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Biomedical subjects

A G Porter

Publications and source records attributed to A G Porter.

At least 73 records · Page 4Linked to original sources
PubMed1990

Site-directed mutagenesis suggests close functional relationship between a human rhinovirus 3C cysteine protease and cellular trypsin-like serine proteases.

Human rhinoviruses, like other picornaviruses, encode a cysteine protease (designated 3C) which cleaves mainly at viral Gln-Gly pairs. There are significant areas of homology between picornavirus 3C cysteine proteases and cellular serine proteases (e.g. trypsin), suggesting a functional relationship between their catalytic regions. To test this functional relationship, we made single substitutions in human rhinovirus type 14 protease 3C at seven amino acid positions which are highly conserved in the 3C proteases of animal picornaviruses. Substitutions at either His-40, Asp-85, or Cys-146, equivalent to the trypsin catalytic triad His-57, Asp-102, and Ser-195, respectively, completely abolished 3C proteolytic activity. Single substitutions were also made at either Thr-141, Gly-158, His-160, or Gly-162, which are equivalent to the trypsin specificity pocket region. Only the mutant with a conservative Thr-141 to Ser substitution exhibited proteolytic activity, which was much reduced compared with the parent. These results, together with immunoprecipitation data which indicate that Asp-85, Thr-141, and Cys-146 lie in accessible surface regions, suggest that the catalytic mechanism of picornavirus 3C cysteine proteases is closely related to that of cellular trypsin-like serine proteases.

Amino Acid Sequence↗
PubMed1990

Human tumour necrosis factors-alpha and -beta: differences in their structure, expression and biological properties.

Tumour necrosis factor-alpha (TNF-alpha) and lymphotoxin (LT or TNF-beta) are cytokines, best known for their cytotoxic or cytostatic effects on some tumour cells. They are structurally related, compete for a common receptor, and are potent inducers of similar biological responses. TNF-alpha and LT appear to have distinct three-dimensional structures because they differ greatly in their sensitivity to various proteases and chemical agents, and antibodies raised against one cytokine do not cross-react with the other cytokine. The closely linked TNF-alpha and LT genes are independently regulated since many cell types produce only TNF-alpha or LT. Expression of the TNF-alpha gene can be controlled either at the transcriptional or at the post-transcriptional level. In some cell types, TNF-alpha and LT induce qualitatively or quantitatively different biological responses, and LT can antagonize the action of TNF-alpha. The disparate biological activities of TNF-alpha and LT may be related to their different interactions with a common receptor. It is possible that TNF-alpha and LT have different physiological roles.

Gene Expression↗
PubMed1989

Antisense oligonucleotide inhibition of encephalomyocarditis virus RNA translation.

We report the inhibition of encephalomyocarditis virus (EMCV) RNA translation in cell-free rabbit reticulocyte lysates by antisense oligonucleotides (13-17-base oligomers) complementary to (a) the viral 5' non-translated region, (b) the AUG start codon and (c) the coding sequence. Our results demonstrate that the extent of translation inhibition is dependent on the region where the complementary oligonucleotides bind. Non-complementary and 3'-non-translated-region-specific oligonucleotides had no effect on translation. A significant degree of translation inhibition was obtained with oligonucleotides complementary to the viral 5' non-translated region and AUG initiation codon. Digestion of the oligonucleotide:RNA hybrid by RNase H did not significantly increase translation inhibition in the case of 5'-non-translated-region-specific and initiator-AUG-specific oligonucleotides; in contrast, RNase H digestion was necessary for inhibition by the coding-region-specific oligonucleotide. We propose that (a) 5'-non-translated-region-specific oligonucleotides inhibit translation by affecting the 40S ribosome binding and/or passage to the AUG start codon, (b) AUG-specific oligonucleotides inhibit translation initiation by inhibiting the formation of an active 80S ribosome and (c) the coding-region-specific oligonucleotide does not prevent protein synthesis because the translating 80S ribosome can dislodge the oligonucleotide from the EMCV RNA template.

Animals↗
PubMed1989

Overlapping sites for constitutive and induced DNA binding factors involved in interferon-stimulated transcription.

A 14 bp interferon (IFN)-stimulated response element (ISRE) from 6-16, a human gene regulated by alpha-IFN, confers IFN inducibility on a heterologous thymidine kinase promoter. A 39 bp double-stranded oligonucleotide corresponding to a 5' region of 6-16 which includes the ISRE competes for factors required for gene expression by alpha-IFN in transfected cells and a single base change (A-11 to C) within the ISRE (GGGAAAATGAAACT) abolishes this competition. Band-shift assays performed with whole-cell extracts and the 39 bp oligonucleotide reveal specific complexes formed by rapidly induced and constitutive factors, both of which fail to bind to the A-11 to C oligonucleotide. A detailed footprinting analysis reveals that these two types of factors bind to overlapping sites within the ISRE, but in very different ways. These data were used to design oligonucleotides which decreased the formation of the inducible complex without affecting the constitutive one. Changes at the 5' margin of the ISRE and upstream of it markedly decrease formation of the induced but not the constitutive complex and also abolish the ability of the 39 bp sequence to function as an inducible enhancer with the thymidine kinase promoter. Thus, induction of 6-16 transcription in IFN-treated cells is likely to be stimulated by binding of the induced factor to the ISRE and upstream sequences, while the subsequent suppression of transcription may involve competition for the ISRE by the other class of factors.

Alkylation↗
PubMed1988

Expression and processing of human rhinovirus type 14 polypeptide precursors in Escherichia coli maxicells.

Human rhinovirus serotype-14 (HRV-14) cDNA, encompassing 87.9% of the coding region, was subcloned in an Escherichia coli expression vector, generating plasmid pKCC101. HRV-14 polypeptides encoded by pKCC101 were synthesized in E. coli maxicells. Pulse-chase experiments with pKCC110, a smaller derivative of pKCC101 containing the protease 3C coding region, have clearly demonstrated the proteolysis of a 55-kDa precursor to several polypeptides, including a doublet with the expected size of protease 3C (20 kDa). The proteolysis of the 55-kDa precursor polypeptide was prevented by ZnCl2, a known inhibitor of picornavirus 3C proteases. Results with a derivative of pKCC110 (pKCC115) which is partially deleted for the protease 3C sequence, support the idea that the doublet proteins are specified by the protease 3C coding region. Taken together, our investigations indicate that the precursor form of protease 3C must be responsible for its own cleavage.

Amino Acid Sequence↗
PubMed1988

BBC microcomputer controlled field inversion gel electrophoresis.

Agarose gel electrophoresis to separate DNA molecules is a widely used technique in molecular biology but there is an upper limit to the sizes that can be resolved. Pulsed field techniques have extended this limit but require expensive equipment. Here we describe a home-made control unit to interface conventional electrophoresis equipment to a BBC microcomputer for the purposes of field inversion gel electrophoresis.

Electrophoresis↗
PubMed1987

Chemical mutagenesis of human interferon-beta: construction, expression in E. coli, and biological activity of sodium bisulfite-induced mutations.

A series of modified human interferon-beta (IFN-beta) genes was produced by sodium bisulfite treatment of the IFN-beta gene cloned in M13. A library of mutated sequences was generated from which subgenomic fragments containing one or a small number of coding alterations were isolated and substituted into the IFN-beta gene in an E. coli expression vector. A number of modified genes and their expression products were evaluated. In several instances levels of expression and biological activity profiles are altered compared to the parental gene product. A number of key amino acids can be identified, whose substitutions have marked effects on biological activity of IFN-beta.

Amino Acid Sequence↗
PubMed1986

Novel modified beta-interferons: gene cloning, expression, and biological activity in bacterial extracts.

A series of novel, modified interferons based on the structure of human beta-interferon have been expressed in Escherichia coli. Modified interferon genes were constructed from sequences derived from the natural beta-interferon gene, a synthetic beta-interferon gene, or a specific combination of the two. A total of 23 out of the 25 novel interferons exhibited antiviral (AV) and antiproliferative (AP) activity which varied from 3 to 230% and 8 to 490% of the values for beta-interferon, respectively. None of the novel interferons had only AV or AP activity, although one had a much reduced ratio of AV/AP activity compared with beta-interferon. Substitution of beta-interferon amino acids 2-7 or 28-46 resulted in interferons with significantly increased AP activity on Daudi lymphoblastoid cells (four- to fivefold). All the novel interferons except two with modifications in the 82-105 region reacted with a neutralizing beta-interferon monoclonal antibody.

Amino Acid Sequence↗
PubMed1983

Increased expression of a cloned gene by local mutagenesis of its promoter and ribosome binding site.

A strategy for local mutagenesis of DNA has been developed. The lac promoter in phage M13mp9 was replaced with the E. coli trp promoter. A restriction fragment bearing only the trp promoter region was mutagenized with nitrous acid, religated to the unmutagenized vector and transfected into E.coli. Several clones which give darker blue plaques on indicator media, suggesting increased beta-galactosidase synthesis, were selected for DNA sequencing. One clone has a G leads to A transition on the 3' side of the 'Pribnow box' which results in a constitutive promoter. Two clones have different point mutations (C leads to T and T leads to C) between the Shine-Dalgarno sequence and initiation codon which raise expression of beta-galactosidase two-fold. A secondary structure model suggests that the latter two mutations could exert their effect by destabilizing base-pairing of the lac Z coding region with the ribosome binding site (RBS), thereby allowing easier access to ribosomes. Support for the model comes from the finding that neither of the RBS mutations increase expression of a different downstream gene which forms no obvious secondary structure with the RBS region, whether or not the mutations are present. These results strengthen the hypothesis that secondary structure masking is a major determinant of RBS strength.

Base Sequence↗
PubMed1981

The absence of introns within a human fibroblast interferon gene.

Experiments in which immobilised restriction fragments of genomic DNA were hybridised with a cloned human fibroblast interferon cDNA indicate that the homologous chromosomal genes exist in only one basic arrangement. This is in marked contrast to recent studies by Nagata et al. (1) showing that there are at least eight gene arrangements for human leukocyte interferon. Having isolated a chromosomal human fibroblast interferon gene from a gene bank, we conclude from nucleotide sequencing studies that there is a complete absence of introns within the RNA-coding region. In view of a similar observation recently made for a human leukocyte interferon gene (1), it would appear as if interferon genes in general are unlike the vast majority of eukaryote genes in this respect.

Base Sequence↗
PubMed1980

The complete amino acid sequence of human fibroblast interferon as deduced using synthetic oligodeoxyribonucleotide primers of reverse transcriptase.

Using synthetic oligodeoxyribonucleotides to prime the transcription of interferon mRNA and cDNA, we recently determined the mRNA sequence coding for the 47 amino-terminal amino acids of mature human fibroblast interferon (1). From this sequence, we have now synthesised an oligodeoxyribonucleotide that is homologous with the mRNA sequence coding for amino acids 42-45 and used it as a primer to selectively transcribe an interferon cDNA template. The sequence of the newly synthesised DNA predicted the sequence of amino acids 48-109 in the interferon polypeptide. By repeating this process with one more primer, we have determined the complete amino acid sequence of mature human fibroblast interferon, a polypeptide of 166 amino acids.

Amino Acid Sequence↗
PubMed1980

The amino-terminal sequence of human fibroblast interferon as deduced from reverse transcripts obtained using synthetic oligonucleotide primers.

From recently published data on the amino-terminal structures of human and mouse interferons, we have predicted and synthesised an oligonucleotide capable of priming specifically the reverse transcription of human fibroblast interferon mRNA present within a total mRNA population. From these transcripts we determined the sequence of the 5'-terminus of the mRNA and identified a putative pre-peptide signal sequence. This enabled us to predict the sequence of another primer capable of directing the synthesis of interferon double-stranded cDNA corresponding to the entire coding region of the mRNA. Further sequencing studies also enabled us to establish the identity of 47 consecutive amino acids beginning with the methionine residue at the amino-terminus of the mature protein.

Amino Acid Sequence↗
PubMed1980

Influenza antigenic determinants are expressed from haemagglutinin genes cloned in Escherichia coli.

A gene sequence for the fowl plague virus (FPV) haemagglutinin molecule has been inserted into a bacterial plasmid such that its transcription is under the control of a promoter derived from the tryptophan operon. Such plasmids direct the synthesis of a protein that reacts specifically with antisera to FPV haemagglutinin. Evidence is also presented that in some cases DNA inserted at the HindIII site of pBR322 is expressed.

Antigens, Viral↗
PubMed1980

Nucleotide sequence of influenza virus RNA segment 8 indicates that coding regions for NS1 and NS2 proteins overlap.

The smallest RNA segment of influenza A viruses (vRNA segment 8) has recently been shown to code for two unrelated nonstructural proteins (NS1 and NS2) translated from separate mRNAs. Molecular weight considerations indicated that there might not be enough space on vRNA segment 8 for the two coding regions unless they overlap. We have recently cloned in bacterial plasmids several genes of an avian influenza A virus, fowl plague virus (EPV), and now present the complete nucleotide sequence of FPV RNA segment 8 largely determined from the cloned DNA. The DNA sequence predicts two open protein synthesis reading frames that can be translated into polypeptides of sizes similar to those of NS1 and NS2. The coding regions for these polypeptides overlap by the equivalent of 43-60 amino acids, the exact amount depending on which of several possible methionines initiates the synthesis of NS2.

Amino Acid Sequence↗
PubMed1979

Complete nucleotide sequence of an influenza virus haemagglutinin gene from cloned DNA.

A synthetic fowl plague virus (FPV) haemagglutinin gene has been cloned in bacteria and the complete sequence of the RNA gene deduced. It is 1,742 nucleotides long and the mRNA codes for 56.3 amino acids in an uninterrupted sequence. The nature of some of the important domains in the haemagglutinin has been established, and their structure is discussed in relation to their function. Extensive amino acid sequence homologies exist between FPV and human influenza haemagglutinins.

Amino Acid Sequence↗
PubMed1978

Sequence of 129 nucleotides at the 3'-terminus of encephalomyocarditis virus RNA.

The sequence of 129 nucleotides next to the poly(A) tail of encephalomyocarditis virus RNA has been determined by rapid gel sequencing of cDNA synthesized with DNA polymerase I or reverse transcriptase and a phasing primer, [5'-32P]p(dT)8dC. The sequence is in accord with (a) the pyrimidine tracts which were mapped in blocks along the cDNA, (B) the sequences of seven characteristic T1 RNase oligonucleotides in the RNA transcribed from the cDNA with RNA polymerase, and (c) a limited amount of sequence deduced by partial spleen phosphodiesterase digestion and depurination of endonuclease IV oligonucleotides. The 3' end shows little secondary structure on its own. Ten nonsense codons block all three reading frames such that at least 26 nucleotides do not code for protein. The possible function of a homology A-A-U-A-A-A with other polyadenylated RNAs is discussed.

Base Sequence↗
PubMed1975

The determination of secondary structure in the poly(C) tract of encephalomyocarditis virus RNA with sodium bisulphite.

The degree of secondary structure in the poly(C) tract of encephalomyocarditis virus (EMCV) RNA has been investigated using sodium bisulphite, which brings about the hydrolysis of non-base-paired cytidylic acid to uridylic acid in RNA. The percentage conversion of C to U in the poly(C) region of native EMCV RNA was similar to that found in a synthetic polynucleotide lacking secondary structure [poly(C)]. When poly(I) was annealed to either native or denatured EMCV RNA, it protected the poly(C) tract from the action of bisulphite. It is concluded that the poly(C) tract of EMCV RNA in solution is very largely single-stranded.

Chemical Phenomena↗