Types of Listeria monocytogenes predicted by the positions of EcoRI cleavage sites relative to ribosomal RNA sequences.

By using taxonomic characters derived from EcoRI restriction endonuclease digestion of genomic DNA and hybridization with a labeled rRNA operon from Escherichia coli, a polymorphic structure of Listeria monocytogenes, characterized by fragments with different frequencies of occurrence, was observed. This structure was expanded by creating predicted patterns through a recursive process of observation, expectation, prediction, and assessment of completeness. This process was applied, in turn, to normalized strain patterns, fragment bands, and positions of EcoRI recognition sites relative to rRNA regions. Analysis of 1346 strains provided observed patterns, fragment sizes, and their frequencies of occurrence in the patterns. Fragment size statistics led to the creation of unobserved combinations of bands, predicted pattern types. The observed fragment bands revealed positions of EcoRI sites relative to rRNA sequences. Each EcoRI site had a frequency of occurrence, and unobserved fragment sizes were postulated on the basis of knowing the restriction site locations. The result of the recursion process applied to the components of the strain data was an extended classification with observed and predicted members.

Regulation of ribosomal RNA expression across the lifespan is fine-tuned by maternal diet before implantation

Cells and organisms respond to nutrient deprivation by decreasing global rates of transcription, translation and DNA replication. To what extent such changes can be reversed is largely unknown. We examined the effect of maternal dietary restriction on RNA synthesis in the offspring. Low protein diet fed either throughout gestation or for the preimplantation period alone reduced cellular RNA content across fetal somatic tissues during challenge and increased it beyond controls in fetal and adult tissues after challenge release. Changes in transcription of ribosomal RNA, the major component of cellular RNA, were responsible for this phenotype as evidenced by matching alterations in RNA polymerase I density and DNA methylation at ribosomal DNA loci. Cellular levels of the ribosomal transcription factor Rrn3 mirrored the rRNA expression pattern. In cell culture experiments, Rrn3 overexpression reduced rDNA methylation and increased rRNA expression; the converse occurred after inhibition of Rrn3 activity. These observations define novel mechanism where poor nutrition before implantation irreversibly alters basal rates of rRNA transcription thereafter in a process mediated by rDNA methylation and Rrn3 factor.

Identification and characterization of rhizospheric microbial diversity by 16S ribosomal RNA gene sequencing

In the present study, samples of rhizosphere and root nodules were collected from different areas of Pakistan to isolate plant growth promoting rhizobacteria. Identification of bacterial isolates was made by 16S rRNA gene sequence analysis and taxonomical confirmation on EzTaxon Server. The identified bacterial strains were belonged to 5 genera i.e. Ensifer, Bacillus, Pseudomona, Leclercia and Rhizobium. Phylogenetic analysis inferred from 16S rRNA gene sequences showed the evolutionary relationship of bacterial strains with the respective genera. Based on phylogenetic analysis, some candidate novel species were also identified. The bacterial strains were also characterized for morphological, physiological, biochemical tests and glucose dehydrogenase (gdh) gene that involved in the phosphate solublization using cofactor pyrroloquinolone quinone (PQQ). Seven rhizoshperic and 3 root nodulating stains are positive for gdh gene. Furthermore, this study confirms a novel association between microbes and their hosts like field grown crops, leguminous and non-leguminous plants. It was concluded that a diverse group of bacterial population exist in the rhizosphere and root nodules that might be useful in evaluating the mechanisms behind plant microbial interactions and strains QAU-63 and QAU-68 have sequence similarity of 97 and 95% which might be declared as novel after further taxonomic characterization.

Phylogeny of east Asian bufonids inferred from mitochondrial DNA sequences (Anura : Amphibia)

We investigated the relationships of Asian bufonids using partial sequences of mitochondrial DNA genes. Twenty-six samples representing 14 species of Bufo from China and Vietnam and 2 species of Torrentophryne from China were examined. Three samples of Bufo viridis from Armenia and Georgia were also sequenced to make a comparison to its sibling tetraploid species B. danatensis. Bufo americanus, from Canada, was used as the outgroup. Sequences from the 12S ribosomal RNA, 16S ribosomal RNA, cytochrome b, and the control region were analyzed using parsimony. East Asian bufonids were grouped into two major clades. One clade included B. andrewsi, B. bankorensis, B. gargarizans, B. tibetanus, B. tuberculatus, its sister clade B. cryptotympanicus, and the 2 species of Torrentophryne. The second clade consisted of B. galeatus, B. himalayanus, B. melanostictus, and a new species from Vietnam. The placement of three taxa (B. raddei B. viridis, and its sister species, B. danatensis) was problematic. The genus Torrentophryne should be synonymized with Bufo to remove paraphyly. Because B. raddei does not belong to the clade that includes B. viridis and B. danatensis, it was removed from the viridis species group. The species status of B bankorensis from Taiwan is evaluated. (C) 2000 Academic Press.

Phylogeny, species limits, and biogeography of the Brazilian lizards of the genus Eurolophosaurus (Squamata : Tropiduridae) as inferred from mitochondrial DNA sequences

Phylogenetic relationships and divergence times for 10 populations of the three recognized ""species"" of Brazilian lizards of genus Eurolophosaurus were estimated from 1229 bp of cyt b, COI, 12S, and 16S rRNA mitochondrial gene segments. Eurolophosaurus is monophyletic and the basal split within the genus separates E divaricatus from a clade comprising E amathites and E nanuzae. Three populations of E divaricatus, which occurs along the western bank of Rio S (a) over tildeo Francisco, were consistently grouped together. Oil the east bank of the river, E amathites and E nanuzae from state of Bahia were recovered as the sister group of E nanuzae populations from state of Minas Gerais. The paraphyly of E nanuzae and the high divergence levels among populations of E divaricatus strongly suggest that species limits in Eurolophosaurus should be revised. Even considering an extreme evolutionary rate of 2.8% sequence divergence per million years for the four gene segments analyzed together, E. divaricatus would have separated from the two other species by at least 5.5 my ago, and E. amathites from E nanuzae populations from Bahia and Minas Gerais, respectively, by 1.5 and 3.5 my. The paleolacustrine hypothesis and changes in the course of the river potentially explain faunal divergence in the area, but divergences are much older than previously admitted. (C) 2007 Elsevier Inc. All rights reserved.

The search of a genetic basis for noise-induced hearing loss (NIHL)

Background and aim: Knowledge about the genetic factors responsible for noise-induced hearing loss (NIHL) is still limited. This study investigated whether genetic factors are associated or not to susceptibility to NIHL. Subjects and methods: The family history and genotypes were studied for candidate genes in 107 individuals with NIHL, 44 with other causes of hearing impairment and 104 controls. Mutations frequently found among deaf individuals were investigated (35delG, 167delT in GJB2, Delta(GJB6- D13S1830), Delta(GJB6- D13S1854) in GJB6 and A1555G in MT-RNR1 genes); allelic and genotypic frequencies were also determined at the SNP rs877098 in DFNB1, of deletions of GSTM1 and GSTT1 and sequence variants in both MTRNR1 and MTTS1 genes, as well as mitochondrial haplogroups. Results: When those with NIHL were compared with the control group, a significant increase was detected in the number of relatives affected by hearing impairment, of the genotype corresponding to the presence of both GSTM1 and GSTT1 enzymes and of cases with mitochondrial haplogroup L1. Conclusion: The findings suggest effects of familial history of hearing loss, of GSTT1 and GSTM1 enzymes and of mitochondrial haplogroup L1 on the risk of NIHL. This study also described novel sequence variants of MTRNR1 and MTTS1 genes.

Role of the Mitochondrial Mutations, m. 827A > G and the Novel m. 7462C > T, in the Origin of Hearing Loss

Samples from 30 deaf probands exhibiting features suggestive of syndromic mitochondrial deafness or from families with maternal transmission of deafness were selected for investigation of mutations in the mitochondrial genes MT-RNR1 and MT-TS1. Patients with mutation m. 1555A>G had been previously excluded from this sample. In the MT-RNR1 gene, five probands presented the m. 827A>G sequence variant, of uncertain pathogenicity. This change was also detected in 66 subjects of an unaffected control sample of 306 Brazilian individuals from various ethnic backgrounds. Given its high frequency, we consider it unlikely to have a pathogenic role on hereditary deafness. As to the MT-TS1 gene, one proband presented the previously known pathogenic m. 7472insC mutation and three probands presented a novel variant, m. 7462C>T, which was absent from the same control sample of 306 individuals. Because of its absence in control samples and association with a family history of hearing impairment, we suggest it might be a novel pathogenic mutation.

Genetic structure, phylogeny, and biogeography of Brazilian eyelid-less lizards of genera Calyptommatus and Nothobachia (Squamata, Gymnophthalmidae) as inferred from mitochondrial DNA sequences

Calyptommatus and Nothobachia genera of gymnophthalmid lizards are restricted to sandy open habitats on Sao Francisco River margins, northeastern Brazil. Phylogenetic relationships and geographic distribution of the four recognized species of Calyptommatus were analyzed from partial mitochondrial cyt b, 12S, and 16S rRNA genes sequencing, taking allopatric populations of the monotypic Nothobachia ablephara as the outgroup. In Calyptommatus a basal split separated C. sinebrachiatus, a species restricted to the eastern bank of the river, from the three other species. In this clade, C. confusionibus, found on western margin, was recovered as the sister group of the two other species, C. leiolepis and C. nicterus, from opposite margins. According to approximate date estimations, C. sinebrachiatus would have separated from the other congeneric species by 4.4-6.5 my, and C. nicterus, also from eastern bank, would be diverging by 1.8-2.6 my from C. leiolepis, the sister species on the opposite margin. C. confusionibus and C. leiolepis, both from western sandy areas, would be differentiating by 2.8-5.0 my. Divergence times of about 3.0-4.0 my were estimated for allopatric populations of Nothobachia restricted to western margin. Significant differences in 16S rRNA secondary structure relatively to other vertebrates are reported. Distinct evolutionary patterns are proposed for different taxa in those sandy areas, probably related to historical changes in the course of Sao Francisco River. (C) 2010 Elsevier Inc. All rights reserved.

The evolution of behavioural systems: a study of grooming in rodents

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Phylogenetic relationships of the Chinese brown frogs (Genus rana) inferred from partial mitochondrial 12S and 16S rRNA gene sequences

Based on partial sequences of the 12S and 16S ribosomal RNA genes, we estimated phylogenetic relationships among brown frogs of the Rana temporaria group from China. From the phylogenetic trees obtained, we propose to include Rana zhengi in the brown frog

The transcript release factor PTRF augments ribosomal gene transcription by facilitating reinitiation of RNA polymerase I

Termination of murine rDNA transcription by RNA polymerase I (Pol I) requires pausing of Pol I by terminator-bound TTF-I (transcription termination factor for Pol I), followed by dissociation of the ternary complex by PTRF (Pol I and transcript release factor). To examine the functional correlation between transcription termination and initiation, we have compared transcription on terminator-containing and terminator-less rDNA templates. We demonstrate that terminated RNA molecules are more efficiently synthesized than run-off transcripts, indicating that termination facilitates reinitiation. Transcriptional enhancement is observed in multiple- but not single-round transcription assays measuring either promoter-dependent or promoter-independent Pol I transcription. Increased synthesis of terminated transcripts is observed in crude extracts but not in a PTRF-free reconstituted transcription system, indicating that PTRF-mediated release of pre-rRNA is responsible for transcriptional enhancement. Consistent with PTRF serving an important role in modulating the efficiency of rRNA synthesis, PTRF exhibits pronounced charge heterogeneity, is phosphorylated at multiple sites and fractionates into transcriptionally active and inactive forms. The results suggest that regulation of PTRF activity may be an as yet unrecognized means to control the efficiency of ribosomal RNA synthesis.

A universal test to determine the integrity of RNA, and its suitability for reverse transcription, in animal tissue laboratory specimens

Degradation of RNA in diagnostic specimens can cause false-negative test results and potential misdiagnosis when tests rely on the detection of specific RNA sequence. Current molecular methods of checking RNA integrity tend to be host species or group specific, necessitating libraries of primers and reaction conditions. The objective here was to develop a universal (multi-species) quality assurance tool for determining the integrity of RNA in animal tissues submitted to a laboratory for analyses. Ribosomal RNA (16S rRNA) transcribed from the mitochondrial 16S rDNA was used as template material for reverse transcription to cDNA and was amplified using polymerase chain reaction (PCR). As mitochondrial DNA has a high level of conservation, the primers used were shown to reverse transcribe and amplify RNA from every animal species tested. Deliberate degradation of rRNA template through temperature abuse of samples resulted in no reverse transcription and amplification. Samples spiked with viruses showed that single-stranded viral RNA and rRNA in the same sample degraded at similar rates, hence reverse transcription and PCR amplification of 16S rRNA could be used as a test of sample integrity and suitability for analysis that required the sample's RNA, including viral RNA. This test will be an invaluable quality assurance tool for determination of RNA integrity from tissue samples, thus avoiding erroneous test results that might occur if degraded target RNA is used unknowingly as template material for reverse transcription and subsequent PCR amplification.

Interactions of 3' terminal and 5' terminal regions of physalis mottle virus genomic RNA with its replication complex

Physalis mottle virus (PhMV) belongs to the tymogroup of positive-strand RNA viruses with a genome size of 6 kb. Crude membrane preparations from PhMV-infected Nicotiana glutinosa plants catalyzed the synthesis of PhMV genomic RNA from endogenously bound template. Addition of exogenous genomic RNA enhanced the synthesis which was specifically inhibited by the addition of sense and antisense transcripts corresponding to 3' terminal 242 nucleotides as well as the 5' terminal 458 nucleotides of PhMV genomic RNA while yeast tRNA or ribosomal RNA failed to inhibit the synthesis. This specific inhibition suggested that the 5' and 3' non-coding regions of PhMV RNA might play an important role in viral replication.

A key to selected rockfishes (Sebastes spp.) based on mitochondrial DNA restriction fragment analysis

Larval and juvenile rockfishes (Sebastes spp.) are difficult to identify using morphological characters. We developed a key based on sizes of restriction endonuclease fragments of the NADH dehydrogenase-3 and -4 (ND3/ND4) and 12S and 16S ribosomal RNA (12S/16S) mitochondrial regions. The key makes use of variation in the ND3/ND4 region. Restriction endonuclease Dde I variation can corroborate identifications, as can 12S/16S variation. The key, based on 71 species, includes most North American taxa, several Asian species, and Sebastolobus alascanus and Helicolenus hilgendorfi that are closely related to rockfishes. Fifty-eight of 71 rockfish species in our database can be distinguished unequivocally, using one to five restriction enzymes; identities of the remaining species are narrowed to small groups: 1) S. polyspinis, S. crameri, and S. ciliatus or variabilis (the two species could not be distinguished and were considered as a single species) ; 2) S. chlorostictus, S. eos, and S. rosenblatti; 3) S. entomelas and S. mystinus; 4)S. emphaeus, S. variegatus, and S. wilsoni; and 5) S. carnatus and S. chrysomelas.