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.     

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.     

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.     

A 5 S rRNA gene is present in the mitochondrial genome of the protist Reclinomonas americana but is absent from red algal mitochondrial DNA

Except in the case of land plants, mitochondrial ribosomes apparently lack a 5 S rRNA species, even though this small RNA is a component of all prokaryotic, chloroplast and eukaryotic cytosol ribosomes. In plants, the mitochondrial 5 S rRNA is encoded by mtDNA and differs in sequence from the 5 S rRNA specified by plant nuclear and chloroplast genomes. A distinctive 5 S rRNA component has not been found in the mitochondrial ribosomes of non-plant eukaryotes and, with the notable exception of the chlorophycean alga, Prototheca wickerhamii, a 5 S rRNA gene has not been identified in those non-plant mtDNAs characterized to date. Here, we report the presence of a 5 S rRNA gene in the mtDNA of the heterotrophic flagellate Reclinomonas americana. This unicellular eukaryote is a member of the jakobid flagellates, an early-diverging group of protists that share ultrastructural characteristics with the retortamonads, primitive protists that lack mitochondria. We report sequence data from the mtDNAs of the red algae Porphyra purpurea and Gracilariopsis lemaneiformis, which we use to evaluate a recent claim that a 5 S rRNA gene exists in the mtDNA of a third rhodophyte alga, Chondrus crispus. Our results lead us to the opposite conclusion: that a 5 S rRNA gene is not encoded by red algal mtDNA. In view of the accumulating evidence favoring a monophyletic origin of the mitochondrial genome, it is likely that a 5 S rRNA gene was present in an ancestral proto-mitochondrial genome, and that contemporary mtDNA-encoded 5 S rRNA genes have all descended from this ancestral gene. Considering the highly restricted phylogenetic distribution of identified mtDNA-encoded 5 S rRNA genes, it follows that the mitochondrial 5 S rRNA gene must have been lost multiple times during evolutionary diversification of the eukaryotic lineage.     

Phylogenetic relationships of artiodactyls and cetaceans as deduced from the comparison of cytochrome b and 12S rRNA mitochondrial sequences

Functional neuroimaging studies in schizophrenia have often been confounded by various factors including medication status. To explore the effects of antipsychotic medications on relative regional cerebral perfusion, we scanned a group of 33 persons with schizophrenia twice, while receiving a stable dose of antipsychotic and after being off antipsychotics for 3 weeks, using technetium-99m hexamethyl-propyleneamine oxime single photon emission computed tomography (Tc-99m HMPAO-SPECT. We found that antipsychotic significantly increased the mean relative cerebral perfusion in the left basal ganglia. Additionally, patients receiving thiothixene (n = 9) had a significantly greater increase in relative cerebral perfusion in the basal ganglia than patients receiving haloperidol (n = 12). These findings indicate that antipsychotics lead to regional increases in cerebral perfusion and that antipsychotic status must be controlled for in functional neuroimaging studies. Functional neuroimaging techniques such as SPECT may be useful in furthering our understanding of the mechanism of antipsychotics.     

A molecular scheme based on 23S rRNA gene polymorphisms for rapid identification of Campylobacter and Arcobacter species

23S rRNA gene PCR amplicons from 118 strains representing 15 species of Campylobacter and four species of Arcobacter were consecutively digested with HpaII, CfoI, and HinfI. A reproducible and discriminatory identification scheme based on combined restriction patterns was developed by using reference strains and was applied to the identification of a variety of isolates.     

Characterization and structure of the mitochondrial small rRNA gene of the entomopathogenic fungus Beauveria bassiana

HCV infection and interferon-alpha (IFN-alpha) therapy have been associated with autoimmunity. To assess whether chronic liver disease (CLD) due to HCV infection or its treatment with IFN-alpha cause autoimmune manifestations, the prevalence of tissue autoantibodies in 51 children with chronic HCV infection and 84 with other CLD was analysed by standard techniques. Sixty-five percent of patients with chronic HCV infection, 66% with chronic hepatitis B infection and 60% with Wilson's disease were positive for at least one autoantibody. In the 51 subjects with chronic HCV infection (29 treated with IFN-alpha, 22 untreated), tested on 165 occasions over a median of 9 months (range 5-42 months), autoantibodies to nuclei (ANA), smooth muscle (SMA), gastric parietal cell (GPC) and/or liver kidney microsomal type 1 (LKM-1) were similarly prevalent in treated and untreated patients (90% versus 68%, P = 0.12). Positivity for SMA was present in 67%, GPC in 32%, ANA in 10%, LKM-1 in 8% of cases. Treatment with IFN-alpha had to be suspended due to transaminase elevation in one SMA-positive, one ANA-positive but in three of four LKM-1-positive patients. Our results show that: (i) autoantibodies are common in viral-induced hepatitis and Wilson's disease; (ii) positivity for SMA, GPC, ANA is part of the natural course of chronic HCV infection, their prevalence being unaffected by IFN-alpha; and (iii) IFN-alpha should be used cautiously in the treatment of LKM-1/HCV-positive patients.     

Going for the old: ancient DNA draws a crowd

Approximately 170 researchers interested in reconstructing the past convened for the 2nd International Conference on Ancient DNA, held in Washington, D.C., from 7 to 9 October. While rejuvenated celluloid dinosaurs have grabbed headlines this year, these scientists were more concerned with topics such as tracing ancient human populations and understanding how DNA can survive the millennia.     

Among-site rate variation and phylogenetic analysis of 12S rRNA in sigmodontine rodents

We analyze sequences from two mitochondrial genes, cytochrome b (cyt b) and 12S rRNA (12S), for a group of sigmodontine rodents among which phylogenetic relationships are well understood based on concordance of morphological, chromosomal, allozyme, and other DNA data sets. Because these two genes are physically linked on the nonrecombining mitochondrial genome, they necessarily share the same history. Phylogenetic analysis of the cyt b gene recovers the well-corroborated relationships, generally with strong support. None of the methods that we employed, including variously weighted parsimony, neighbor joining on both single-rate and gamma-corrected distances, and maximum likelihood, were able to recover these relationships for the 12S gene. Parsimony analyses of the 12S data resulted in a relatively strongly supported placement of Peromyscus eremicus that conflicts with that suggested by cyt b and all other data. There is extreme among-site rate variation in the 12S sequences and moderate levels in the cyt b sequences. This highly skewed distribution of rates in the 12S gene makes phylogenetic analyses of these sequences particularly susceptible to the misleading effects of nonindependence and other nonrandom noise, suggesting that phylogenetic analyses of data sets that contain a great deal of among-site rate variation be interpreted with caution.     

Evaluating multiple alternative hypotheses for the origin of Bilateria: an analysis of 18S rRNA molecular evidence

Six alternative hypotheses for the phylogenetic origin of Bilateria are evaluated by using complete 18S rRNA gene sequences for 52 taxa. These data suggest that there is little support for three of these hypotheses. Bilateria is not likely to be the sister group of Radiata or Ctenophora, nor is it likely that Bilateria gave rise to Cnidaria or Ctenophora. Instead, these data reveal a close relationship between bilaterians, placozoans, and cnidarians. From this, several inferences can be drawn. Morphological features that previously have been identified as synapomorphies of Bilateria and Ctenophora, e.g., mesoderm, more likely evolved independently in each clade. The endomesodermal muscles of bilaterians may be homologous to the endodermal muscles of cnidarians, implying that the original bilaterian mesodermal muscles were myoepithelial. Placozoans should have a gastrulation stage during development. Of the three hypotheses that cannot be falsified with the 18S rRNA data, one is most strongly supported. This hypothesis states that Bilateria and Placozoa share a more recent common ancestor than either does to Cnidaria. If true, the simplicity of placozoan body architecture is secondarily derived from a more complex ancestor. This simplification may have occurred in association with a planula-type larva becoming reproductive before metamorphosis. If this simplification took place during the common history that placozoans share with bilaterians, then placozoan genes that contain a homeobox, such as Trox2, should be explored, for they may include the gene or genes most closely related to Hox genes of bilaterians.     

MitBASE: a comprehensive and integrated mitochondrial DNA database

The mechanisms underlying the increase in energy expenditure during leptin treatment are not clear. We recently showed that a 5-h intravenous or intracerebroventricular infusion of leptin elevated basal glucose uptake in skeletal muscle (SM) and brown adipose tissue and increased whole-body glucose turnover in C57Bl/6J mice (Kamohara S, Burcelin R, Halaas JL, Friedman JM, Charron MJ: Acute stimulation of glucose metabolism in mice by leptin treatment. Nature 389:374-377, 1997). We extended the previous study by measuring steady-state levels of uncoupling protein (UCP)-2 mRNA and UCP-3 mRNA in white adipose tissue (WAT) and SM. Leptin by intravenous or intracerebroventricular infusion for 5 h was associated with a decrease in UCP-2 mRNA in WAT (47-52%) and UCP-3 mRNA in SM (33-37%). Because overexpression of UCP-2 or UCP-3 can depolarize the inner mitochondrial membrane, suppression of UCP-2 mRNA and UCP-3 mRNA may in fact lower respiratory demands in WAT and SM. This is consistent with the parallel suppression of cytochrome oxidase subunit IV (COX-IV) mRNA in WAT (35-39%) after leptin infusion. COX-IV mRNA in SM did not respond to acute leptin treatment. Mitochondrial inorganic phosphate carrier (P1C) mRNA was also suppressed in WAT (33-35%) by either method of leptin infusion, but only intravenous infusion of leptin reduced P1C mRNA in SM (40%). Denervation suppressed mRNA levels for UCP-2 (49%), UCP-3 (36%), and COX-IV (59%) and eliminated the acute response to leptin in SM. The comparable response to leptin under intravenous or intracerebroventricular infusion and the loss of responsiveness after denervation strongly suggest that the acute effects of leptin involve central signaling pathways.     

The sorting of mitochondrial DNA and mitochondrial proteins in zygotes: preferential transmission of mitochondrial DNA to the medial bud

Green fluorescent protein (GFP) was used to tag proteins of the mitochondrial matrix, inner, and outer membranes to examine their sorting patterns relative to mtDNA in zygotes of synchronously mated yeast cells in rho+ x rho0 crosses. When transiently expressed in one of the haploid parents, each of the marker proteins distributes throughout the fused mitochondrial reticulum of the zygote before equilibration of mtDNA, although the membrane markers equilibrate slower than the matrix marker. A GFP-tagged form of Abf2p, a mtDNA binding protein required for faithful transmission of rho+ mtDNA in vegetatively growing cells, colocalizes with mtDNA in situ. In zygotes of a rho+ x rho+ cross, in which there is little mixing of parental mtDNAs, Abf2p-GFP prelabeled in one parent rapidly equilibrates to most or all of the mtDNA, showing that the mtDNA compartment is accessible to exchange of proteins. In rho+ x rho0 crosses, mtDNA is preferentially transmitted to the medial diploid bud, whereas mitochondrial GFP marker proteins distribute throughout the zygote and the bud. In zygotes lacking Abf2p, mtDNA sorting is delayed and preferential sorting is reduced. These findings argue for the existence of a segregation apparatus that directs mtDNA to the emerging bud.     

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.     

Molecular evolution of the family Camelidae: a mitochondrial DNA study

We report the first molecular evolutionary analysis of the family Camelidae by analysing the full DNA sequence of the mitochondrial cytochrome b gene. Estimates for the time of divergence of the Old World (Camelini) and New World (Lamini) tribes obtained from sequence data are in agreement with those derived from the fossil record. The DNA sequence data were also used to test current hypotheses concerning the ancestors of the domesticated llama and alpaca. The results show that hybridization has occurred in the ancestry of both domesticated camelids, obscuring the origin of the domestic species.     

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.     

Evaluation of a new method for identification of bacteria based on sequence homology of 16S rRNA gene

A new identification method for bacteria based on partial sequences of divergent regions of the 16S rRNA gene was evaluated. The method involves PCR-based amplification of 16S rRNA gene fragments, followed by sequencing and comparison of sequences of about 300 nucleotides with those in the database of NCBI (National Center for Biotechnology Information) via the Internet. Most of the bacteria tested could be identified at the species level even if some unread nucleotides were present in the sequence. Although this method still requires improvement, it has the potential to be a highly reliable and practical identification method for bacteria.