Cloning and nucleotide sequencing of the S4 genome segment of avian reovirus S1133

The sequence of RNA genome segment S4 of the avian reovirus (ARV) strain S1133 was determined. S4 RNA is 1185 base pairs long and contains one open reading frame encoding a protein of 367 amino acid residues (40.6 kDa), the similar size as the known S4 gene product (sigma NS), with a net charge of -1 at neutral pH. The S4 RNA sequence possesses a pentanucleotide sequence UCAUC at the 3'-terminus of its plus strand like in ARV S1 and S3 segments and ten segments of mammalian reovirus (MRV). The predicted amino acid sequence comparison revealed that the homology is 44.02%, 45.71%, and 42.33% for ARV sigma NS and three serotypes of MRV sigma NS, respectively. The relatively high content of alpha-helix structure in the C-terminal portion of ARV sigma NS suggests that this protein may functionally relate to MRV sigma NS. Northern blot hybridization showed that a 32P-labeled cDNA insert S4-49 from ARV S4 RNA cross-hybridized with the corresponding RNA segments of all seven strains of ARV tested.     

Reovirus growth in cell culture does not require the full complement of viral proteins: identification of a sigma1s-null mutant

The reovirus sigma1s protein is a 14-kDa nonstructural protein encoded by the S1 gene segment. The S1 gene has been linked to many properties of reovirus, including virulence and induction of apoptosis. Although the function of sigma1s is not known, the sigma1s open reading frame is conserved in all S1 gene sequences determined to date. In this study, we identified and characterized a variant of type 3 reovirus, T3C84-MA, which does not express sigma1s. To facilitate these experiments, we generated two monoclonal antibodies (MAbs) that bind different epitopes of the sigma1s protein. Using these MAbs in immunoblot and immunofluorescence assays, we found that L929 (L) cells infected with T3C84-MA do not contain sigma1s. To determine whether sigma1s is required for reovirus infection of cultured cells, we compared the growth of T3C84-MA and its parental strain, T3C84, in L cells and Madin-Darby canine kidney (MDCK) cells. After 48 h of growth, yields of T3C84-MA were equivalent to yields of T3C84 in L cells and were fivefold lower than yields of T3C84 in MDCK cells. After 7 days of growth following adsorption at a low multiplicity of infection, yields of T3C84-MA and T3C84 did not differ significantly in either L cells or MDCK cells. To determine whether sigma1s is required for apoptosis induced by reovirus infection, T3C84-MA and T3C84 were tested for their capacity to induce apoptosis, using an acridine orange staining assay. In these experiments, the percentages of apoptotic cells following infection with T3C84-MA and T3C84 were equivalent. These findings indicate that nonstructural protein sigma1s is not required for reovirus growth in cell culture and does not influence the capacity of reovirus to induce apoptosis. Therefore, reovirus replication does not require the full complement of virally encoded proteins.     

Effects of hypothermia or anesthetics on hippocampal glutamate and glycine concentrations after repeated transient global cerebral ischemia

The reovirus group C temperature-sensitive mutant tsC447, whose defect maps to the S2 gene, which encodes the major core protein sigma 2, fails to assemble core particles at the nonpermissive temperature. To identify other proteins that may interact with sigma 2 during assembly, we generated and examined 10 independent revertants of the mutant. To determine which gene(s) carried a compensatory suppressor mutation(s), we generated intertypic reassortants between wild-type reovirus serotype 1 Lang and each revertant and determined the temperature sensitivities of the reassortants by efficiency-of-plating assays. Results of the efficiency-of-plating analyses indicated that reversion of the tsC447 defect was an intragenic process in all revertants. To identify the region(s) of sigma 2 that had reverted, we determined the nucleotide sequences of the S2 genes. In all revertant sequences examined, the G at nucleotide position 1166 in tsC447 had reverted to the A present in the wild-type sequence. This reversion leads to the restoration of a wild-type asparagine (in place of a mutant aspartic acid) at amino acid 383 in the sigma 2 sequence. These results collectively indicate that the functional lesion in tsC447 is Asp-383 and that this lesion cannot be corrected by alterations in other core proteins. These observations suggest that this region of sigma 2, which may be important in mediating assembly of the core particle, does not interact significantly with other reovirus proteins.     

Localization of a C-terminal region of lambda2 protein in reovirus cores

The 144-kDa lambda2 protein is a structural component of mammalian reovirus particles and contains the guanylyltransferase activity involved in adding 5' caps to reovirus mRNAs. After incubation of reovirus T3D core particles at 52 degrees C, the lambda2 protein became sensitive to partial protease degradation. Sequential treatments with heat and chymotrypsin caused degradation of a C-terminal portion of lambda2, leaving a 120K core-associated fragment. The four other proteins in cores--lambda1, lambda3, mu2, and sigma2--were not affected by the treatment. Purified cores with cleaved lambda2 were subjected to transmission cryoelectron microscopy and image reconstruction. Reconstruction analysis demonstrated that a distinctive outer region of lambda2 was missing from the modified cores. The degraded region of lambda2 corresponded to the one that contacts the base of the sigma1 protein fiber in reovirus virions and infectious subvirion particles, suggesting that the sigma1-binding region of lambda2 is near its C terminus. Cores with cleaved lambda2 were shown to retain all activities required to transcribe and cap reovirus mRNAs, indicating that the C-terminal region of lambda2 is dispensable for those functions.     

Cloning and nucleotide sequence of the gene encoding dinitrogenase reductase (nifH) from the cyanobacterium Nostoc 6720

The nucleotide sequence of the 3' end of the nifU coding sequence, the complete coding sequence of nifH and a substantial part of the 5' end of nifD coding sequence from Nostoc 6720 is presented. The coding sequences are highly conserved with those of Anabaena 7120 and Anabaena sp. L31. However the intergenic region between nifU and nifH contains two segments of short tandemly repetitive repeat sequences (STRRs) that differ from the STRR that is common to both Anabaena7120 and Anabaenasp. L31. Various sequence structures that are common to Nostoc 6720, the Anabaena strains and Plectonema boryanum are discussed.     

Evolutionary genetics of the isocitrate dehydrogenase gene (icd) in Escherichia coli and Salmonella enterica

Sequences of the icd gene, encoding isocitrate dehydrogenase (IDH), were obtained for 33 strains representing the major phylogenetic lineages of Escherichia coli and Salmonella enterica. Evolutionary relationships of the strains based on variation in icd are generally similar to those previously obtained for several other housekeeping and for invasion genes, but the sequences of S. enterica subspecies V strains are unusual in being almost intermediate between those of the other S. enterica subspecies and E. coli. For S. enterica, the ratio of synonymous (silent) to nonsynonymous (replacement) nucleotide substitutions between pairs of strains was larger than comparable values for 12 other housekeeping and invasion genes, reflecting unusually strong purifying selection against amino acid replacement in the IDH enzyme. All amino acids involved in the catalytic activity and conformational changes of IDH are strictly conserved within and between species. In E. coli, the level of variation at the 3' end of the gene is elevated by the presence in some strains of a 165-bp replacement sequence supplied by the integration of either lambdoid phage 21 or defective prophage element e14. The 72 members of the E. coli Reference Collection (ECOR) and five additional E. coli strains were surveyed for the presence of phage 21 (as prophage) by PCR amplification of a phage 21-specific fragment in and adjacent to the host icd, and the sequence of the phage 21 segment extending from the 3' end of icd through the integrase gene (int) was determined in nine strains of E. coli. Phage 21 was found in 39% of E. coli strains, and its distribution among the ECOR strains is nonrandom. In two ECOR strains, the phage 21 int gene is interrupted by a 1,313-bp insertion element that has 99.3% nucleotide sequence identity with IS3411 of E. coli. The phylogenetic relationships of phage 21 strains derived from sequences of two different genomic regions were strongly incongruent, providing evidence of frequent recombination.     

Ribosomal S27a coding sequences upstream of ubiquitin coding sequences in the genome of a pestivirus

Molecular characterization of cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strain CP Rit, a temperature-sensitive strain widely used for vaccination, revealed that the viral genomic RNA is about 15.2 kb long, which is about 2.9 kb longer than the one of noncytopathogenic (noncp) BVDV strains. Molecular cloning and nucleotide sequencing of parts of the genome resulted in the identification of a duplication of the genomic region encoding nonstructural proteins NS3, NS4A, and part of NS4B. In addition, a nonviral sequence was found directly upstream of the second copy of the NS3 gene. The 3' part of this inserted sequence encodes an N-terminally truncated ubiquitin monomer. This is remarkable since all described cp BVDV strains with ubiquitin coding sequences contain at least one complete ubiquitin monomer. The 5' region of the nonviral sequence did not show any homology to cellular sequences identified thus far in cp BVDV strains. Databank searches revealed that this second cellular insertion encodes part of ribosomal protein S27a. Further analyses included molecular cloning and nucleotide sequencing of the cellular recombination partner. Sequence comparisons strongly suggest that the S27a and the ubiquitin coding sequences found in the genome of CP Rit were both derived from a bovine mRNA encoding a hybrid protein with the structure NH2-ubiquitin-S27a-COOH. Polyprotein processing in the genomic region encoding the N-terminal part of NS4B, the two cellular insertions, and NS3 was studied by a transient-expression assay. The respective analyses showed that the S27a-derived polypeptide, together with the truncated ubiquitin, served as processing signal to yield NS3, whereas the truncated ubiquitin alone was not capable of mediating the cleavage. Since the expression of NS3 is strictly correlated with the cp phenotype of BVDV, the altered genome organization leading to expression of NS3 most probably represents the genetic basis of cytopathogenicity of CP Rit.     

A comparison of nucleotide sequences of measles virus L genes derived from wild-type viruses and SSPE brain tissues

The nucleotide sequences of the large protein (L) gene derived from two wild-type measles viruses (MV) and two SSPE brain-derived viruses have been determined. All sequences have single large open reading frames encoding 2183 amino acid residues. The deduced L proteins are well conserved and the proposed functional domains which have been identified for rhabdo- and paramyxoviruses are completely conserved in all strains. The degree of variability of L proteins is the lowest of all structural proteins of MV, reflecting its role in virus reproduction and persistence. Biased hypermutation was not observed in the L genes derived from SSPE brain tissue. None of the nucleotide changes can be associated with the attenuated phenotype of the Edmonston vaccine viruses.     

Recombination in Tula hantavirus evolution: analysis of genetic lineages from Slovakia

To examine the evolution of Tula hantavirus (TUL), carried by the European common vole (Microtus arvalis and M. rossiaemeridionalis), we have analyzed genetic variants from Slovakia, the country where the virus is endemic. Phylogenetic analysis (PHYLIP) based on either partial (nucleotides [nt] 441 to 898) or complete N-protein-encoding sequences divided Slovakian TUL variants into two main lineages: (i) strains from eastern Slovakia, which clustered with Russian strains, and (ii) strains from western Slovakia situated closer to those from the Czech Republic. We found genetic diversity of 19% between the two groups and 4% within the western Slovakian TUL strains. Phylogenetic analysis of the 3' noncoding region (3'-NCR), however, placed the eastern Slovakian strains closer to those from western Slovakia and the Czech Republic, with a greater distance to the Russian strains, suggesting a recombinant nature of the S segment in the eastern Slovakian TUL lineage. A bootscan search of the S-segment sequences of TUL strains revealed at least two recombination points in the S sequences of eastern Slovakian TUL strains (nt 400 to 415 and around 1200) which agreed well with the pattern of amino acid substitutions in the N protein and deletions/insertions in the 3'-NCR of the S segment. These data suggest that homologous recombination events occurred in the evolution of hantaviruses.     

Sequence elements required for apolipoprotein B mRNA editing enhancement activity from chicken enterocytes

Mammalian intestinal apolipoprotein B (apoB) mRNA edits codon 2153 from CAA in apoB100 mRNA to a stop codon (UAA) in apoB48 mRNA. By contrast, chicken intestinal apoB mRNA contains a CAA codon at the corresponding site, but is not edited. Chicken enterocyte S100 extracts fail to edit mammalian apoB RNA, but contain factor(s) which enhance the mammalian enterocytes editing activity. By converting the chicken apoB mooring sequences to the conserved mammalian sequences, the study confirmed that this 11-nucleotide stretch was necessary and sufficient for minimal RNA editing. Using rat and chicken apoB chimeric constructs, the study revealed that mammalian apoB sequences were required for editing enhancement. In concert with the 29-nucleotide conserved cassette, the 5' rat apoB element (nucleotides 6615-6629) increased editing at C-6666, and was necessary for editing enhancement of chicken enterocyte S100 extracts. Similarly, the 3' rat apoB element (nucleotides 6726-6752) was required for editing enhancement of chicken enterocyte S100 extracts, but to a lesser extent in efficiency, compared to the 5' region. In conclusion, this study identified the sequences required for editing enhancement activity from chicken enterocyte S100 extracts.     

Neutralization of reovirus: the gene responsible for the neutralization antigen

The S1 genome segment of reovirus is linked to type specificity as determined by neutralization antibody. This gene segment codes for a minor outer capsid polypeptide (sigma1). Therefore, sigma1 is the peptide responsible for induction of neutralization antibody and confers type specificity. This biologic property of reovirus was defined using hybrid recombinants clones between reovirus types 1 and 3 and 2 and 3.     

High-level genetic diversity in the vapD chromosomal region of Helicobacter pylori

Helicobacter pylori isolates from different patients are characterized by diversity in the nucleotide sequences of individual genes, variation in genome size, and variation in gene order. Genetic diversity is particularly striking in vacuolating cytotoxin (vacA) alleles. In this study, five open reading frames (ORFs) were identified within a 4.2-kb region downstream from vacA in H. pylori 60190. One of these ORFs was closely related to the virulence-associated protein D (vapD) gene of Dichelobacter nodosus (64.9% nucleotide identity). A probe derived from vapD of H. pylori 60190 hybridized with only 19 (61.3%) of 31 H. pylori strains tested. Sequence analysis of the vapD region in vapD-negative H. pylori strains revealed that there were two different families of approximately 0.5-kb DNA segments, which were both unrelated to vapD. The presence of vapD was not associated with any specific family of vacA alleles. These findings are consistent with a recombinational population structure for H. pylori.     

Cloning and characterization of the rpoH gene of Rhodobacter capsulatus

By using an oligonucleotide mixture corresponding to a region highly conserved among alternative sigma factors we identified a new sigma factor gene (rpoH) from Rhodobacter capsulatus. This gene encodes a protein of 34 kDa with strong similarity to the RpoH (sigma32) factors from other bacterial species. It was not possible to inactivate the R. capsulatus rpoH gene by introducing a resistance cassette, implying that it is essential for growth. The 5' ends of the mRNAs were mapped to two sequences with similarity to an rpoH- and an rpoD-dependent promoter, respectively. The amounts of both these mRNAs increased after heat shock, but were unaffected by a decrease in oxygen tension. Western analysis using a sigma factor-specific antibody revealed the accumulation of a protein of about 34 kDa after heat shock, and an increase in the amounts of a protein with the same size after reduction of oxygen tension in R. capsulatus cultures.     

Isolation and characterization of human fast skeletal beta troponin T cDNA: comparative sequence analysis of isoforms and insight into the evolution of members of a multigene family

A cDNA encoding human fast skeletal beta troponin T (beta TnTf) has been isolated and characterized from a fetal skeletal muscle library. The cDNA insert is 1,000 bp in length and contains the entire coding region of 777 bp and 5' and 3' untranslated (UT) segments of 12 and 211 bp, respectively. The 3' UT segment shows the predicted stem-loop structure typical of eukaryotic mRNAs. The cDNA-derived amino acid sequence is the first available sequence for human beta TnTf protein. It is encoded by a single-copy gene that is expressed in a tissue-specific manner in fetal and adult fast skeletal muscles. Although the human beta TnTf represents the major fetal isoform, the sequence information indicates that this cDNA and the coded protein are quite distinct from the fetal and neonatal TnTf isoforms reported in other mammalian fetal muscles. The hydropathy plot indicates that human beta TnTf is highly hydrophilic along its entire length. The protein has an extremely high degree of predicted alpha-helical content involving the entire molecule except the carboxy-terminal 30 residues. Comparative sequence analysis reveals that the human beta TnTf shares a high level of sequence similarity in the coding region with other vertebrate TnTf and considerably reduced similarity with slow skeletal and cardiac TnT cDNAs. The TnT isoforms have a large central region consisting of amino acid residues 46-204 which shows a high sequence conservation both at the nucleotide and amino acid levels. This conserved region is flanked by the variable carboxy-terminal and an extremely variable amino-terminal segment. The tropomyosin-binding peptide of TnT, which is represented by amino acid residues 47-151 and also includes a part of troponin I binding region, is an important domain of this central segment. It is suggested that this conserved segment is encoded by an ancestral gene. The variable regions of vertebrate striated TnT isoforms reflect the subsequent addition and modification of genomic sequences to give rise to members of the TnT multigene family.     

Evidence that whitefly-transmitted cowpea mild mottle virus belongs to the genus Carlavirus

Two strains of whitefly-transmitted cowpea mild mottle virus (CPMMV) causing severe (CPMMV-S) and mild (CPMMV-M) disease symptoms in peanuts were collected from two distinct agro-ecological zones in India. The host-range of these strains was restricted to Leguminosae and Chenopodiaceae, and each could be distinguished on the basis of symptoms incited in different hosts. The 3'-terminal 2500 nucleotide sequence of the genomic RNA of both the strains was 70% identical and contains five open reading frames (ORFs). The first three (P25, P12 and P7) overlap to form a triple gene block of proteins, P32 encodes the coat protein, followed by P12 protein located at the 3' end of the genome. Genome organization and pair-wise comparisons of amino acid sequences of proteins encoded by these ORFs with corresponding proteins of known carlaviruses and potexviruses suggest that CPMMV-S and CPMMV-M are closely related to viruses in the genus Carlavirus. Based on the data, it is concluded that CPMMV is a distinct species in the genus Carlavirus.     

Non-congruent relationships between variation in emm gene sequences and the population genetic structure of group A streptococci

To examine the molecular population genetics of the M protein family of Streptococcus pyogenes (group A Streptococcus), the 5' regions of polymerase chain reaction-amplified emm products from 79 M serotypes were sequenced and the phylogeny was compared to estimates of overall genetic relationships among strains determined by multilocus enzyme electrophoresis. Although the 5' emm sequences from several strains designated as distinct M types were identical or almost identical, the overall pattern is characterized by very extensive variation. The composition of distinct emm sequence clusters generally parallels the ability of strains to express serum opacity factor and in some cases historical associations of certain M types with acute rheumatic fever, but not with M types classified as nephritogenic. For many strains there is a lack of congruency between variation in 5' emm sequences and estimates of overall chromosomal relationships, which is undoubtedly due to horizontal transfer and recombination of emm sequences. The results of these studies provide insights into the nature and extent of emm sequence variation and describe how this variation 'maps' onto the population genetic structure of extant S. pyogenes lineages. The complexity of emm sequence and streptococcal cell lineage relationships revealed by this analysis has significant implications for understanding evolutionary events generating strain diversity and the epidemiology of S. pyogenes diseases.