes were found only in one isolate of Enterobacter aerogenes which carried a bla DHA-1 -like gene. Conclusions: bla CMY-2 is the most prevalent plasmid-mediated ampC gene among Enterobacteriaceae. All the bla CMY-2 genes identified in the present study were associated with a specific transposon-like element that may be responsible for the spread of bla CMY-2 among Enterobacteriaceae.

Salmonella enterica serovar Choleraesuis generally causes systemic human salmonellosis without diarrhea, and therefore, antimicrobial treatment is essential for such patients. The drug resistance information on this organism is thus of high value. Serovar Choleraesuis usually harbors a virulence plasmid (pSCV) of 50 kb in size. Of the 16 clinical isolates identified to be serovar Choleraesuis, all except one harbored a pSCV and seven of them carried a pSCV of more than 125 kb in size. A pSCV was defined as a plasmid carrying spvC and characteristic deletions detected by PCR and by DNA-DNA hybridization (for the former criterion). The results of PCR, restriction fragment profiles, and Southern DNA-DNA hybridizations of the profiles all indicated that such larger pSCVs were derived from the 50-kb plasmid recombined with non-pSCVs found in some clinical isolates. Fifteen of the 17 strains, including a laboratory strain, were then tested for drug resistance against 16 antibiotics with E-test and the dilution method. The laboratory strain, which harbored a 50-kb pSCV and a 6-kb non-pSCV, was resistant only to sulfonamides (SUL), and its resistance gene, sulII, checked with PCR and DNA-DNA hybridization, was located on the 6-kb non-pSCV. All 14 clinical strains were resistant to multiple drugs. Of the 14, 7 were resistant to SUL, and the resistance gene was located on a plasmid. The sulII gene, but not bla TEM-1 , was carried only on the 6-kb non-pSCV. Of the remaining six large plasmids, three of 90 kb, two of 136 kb, and one of 140 kb, the last three were pSCVs and carried the other SUL gene (sulI) and the bla TEM-1 gene. The six strains were also resistant to trimethoprim-sulfamethoxazole. None of the 50-kb pSCVs carried resistance genes. These drug resistance genes on the large pSCVs were apparently also acquired through recombination.

The emergence of resistance to antimicrobial agents within the salmonellas is a worldwide and severe problem. A case of treatment failure due to the emergence of resistance to ceftriaxone in Salmonella enterica serotype Anatum was studied. S. enterica serotype Anatum and Escherichia coli, both of which are susceptible to ceftriaxone, were initially isolated from a diabetic patient hospitalized for the treatment of wound and urinary tract infections. Resistant S. enterica serotype Anatum and E. coli strains were isolated concomitantly 2 weeks after the initiation of ceftriaxone therapy. The patient eventually died of a sepsis caused by the ceftriaxone-resistant salmonella. PCR, nucleotide sequence analysis, and DNA-DNA hybridization identified a bla CTX-M-3 gene located on a 95.1-kb plasmid from the ceftriaxone-resistant isolates of S. enterica serotype Anatum and E. coli. The plasmid was proved to be conjugative. Molecular fingerprinting showed that the susceptible and resistant strains were genetically indistinguishable. The emergence of resistance to ceftriaxone in S. enterica serotype Anatum was due to the in vivo acquisition of a plasmid containing the bla CTX-M-3 gene and was the cause for treatment failure in this patient.

A genomic library from Burkholderia cepacia IS-16 was constructed in Escherichia coli by partial Sau3AI digestion of the chromosomal DNA, with the plasmid vector Bluescript SK. This library was screened for clones able to grow as green stained colonies on selective medium developed for detecting phosphatase-positive colonies. Three green-stained clones (pFS1, pFS2, and pFS3) carried recombinant plasmids harboring DNA inserts of 5.0, 8.0, and 0.9 kb, respectively. DNA hybridization experiments demonstrated the presence of overlapping DNA fragments in the three clones and that these three clones were all derived from Burkholderia cepacia IS-16 genomic DNA. DNA sequence analysis, together with polyacrylamide gels of proteins encoded by E. coli containing pFS3, suggested that the isolated 0.9-kb DNA fragment encodes the functional portion of a phosphate transport protein.

An actinomycete strain, designated HBUM 170018 T , was isolated from soil from Hebei Province, China, and subjected to a polyphasic taxonomic analysis. This study included morphological and physiological investigations and analyses concerning cell chemistry, genomic DNA G+C content, DNA-DNA hybridization and phylogeny. The data obtained were consistent with the assignment of strain HBUM 170018 T to the genus Streptosporangium and were confirmed by the results of 16S rRNA gene sequence analysis. Strain HBUM 170018 T can be differentiated from all Streptosporangium species with validly published names with reference to phenotypic characteristics, phylogenetic data and DNA-DNA hybridization results. On the basis of 16S rRNA gene sequencing, DNA-DNA hybridization and phenotypic data, strain HBUM 170018 T represents a novel species of the genus Streptosporangium, for which the name Streptosporangium canum sp. nov. is proposed. The type strain is HBUM 170018 T (57177 T 5DSM 45034 T 5CGMCC 4.2126 T ).

Purpose: The aim of this study was to identify microorganisms which might be present in closed periapical lesions and to determine their relationship to conventional clinical parameters. - Methods: In 11 patients, samples were taken with paper points from 14 teeth with periapical radiolucency, in seven cases two samples were taken from two different sites of the same tooth (n=21). Clinical parameters were determined. The 16S rDNA of eleven bacterial species could be simultaneously detected via a modified polymerase chain reaction (PCR) based technique. - Results: All samples contained more than one bacterial species. Most frequently, M. micros was detected alone or in combination with E. faecalis, P. aeruginosa, E. coli, F. nucleatum or S. sanguinis. When apical palpation was positive, often M. micros and F. nucleatum were both present. Other clinical symptoms like tenderness to percussion were not associated with a particular microflora. - Conclusions: In closed periapical lesions detected by radiography, specific bacteria were identified by means of a modified PCR technique. No clear associations between clinical symptoms and these bacteria were found. Surgical intervention might be indicated for some of the persistent lesions.

An approach for building DNA hybridization arrays has been developed, in which silica aquogel arrays are produced using micropiezoelectric printheads. When supercritically dried, the pads in these gel arrays have a footprint size of 0.4 ± 0.1 mm in diameter in which the porous silica has a density of 0.045 g cm À3 , pore diameters ranging between a few nanometers and 0.5 lm, and internal surface areas >800 m 2 g À1 (5 • 10 À4 m 2 per pad). This enormous internal surface area, combined with reasonable accessibility to internal binding sites, makes aquogel pads ideally suited for hybridization arrays. Silanized probe DNA was immobilized within the gel pads during gelation, and hybridization was carried out with fluorescently labeled target DNA. The array sites containing probe DNA and the control sites without probes are readily distinguishable using laser-assisted fluorescent scanning.

Seven strains of Borrelia burgdorferi isolated from ticks and from human beings in Europe and U.S.A. were analyzed for DNA restriction patterns with several enzymes and for DNA homology in Southern blot hybridizations. The restriction patterns showed a moderately high variability. In Southern blot hybridization, strain B31 (U.S.A.) DNA gave a strong signal with itself, strain Bsf (U.S.A.) and Alcaide (isolated in Italy but presumably contracted in Venezuela). Strain B45 (F.R.G.) hybridized to itself, strain BITS (Italy) and to strain D.A. (Italy). Strain Nancy (Italy) gave a signal only when hybridized to itself, although it was classified as Borrelia on the basis of the clinical manifestations, SDS-PAGE protein pattern and antigenic determinants. No hybridization differences were observed for strains isolated from different hosts in the same continental geographical area.

Xanthomonas axonopodis pv. vignicola is the causative agent of bacterial blight, a destructive disease of cowpea in Africa. Laboratory studies were conducted with the objective of detecting variations among X. axonopodis pv. vignicola isolates which were collected from three different cowpea growing zones (Makurdi, Guma and Gboko Local Government Areas) in Benue state. The isolates were designated MKD388-1, GUM391 and GBK205-8 respectively. Isolates were cultured on Nutrient agar, tested for their Gram-staining reaction and their capability to utilize asparagine as a sole source of carbon and nitrogen. They were also subjected to biochemical tests. The genetic diversity of the isolates was assessed using Single Sequence Repeat (SSR) primers. Four primer combinations selected based on their reproducibility and amplification were used to differentiate the X. axonopodis pv. vignicola strains. Following these tests, the three isolates showed variations in color and growth character of colonies ranging from yellow to creamy colony color with mucoid growth. Variations to different biochemical tests were also observed among isolates. Primer assay showed genetic variation among the isolates as strains from Makurdi and Guma Local Governments showed high levels of molecular similarity while Gboko strain was distant. This study has lead to an understanding of the dynamics of pathogen variability that can be used to develop resistance gene pyramiding or gene deployment strategies. This will prevent selection for new virulence, which are effective against the currently available genetic sources of resistances genes.

This study presents new comparative sequence data from the nuclear RAG-1 gene for an increased taxon sample in order to investigate phylogenetic relationships among a diverse songbird superfamily, the Muscicapoidea, which has variously included the waxwings, silky flycatchers, Palm Chat, dippers, starlings, mockingbirds, thrushes, chats, and Old World flycatchers. At the same time, our results provide a test of the often-cited relationships inferred from the phenetic studies of Sibley and Ahlquist [Phylogeny and Classification of Birds: A Study in Molecular Evolution. Yale University Press, New Haven, 1990] using DNA hybridization distances. Nuclear DNA sequences confirm the monophyly of the ''core muscicapoid'' group, as defined by Barker et al. [Proc. R. Soc. Lond. B 269 (2002) 295] and also support the sister-group relationship of the Sturnidae and Mimidae, on the one hand, and the large-bodied thrushes (Turdini) + the Old World flycatchers and robins, on the other. The results of the phylogenetic analysis allow preliminary inferences about muscicapoid biogeographic history.

Sodium butyrate (3 mM) inhibited the entry into the S phase of quiescent 3T3 cells stimulated by serum, but had no effect on the accumulation of cellular ribonucleic acid. Simian virus 40 infection or manual microinjection of cloned fragments from the simian virus 40 A gene caused quiescent 3T3 cells to enter the S phase even in the presence of butyrate. NGI cells, a line of 3T3 cells transformed by simian virus 40, grew vigorously in 3 mM butyrate. Homokaryons were formed between G1 and S-phase 3T3 cells, Butyrate inhibited the induction of deoxyribonucleic acid synthesis that usually occurs in B1 nuclei when G1 cells are fused with S-phase cells. However, when G1 3T3 cells were fused with exponentially growing NGI cells, the 3T3 nuclei were induced to enter deoxyribonucleic acid synthesis. In tsAF8 cells, a ribonucleic acid polymerase II mutant that stops in the G1 phase of the cell cycle, no temporal sequence was demonstrated between the butyrate block and the temperature-sensiti...

and Implications Sensitivity of a polymerase chain reaction (PCR) procedure was evaluated for Escherichia coli O157:H7 and Shiga toxin gene (stx) detection in ground beef and ground pork at contamination levels of 0.14, 1.4, and 14 colony forming units per gram (CFU/g) of meat. The PCR procedure, developed during our previous research, amplifies three target genes simultaneously: uidA that is specific for E. coli O157:H7, and stx1 and stx2, the genes for Shiga toxins 1 and 2, respectively. Detection of the uidA gene by the PCR was 91% sensitive in beef and 55% sensitive in pork, and 90% and 71% sensitive for stx gene detection in beef and pork, respectively. In comparison, detection of E. coli O157:H7 by culture, done simultaneously with PCR, was 53% sensitive in beef and 11% sensitive in pork. Results indicate that this PCR procedure is a rapid and sensitive method for STEC and E. coli O157:H7 detection in meat at contamination levels less than 1 CFU/g.

We are developing distance-restrained docking strategies for modeling macromolecular complexes that combine available high-resolution structures of the components and intercomponent distance restraints derived from systematic fluorescence resonance energy transfer (FRET) measurements. In this article, we consider the problem of docking smallmolecule ligands within macromolecular complexes. Using simulated FRET data, we have generated a series of benchmarks that permit estimation of model accuracy based on the quantity and quality of FRET-derived distance restraints, including the number, random error, systematic error, distance distribution, and radial distribution of FRET-derived distance restraints. We find that expected model accuracy is 10 A ˚or better for models based on: i), $20 restraints with up to 15% random error and no systematic error, or ii), $20 restraints with up to 15% random error, up to 10% systematic error, and a symmetric radial distribution of restraints. Model accuracies can be improved to 5 A ˚or better by increasing the number of restraints to $40 and/or by optimizing the distance distribution of restraints. Using experimental FRET data, we have defined the positions of the binding sites within bacterial RNA polymerase of the small-molecule inhibitors rifampicin (Rif) and rifamycin SV (Rif SV). The inferred binding sites for Rif and Rif SV were located with accuracies of, respectively, 7 and 10 A ˚relative to the crystallographically defined binding site for Rif. These accuracies agree with expectations from the benchmark simulations and suffice to indicate that the binding sites for Rif and Rif SV are located within the RNA polymerase active-center cleft, overlapping the binding site for the RNA-DNA hybrid.

Ca2÷-dependent epithelial cell-cell adhesion molecule. Downregulation of E-cadherin expression often correlates with strong invasive potential and poor prognosis of human carcinomas. By using recombinant k phage, cosmid, and P1 phage clones, we isolated the full-length human E-cadherin gene (CDH1). The gene spans a region of approximately 100 kb, and its location on chromosome 16q22.1 was confirmed by FISH analysis. Detailed restriction mapping and partial sequence analysis of the gene allowed us to identify 16 exons and a 65-kb-long intron 2. The intron-exon boundaries are highly conserved in comparison with other "classical cadherins." In intron 1 we identified a 5' high-density CpG island that may be implicated in transcription regulation during embryogenesis and malignancy.

It is with regret that I accept the request of John L. Hamerton of Winnipeg to step-down from his position of Associate Editor for the Human Cytogenetics section of this Journal. John is one of the founding editors of this Journal. Since then (1962) he has rendered immeasurable services to the Journal. John is one of the pioneers in human cytogenetics. In a now classic paper he and Charles E. Ford, also a founding Editor, firmly established the correct somatic chromosome number for man as 46 . This was very shortly after Tjio and Levan (1956) reported a provisional number of 46. John went on to make valuable contributions to human cytogenetic knowledge. In some 170 papers and 9 books he authored or edited, he provided information that was critical for the rapid development and expansion of this field. His two volume book. Human Cytogenetics (Academic Press, 1971) served as a key source of information for students and workers in the field for a long time. John's interests range beyond human cytogenetics. He made a number of important contributions to animal cytogenetics and cytotaxonomy. As an example, I had the pleasure to collaborate with him and several others in 1961 in determining that the correct somatic chromosome num berof the gorilla was 48, the number that before 1956 was believed to be that of man . We later extended these studies to all the Hominoidea Hamerton and Klinger, 1963;. John also made important contributions to our knowledge of Down syndrome; of the correlation between X inactivation and late replication of the mammalian X chromosome; and of intersexuality in the goat, to give some additional examples. He was among the first to pursue human gene mapping and organized and hosted the Human Gene ¡Mapping 4 conference in Winnipeg Canada in 1977. After this conference he organized a canoe trip on the lakes of Manitoba. Without informing John that most had never set foot in a canoe before, twelve partici pants1 signed up for this trip that covered several large lakes with portages between them. A series of unex pected unfavorable and unusually favorable climatic and other events and coincidences followed. This resulted in a very remarkable and unforgettable experience. In contrast to what usually happens the story is frequently retold without embellishment by those of us fortunate to have been there, probably because it is an almost unbelievable story even when told accurately.

Coryneforms have emerged as important pathogens causing severe human infections in immunosuppressed patients. However, it is increasingly being perceived that the pathogenic potential of Coryneform bacteria is being underestimated and it is not limited to merely this group of patients. These organisms are reported to cause serious infections such as bacteremia, valvular endocarditis, neurosurgical shunt infections, meningitis, brain abscess, peritonitis, osteomyelitis, septic arthritis, pneumonia, empyema and urinary tract infections. The study was conducted in the Department of Microbiology, Jawaharlal Nehru Medical College and Hospital, Aligarh, INDIA for a period of eight months from August 2007 to March 2008. All the patients in the study were divided into two groups: (i) Immunocompromised (ii) Immunocompetent. Direct microscopic examination was done by Gram staining, followed by culture. All the isolated Coryneform species were subjected to antimicrobial susceptibility testing using Kirby bauer disc diffusion method on Mueller Hinton agar incorporated with 5% sheep blood. A total of 2508 samples (68.6%) showed growth on culture. Coryneforms were isolated and considered as pathogens in 5.5% samples. Monomicrobial infection of Corynebcaterium species was more common (69.1%) with surgical site infection (27.3%) as the commonest presentation. Coryneforms isolates from the immunocompetent patients accounted for 98(70.5%) isolates, whereas 41(29.5%) isolates were from patients with an immunocompromised status. C. minutissimum was the commonest Coryneform isolated in both the groups. The other Coryneform species isolated were C. jeikium, Microbacterium species, C. xerosis, C. striatum, C. urealyticum, C. afermentans, C. amycolatum and C. ulcerans. Alarmingly widespread antibiotic resistance was observed among most of the Coryneforms. The average sensitivity of the -lactam antimicrobials being just 55% and the aminoglycosides even less than 50% (48%). Only 67(47.5%) strains were sensitive to flouroquinolones. Linezolid was the only antimicrobial to which all the Coryneform isolates included in this study were uniformly sensitive. This study highlights the need for maintaining a high degree of suspicion as far as Coryneform infections are concerned. They should not be dismissed as skin commensals in immunocompetent patients without determining their clinical relevance. Sensitizing clinicians about these emerging pathogens and their multidrug resistant nature is also essential.

Down syndrome (DS) is a major cause of mental retardation and congenital heart disease. Besides a characteristic set of facial and physical features, DS is associated with congenital anomalies of the gastrointestinal tract, an increased risk of leukemia, immune system defects, and an Alzheimer-like dementia. Moreover, DS is a model for the study of human aneuploidy. Although usually caused by the presence of an extra chromosome 21, subsets of the phenotypic features of DS may be caused by the duplication of small regions of the chromosome. The physical map of chromosome 21 allows the molecular definition of the regions duplicated in these rare cases of partial trisomy. As a first step in identifying the genes responsible for individual DS features and their pathophysiology, a panel of cell lines derived from 16 such individuals has been established and the molecular break points have been determined using fluorescence in situ hybridization and Southern blot dosage analysis of 32 mar...

BackgroundCytolethal distending toxin (CDT)-producing Escherichia coli (CTEC) has been isolated from patients with gastrointestinal or urinary tract infection, and sepsis. However, the source of human infection remains unknown. In this study, we attempted to detect and isolate CTEC strains from fecal specimens of healthy farm animals and characterized them phenotypically and genotypically.ResultsBy PCR analysis, the cdtB gene was detected in 90 and 14 out of 102 and 45 stool specimens of healthy cattle and swine, respectively, and none from 45 chicken samples. Subtypes of the cdtB genes (I to V) were further examined by restriction fragment length polymorphism analysis of the amplicons and by type-specific PCRs for the cdt-III and cdt-V genes. Of the 90 cdtB gene-positive cattle samples, 2 cdt-I, 25 cdt-III, 1 cdt-IV, 52 cdt-V and 1 both cdt-III and cdt-V gene-positive strains were isolated while 1 cdt-II and 6 cdt-V gene-positive were isolated from 14 cdtB positive swine samples. S...

Objective: The present clinical study investigated the microbiota of teeth with persistent secondary endodontic infection in the different phases of root canal retreatment. Methods: Twenty filled single-rooted teeth with apical periodontitis were included. Samples were collected with sterile paper points before chemo-mechanical preparation (CMP) (S1), after CMP (S2) and after 30 days of calcium hydroxide-based intracanal medication (ICM) (S3). Cultivable bacteria were assessed by colony forming units count (CFU/mL). DNA was extracted and assessed by using nested PCR. Paired t-test and repeated measures ANOVA were applied for intragroup analysis in the stages of endodontic therapy at a significance level of 5%. Results: Cultivable bacteria were detected in all initial samples. CMP reduced bacteria by 99.4% and ICM by 99.5%. The most prevalent species found in the initial samples were E. faecalis (20/20), P. gingivalis (20/20), F. nucleatum (17/20) and A. actinomycetemcomitans (10/20)...

Due to the clinical importance of differentiating the two species of the Entamoeba histolytica/Entamoeba dispar complex, we developed a multiplex polymerase chain reaction (PCR) method that overcomes time-consuming and laborious procedures. We report here a DNA extraction protocol using non-fixed stool samples that avoid long lysis-incubation periods through the combined use of zirconium beads and a lysis-supporting buffer. We characterized 49 of 52 stool specimens from Cuban patients with amoebiosis. Among them, 36 (75.5%) were infected only with E. dispar (the nonpathogenic species), while 13 (24.5%) displayed a mixed infection with both E. dispar and E. histolytica. The multiplex PCR protocol showed a specificity of 1.00 and a sensitivity of 0.94. Furthermore, the entire procedure can be performed in one day. This approach is therefore reliable and applicable in the field for epidemiologic studies.

Two single-copy DNA segments of 6 kilobases (kb) and 2.3 kb were labeled with biotin-labeled dUTP (Bio11-dUTP) and hybridized to human chromosomes. These probes were detected by immunofluorescence and directly mapped on chromosomes by using classical fluorescence microscopy and a microchannel-plate-intensified video camera. By a subsequent R-banding, the 6-kb and 2.3-kb fragments were precisely localized to the 18p11.3 band and to the 22q11.2 band, respectively, in agreement with previous results obtained with radioactive probes. The adaptation of fluorescence intensification and digital image processing (frame integration to enhance signal-to-noise ratio and linear contrast stretching) to microscopy makes it possible to detect very weak fluorescent spots on chromosomes. This system allows a high spatial resolution (less than 0.6 micron), even at very low fluorescence levels. The efficiency and the specificity of the hybridization and detection methodology give a direct and precise ...

Spontaneously hypertensive rats were given an angiotensin-converting enzyme (ACE) inhibitor (benazepril or quinapril) or hydralazine and were left for up to 6 hr. To examine whether administration of antihypertensive agents affects expression of immediate early genes in left ventricular myocardium, groups of rats were sacrificed at 1,3, and 6 hr after dosing; total RNA was extracted from left ventricular tissue and analyzed by blot hybridization technique using labeled probes for c-myc, c-fos, and GAPDH mRNA. All three antihypertensive agents reduced pressure similarly, and treatment with the two ACE inhibitors increased c-fos and c-myc mRNA expression in left ventriculum. By contrast, hydralazine did not increase steady-state mRNA expression of either proto-oncogene. Thus, in parallel with the pressure fall, acute administration of the ACE inhibitors induced expression of c-fos and c-myc mRNAs in the left ventricle. Since the equidepressor dose of hydralazine did not affect expression of these proto-oncogenes, this effect of ACE inhibitors is independent of their hemodynamic action.