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PubMed1989

Detailed structural analysis of a genome rearrangement in bovine rotavirus.

A genome rearrangement involving RNA segment 11 of a bovine rotavirus has been analysed by molecular cloning and sequencing. This revealed that the rearranged genome segment was generated by a head to tail concatemerisation of two almost full length copies of segment 11. The upstream copy of the gene has lost its 3' end and the downstream copy its 5' end. The truncation of the upstream copy of the gene occurs within the termination codon for VP11 converting it from a UAG to a UGA, the rearranged gene is therefore still able to encode a normal VP11. The possible mechanisms by which this rearrangement may have been generated are discussed.

Amino Acid Sequence↗

PubMed1992

Evolutionary analyses of five US bluetongue viruses using the cognate S2 genes.

Full-length cDNA copies of S2 genes (segment 8), coding for non-structural protein 2 (NS2) of bluetongue virus serotypes 2, 11, 13 and 17 were selectively synthesized by a modified polymerase chain reaction (Clamp-R) and cloned into the PstI site of the pUC19 plasmid. Each of these S2 cognate genes was 1125 nucleotides in length with an initiation and a termination codon at nucleotides 20-22 and 1082-1084, respectively, resulting in a long open reading frame capable of coding for a protein of 354 amino acids. The deduced amino acid sequence of NS2 protein had a high concentration of lysine and contained a relatively low number of tryptophan and histidine residues. There was a highly conserved hydrophilic region at the carboxyl termini of predicted NS2 proteins in all five BTV serotypes, even though the amino acid sequence in this region in BTV-2 was more variable than in the other four serotypes. There was significant sequence homology of the cognate S2 genes at both the nucleotide and the amino acid levels. Phylogenetic analyses using the S2 gene sequences indicated that BTV-10, -11, -13 and -17 were more closely related and BTV-2 was the most distantly related serotype among the five US bluetongue viruses.

Amino Acid Sequence↗

PubMed1995

Sequence analysis of putative pVIII, E3 and fibre regions of porcine adenovirus type 3.

Sequence analysis of a region of the genome of porcine adenovirus type 3 from map unit 79.5 to map unit 92 was performed. Homology studies revealed genes coding for the hexon-associated protein pVIII on the left and for the fibre protein on the right of the sequenced region. By analogy with the genomic organization of other adenoviruses, the 1179 bp sequence between the pVIII and fibre open reading frames, extending from map unit 81.3 to map unit 84.7, was identified as the equivalent of the E3 region of human adenoviruses. The deduced amino acid sequence of one of the three open reading frames of the putative E3 region showed homology with the 13.3K E3 protein of canine adenovirus type 2. The primary structure of the putative fibre protein was similar to that described for human adenovirus types 2 and 5, with a 14 pseudorepeat motif in the shaft region of the fibre. A 742 bp tandem repeat starting in the middle of the fibre gene and extending beyond the termination codon of this gene was observed.

Adenoviridae↗

PubMed1990

A new hepatitis B virus strain in patients with severe anti-HBe positive chronic hepatitis B.

In hepatitis B virus carriers who are anti-HBe positive despite ongoing viral replication (HBcAg in liver and HBV-DNA in serum) the natural course of hepatitis is severe and the response to interferon is low. We investigated whether a new hepatitis B virus (HBV) strain could be involved. A translational termination codon at the carboxyterminal end of the pre-C region responsible for the lack of HBeAg secretion was found in 18 of 19 HBV clones isolated from seven pedigreed patients with this clinical syndrome. The same findings were confirmed by direct sequencing. One of these patients underwent a liver transplant and HBV infection of the new liver resulted in high titered viremia and intrahepatic expression of HBcAg, without detectable HBeAg in serum. Another patient was superinfected by hepatitis delta virus (HDV) and developed high titres of total and IgM anti-HD. In spite of this, chronic hepatitis remained unchanged during 7 years of follow-up. These data strongly suggest that a viable precore minus mutant of hepatitis B virus is responsible for the lack of HBeAg in the serum of these patients. The HBV variant may explain the peculiar geographic distribution of anti-HBe positive hepatitis. The variations in the virus genome sequence may cause the more severe form of liver disease and modify the pathogenicity in the case of HDV superinfection.

Adult↗

PubMed1991

The roles of the delta antigen in the structure and replication of hepatitis delta virus.

The delta antigen is the only known protein encoded by hepatitis delta virus (HDV). The predicted protein is 195 amino acids in length, but for reasons that are not yet clear, there occurs during the replication of the HDV genome, a specific base change in the termination codon of this open reading frame. This leads to the synthesis of a form of the delta antigen that is 19 amino acids longer, with a total length of 214 amino acids. Studies are described which relate to the roles of these two forms of the delta antigen in genome replication and subsequent particle assembly.

Animals↗

PubMed1991

UAG readthrough is not increased in vivo by Moloney murine leukemia virus infection.

Expression of the pol gene of the murine leukemia viruses is subject to translational control at the UAG termination codon of the upstream gene gag. Previous experiments have suggested that: i) Moloney murine leukemia virus infection induces a tRNA(Gln)iii) in an in vitro system using the tobacco mosaic virus as template, this tRNA is able to increase readthrough at the UAG codon [1]. Here we demonstrate that, in vivo, Moloney murine leukemia virus infection does not increase translational readthrough at either the tobacco mosaic virus or the Moloney murine leukemia virus UAG stop codons.

3T3 Cells↗

PubMed1978

The nucleotide sequence of the transforming HpaI-E fragment of adenovirus type 5 DNA.

The primary structure of the HpaI-E fragment of adenovirus type 5 (Ad5) DNA has been determined, mainly by the method of Maxam and Gilbert (1977). This fragment comprises the leftmost 4.5% of the Ad5 genome, and has been shown to be the shortest DNA fragment capable of transforming cells. The identification of potential initiation and termination codons in the determined sequence indicates that two small polypeptides consisting of 186, and 81 amino acids, respectively, could be synthesized. Taking into account recent data on RNA splicing, a possibility is considered that this DNA may code also for larger polypeptides.

Adenoviruses, Human↗

PubMed1980

Site-specific mutagenesis using oligodeoxyribonucleotides: isolation of a phenotypically silent phi X174 mutant, with a specific nucleotide deletion, at very high efficiency.

A decadeoxyribonucleotide, pAAATCCCTCA, was synthesized to complement nucleotides 2920--2930 of coliphage phi X174 viral DNA except that the nucleotide corresponding to position 2925 is deleted. The phage DNA sequence in this region codes for the ribosome-binding site of gene H. The oligodeoxyribonucleotide was integrated into double-stranded phi X174 DNA by using it as a primer for DNA polymerase with wild-type DNA template followed by ligation. The resultant heteroduplex DNA was used to transfect Escherichia coli spheroplasts and progeny bacteriophage were isolated. The synthetic decadeoxyribonucleotide was then used to enrich mutant viral DNA from which nucleotide 2925 had been deleted. After three cycles of enrichment and spheroplast transfection only mutant DNA was detectable. The deletion of nucleotide 2925 from the phi X174 genome seriously disrupts a sequence complementary to the 3'-terminus of the 16S rRNA of E. coli and also eliminates a translation termination codon from that sequence. However, the mutant grows normally with no readily perceptible phenotype. Thus, a short synthetic oligodeoxyribonucleotide has been used to construct, and to isolate with 100% efficiency, a mutant for which there is no biological selection procedure.

Bacteriophage phi X 174↗

PubMed1980

Sequence of a yeast DNA fragment containing a chromosomal replicator and the TRP1 gene.

The DNA sequence of a 1.45 kb EcoRI fragment from the yeast (Saccharomyces cerevisiae) TRP1 region has been determined. The fragment contains the TRP1 gene and a yeast chromosomal replicator. The TRP1 gene has been located on the fragment by analysis of potential initiation and termination codons in the DNA sequence. This location has been confirmed by subcloning portions of the fragment. Both the 5' and 3' noncoding regions of the TRP1 gene contain sequence homologies with analogous areas surrounding other yeast genes. The yeast replicator has been localized in a region near the 3' end of the TRP1 gene. The DNA sequence in this region contains several structural features which may be involved in the initiation of DNA replication.

Aldose-Ketose Isomerases↗

PubMed1981

Nucleotide sequence of the argF regulatory region of Escherichia coli K-12.

The deoxyribonucleotide sequence has been determined for the regulatory region of the arginine F gene (argF) of Escherichia coli K-12. The location of the argF coding region was deduced by comparison of the DNA sequence to the sequence predicted from the primary structure of the N-terminus of the argF gene product, the subunit of the "F" isoenzyme of ornithine transcarbamylase. Transcription of the argF gene was found to initiate at a position approx. 40 bp preceding the N-terminal codon for OTCase. Comparison of the region surrounding the origin of transcription with a computer-generated "model promoter sequence" revealed structural similarities between the two sequences, in particular, the promoter-associated stretches known as the "Pribnow box" and "minus 35 contact site". Another feature noted for the argF promoter region was its extreme abundance of A : T nucleotide pairs. In the region preceding the start site for argF translation, a sequence was observed to be complementary to the 3' end of the 16S RNA component of the E. coli ribosome. Both the length and the nucleotide sequence of the argF leader region indicate that the argF gene does not contain an attenuator proposed to exist in other operons concerned with amino acid biosynthesis.

Bacterial Proteins↗

PubMed1982

The nucleotide sequence of the HIS4 region of yeast.

We have determined the nucleotide sequence of the yeast HIS4 gene and its 5' and 3' flanking sequences. The protein chain has a calculated Mr-value of 87 935. Th 5' end of the HIS4 transcript maps at a position 63 bp upstream from the site of initiation of protein synthesis. The 3' end of the HIS4 transcript maps at a position approx. 118 bp after the UAG termination codon. There is no evidence for intervening sequences within the transcription unit. Functional sub-regions within the HIS4 coding frame have been identified by determining the sequence changes for various his4 mutations.

Base Sequence↗

PubMed1983

Comparison of the nucleotide sequences of early region E1b DNA of human adenovirus types 12, 7 and 5 (subgroups A, B and C).

The nucleotide sequences of human adenovirus serotypes 12 (Ad12, oncogenic subgroups A), 7 (Ad7, weakly oncogenic subgroup B) and 5 (Ad5, non-oncogenic subgroup C) DNAs have been compared. The region studied stretches from the termination codon of region E1a (m.p. 4.2) and comprises the entire region of the three viral DNAs, ending at the polyadenylation signal of the gene for viral polypeptide IVa2 (m.p. 11.2). The homology in the sequences encoding the E1b proteins of Mr 20 000 and Mr 55 000 is 55-60%, when two serotypes are compared, and about 45% in a three-strain comparison. However, a short internal segment encoding the C terminus of the Mr 20 000 protein, and at the same time amino acids 23-120 of the Mr 55 000 protein show a much higher degree of divergence in the three strains, as do the noncoding areas. The present study does not reveal how the three serotypes are phylogenetically related.

Adenoviruses, Human↗

PubMed1983

Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3'5"-aminoglycoside phosphotransferase type III.

We have cloned in Escherichia coli and sequenced a 1489-bp DNA fragment conferring resistance to kanamycin and originating from the streptococcal plasmid pJH1. The resistance gene was located by analysis of the initiation and termination codons in an open reading frame (ORF) of 792 bp. The deduced gene product, a 3'5''-aminoglycoside phosphotransferase of type III, has an Mr of 29,200. Comparison of its amino acid sequence with those of type I (Oka et al., 1981) and type II (Beck et al., 1982) 3' phosphotransferase, from transposable elements Tn903 and Tn5, respectively, indicated a statistically significant structural relationship between these enzymes from phylogenetically remote bacterial genera. The degree of homology observed indicate that phosphotransferase type III and type I genes have diverged from a common ancestor and that the phosphotransferase type II gene has emerged more recently from the type I evolutionary pathway.

Amino Acid Sequence↗

PubMed1984

Nucleotide sequence analysis of a zein genomic clone with a short open reading frame.

The nucleotide sequence of the zein genomic clone (W22)Z7 and its flanking sequences from the W22 inbred line of maize is reported. The sequence is 1587 bp long and contains 444 bp of 5' noncoding sequence and 342 bp of 3' noncoding sequence. The Z7 sequence belongs to a large complex multigene family and is a member of the B49 subfamily. It is 86% homologous to other known sequences from the same subfamily, but contains four in-frame termination codons caused by single base changes. Its flanking regions contain the usual eukaryotic transcriptional signals.

Base Sequence↗

PubMed1984

The coding region of the human c-mos pseudogene contains Alu repeat insertions.

We have determined the nucleotide sequence of an 841-bp fragment derived from a segment of the human genome previously cloned by Chumakov et al. [Gene 17 (1982) 19-26] and Zabarovsky et al. [Gene 23 (1983) 379-384] and containing regions homologous to the viral mos gene probe. This sequence displays homology with part of the coding region of the human and murine c-mos genes, contains several termination codons, and is interrupted by two Alu-family elements flanked by short direct repeats. Probably, the progenitor of the human c-mos gene was duplicated approximately at the time of mammalian divergence, was converted to a pseudogene, and acquired insertions of two Alu elements.

Base Sequence↗

PubMed1984

Human metallothionein MT-I and MT-II processed genes.

Two intronless pseudogenes, corresponding to the human metallothionein MT-I and MT-II processed genes, have been isolated from a human genomic library. MT-I processed gene has accumulated a number of mutations including a nonsense mutation giving rise to a termination codon at amino acid position 21, and a single base deletion at amino acid position 47 causing a shift in the reading frame. MT-II processed gene is a full-length perfect copy of its corresponding mRNA except for a few mutations. Most of the mutations in MT-II processed gene are silent except that the amino acid glycine (GGT) at position 10 is changed to serine (AGT) due to a transition. Both MT-I and MT-II processed genes possess poly(A) sequences of 21 and 17 nucleotides, respectively, 3' to the consensus AATAAA sequence. While these genes are quite similar in their sequences at the 3'-untranslated region, they show less than 50% homology in the 5'-untranslated sequences. Two direct repeats of 16 and 18 nucleotides in length define the limits of the MT-I and MT-II processed genes, respectively, and have been confirmed by S1 nuclease mapping analysis. In both MT-I and MT-II processed genes these direct repeats towards the 5' end of the gene start with an AhaIII (TTTAAA) restriction site. Our studies suggest that these direct repeats are the results of the insertion site duplication.

Amino Acid Sequence↗

PubMed1985

Nucleotide sequence of the gene ereA encoding the erythromycin esterase in Escherichia coli.

We have cloned and determined the nucleotide sequence of the gene ereA of plasmid pIP1100 which confers high-level resistance to erythromycin (Em) in Escherichia coli. The gene was defined by initiation and termination codons and by in vitro insertion-inactivation into an open reading frame (ORF) of 1032 bp corresponding to a product with an Mr of 37 765. However, the enzyme, an Em esterase, displayed an apparent Mr of 43 000 upon electrophoresis of a minicell extract on the SDS-polyacrylamide gels. The G + C content (50.5%) of the gene ereA and the preferential codon usage in its ORF suggest that this resistance determinant should be indigenous to E. coli.

Amino Acid Sequence↗

PubMed1986

A plasmid vector for cloning and expression of gene segments: expression of an HTLV-I envelope gene segment.

We describe a cloning-expression vector system for selecting DNA fragments containing open reading frames (ORFs) and expressing them as beta-galactosidase (beta Gal) hybrid fusion proteins. The plasmid vector, pWS50, utilizes the very strong and easily regulated bacteriophage lambda promoter pL, and the efficient translation initiation signals of the N-terminal segment of the lambda cII gene. Fused distally to and out of translational phase with cII is the E. coli lacZ gene, lacking its own transcriptional and translational initiating signals. A unique restriction enzyme site (NruI) is located between the upstream regulatory sequences and the lacZ gene, which provides a cloning site for the insertion of blunt ended DNA fragments. In addition, there are two other unique restriction sites (NheI and BamHI) located in this region which can also be used as closing sites. If a DNA fragment does not contain any translation termination codons (i.e., an ORF), and is inserted correctly into the vector, the translational reading frame between cII and lacZ can be restored. Colonies containing these recombinants can be easily screened as LacZ+ on lactose indicator media. The beta-galactosidase fusion proteins produced from the LacZ+ recombinants are identified on sodium dodecyl sulfate polyacrylamide gels by their large size and high level of production. To test the ORF cloning-expression system, a segment of the human T-cell lymphotrophic virus type I envelope gene was cloned and expressed at high levels. The envelope-beta Gal fusion protein was recognized by antibodies in serum from a patient with adult T-cell leukemia.

Antibodies, Neoplasm↗