Search PubMed⌕ Search

Biomedical subjects

G W Both

Publications and source records attributed to G W Both.

At least 73 records · Page 4Linked to original sources
PubMed1984

Nucleotide sequence of the dsRNA genomic segment 7 of Simian 11 rotavirus.

A full length cDNA copy of dsRNA segment seven of Simian 11 rotavirus has been obtained by standard molecular cloning techniques. Segment seven codes for the non-structural viral protein NCVP4 and is 1104 nucleotides in length with putative 5'- and 3'- terminal non-coding regions of 25 and 134 residues respectively. The longest open reading frame of 315 codons extends from nucleotide 26 to 970 inclusive. However, the presence nearby of two other AUG codons makes it unclear which codon is used for initiation. The second AUG conforms to the Kozak consensus sequence and if utilised, would yield a protein 312 amino acids in length with a nett charge at pH7 of -2.5. Determination of the gene 7 sequence indicates that terminal sequence conservation among rotavirus gene segments is limited to three and two nucleotides at the 5' and 3' ends of the plus strand, respectively.

Amino Acid Sequence↗
PubMed1984

Comparative sequence analysis of rotavirus genomic segment 6--the gene specifying viral subgroups 1 and 2.

Cloned DNA copies of rotavirus genomic segment 6 from simian 11 (subgroup 1) and human strain Wa (subgroup 2) rotaviruses have been used to determine the nucleotide sequences of the gene that determines viral subgroup specificity. Both genomic segments are 1,356 nucleotides in length and possess 5'- and 3'-terminal untranslated regions of 23 and 142 nucleotides, respectively. The inferred amino acid sequence reveals VP6 to be a polypeptide of 397 amino acids in which more than 90% of the amino acid sequence is conserved between the two viruses. There are 34 amino acid changes between the subgroup 1 and 2 polypeptides, most clustered in three regions of the molecule at residues 39 through 62, 80 through 122, and 281 through 315.

Amino Acid Sequence↗
PubMed1983

Serotype-specific glycoprotein of simian 11 rotavirus: coding assignment and gene sequence.

Cloned DNA copies of the double-stranded RNA genomic segments of simian 11 rotavirus have been used to determine the coding assignment for VP7, the type-specific antigen of this virus. Translation of hybrid-selected mRNAs in an in vitro system supplemented with canine pancreatic microsomes permitted VP7 to be assigned to segment 9 and the two nonstructural viral proteins NCVP4 and NCVP3, to segments 7 and 8, respectively. Hybridization of cloned DNA probes for segments 7-9 with the corresponding segments of human rotavirus Wa confirmed these assignments. The complete nucleotide sequence of gene 9 has been determined. The deduced amino acid sequence reveals VP7 to be 326 amino acids in length with two NH2-terminal hydrophobic regions and a single glycosylation site at residues 69-71.

Amino Acid Sequence↗
PubMed1983

Hemagglutinin of swine influenza virus: a single amino acid change pleiotropically affects viral antigenicity and replication.

The complete nucleotide sequence has been obtained of the H1 hemagglutinin (HA) gene of a high-yielding (H) mutant of the A/NJ/11/76(H1N1) strain of swine influenza virus in studies of a viral reassortant (X-53a) bearing this gene. This determination has permitted comparison with human influenza H1N1 prototype viruses A/WSN/33 and A/PR/8/34, with which 80% and 94% amino acid homology was found between HA1 and HA2, respectively. Partial sequences have been determined for other viral reassortants containing either H or L (low-yielding phenotype) genes derived from A/NJ/11/76. Sequence of the HA1 region of an L mutant prototype was virtually completed and differed from that of the H mutant by only four amino acid changes. Sequence analysis of four other viruses was restricted to regions of the HA with which monoclonal antibodies capable of distinguishing L and H mutants are presumed to react. Therefore, changes in these sequences are relevant to changes in viral phenotype. Change at residue 155 from Gly to Glu is associated with change from L to H HA phenotype. This site, structurally equivalent to amino acid 158 on the Wiley et al. HA model [Wiley, D. C., Wilson, I. A. & Skehel, J. J. (1981) Nature (London) 289, 373-378] is near the tip of the HA monomer adjacent to the proposed receptor binding site and therefore credibly could influence both viral antigenicity and replication. Because both L and H variants exist in nature and because revertants may be selected in the laboratory as replication variants in the absence of immunoselection, these studies provide evidence for fortuitous antigenic change in association with change in biological function, which is determined by a single base change.

Antigens, Viral↗
PubMed1983

Antigenic drift in influenza virus H3 hemagglutinin from 1968 to 1980: multiple evolutionary pathways and sequential amino acid changes at key antigenic sites.

Surveys of the antigenic properties of a wide range of variants of the H3N2 (Hong Kong) influenza virus subtype have revealed complex patterns of variants cocirculating during each of the main epidemic eras of the subtype. We determined hemagglutinin (HA) gene sequences for 14 isolates chosen to give the wildest possible spread of variant types. The addition of these data to existing HA gene sequence information for other variants provides a comprehensive picture of HA gene evolution during antigenic drift among H3N2 subtype viruses. The data reveal the existence of multiple evolutionary pathways during at least one period of development of the subtype and strikingly demonstrate that amino acid changes are limited to a small number of locations on the HA molecule during antigenic drift. The occurrence of sequential amino acid changes at key positions within these variable regions suggests that the HA structure has remained constant during subtype evolution so that only limited possibilities remain for further antigenic drift among H3N2 viruses.

Amino Acid Sequence↗
PubMed1983

Coding assignment and nucleotide sequence of simian rotavirus SA11 gene segment 10: location of glycosylation sites suggests that the signal peptide is not cleaved.

A cloned DNA copy of simian rotavirus SA11 genomic segment 10 was used to confirm the assignment of the nonstructural glycoprotein NCVP5 to this gene. Determination of the nucleotide sequence for gene 10 indicated that NCVP5 is 175 amino acids in length and has an N-terminal hydrophobic region with the characteristics of a signal sequence for membrane translocation. Unexpectedly, this region was also the location for the only two potential glycosylation sites within the molecule, asparagine residues 8 and 18. The carbohydrates carried by NCVP5 were of the high-mannose type, Man9GlcNAc and Man8GlcNAc, with the mannose 9 species predominating; no complex oligosaccharides were present. If these asparagine residues are the sites for carbohydrate attachment, this implies that cleavage of the putative signal peptide does not occur during the maturation of this nonstructural glycoprotein.

Amino Acid Sequence↗
PubMed1982

A general strategy for cloning double-stranded RNA: nucleotide sequence of the Simian-11 rotavirus gene 8.

Using Simian-11 rotavirus RNA, a strategy has been developed for the production of full length cloned copies of the genes of double-stranded (dsRNA) viruses. Genomic RNA segments were polyadenylated and reverse transcribed to yield a mixture of full length cDNA copies of both possible polarities. The cDNAs were annealed, filled in to complete any partial copies, tailed and inserted into the PstI site of pBR322 using dG/dC tailing. Cloned rotavirus cDNA gene copies were assigned to genomic RNA segments by Northern hybridization. The complete sequence of gene 8 which codes for NCVP3, a non-structural protein of SA11 rotavirus, was determined from a cloned gene copy. It is 1059 bases in length and has an open reading frame which could code for a protein containing 317 amino acids. The apparent 5' and 3' terminal non coding regions are 46 and 59 bases in length, respectively. The sequence ATGTGACCOH at the 3' end of the plus strand is conserved in four of the eleven genes examined. The cloning procedures used should be generally applicable to viruses with segmented dsRNA genomes.

Base Sequence↗
PubMed1981

Antigenic drift in the hemagglutinin of the Hong Kong influenza subtype: correlation of amino acid changes with alterations in viral antigenicity.

The nucleotide sequence of the gene coding for the large subunit of influenza virus hemagglutinin (HA1) was determined for strains A/NT/60/68, A/Eng/878/69, and A/Qu/7/70, three early isolates of the Hong Kong subtype. Sequences were obtained by the dideoxy chain termination method, using reverse transcriptase to synthesize partial DNA copies of the RNA gene. HA1 amino acid sequences predicted from the gene sequences were compared with published data for strains A/Aichi/2/68 and A/Vic/3/75. Compared with earlier strains, the HA1s of A/eng/878/69 and A/Qu/7/70 each contained three amino acid changes. Some of these were also found in A/Vic/3/75, but some were unique to the particular strain. When all of the strains were titrated with a panel of monoclonal antibodies directed against A/NT/60/68, alterations in viral antigenicity could be correlated with particular amino acid changes. The existence of multiple pathways for viral evolution during antigenic drift is discussed.

Amino Acid Sequence↗
PubMed1981

Conservation and variation in the hemagglutinins of Hong Kong subtype influenza viruses during antigenic drift.

The nucleotide sequence was determined for the hemagglutinin gene of the Hong Kong subtype influenza strain A/Bangkok/1/79. The amino acid sequence predicted from these data shows a total of 36 amino acid changes as compared with hemagglutinin for a 1968 Hong Kong strain, 11 more than had occurred in a 1975 strain. The distribution of these changes confirmed that there are conserved and highly variable regions in hemagglutinin as the viral gene evolves during antigenic drift in the Hong Kong subtype. Of the four variable regions found in this study, only two have been seen previously. Correlation of highly variable areas in the hemagglutinins of Hong Kong subtype field strains with sites of amino acid changes in antigenically distinct influenza variants enabled us to predict likely antigenic regions of the protein. The results support and extend similar predictions made recently, based on the three-dimensional arrangement of hemagglutinin from a 1968 influenza strain.

Amino Acid Sequence↗
PubMed1980

Complete nucleotide sequence of the haemagglutinin gene from a human influenza virus of the Hong Kong subtype.

The complete nucleotide sequence has been determined for a cloned double-stranded DNA copy of the haemagglutinin gene from the human influenza strain A/NT/60/68/29C, a laboratory-isolated variant of A/NT/60/68, an early strain of the Hong Kong subtype. The gene is 1765 nucleotides long and contains information sufficient to code for a protein of 566 amino acids, which includes a hydrophobic leader peptide (16 residues), HA1 (328), HA2 (221) and an arginine residue which joins the HA subunits. Comparison of the predicted amino acid sequence for 29C haemagglutinin with protein sequence data available for HA from other influenza strains shows that no potential coding information is lost by processing of the mRNA. A comparison of the amino acid sequences predicted from the gene sequences for 29C and fowl plague virus haemagglutinins, (1) indicates the extent to which changes can occur in the primary sequence of different regions of the protein, while maintaining essential structure and function.

Amino Acid Sequence↗
PubMed1979

The influenza virus haemagglutinin gene: cloning and characterisation of a double-stranded DNA copy.

A protocol has been developed for the synthesis of a double-stranded DNA (dsDNA) copy of the influenza virus RNA genome segment which codes for the major surface antigen, haemagglutinin (HA). This dsDNA copy was inserted, after digestion with S1 nuclease and poly (dC) tailing with terminal transferase, into poly(dG)-tailed, PstI-cut, pBR322 DNA, and used to transform E. coli RR1. Tetracycline-resistant bacterial colonies were screened for the presence of plasmid containing the copied HA gene by testing their ability to hybridise to a specific, 32P-labelled, single-stranded DNA probe. Four cloned hybrid plasmids, containing DNA complementary to the HA gene of the influenza strain 29C (a laboratory derivative of influenza A/NT/60/68 (1)) were analysed by restriction enzyme mapping. Each contained a dsDNA insert equivalent to a full length copy of the HA gene. The nucleotide sequence of a selected restriction fragment from the DNA inserted in one of these cloned plasmids (C89) was determined. The amino acid sequence deduced from these data agreed with the amino acid sequence determined for the corresponding region of HA from the influenza strain A/Mem/102/72, another member of the Hong Kong subtype, identifying the inserted dsDNA of C89 as an authentic copy of the influenza HA gene.

Agglutinins↗
PubMed1979

Nucleotide sequence coding for the N-terminal region of the matrix protein influenza virus.

After polyadenylation in vitro of the influenza virus RNA segment which contains the coding information for the matrix protein, a cDNA copy can be made using the primer p(dT)8-dA and reverse transcriptase. The sequence of 166 nucleotides of the cDNA was determined by a modification [Brownlee, G. G. & Cartwright, E. M. (1977) J. Mol. Biol, 114, 93--117] of the plus/minus method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441--481] and adaptation of the "dideoxy" method [Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5463--5467] for sequencing DNA. The cDNA sequences is of the same sense as the mRNA for matrix protein and contains a potential initiating codon, d(ATG), at position 26--28. When matrix protein purified from virus particles was digested with chymotrypsin or trypsin and the amino acid compositions of separated peptides determined, one peptide containing nine amino acids found which had a composition corresponding to that predicted by the cDNA sequence following the first methionine codon, confirming that protein synthesis initiates at this position. The compositions of four other peptides matches those predicted from the nucleotide sequence. There is no processing of the N terminus of the protein before incorporation into the virus particle except for removal of the N-terminal methionine and addition of a "blocking" group on the resulting N-terminal serine residue.

Amino Acid Sequence↗
PubMed1979

A new method for the size estimation of the RNA genome segments of influenza virus.

Previous estimates of the size of the RNA genome segments of influenza virus have been unreliable because of a lack of suitable RNA species as size markers. We have attempted to overcome this problem by utilising the ability of AMV reverse transcriptase to synthesise full length DNA copies of RNA molecules in the presence of a suitable primer. By comparing such DNA copies of the RNA segments of the influenza virus genome with sequenced restriction fragments from the E. coli plasmid pBR322, we have made more reliable estimates of the sizes of the eight genome segments from influenza virus A/NT/60/68.

Avian Myeloblastosis Virus↗