Higher-level snake phylogeny inferred from mitochondrial DNA sequences of 12S rRNA and 16S rRNA genes

N-Nitrosomethylbenzylamine (NMBA) is a potent esophagus-specific carcinogen that has been utilized extensively in the study of esophageal carcinogenesis in rats. While many studies have focused on the pathogenesis of NMBA-induced esophageal tumors, the tumorigenicity of NMBA itself has not been thoroughly investigated in any single, systematic dose-response study. Therefore, in this study we evaluated NMBA tumorigenicity in rats following various short-term s.c. treatment regimens with the aim of developing an abbreviated treatment protocol which could be used in future studies. To assess the possible correlation of basal cell proliferation with NMBA tumorigenicity, we evaluated the expression of proliferating cell nuclear antigen (PCNA) in both control and NMBA-treated rats. In rats which received a cumulative NMBA dosage of 7.5 mg/kg over the course of 5 weeks, tumor incidence and multiplicity were as follows: 40% with 0.4 +/- 0.3 tumors/rat at week 10; 100% with 2.2 +/- 1.0 tumors/rat at week 20; and 100% with 2.3 +/- 1.0 tumors/rat at week 30. These rats exhibited marked increases in basal cell labeling, with indices that were 1.5- to 1.8-fold higher than controls. NMBA treatment regimens of shorter duration with equivalent or higher cumulative dosages were generally ineffective in producing esophageal tumors, even though significantly elevated levels of basal cell proliferation occurred. Together, these findings indicate that the duration of NMBA treatment is of critical importance in the tumorigenic potential of the carcinogen.     

A qualitative evaluation of the published oligonucleotides specific for the 16S rRNA gene sequences of the ammonia-oxidizing bacteria

Over the past few years, there has been an increasing interest in making oligonucleotides specific for ammonia-oxidizing bacteria (AOB), in order to detect and monitor these slow growing bacteria in environmental samples, in enrichment cultures and in wastewater treatment plants. Based on 16S rDNA sequences, a broad selection of oligonucleotides have been designed, either encompassing all known AOB in the beta-subgroup of the Proteobacteria (beta AOB), or subclasses within beta AOB. Thirty different oligonucleotides have so far been published, with varying specificity. The first AOB-specific oligonucleotides published were obtained as a result of an alignment of only eleven 16S rDNA sequences from AOB. Including the present study, there are now forty nearly full length 16S rDNA sequences available from these bacteria, in addition to a number of partial sequences, so that an improved evaluation of the published oligonucleotides can be done. Two new 16S rRNA gene sequences from Nitrosospira are presented here, in a phylogenetic analysis containing every 16S rRNA gene sequences (> 1 kb) available from AOB. On the basis of an alignment of all these sequences, combined with searches in the nucleotide sequence databases, an evaluation of the thirty published oligonucleotides is presented. The analysis expose the strength and weakness of each oligonucleotide and discuss the use of oligonucleotides specific for 16S rRNA genes in future studies of AOB. The present work also identifies one new, broad range primer, specific for the AOB in the beta-subgroup of the Proteobacteria.     

Phylogenetic positions of Clostridium chauvoei and Clostridium septicum based on 16S rRNA gene sequences

The sequences of the 16S rRNA genes (rrs genes) of Clostridium chauvoei, the causative agent of blackleg in cattle, and the phenotypically related organism Clostridium septicum were determined. After amplification of 1,507-bp PCR fragments from the corresponding rrs genes, the sequences were determined in a single round of sequencing by using conserved region primers. A sequence similarity analysis of the sequences revealed the close phylogenetic relationship of C. chauvoei and C. septicum in Clostridium cluster I (M. D. Collins, P. A. Lawson, A. Willems, J. J. Cordoba, J. Fernandez-Garayzabal, P. Garcia, J. Cai, H. Hippe, and J. A. E. Farrow, Int. J. Syst. Bacteriol. 44:812-826, 1994), which includes Clostridium carnis, Clostridium perfringens, Clostridium botulinum, and Clostridium tetani. We found that 99.3% of the nucleotides in the genes of C. chauvoei and C. septicum are identical.     

A new computational method for detection of chimeric 16S rRNA artifacts generated by PCR amplification from mixed bacterial populations

A new computational method (chimeric alignment) has been developed to detect chimeric 16S rRNA artifacts generated during PCR amplification from mixed bacterial populations. In contrast to other nearest-neighbor methods (e.g., CHECK_CHIMERA) that define sequence similarity by k-tuple matching, the chimeric alignment method uses the score from dynamic programming alignments. Further, the chimeric alignments are displayed to the user to assist in sequence classification. The distribution of improvement scores for 500 authentic, nonchimeric sequences and 300 artificial chimeras (constructed from authentic sequences) was used to study the sensitivity and accuracy of both chimeric alignment and CHECK_CHIMERA. At a constant rate of authentic sequence misclassification (5%), chimeric alignment incorrectly classified 13% of the artificial chimeras versus 14% for CHECK_CHIMERA. Interestingly, only 1% of nonchimeras and 10% of chimeras were misclassified by both programs, suggesting that optimum performance is obtained by using the two methods to assign sequences to three classes: high-probability nonchimeras, high-probability chimeras, and sequences that need further study by other means. This study suggests that k-tuple-based matching methods are more sensitive than alignment-based methods when there is significant parental sequence similarity, while the opposite becomes true as the sequences become more distantly related. The software and a World Wide Web-based server are available at http://www-hto.usc.edu/software/mglobal CHI.     

Molecular systematics and evolution of reproductive traits of North American freshwater unionacean mussels (Mollusca: Bivalvia) as inferred from 16S rRNA gene sequences

North American freshwater unionacean bivalves are a diverse group of nearly 300 species. Unionaceans exhibit an array of conchological, anatomical, life history, and reproductive characteristics that have figured prominently in proposed classification schemes. Recently, two very different classifications of North American unionaceans have been proposed. Depending on the classification system utilized, a very different evolutionary trajectory of anatomical and reproductive features is obtained. The lack of a robust, well corroborated phylogeny of North American unionacean bivalves hinders the progress of evolutionary and ecological studies involving these species. Here we present a mitochondrial DNA (mtDNA) based phylogeny for North American unionacean mussels and compare it to previously proposed classifications. In addition, we present a 'total evidence' phylogeny which incorporates both the mtDNA sequence data and available morphological data. The molecular and total evidence phylogenies agree largely with the conclusions of a previous study based largely on immunoelectrophoretic data. North American unionaceans can be divided into two families: the Unionidae, which is comprised of most of the species and the Margaritiferidae. Within the Uniondae are two subfamilies, the Anodontinae and Ambleminae. The resultant phylogeny was used to examine the evolution of several key anatomical features including the number of gills (demibranchs) used by females to brood developing embryos, incubation length (bradytictic vs tachytictic), larval (glochidial) tooth structures, and shell texture. Both molecular and total evidence phylogenies indicate several of the aforementioned characters evolved independently or were subsequently lost or gained in several lineages.     

Binding of neomycin-class aminoglycoside antibiotics to the A-site of 16 S rRNA

A major feature of macrophage metabolism is its capacity to produce and export cholesterol. Several reports have shown that the manipulation of lymphocyte cholesterol content elicits important changes in lymphocyte proliferation. These findings lead to an inquiry as to whether macrophage-derived cholesterol released into the lymphocyte surroundings may be transferred to the latter thus affecting lymphocyte function. In this study, cholesterol transfer from macrophages to lymphocytes was examined in vitro using rat cells in culture. The findings indicate that there may be a significant transfer of cholesterol from [4-14C]cholesterol labeled resident peritoneal macrophages to mesenteric lymph node resting lymphocytes (up to 173.9 +/- 2.7 pmol/10(7) lymphocytes/10(7) macrophages when co-cultivated for 48 h), in a lipoprotein-dependent manner. This represents the mass transfer of ca. 17 nmoles of cholesterol molecules per 10(7) lymphocytes from 10(7) macrophages (calculated on the basis of specific radioactivity incorporated into macrophages after the pre-labelling period), which suggests that macrophages are capable of replacing the whole lymphocyte cholesterol pool every 21 h. Moreover, an 111%-increase in the total cholesterol content of lymphocytes was found after co-cultivation with macrophages for 48 h. When compared to peritoneal cells, monocytes/macrophages obtained from circulating blood leukocytes presented a much higher cholesterol transfer capacity to lymphocytes (3.06 +/- 0.10 nmol/10(7) lymphocytes/10(7) macrophages co-cultivated for 24 h). Interestingly, inflammatory macrophages dramatically reduced their cholesterol transfer ability (by up to 91%, as compared to resident macrophages). Cholesterol transfer may involve a humoral influence, since it is not only observed when cells are co-cultivated in a single-well chamber system (cells in direct contact), but also in a two-compartment system (where cells can communicate but not by direct contact). Co-cultivation with macrophages decreased the basal incorporation of [2-14C]thymidine into lymphocyte DNA and the addition of cholesterol to lymphocyte culture media also impaired the lymphocyte proliferative response to the mitogens concanavalin A (Con A) and bacterial lipopolysaccharide (LPS). The above results suggest that macrophages may transfer cholesterol to lymphocytes (from both lymph nodes and blood), thus regulating lymphocyte function by raising the intracellular cholesterol content and suppressing lymphocyte proliferative activity. If this is so, a modulatory role for the transfer of cholesterol in both physiological (e.g. immune response) and pathological conditions (e.g. atherosclerosis) may be postulated. This hypothesis is currently under investigation in our laboratory.     

Hierarchical analysis of variation in the mitochondrial 16S rRNA gene among Hymenoptera

Nucleotide sequences from a 434-bp region of the 16S rRNA gene were analyzed for 65 taxa of Hymenoptera (ants, bees, wasps, parasitoid wasps, sawflies) to examine the patterns of variation within the gene fragment and the taxonomic levels for which it shows maximum utility in phylogeny estimation. A hierarchical approach was adopted in the study through comparison of levels of sequence variation among taxa at different taxonomic levels. As previously reported for many holometabolous insects, the 16S data reported here for Hymenoptera are highly AT-rich and exhibit strong site-to-site variation in substitution rate. More precise estimates of the shape parameter (alpha) of the gamma distribution and the proportion of invariant sites were obtained in this study by employing a reference phylogeny and utilizing maximum-likelihood estimation. The effectiveness of this approach to recovering expected phylogenies of selected hymenopteran taxa has been tested against the use of maximum parsimony. This study finds that the 16S gene is most informative for phylogenetic analysis at two different levels: among closely related species or populations, and among tribes, subfamilies, and families. Maximization of the phylogenetic signal extracted from the 16S gene at higher taxonomic levels may require consideration of the base composition bias and the site-to-site rate variation in a maximum-likelihood framework.     

The 16S-23S rRNA intergenic spacer region of Bartonella (Rochalimaea) species is longer than usually described in other bacteria

The isozymes of prostaglandin G/H synthase (PGHS) are shown to be differentially inhibited in vitro by currently marketed nonsteroidal anti-inflammatory drugs (NSAIDs) using microsomal rhPGHS-1 and rhPGHS-2. Comparison of selectivity ratios (IC50 rhPGHS-1/IC50 rhPGHS-2) demonstrated a 10-fold selectivity of etodolac (Lodine) for rhPGHS-2, whereas the other NSAIDs evaluated demonstrated no preference or a slight preference for inhibition of rhPGHS-1. In vitro enzyme results were supported by a human whole blood assay where etodolac also demonstrated a 10-fold selectivity for inhibition of PGHS-2 mediated TxB2 production. Taken together, these data may be key to explaining the clinically observed gastrointestinal safety of etodolac versus other marketed NSAIDs.     

Evolutionary relationships among the Braconidae (Hymenoptera: Ichneumonoidea) inferred from partial 16S rDNA gene sequences

Phylogenetic relationships among the Braconidae were examined using homologous 16S rDNA gene sequence data. Analyses recovered the few well-supported relationships evident in this family from morphological analyses, viz the monophyly of the microgastroid complex of subfamilies, the monophyly of the cyclostome complex of subfamilies (= braconoids), a sister-group relationship between the Alysiinae and Opiinae, and a close relationship between the Helconinae and Blacinae. With respect to the braconoid complex of subfamilies, a sister-group relationship was recovered between Aphidiinae and Mesostoinae, and a clade composed of Gnamptodontinae + Histeromerinae + Rhyssalinae + Aphidiinae + Mesostoinae was also recovered. The Doryctinae and Rogadinae sensu lato (s.l.) were generally not resolved as monophyletic. With respect to the helconoid complex of subfamilies, a sister-group relationship was recovered between Sigalphinae and Agathidinae, whereas Neoneurinae fell out among other helconoid subfamilies. Other relationships among the helconoid subfamilies were unclear from these analyses. With respect to the microgastroid complex of subfamilies, our data conform to morphological estimates, recovering ((Microgastrinae + Miracinae) + Cardiochilinae) + Cheloninae. The topology of our trees suggests that the cyclostome subfamilies are a natural derived group, inferring that endoparasitism (not ectoparasitism) is the ancestral state for the Braconidae, unless all of the ectoparasitic ancestors of the helconoid + microgastroid subfamilies are now extinct.     

Occurrence of introns in the 16S rRNA genes of members of the genus Thermoproteus

Cervical smears (n = 150) from five departments showing high-grade dyskaryosis were examined by three cytologists. All the smears came from patients with biopsy-proven CIN III. One hundred had been correctly reported (true positives) but 50 had originally been reported as negative and had been found to be positive only on review (false negatives). There were significant differences between the two sets in the characteristics of the dyskaryotic cell population. The false-negative smears tended to have fewer than 200 dyskaryotic cells. The nuclei of the dyskaryotic cells tended to have fine rather than coarse nuclear chromatin. A smear with fewer than 50 dyskaryotic cells is 26 times more likely to be reported as negative than one with more than 200 dyskaryotic cells. The results suggest that there is a type of severely dyskaryotic smear that is inherently likely to be missed on routine screening.     

Paromomycin binding induces a local conformational change in the A-site of 16 S rRNA

Site-directed mutagenesis studies have shown that the isopenicillin N synthase of Cephalosporium acremonium (cIPNS) requires two essential histidine residues (H216, H272) for activity. The determination of iron bound to the wildtype cIPNS and its absence in the mutants lacking histidine at positions 216 and 272 clearly supports the essential role these two histidines play in iron binding. However, nuclear magnetic resonance (NMR) studies have indicated that there could be three histidine residues that possibly coordinate the essential iron at the active site. To search for a presumed third histidine ligand, mutant cIPNS genes containing mutations at two histidine codons were created by in vitro cloning of fragments from the expression vectors bearing the respective cIPNS genes each with a single histidine mutation at positions H49, H64, H116, H126 and H137. All ten possible double histidine mutant cIPNS constructs were subsequently expressed in Escherichia coli. If a third histidine had a participatory role in the iron active centre of cIPNS, then one of the constructed double histidine mutants would have lost its enzymatic activity. However, analysis of the cIPNS activities of these recombinant double histidine mutants indicated that none of them was totally inactivated. Thus, the involvement of a third histidine can be repudiated.     

Detection of kanamycin-resistant Mycobacterium tuberculosis by identifying mutations in the 16S rRNA gene

In Mycobacterium smegmatis and a limited number of Mycobacterium tuberculosis strains, the involvement of alterations of the 16S rRNA gene (rrs) in resistance to kanamycin has been shown. To investigate the extent to which mutations in a specific region of the rrs gene and the kanamycin-resistant phenotype in clinically isolated M. tuberculosis strains were correlated, 43 kanamycin-resistant strains (MICs, > or =200 microg/ml), 71 kanamycin-susceptible strains, and 4 type strains were examined. The 300-bp DNA fragments carrying the rrs gene and the intervening sequence between the rrs gene and 23S rRNA (rrl) gene fragments were amplified by PCR and were subjected to PCR-based direct sequencing. By comparing the nucleotide sequences, substitutions were found in 29 of 43 (67.4%) kanamycin-resistant clinical isolates at positions 1400, 1401, and 1483 but in none of the 71 sensitive isolates or the 4 type strains. The most frequent substitution, from A to G, occurred at position 1400. A substitution from C to T at position 1401 was found once. Two clinical isolates carried the double mutation from C to A at position 1401 and from G to T at position 1483. In addition, we found that these mutants can be distinguished from wild-type strains by digestion with the restriction endonucleases TaiI and Tsp45I. Furthermore, we found that the genotypes of kanamycin-resistant strains can be discriminated from each other by digestion with a restriction endonuclease, BstUI or DdeI.     

Nearly identical 16S rRNA sequences recovered from lakes in North America and Europe indicate the existence of clades of globally distributed freshwater bacteria

Negative pressure transients (NPT) recorded in a single closing event of mechanical valves in the mitral position in an in vitro setup are compared with data recorded in the left atrium in vivo with the valves implanted in the mitral position in an animal model. The loading at valve closure (dP/dtCL) computed from the in vivo ventricular pressure recording (ranging from 700 to 2300 mm Hg/s) agreed with the magnitudes predicted in our earlier in vitro experiments (750-3000 mm Hg/s). The NPT signals and the corresponding power spectral density plots from the in vivo data were in qualitative agreement with those recorded in vitro. The NPT magnitudes were found to be below the vapor pressure for blood in mechanical valves with rigid occluders suggesting a potential for the valve to cavitate in vivo. Our in vivo results also suggest that the valves with flexible occluders are less likely to cavitate. The correlation of the in vitro and in vivo data also suggests that the flexibility of valve housing used in the in vitro studies is not an important factor in the dynamics of mechanical valve closure in vivo.     

Toward the phylogeny of the family Lacertidae: implications from mitochondrial DNA 12S and 16S gene sequences (Reptilia: Squamata)

A phylogeny of the family Lacertidae was derived from mtDNA gene sequence data. Seventeen species, representing 16 currently recognized genera and subgenera, were included in the analysis. A total of 954 bp was obtained and aligned from 12S and 16S partial gene sequences. A preferred tree was selected based on weighted parsimony and functional ingroup and outgroup analyses. Decay analysis, bootstrapping, and permutation tail probability were used to evaluate support for the recovered nodes. The genus Gallotia was resolved as the basal taxon and the sister group of all remaining lacertids. Takydromus branched off next. All African lacertids grouped together and formed a monophyletic clade with the Eurasian genera Eremias and Ophisops. The remaining Eurasian lacertids sequentially branched off near the base of the tree in a "comb-like" fashion. The basal position of Gallotia and the monophyly of African lacertids are consistent with previous hypotheses. The European-origin hypothesis of lacertids is favored, and the distribution of lacertids in Africa is likely a Miocene dispersal event. Most of the extant European lacertids probably arose after the Eocene. The classification of the family needs to be revised.     

Estimation of the relative abundance of different Bacteroides and Prevotella ribotypes in gut samples by restriction enzyme profiling of PCR-amplified 16S rRNA gene sequences

We describe an approach for determining the genetic composition of Bacteroides and Prevotella populations in gut contents based on selective amplification of 16S rRNA gene sequences (rDNA) followed by cleavage of the amplified material with restriction enzymes. The relative contributions of different ribotypes to total Bacteroides and Prevotella 16S rDNA are estimated after end labelling of one of the PCR primers, and the contribution of Bacteroides and Prevotella sequences to total eubacterial 16S rDNA is estimated by measuring the binding of oligonucleotide probes to amplified DNA. Bacteroides and Prevotella 16S rDNA accounted for between 12 and 62% of total eubacterial 16S rDNA in samples of ruminal contents from six sheep and a cow. Ribotypes 4, 5, 6, and 7, which include most cultivated rumen Prevotella strains, together accounted for between 20 and 86% of the total amplified Bacteroides and Prevotella rDNA in these samples. The most abundant Bacteroides or Prevotella ribotype in four animals, however, was ribotype 8, for which there is only one known cultured isolate, while ribotypes 1 and 2, which include many colonic Bacteroides spp., were the most abundant in two animals. This indicates that some abundant Bacteroides and Prevotella groups in the rumen are underrepresented among cultured rumen Prevotella isolates. The approach described here provides a rapid, convenient, and widely applicable method for comparing the genotypic composition of bacterial populations in gut samples.     

The importance of base pairing in the penultimate stem of Escherichia coli 16S rRNA for ribosomal subunit association

The influence of base pairing in the penultimate stem of Escherichia coli 16S rRNA (defined as nt 1409-1491) on ribosome function has been addressed by the construction of mutations in this region of rRNA. Two sets of mutations were made on either side of a structurally conserved region in the penultimate stem that disrupted base pairing, while a third set of mutations replaced the wild-type sequence with other base pair combinations. The effects of these mutations were analyzed in vivo and in vitro . The mutations that disrupted base pairing caused significant increases in cell doubling times as well as a severe subunit association defect and a modest increase in frame shifting and stop codon read-through. Restoration of base pairing restored wild-type growth rates, decoding and subunit association, indicating that base pairing in this region is essential for proper ribosome function.     

Isolation of new bacterial species from drinking water biofilms and proof of their in situ dominance with highly specific 16S rRNA probes

A polyphasic approach involving cultivation, direct viable counts, rRNA-based phylogenetic classification, and in situ probing was applied for the characterization of the dominant microbial population in a municipal drinking water distribution system. A total of 234 bacterial strains cultivated on R2A medium were screened for bacteria affiliated with the in situ dominating beta subclass of Proteobacteria. The isolates were grouped according to common features of their cell and colony morphologies, and eight representative strains were used for 16S rRNA sequencing and the development of a suite of strain-specific oligonucleotide probes. Phylogenetic analysis indicated that all of the isolates were hitherto unknown bacteria. Three of them, strains B4, B6, and B8, formed a separate cluster of closely related organisms within the beta 1 subclass of Proteobacteria. In situ probing revealed that (i) 67 to 72% of total bacteria, corresponding to more than 80% of beta-subclass bacteria, could be encompassed with the strain-specific probes and (ii) the dominating bacterial species were culturable on R2A medium. Additionally, two-thirds of the autochthonous drinking water population could be shown to be in a viable but nonculturable (VBNC) state by using a direct viable count approach. The comparison of isolation frequencies with the in situ abundances of the eight investigated strains revealed differences in their culturability, indicating variable ratios of culturable to VBNC cells among the strains. The further characterization of biofilms throughout the distribution network demonstrated strains B6 and B8 to be dominant bacterial strains in groundwater and distribution system biofilms. The other strains could be found at various frequencies in the different parts of the distribution system; several strains appeared exclusively in drinking water biofilms obtained from a house installation system.