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PubMed1996

Molecular characterization of the segment 2 gene of epizootic hemorrhagic disease virus serotype 2: gene sequence and genetic diversity.

The complete nucleotide sequence of the gene encoding the major outer capsid protein VP2 from the Alberta isolate of epizootic hemorrhagic disease virus serotype 2 (EHDV-2) was determined. Complementary DNA (cDNA) corresponding to segment 2 was 3002 nucleotides in length with a single open reading frame that encoded a VP2 of 982 amino acids. Although the VP2 from EHDV-2 was only 34% homologous to the cognate protein from EHDV-1, their predicted hydropathic profiles were similar, suggesting that conservation of structure is important biologically to these capsid proteins. Sequence analysis of six North American EHDV-2 field isolates showed a high degree of comparative genetic identity (> 97%). Phylogenetic profiles constructed suggest that regionalization of the viruses within the North American continent has contributed to the genetic diversity.

Amino Acid Sequence↗
PubMed1992

Nucleotide sequence of messenger RNA encoding VHDJH and VKJK of a highly conserved idiotype-defined primary response anti-hapten antibody.

The primary humoral immune response of mice to the hapten phthalate (Xmp) is focused upon two adjacent immunodominant negatively charged carboxyl groups on a benzene ring that are in positions meta and para to the azolinkage (i.e., Xmp) to the protein carrier keyhole limpet hemocyanin. A significant fraction of the anti-Xmp antibodies raised in several different inbred mouse strains (BALB/c, DBA/2, A/HeHa; C3H, and SM/J), and many wild mouse populations express a cross-reactive Id, CRIXmp-1. This CRIXmp-1 is conspicuously absent in C57BL/6 mice. In order to obtain a better understanding of the events and parameters that influence the selection and regulation of the primary response B cell repertoire, and to explore the structural basis of Ag binding, we have determined the nucleotide sequence of the entire V region gene complexes, which encode the H and L chains of these highly conserved and dominant CRIXmp-1+ antibodies. Our data establish that the H chain gene complex consists of a single VH germ-line gene that is identical to VH Oxazolone-1, encoding the H chain of another highly conserved and dominant cross-reactive Id family associated with the primary response to Oxazolone. In CRIXmp-1+ Xmp-specific hybridomas this gene is joined to a limited set of D region sequences that express a conserved amino acid motif-GLR. At least three of the five D regions examined are coded for by DFL16.2. This VHD complex can be utilized with one of three different JH region genes (JH1, JH2, and JH4) without any significant effect upon antibody fine specificity or Id. In spite of this lack of JH fidelity all of the CRIXmp-1+ hybridomas have precisely maintained the same length in the H chain CDR3 and FRW4 by altering either the length of the D segment or the length of JH. Nucleotide sequence analysis of the VL gene complex of CRIXmp-1+ anti-Xmp antibodies indicates that the L chain V region is also encoded by a single germ-line gene. The amino acid sequence predicted from the nucleotide sequence of the VKJK from Xmp-specific CRIXmp-1+ hybridomas is identical to the sequence of the anti-arsonate antibody 1210.7, which is the prototype of another Id family (CRI) that is conserved and dominant in BALB/c mice.

Acute-Phase Reaction↗
PubMed1999

Comparative sequence analysis of tmRNA.

Minimal secondary structures of the bacterial and plastid tmRNAs were derived by comparative analyses of 50 aligned tmRNA sequences. The structures include 12 helices and four pseudoknots and are refinements of earlier versions, but include only those base pairs for which there is comparative evidence. Described are the conserved and variable features of the tmRNAs from a wide phylogenetic spectrum, the structural properties specific to the bacterial subgroups and preliminary 3-dimensional models from the pseudoknotted regions.

Bacteria↗
PubMed1995

Evidence of transmission of hepatitis D virus to spouses from sequence analysis of the viral genome.

To study sexual transmission of hepatitis D virus (HDV), 52 spouses of 56 index patients were observed and HDV genomes from antibody to HDV (anti-HDV)-positive couples were sequenced. Of the spouses, 11 (21%) were serum HBsAg positive, 3 (27%) of whom were also anti-HDV positive. The HDV sequences between spouses were found to be nearly identical (98% to 98.8%) in the region analyzed (nt 911 to nt 1260). Only one couple showed an identity > 90% with the genotype I HDV strains. The HDV sequences of the remaining two couples showed > 95% identity with each other and > 91% homology with genotype II, but they shared only a 73.1% to 73.7% homology with those of the first couple. The regions corresponding to the autocatalytic cleavage sites, the junction between the middle and the carboxyl terminal one-third domains, and the middle domain of the open reading frame for delta antigen on the antigenomic HDV RNA were more conserved with < 19% divergence among the three couples. Interestingly, there was a 56% divergence in the region corresponding to the carboxyl end of the open reading frame for the large delta antigen on the antigenomic HDV RNA. In summary, this study provides a direct nucleotide evidence of a common source of HDV infection in each couple. Despite divergence in the viral nucleotide sequence, both genotypes I and II were found in Taiwan and were transmitted from patients with a history of prostitute contact to spouses through sexual contact.

Base Sequence↗
PubMed1994

Two unique CYP1 genes are expressed in response to 3-methylcholanthrene treatment in rainbow trout.

In animals, exposure to polyaromatic hydrocarbons (PAHs) such as 3-methylcholanthrene (3-MC) is known to induce the expression of two unique cytochrome P450 genes, CYP1A1 and CYP1A2. These genes are thought to have originated by a gene duplication event and diverged no more than 250 million years ago (D. W. Nerbert and F. J. Gonzalez, 1987, Annu. Rev. Biochem. 56, 945-993). Lower vertebrates, such as fish, diverged from land animals before this time and are thought to express only a single CYP1 gene. In this paper, we present evidence to refute this hypothesis and report the isolation and complete genomic nucleotide sequence of two distinct CYP1 genes in rainbow trout. Genomic clones encoding the entire CYP1A1 and CYP1A2 genes were characterized. DNA sequence analysis revealed that both genes contained seven exons and six introns. Exons 1-7 of CYP1A1 and CYP1A2 were highly similar in length and nucleotide sequence. In contrast, the 5'-flanking region and introns 1, 2, 5, and 6 of both genes were significantly less conserved. Two xenobiotic regulatory elements (XREs) were identified in the 5'-flanking region of CYP1A1 but not in that of CYP1A2. The 5'-most start site of transcription was determined to begin at a cytosine residue 27 bases downstream of the putative TATA box of both genes. Northern blot analysis demonstrated that exposure to 3-MC resulted in an increase in CYP1 mRNA levels in the liver. RNase protection assays conducted with riboprobes specific for either CYP1A1 or CYP1A2 confirmed that the transcripts of both genes were expressed in rainbow trout liver in response to 3-MC treatment.

Animals↗
PubMed1993

Sequence analysis of the Ebola virus genome: organization, genetic elements, and comparison with the genome of Marburg virus.

Sequence analysis of the second through the sixth genes of the Ebola virus (EBO) genome indicates that it is organized similarly to rhabdoviruses and paramyxoviruses and is virtually the same as Marburg virus (MBG). In vitro translation experiments and predicted amino acid sequence comparisons showed that the order of the EBO genes is: 3'-NP-VP35-VP40-GP-VP30-VP24-L. The transcriptional start and stop (polyadenylation) signals are conserved and all contain the sequence 3'-UAAUU. Three base intergenic sequences are present between the NP and VP35 genes (3'-GAU) and VP40 and GP genes (3'-AGC), and a large intergenic sequence of 142 bases separates the VP30 and VP24 genes. Novel gene overlaps were found between the VP35 and VP40, the GP and VP30, and the VP24 and L genes. Overlaps are 20 or 18 bases in length and are limited to the conserved sequences determined for the transcriptional signals. Stem-and-loop structures were identified in the putative (+) leader RNA and at the 5' end of each mRNA. Hybridization studies showed that a small second mRNA is transcribed from the glycoprotein gene, and is produced by termination of transcription at an atypical polyadenylation signal located in the middle of the coding region. The predicted amino acid sequence of the glycoprotein contains an N-terminal signal peptide sequence, a hydrophobic anchor sequence, and 17 potential N-linked glycosylation sites. Alignment of predicted amino acid sequences showed that the structural proteins of EBO and MBG contain large regions of homology despite the absence of serologic cross-reactivity.

Amino Acid Sequence↗
PubMed1993

Codon reading scheme in Mycoplasma pneumoniae revealed by the analysis of the complete set of tRNA genes.

The 33 genes encoding the complete set of tRNA species in Mycoplasma pneumoniae have been cloned and sequenced. They are organized into 5 clusters in addition to 9 single genes. No redundant gene was found, indicating that 33 tRNAs correspond to 32 different anticodons and decode all 62 codons used in this organism. There is only one single tRNA for each of the Ala, Leu, Pro, and Val family boxes. Therefore, a simplified decoding system resembling that recently described for Mycoplasma capricolum (1) has to also exist in M.pneumoniae. However, analysis of the anticodon set and codon usage revealed features characteristic of the latter: (i) there is no obvious preference toward AT rich synonymous codons, (ii) CGG codons are assigned for arginine and are translated by tRNA Arg(UCG), and (iii) CNN or GNN anticodons are encountered in the Ser, Thr, Arg, and Gly family boxes. We thus propose that this codon-anticodon recognition pattern has emerged in the 'M.pneumoniae cluster' under a genomic economization strategy but without the influence of AT pressure.

Amino Acid Sequence↗
PubMed1997

Proviral organization and sequence analysis of feline immunodeficiency virus isolated from a Pallas' cat.

The nucleotide sequence and genomic organization have been determined for a highly cytopathic feline immunodeficiency virus (FIV) isolated from a Pallas' cat. The 9747-bp provirus of this virus, FIV-Oma, has typical lentivirus organization with LTRs, gag, pol, and env open reading frames (ORFs), putative vif and rev ORFs, and an ORF similar to ORF2/ORFA of domestic cat FIV isolates. Although the FIV-Oma provirus is 300 to 600 bp longer than other FIV proviruses, these additional bases are distributed throughout the genome. Phylogenetic analysis of a conserved region of the pol gene suggests that FIV-Oma is more closely related to some of the puma and lion lentiviruses than it is to domestic cat FIV isolates; however, many regions of the genome exhibit extensive nucleotide sequence divergence. None of the eight molecular proviral clones isolated from a genomic library are infectious, but we have constructed an infectious, cytopathic clone of FIV-Oma from subcloned and PCR-amplified fragments of these proviral clones. This clone will be useful for identifying the genetic determinants of FIV-Oma's biological activities.

Amino Acid Sequence↗
PubMed1999

Sequence analysis and expression of a mRNA for a larval-specific cuticular protein, LCP1, from Helicoverpa armigera.

Several cDNA clones for a larval cuticular protein from Helicoverpa armigera were isolated and sequenced. The cDNA clones contain an open reading frame encoding a 109 residue protein which is homologous to other known cuticular proteins. The predicted protein appears to have a signal peptide which would be removed to give a mature protein of 91 amino acid residues with an Mr of 10 127. The mature protein, LCP1 (Larval Cuticular Protein), would be highly acidic, as is found for other cuticular proteins from flexible insect cuticles. The mRNA appears to be expressed throughout larval development although it is more highly expressed in the integument of the late final instar.

Amino Acid Sequence↗
PubMed1997

Genotypic analysis of the 5'-untranslated region of a pestivirus strain isolated from human leucocytes.

The 5'-untranslated genomic region of the pestivirus strain Europa, originated in human leucocytes and previously identified as bovine diarrhea virus (BVDV), was amplified by reverse transcription-PCR and sequenced. Analyses based on primary nucleotide sequence homology and on secondary palindromic sequence structures characteristic to genotypes revealed that this human isolate should be assigned to a novel genotype of pestivirus, type Ic. This newly emerged genotype was related to, but distinguishable from the three known BVDV genotypes, Ia, Ib and II. Three other bovine field isolates of BVDV originated from Germany were also found to belong to this new genotype Ic. Within pestivirus genotype Ic strains, the overall nucleotide sequence homology was 95-96%, and 88-92%, 88-90% and 77-79% with the other BVDV genotypes Ia, Ib and II, respectively. With the strains from border disease virus (genotype III) and hog cholera virus (genotype IV), homologies were less than 75%.

Animals↗
PubMed1997

Nucleotide sequence analysis of the large (L) genes of phocine distemper virus and canine distemper virus (corrected sequence).

This paper corrects the previously published sequence of the L gene of canine distemper virus (CDV). Errors in the published sequence (M. S. Sidhu et al., 1993, Virology 193, 50-65) led to frame shifts between residues 1021-1032, 1190-1219 and 1645-1650; a deletion of 21 amino acids between residues 1684-1705, and a single residue deletion at residue 1478. Residue 237 is now found to be glycine rather than tryptophan and residue 1626 proline instead of threonine. The sequence of the L gene of phocine distemper virus (PDV) was also determined. Alignment of the morbillivirus L proteins showed that PDV and CDV are more closely related to each other than to rinderpest virus and measles virus. Two regions of low identity are proposed to function as hinge regions between three highly conserved domains (I-III) in the morbillivirus L proteins. New sequence motifs have been identified on the basis of conservation in the morbilliviruses and the Paramyxovirinae.

Amino Acid Sequence↗
PubMed1998

The simian T-lymphotropic virus STLV-PP1664 from Pan paniscus is distinctly related to HTLV-2 but differs in genomic organization.

We have isolated a highly divergent simian T-lymphotropic virus, STLV-PP1664, from a wild-caught bonobo (Pan paniscus). Previous phylogenetic analysis suggested that this virus represents an additional type of STLV but this has now become a matter of discussion. We have now obtained and analyzed the entire genome of STLV-PP1664. All major genes and their corresponding viral messengers were identified. Sequence comparison and phylogenetic analysis indicated that this virus, together with the closely related panp isolate, belongs to an early lineage within the PTLV-2 clade, differing from HTLV-2 by about 25%. In contrast to the HTLV-1 and HTLV-2 LTR, only two 21-bp repeats instead of three were found in the STLV-PP1664 LTR. Additional messengers, resulting from alternative splicing, potentially encode five different accessory proteins from open reading frames in the pX region: prorfI, porfII, ptorfV', and two isoforms of Rex. The amino acid sequences of these proteins are only distinctly related to the accessory proteins from HTLV-2. These data suggest a different genomic organization of the STLV-PP1664 pX region than that of HTLV-2. We conclude that STLV-PP1664, although related to HTLV-2, has some distinct features in the LTR and the pX regions, the impact of which needs further investigation. Although arguments pro and contra a distinct classification are nearly equally balanced, we propose to classify this virus as an STLV-2, designated STLV-2PP1664.

Amino Acid Sequence↗
PubMed1997

Nucleotide sequence and genomic organization of Acyrthosiphon pisum virus.

The nucleotide sequence of the genomic RNA of Acyrthosiphon pisum virus was determined. The APV genome is 10,016 nucleotides in length, excluding the 3'-end poly(A) track, and contains two large open reading frames (ORFs), encoding proteins of 296,340 and 63,279 Da. The ORF1 is preceded by an untranslated leader sequence of 267 nucleotides. The ORF1 product contains sequence motifs characteristic of RNA-dependent RNA polymerases, chymotrypsin-like proteases, and helicases. Interviral sequence comparison revealed significant similarities with viruses belonging to the so-called picornavirus superfamily. The ORF2 is most likely expressed by a -1 translational frameshift and is followed by an untranslated sequence of 222 nucleotides. Internal amino acid sequences of three capsid proteins (66K, 34K, 23/24K) were determined. Comparison of the obtained amino acid sequences with the APV sequence disclosed that the structural proteins are located in the 3'-terminal half of the genome. The 34K protein is encoded by the ORF1, while the 66K protein contains both ORF1-(34K) and ORF2-derived sequences and is probably expressed by a translational frameshift. The 23/24K proteins most likely arise by proteolytic breakdown of the 34K protein. Although the deduced APV genomic organization in some aspects resembles that of the picornaviruses, its overall genomic organization indicates that APV is a distinct species only distantly related to the Picornaviridae.

Amino Acid Sequence↗
PubMed1994

Predicted stem-loop structures and variation in nucleotide sequence of 3' noncoding regions among animal calicivirus genomes.

Caliciviruses are nonenveloped with a polyadenylated genome of approximately 7.6 kb and a single capsid protein. The "RNA Fold" computer program was used to analyze 3'-terminal noncoding sequences of five feline calicivirus (FCV), rabbit hemorrhagic disease virus (RHDV), and two San Miguel sea lion virus (SMSV) isolates. The FCV 3'-terminal sequences are 40-46 nucleotides in length and 72-91% similar. The FCV sequences were predicted to contain two possible duplex structures and one stem-loop structure with free energies of -2.1 to -18.2 kcal/mole. The RHDV genomic 3'-terminal RNA sequences are 54 nucleotides in length and share 49% sequence similarity to homologous regions of the FCV genome. The RHDV sequence was predicted to form two duplex structures in the 3'-terminal noncoding region with a single stem-loop structure, resembling that of FCV. In contrast, the SMSV 1 and 4 genomic 3'-terminal noncoding sequences were 185 and 182 nucleotides in length, respectively. Ten possible duplex structures were predicted with an average structural free energy of -35 kcal/mole. Sequence similarity between the two SMSV isolates was 75%. Furthermore, extensive cloverleaflike structures are predicted in the 3' noncoding region of the SMSV genome, in contrast to the predicted single stem-loop structures of FCV or RHDV.

Base Sequence↗
PubMed1996

Characterisation of Wongorr virus, an Australian orbivirus.

Sequence analyses of VP3 gene segments of Wongorr virus isolates from the Northern Territory of Australia were compared with the cognate gene segments from Picola and Paroo River viruses. Previous serological investigations had demonstrated some relationships between these viruses, however VP3 gene sequence and phylogenetic analyses placed these viruses within the same serogroup which was distinct from other described orbivirus serogroups. A polymerase chain reaction (PCR) was developed for the detection of this serogroup and used to identify and determine partial sequence data for other isolates of the virus. Wongorr virus and the other tick and mosquito-borne orbiviruses (Kemerovo and Corriparta), were more closely related than the Culicoides transmitted orbiviruses, such as bluetongue (BTV) and African horse sickness virus (AHSV) which were shown to be on a separate branch of the orbivirus phylogenetic tree.

Aedes↗
PubMed1997

Immunoglobulin heavy chain junctional diversity in young and aged humans.

The causes of observed deficiencies to the humoral immune response in aged humans are unknown. Since a major source of antibody diversity is generated at the VH-D-JH junctional regions of the immunoglobulin heavy chain, we determined whether differences in junctional diversity are manifested with aging. We compared the CDR3 regions of IgM heavy chain transcripts isolated from young adult and aged humans. A PCR assay that measures CDR3 length in the majority of mu-heavy chains showed the same average size and normal range of CDR3 length in aged individuals as observed in young adults. To characterize the features of junctional diversity of aged adults in more detail, we determined the CDR3 sequences of a subset of the mu-heavy chain repertoire that utilizes members of the VH 5 family. In general CDR3 length, D family usage, and JH gene usage were similar in aged compared to young adults. Thus, in contrast to dramatic changes in heavy chain junctional diversity associated with fetal to adult development, no major differences were found between young and aged adults. Since the CDR3 repertoire generated in aged individuals appears to be as diverse as that observed in younger adults, the decline in humoral immunocompetence with aging cannot be attributed to a restriction in heavy chain junctional diversification processes.

Adult↗
PubMed1999

In vitro selection of RNA aptamers that bind special elongation factor SelB, a protein with multiple RNA-binding sites, reveals one major interaction domain at the carboxyl terminus.

The SelB protein of Escherichia coli is a special elongation factor required for the cotranslational incorporation of the uncommon amino acid selenocysteine into proteins such as formiate dehydrogenases. To do this, SelB binds simultaneously to selenocysteyl-tRNA(Sec) and to an RNA hairpin structure in the mRNA of formiate dehydrogenases located directly 3' of the selenocysteine opal (UGA) codon. The protein is also thought to contain binding sites allowing its interaction with ribosomal proteins and/or rRNA. SelB thus includes specific binding sites for a variety of different RNA molecules. We used an in vitro selection approach with a pool completely randomized at 40 nt to isolate new high-affinity SelB-binding RNA motifs. Our main objective was to investigate which of the various RNA-binding domains in SelB would turn out to be prime targets for aptamer interaction. The resulting sequences were compared with those from a previous SELEX experiment using a degenerate pool of the wild-type formiate dehydrogenase H (fdhF) hairpin sequence (Klug SJ et al., 1997, Proc. Natl. Acad. Sci. USA 94:6676-6681). In four selection cycles an enriched pool of tight SelB-binding aptamers was obtained; sequencing revealed that all aptamers were different in their primary sequence and most bore no recognizable consensus to known RNA motifs. Domain mapping for SelB-binding aptamers showed that despite the different RNA-binding sites in the protein, the vast majority of aptamers bound to the ultimate C-terminus of SelB, the domain responsible for mRNA hairpin binding.

Bacterial Proteins↗
PubMed1998

Complete nucleotide sequence of the genomic RNA of tobacco mosaic virus strain Cg.

Tobacco mosaic virus (TMV)-Cg is a crucifer-infecting tobamovirus that was isolated from field-grown garlic. We determined the complete nucleotide sequence of the genomic RNA of TMV-Cg. The genomic RNA of TMV-Cg consists of 6303 nucleotides and encodes four large open reading frames, organized basically in the same way as that of other tobamoviruses. The nucleotide and deduced amino acid sequences are very similar to those of the other crucifer-infecting tobamoviruses that have been sequenced so far.

Amino Acid Sequence↗