Sequence and organization of barley yellow dwarf virus genomic RNA

The nucleotide sequence of the genomic RNA of barley yellow dwarf virus, PAV serotype was determined except for the 5′-terminal base, and its genome organization deduced. The 5,677 nucleotide genome contains five large open reading frames (ORFs). The genes for the coat protein (1) and the putative viral RNA-dependent RNA polymerase were identified. The latter shows a striking degree of similarity to that of carnation mottle virus (CarMV). By comparison with corona- and retrovirus RNAs, it is proposed that a translational frameshift is involved in expression of the polymerase. An ORF encoding an Mr 49,797 protein (50K ORF) may be translated by in-frame readthrough of the coat protein stop codon. The coat protein, an overlapping 17K ORF, and a 3′ 6.7K ORF are likely to be expressed via subgenomic mRNAs. © 1988 IRL Press Limited.

Nucleotide sequences of an Australian and a Canadian isolate of potato leafroll luteovirus and their relationships with two European isolates

The genomes of an Australian and a Canadian isolate of potato leafroll virus have been cloned and sequenced. The sequences of both isolates are similar (about 93%), but the Canadian isolate (PLRV-C) is more closely related (about 98% identity) to a Scottish (PLRV-S) and a Dutch isolate (PLRV-N) than to the Australian isolate (PLRV-A). The 5'-terminal 18 nucleotide residues of PLRV-C, PLRV-A, PLRV-N and beet western yellows virus have 17 residues in common. In contrast, PLRV-S shows no obvious similarity in this region. PLRV-A and PLRV-C genomic sequences have localized regions of marked diversity, in particular a 600 nucleotide residue sequence in the polymerase gene. These data provide a world-wide perspective on the molecular biology of PLRV strains and their comparison with other luteoviruses and related RNA plant viruses suggests that there are two major subgroups in the plant luteoviruses.

Sequence and identification of the barley yellow dwarf virus coat protein gene

The nucleotide sequence of the coat protein gene of barley yellow dwarf virus (BYDV, PAV serotype) was determined, and the amino acid sequence was deduced. The open reading frame, encoding a protein of relative molecular mass (Mr) 22,047, was confirmed as the coat protein gene by comparison with amino acid sequences of tryptic peptides derived from dissociated virions. In addition, a fragment of this gene expressed in Escherichia coli produced a product which was recognized by antibodies prepared against purified BYDV virions. An overlapping reading frame encoding an Mr 17,147 protein is contained completely within the coat protein gene. © 1988.

Sequence-dependent conformation of an A-DNA double helix: The crystal structure of the octamer d(G-G-T-A-T-A-C-C)

The crystal structures of the synthetic self-complementary octamer d(G-G-T-A-T-A-C-C) and its 5-bromouracil-containing analogue have been refined to R values of 20% and 14% at resolutions of 1·8 and 2·25 Å, respectively. The molecules adopt an A-DNA type double-helical conformation, which is minimally affected by crystal forces. A detailed analysis of the structure shows a considerable influence of the nucleotide sequence on the base-pair stacking patterns. In particular, the electrostatic stacking interactions between adjacent guanine and thymine bases produce symmetric bending of the double helix and a major-groove widening. The sugar-phosphate backbone appears to be only slightly affected by the base sequence. The local variations in the base-pair orientation are brought about by correlated adjustments in the backbone torsion angles and the glycosidic orientation. Sequence-dependent conformational variations of the type observed here may contribute to the specificity of certain protein-DNA interactions.

Detection,amplification and sequence homology of sodC in clinical isolates of Salmonella sp

Background & objectives: Periplasmic copper and zinc superoxide dismutase (Cu,Zn-SOD or SodC) is an important component of the antioxidant shield which protects bacteria from the phagocytic oxidative burst. Cu,Zn-SODs protect Gram-negative bacteria against oxygen damage which have also been shown to contribute to the pathogenicity of these bacterial species. We report the presence of SodC in drug resistant Salmonella sp. isolated from patients suffering from enteric fever. Further sodC was amplified, cloned into Escherichia coli and the nucleotide sequence and amino acid sequence homology were compared with the standard strain Salmonella Typhimurium 14028. Methods: Salmonella enterica serovar Typhi (S. Typhi) and Salmonellaenterica serovar Paratyphi (S. Paratyphi) were isolated and identified from blood samples of the patients. The isolates were screened for the presence of Cu, Zn-SOD by PAGE using KCN as inhibitor of Cu,Zn-SOD. The gene (sodC) was amplified by PCR, cloned and sequenced. The nucleotide and amino acid sequences of sodC were compared using CLUSTAL X.Results: SodC was detected in 35 per cent of the Salmonella isolates. Amplification of the genomic DNA of S. Typhi and S. Paratyphi with sodC specific primers resulted in 519 and 515 bp amplicons respectively. Single mutational difference at position 489 was observed between thesodC of S. Typhi and S. Paratyphi while they differed at 6 positions with the sodC of S. Typhimurium 14028. The SodC amino acid sequences of the two isolates were homologous but 3 amino acid difference was observed with that of standard strain S. Typhimurium 14028.Interpretation & conclusions: The presence of SodC in pathogenic bacteria could be a novel candidate as phylogenetic marker.

Both Surface Proteins (VP4 and VP7) of an Asymptomatic Neonatal Rotavirus Strain (1321) Have High Levels of Sequence Identity with the Homologous Proteins of a Serotype 10 Bovine Rotavirus

The nucleotide sequence of genes 4 and 9, encoding the outer capsid proteins VP4 and VP7 of a serotype 10 tissue culture-adapted strain, 1321, representative of asymptomatic neonatal rotaviruses isolated from neonates in Bangalore, India, were determined. Comparison of nucleotide and deduced amino acid sequences of 1321 VP4 and VP7 with previously published sequences of various serotypes revealed that both genes were highly homologous to the respective genes of serotype 10 bovine rotavirus, B223. The VP4 of 1321 represents a new human P serotype and the 1321 and related strains represent the first description of neonatal rotaviruses that appear to derive both surface proteins from an animal rotavirus.

Structural proteins of mycobacteriophage I3: cloning, expression and sequence analysis of a gene encoding a 70-kDa structural protein

The structural proteins of mycobacteriophage I3 have been analysed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE), radioiodination and immunoblotting. Based on their abundance the 34- and 70-kDa bands appeared to represent the major structural proteins. Successful cloning and expression of the 70-kDa protein-encoding gene of phage I3 in Escherichia coli and its complete nucleotide sequence determination have been accomplished, A second (partial) open reading frame following the stop codon for the 70-kDa protein was also identified within the cloned fragment. The deduced amino-acid sequence of the 70-kDa protein and the codon usage patterns indicated the preponderance of codons, as predicted from the high G+C content of the genomic DNA of phage I3.

Characterization of complete genome sequence of the spring viremia of carp virus isolated from common carp (Cyprinus carpio) in China

The complete genome of spring viraemia of carp virus (SVCV) strain A-1 isolated from cultured common carp (Cyprinus carpio) in China was sequenced and characterized. Reverse transcription-polymerase chain reaction (RT-PCR) derived clones were constructed and the DNA was sequenced. It showed that the entire genome of SVCV A-1 consists of 11,100 nucleotide base pairs, the predicted size of the viral RNA of rhabdoviruses. However, the additional insertions in bp 4633-4676 and bp 4684-4724 of SVCV A-1 were different from the other two published SVCV complete genomes. Five open reading frames (ORFs) of SVCV A-1 were identified and further confirmed by RT-PCR and DNA sequencing of their respective RT-PCR products. The 5 structural proteins encoded by the viral RNA were ordered 3'-N-P-M-G-L-5'. This is the first report of a complete genome sequence of SVCV isolated from cultured carp in China. Phylogenetic analysis indicates that SVCV A-1 is closely related to the members of the genus Vesiculovirus, family Rhabdoviridae.

A novel invariant and sequence comparison for DNA

We consider numerical characterization of DNA primary sequence based on the positions of bases (a, t, c, g) and the pairs of bases X, Y in DNA (X, Y=a, t, c, g). This leads to a representation of DNA by a numerical sequence. Then, we extract a novel invariant (molecular connectivity index) from the derived numerical sequences. The suitable invariant can offer a characterization of DNA primary sequence. Finally, we provide an illustration of its utility by making a comparison between ten DNA sequences belonging to beta-globin gene in different species. The evolutionary relationships of ten species we have revealed in this contribution accord with phylogenetic tree properly.

Distribution, gene sequence and expression in vivo of the plasmid encoded fimbrial antigen of Salmonella serotype Enteritidis

The pefA gene which encoded the serotype associated plasmid (SAP) mediated fimbrial major subunit antigen of Salmonella enterica serotype Typhimurium shared genetic identity with 128 of 706 salmonella isolates as demonstrated by dot (colony) hybridization. Seventy-seven of 113 isolates of Typhimurium and individual isolates of serotypes Bovis-morbificans, Cholerae-suis and Enteritidis phage type 9b hybridized pefA strongly, whereas 48 isolates of Enteritidis hybridized pefA weakly and one Enteritidis isolate of phage type 14b failed to hybridize. Individual isolates of 294 serotypes and 247 individual isolates of serotype Dublin did not hybridize pefA. Southern hybridization of plasmids extracted from Enteritidis demonstrated that the pefA gene probe hybridized strongly an atypical SAP of 80 kb in size harboured by one Enteritidis isolate of phage-type 9b, whereas the typical SAP of 58 kb in size harboured by 48 Enteritidis isolates hybridized weakly. One Enteritidis isolate of phage type 14b which failed to hybridize pefA in dot (colony) hybridization experiments was demonstrated to be plasmid free. A cosmid library of Enteritidis phage type 4 expressed in Escherichia coli K12 was screened by hybridization for the presence of pef sequences. Recombinant clones which were deduced to harbour the entire pef operon elaborated a PEF-like fimbrial structure at the cell surface. The PEF-like fimbrial antigen was purified from one cosmid clone and used in western blot experiments with sera from chickens infected with Enteritidis phage-type 4. Seroconversion to the fimbrial antigen was observed which indicated that the Enteritidis PEF-like fimbrial structure was expressed at some stage during infection. Nucleotide sequence analysis demonstrated that the pefA alleles of Typhimurium and Enteritidis phage-type 4 shared 76% DNA nucleotide and 82% deduced amino acid sequence identity.

Sequence analysis and distribution of an IS3-like insertion element isolated from Salmonella enteritidis

The nucleotide sequence of a 3 kb region immediately upstream of the sef operon operon of Salmonella enteritidis was determined. A 1230 base pair insertion sequence which shared sequence identity (> 75%) with members of the IS3 family was revealed. This element, designated IS1230, had almost identical (90% identity) terminal inverted repeats to Escherichia coli IS3 but unlike other IS3-like sequences lacked the two characteristic open reading frames which encode the putative transposase. S. enteritidis possessed only one copy of this insertion sequence although Southern hybridisation analysis of restriction digests of genomic DNA revealed another fragment located in a region different from the sef operon which hybridised weakly which suggested the presence of an IS1230 homologue. The distribution of IS1230 and IS1230-like elements was shown to be widespread amongst salmonellas and the patterns of restriction fragments which hybridised differed significantly between Salmonella serotypes and it is suggested that IS1230 has potential for development as a differential diagnostic tool.

Sequence analysis and distribution in Salmonella enterica serovars of IS3-like elements

The genome of Salmonella enterica serovar Enteritidis was shown to possess three IS3-like insertion elements, designated IS1230A, B and C, and each was cloned and their respective deoxynucleotide sequences determined. Mutations in elements IS1230A and B resulted in frameshifts in the open reading frames that encoded a putative transposase to be inactive. IS1230C was truncated at nucleotide 774 relative to IS1230B and therefore did not possess the 3' terminal inverted repeat. The three IS1230 derivatives were closely related to each other based on nucleotide sequence similarity. IS1230A was located adjacent to the sef operon encoding SEF14 fimbriae located at minute 97 of the genome of S. Enteritidis. IS1230B was located adjacent to the umuDC operon at minute 42.5 on the genome, itself located near to one terminus of an 815-kb genome inversion of S. Enteritidis relative to S. Typhimurium. IS1230C was located next to attB, the bacteriophage P22 attachment site, and proB, encoding gamma-glutamyl phosphate reductase. A truncated 3' remnant of IS1230, designated IS1230T, was identified in a clinical isolate of S. Typhimurium DT193 strain 2391. This element was located next to attB adjacent to which were bacteriophage P22-like sequences. Southern hybridisation of total genomic DNA from eighteen phage types of S. Enteritidis and eighteen definitive types of S. Typhimurium showed similar, if not identical, restriction fragment profiles in the respective serovars when probed with IS1230A.

PPARGC1A sequence variation and cardiovascular risk-factor levels: a study of the main genetic effects and gene x environment interactions in children from the European Youth Heart Study.

AIMS/HYPOTHESIS: The PPARGC1A gene coactivates multiple nuclear transcription factors involved in cellular energy metabolism and vascular stasis. In the present study, we genotyped 35 tagging polymorphisms to capture all common PPARGC1A nucleotide sequence variations and tested for association with metabolic and cardiovascular traits in 2,101 Danish and Estonian boys and girls from the European Youth Heart Study, a multicentre school-based cross-sectional cohort study. METHODS: Fasting plasma glucose concentrations, anthropometric variables and blood pressure were measured. Habitual physical activity and aerobic fitness were objectively assessed using uniaxial accelerometry and a maximal aerobic exercise stress test on a bicycle ergometer, respectively. RESULTS: In adjusted models, nominally significant associations were observed for BMI (rs10018239, p = 0.039), waist circumference (rs7656250, p = 0.012; rs8192678 [Gly482Ser], p = 0.015; rs3755863, p = 0.02; rs10018239, beta = -0.01 cm per minor allele copy, p = 0.043), systolic blood pressure (rs2970869, p = 0.018) and fasting glucose concentrations (rs11724368, p = 0.045). Stronger associations were observed for aerobic fitness (rs7656250, p = 0.005; rs13117172, p = 0.008) and fasting glucose concentrations (rs7657071, p = 0.002). None remained significant after correcting for the number of statistical comparisons. We proceeded by testing for gene x physical activity interactions for the polymorphisms that showed nominal evidence of association in the main effect models. None of these tests was statistically significant. CONCLUSIONS/INTERPRETATION: Variants at PPARGC1A may influence several metabolic traits in this European paediatric cohort. However, variation at PPARGC1A is unlikely to have a major impact on cardiovascular or metabolic health in these children.

The systematics of freshwater crayfish of the genus Cherax Erichson (Decapoda : Parastacidae) in eastern Australia re-examined using nucleotide sequences from 12S rRNA and 16S rRNA genes

Nucleotide sequence data were used to re-examine systematic relationships and species boundaries within the genus Cherax from eastern Australia. Partial sequences were amplified from the 12S (~365 bp) and 16S (~545 bp) rRNA mitochondrial gene regions. Levels of intra- and inter-specific divergence for Cherax species were very similar between the two gene regions and similar to that reported for other freshwater crayfish for 16S rRNA. Phylogenetic analyses using the combined data provided strong support for a monophyletic group containing 11 eastern Australian species and comprising three well-defined species-groups: the 'C. destructor' group containing three species, the 'C. cairnsensis' group containing four species and the 'C. cuspidatus' group containing two species. Cherax dispar and C. robustus are distinct from all other species and each other. In addition, two northern Australian and a New Guinean species were placed in the 'Astaconephrops' group, which is the sister-group to the eastern Australian Cherax lineage. Several relationships were clarified, including: the status of northern and southern C. cuspidatus as separate species; a close relationship between C. cairnsensis and C. depressus; the validity of C. rotundus and C. setosus as separate species and their close affinities with C. destructor; and the distinctiveness of the northern forms of Cherax. The analysis of the 12S rRNA and 16S rRNA data is highly concordant with the results of previous allozyme studies.