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.

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.

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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.

1. RNA polymerase requires thiol compounds to maintain optimum activity during isolation, although it is not a thiol enzyme.2. Total thiols have been determined for RNA core polymerase by reaction with p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol and 5,5'-dithio-bis(2-nitrobenzoic acid). Their distribution between the subunits has been determined as 0101,9,9' = 4.4.8.8.3. An arbitrary division of the reaction rate profile shows that of the 18 thiols reacting with 5,5'-dithio-bis(2-nitrobenzoec acid) in 18 h, over 10 react in lo/, of this time. Most of these are buried thiols since only 2 or 3 react with N-[p-(-methyl-2-benzthiazole)phenyl]maleimide.4. Enzyme activity is lost gradually on titration with 6,5'-dithio-bis(2-nitrobenzoic acid), but rapidly with p-chloromercuribenoate until 10 -12 thiols have reacted per mole; polymerase then undergoes a large reversible change in activity, although with 5,5'-dithiobis(2-nitrobenzoic acid) reversibility is slow and depends on the groups/mole that have reacted.5 . This inactivation by 5,5'-dithio-bis(2-nitrobenzoic acid) may be prevented by pre-initiating the enzyme which causes the reaction to stop at 11 thiols/mole, leaving 60-70°/, of the original activity. DNA on its own does not produce this resistance, which is proportional to the amount of initiated complex present.6. Measurements with fluorescent probes show that considerable destabilisation of the tertiary structure occurs on reaction with p-chloromercuribenzoate. Consideration of this together with the known subunit distribution of thiols strongly suggests that DNA acts by stabilising the structure rather than by direct steric hindrance.The DNA-dependent RNA polymerase of Escherichia coli B contains four subunits. The core enzyme (a&3') reversibly associates with sigma to form the holo enzyme (oc2BB') and dissociates during transcription. Both forms are generally found to require the addition of added thiol groups to maintain optimum activity. The enzyme is inhibited by reagents which react with the thiol groups Ll,2] although it is not clear whether the reactive thiol(s) are at the active site, template binding site, or are active in maintaining quaternary structure [3].In the present paper we investigate in detail the subunit distribution, structure, and the function of the thiol groups in the maintenance of enzyme activity. A preliminary communication has appeared MATERIALS AhTD DTHODS RNA Polymerase Core RNA polymerase waa isolated from E. coli B by a modiiied version of the procedure of Chamberlin Enzyme. RNA polymerase or nucleoside triphosphate : RNA nucleotidyltransferase (DNA dependent) (EC 2.7.7.6). 141.and Berg [l] with or without DEAE-cellulose chromatography followed by phosphocellulose chromatography [5] and zonal centrifugation. The initial stages (up to ammonium sulphate fractionation) were adapted to handle large quantities of starting material as follows. 1.8 kg frozen cells were homogenized in two lots with 2 1 0.01 M Tris-HC1 pH 8,O.Ol M MgCl,, 0.1 mM EDTA a t room temperature ...