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.

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.

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.

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.

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.

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.