Synthetic peptides mimic subtype specificity of foot-and-mouth disease virus.

The major immunogen of foot-and-mouth disease virus (FMDV) is located between amino acids 141-160 of the capsid protein VP1. Synthetic peptides corresponding to the major immunogenic region give good neutralising antibody responses and protection in guinea pigs. To define more precisely the immunogenic site of the virus, we have examined serological differences between subtypes of the A serotype using synthetic peptides covering the 141-160 region. We show that these synthetic peptides carry determinants which mimic the subtype specificity of the virus. The correlation between these results and predictive structural models, based on the amino acid sequence, is discussed.

Location of aromatic amino acids and belix content in Escherichia coli ribonucleic acid polymerase.

1. The perturbing effect of glycerol on the direct spectrum of Escherichia coli DNA-dependent RNA polymerase has been studied. 2. By comparison with model compounds and with the unfolded polymerase in 3.8m-urea it was possible to determine the ratio of tyrosine and tryptophan residues present. On reduction of the urea-treated enzyme with 2-mercaptoethanol, no further change in the difference spectrum occurred. 3. The amino acid composition of the enzyme is given. 4. In the intact protein approx. 30% of the tryptophan and 54% of the tyrosine residues were exposed. In conjunction with the extinction value and molecular weight this corresponded to 7 tryptophan residues and 57 tyrosine residues on the surface and 16 tryptophan residues and 48 tyrosine residues ;buried'. 5. The optical rotatory dispersion of the enzyme was unaffected by 20% glycerol. 6. The helix content calculated from Moffit plots over 560-300nm was 13%, and from the 233nm trough 13%.

Structural interpretation of the binding of 9-azidoacridine to D-amino acid oxidase.

D-Amino acid oxidase (EC 1.4.3.3) forms an inhibited complex with the nucleotide- and aromatic-binding-site affinity reagent 9-azido[3H]acridine. Tryptic digestion of the photolysed complex yielded two radioactive peptides, 222-265 (T23) and 298-328 (T29), which core and secondary structure analysis revealed to be exposed, but which also comprised the propargylglycine-binding residues. This suggests that at least parts of the peptides containing these residues are in the active centre and that they are spatially close to the flavin-binding site.

The inhibition of ribonucleic acid polymerase by acridines.

1. The aminoacridines, proflavine (3,6-diaminoacridine) and 9-aminoacridine, and a hydrogenated derivative, 9-amino-1,2,3,4-tetrahydroacridine, were shown to inhibit in vitro the DNA-primed RNA polymerase of Escherichia coli. The inhibition is strong with both proflavine and 9-aminoacridine, but weak with 9-amino-1,2,3,4-tetrahydroacridine. 2. The extent to which the three acridines bind to calf-thymus DNA in the enzyme medium was studied spectrophotometrically. The extent of binding decreases in the order: proflavine, 9-aminoacridine, 9-amino-1,2,3,4-tetrahydroacridine. Some evidence was also obtained for interaction between the nucleoside triphosphate substrates and proflavine or 9-aminoacridine; no such interaction was detectable with 9-amino-1,2,3,4-tetrahydroacridine. 3. Although the amount of acridine bound to DNA increases with increasing inhibition, a stage is reached where an increase in acridine concentration still causes an increase in inhibition, with practically no increase in the amount bound to DNA. 4. Plots of reciprocal rates against the reciprocal of DNA concentration were linear and had a common intercept when proflavine or 9-aminoacridine was present. Similar relations were obtained when the reciprocal concentration of nucleoside triphosphates was plotted. The observations are interpreted kinetically in terms of a competitive inhibition of the enzyme by proflavine or 9-aminoacridine and of a kinetic role for the DNA analogous to ;activation'. 5. This suggests that inhibitory acridine molecules can occupy the sites on the RNA polymerase that are specific for binding the nucleoside triphosphate substrate or the bases of the DNA, when these become accessible during the copying process.

Location of DNA and nucleotide binding sites on wheat germ RNA polymerase II.

We have investigated the roles of the 13 subunits present in wheat germ RNA polymerase II, using the inhibitors; pyridoxal 5'-phosphate and the periodate oxidation product of adenosine (AOP). Pyridoxal 5'-phosphate is shown to interact with at least part of the DNA binding site as well as the nucleotide binding sites, whereas AOP probably binds to the nucleotide binding sites. Reduction of the enzyme:inhibitor complex with sodium [3H] borohydride and identification of labelled subunits shows that in both cases the inhibitors bind primarily to subunits a and b. We conclude that subunits a and b contain at least part of the catalytic site, but do not rule out possible involvement of other subunits in the various steps of transcription.

Cross-linking of wheat germ RNA polymerase II with dithiobis(succinimidyl propionate).

The subunit arrangement of wheat germ RNA polymerase II was examined using the cleavable cross-linking reagent dithiobis(succinimidyl propionate). Conditions were chosen such that the enzyme was active prior to treatment, and that most of the subunits were reactive towards the reagent. Our results indicate that the enzyme is made up from a central core involving the two large subunits, around which the small subunits are independently arranged. Possible relations between the overall structure and the role of individual subunits in transcription are discussed.

9-azidoacridine, a new photoaffinity label for nucleotide- and aromatic-binding sites in proteins.

The effect of the photolytic reagent 9-azidoacridine, optionally 3H-labelled, was studied both kinetically and structurally on nine different enzymes, namely alpha-chymotrypsin, lactate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, alcohol dehydrogenase, alanine dehydrogenase, D-amino acid oxidase, ribonuclease A, alkaline phosphatase and alpha-amylase. Dark inhibition was observed in several cases. The concentration of the inhibitor ranged from 0.2 microM to 0.67 microM and demonstrated competitive kinetics with nucleotide cofactors when present. All concentrations of inhibitor showed increased inhibition on photolysis. Examination of the oligopeptides from hydrolysis of the covalently 3H-labelled derivative in conjunction with known amino acid sequence and tertiary structure established that the primary site of interaction in those cases for which the tertiary structure was available involved the active-site region. The above results in conjunction with those obtained with the structural analogues 9-aminoacridine and 9-amino-1,2,3,4-tetrahydroacridine established that this reagent acts as a molecular probe of aromatic- and, in particular, nucleotide-binding sites. This reagent provides a further additional method for studying the nucleotide cofactor domain.

The interaction of aflatoxin B1 with polynucleotides and its effect on ribonucleic acid polymerase.

1. The interaction of aflatoxin B(1) with different polynucleotides was studied spectrophotometrically. Equations were derived that enable the degree of binding to be determined without first determining the extinction coefficient of the bound form. 2. The interaction with calf thymus DNA obeys first-order relationships with an association constant of 0.40mm(-1), but there is some evidence for a secondary binding process from results obtained at 390nm. 3. The spectral shifts decreased in the order polyadenylic acid+polyuridylic acid>DNA>polyadenylic acid>polyadenylic acid+polyinosinic acid. Polycytidylic acid, polyuridylic acid, polyinosinic acid (both single- and triple-stranded), AMP, CMP, GMP and UMP did not interact with aflatoxin. It was concluded that there is a requirement for the amino group of adenine (or possibly guanine) for binding of aflatoxin to polynucleotides to occur. 4. Binding is reversed by increasing ionic strength, and by Mn(2+) and Mg(2+) in the concentration range studied (0-5mm). The effect of the Mn(2+) or Mg(2+) was far greater than would be expected on the basis of their ionic strength. With both the bivalent cations and sodium chloride the reversal is greatest with double-stranded polynucleotides. 5. Inhibition in vitro of the DNA-dependent RNA polymerase of Escherichia coli by aflatoxin B(1) was detected only in the absence of Mg(2+) and at concentrations of Mn(2+) below the optimum for RNA synthesis in vitro. 6. The degree of inhibition (maximally 30%) was dependent on the concentration of Mn(2+) and decreased during incubation.

The effects of gamma-irradiation on the priming by deoxyribonucleohistone of ribonucleic acid polymerase.

1. The effect of gamma-irradiation of solutions of DNA and deoxyribonucleohistone (DNH) on their ability to prime the synthesis of RNA by DNA-dependent RNA polymerase has been studied. 2. The priming ability of both DNA and DNH decreased continuously with increasing radiation dose, but more rapidly with DNH. 3. These decreases have been compared with decreases in molecular weight and with the breakdown of the specific hydrogen-bonded structure of DNA. 4. It is concluded that a process was occurring during gamma-irradiation of DNH that, although involving a decrease in molecular weight, did not diminish and even enhanced its priming ability. This is consistent with previous physicochemical evidence that gamma-irradiation causes dissociation of histone from DNH.

An orf virus sequence showing homology to the 14K 'fusion' protein of vaccinia virus.

The nucleotide sequence of a region of DNA 30 kb from the right end of the orf virus genome has been determined. Examination of the sequences revealed an open reading frame encoding a 10K peptide with significant amino acid homology to the 14K 'fusion' protein reported in vaccinia virus. The orf virus sequence has a 31% identity with the vaccinia virus protein, but a higher level of homology of core predicted residues. The secondary structure of both proteins is also similar. The occurrence of the TAAAT sequence upstream from the initiation codon indicates that the sequence is likely to be transcribed late in infection.

Chemical basis of antigenic variation in foot-and-mouth disease virus.

One of the difficulties in controlling foot and mouth disease by vaccination is the occurrence of the virus as seven distinct serotypes because immunity conferred by vaccination against one serotype leaves the animals susceptible to infection by the other six. Moreover, the antigenic variation, even within a serotype, can be so great that immunity against the homologous strain of virus need not necessarily ensure protection against infection by other viruses within that serotype. Here we report the separation of three natural antigenic variants, distinguishable in cross-neutralization tests from an isolate of foot-and-mouth disease virus (FMDV). The serological differences could also be demonstrated by antisera elicited by synthetic peptides corresponding to residues 141-160 of the capsid polypeptide VP1, showing that this region contains a major immunogenic site of the virus. The results have practical implications for the choice of viruses for vaccine production.