Nine synthetic pyrimidine homopolyribonucleotides were analyzed as regards their ability to inhibit the DNA polymerase of the avian myeloblastosis virus (AMV). Substitutions in the 4 and 5 position of the ring and the 2' position on the sugar were found to significantly alter inhibitory activity. The most potent class of inhibitors, 2'-halogeno-

Continuous culture was employed to study plasmid instability in an amylase-producing Bacillus subtilis 1A289 that was genetically manipulated. No true steady state could be obtained with 1A289(pEAA)-strain (plasmid)-due to its structural instability, which occurred both with glucose and Maltrin-100 as limiting carbon sources. The plasmid, pEAA (Cm(R), amy(+), i.e., chloramphenicol resistant, amylase positive) degenerated into a smaller plasmid, pEAA1 (CM(R), amy(-)) that was stable. There was a direct correlation between amylase-producing ability and this structural instability since f(amy) (fraction of cells with amylase-producing ability) reached zero at the same time that f (fraction of cells that are resistant to chloramphenicol) reached its maximum level. Since the deletion in pEAA was larger than the original amylase-gene insert, either all of part of the insert is absent from pEAA1. Though on discernible change in 1A289(pHV33), where pHV33 is the vector plasmid, was observed during continuous cultivation, its behavior was different from that of the stable 1A289(pEAA1).

Poly(2'-fluoro-2'-deoxyuridylic acid) is known to be an effective inhibitor ofthe deoxyribonucleic acid polymerase found within the oncornaviruses. This synthetic polynucleotide was found to inhibit the replication of vesicular stomatitis virus in mouse L cells. The polymer was shown to be capable of inhibiting the viral ribonucleic acid (RNA)-dependent RNA polymerase, and it is proposed that this is the mechanism of antiviral activity. The following observations support this viewpoint: (i) the polymer is most active when added after virus adsorption; (ii) the antiviral activity is not species specific; and (iii) the polynucleotide is nontoxic to the host cell. Conventional methodologies designed to increase nucleic acid uptake by cultured cells do not show an increase in antiviral potency.The antiviral properties of synthetic polynucleotides have, in the past, been associated with either their ability to induce interferon (23) or to enhance the immune response against viral antigens (3). Recently, a more direct mechanism of antiviral activity has been attributed to this class of polymers by the demonstration that certain single-stranded polynucleotides can block the activity of virus-associated nucleic acid polymerases (8,16,17,22). The majority of these data was obtained in studies utilizing the ribonuleic acid (RNA)-dependent deoxyribonucleic acid (DNA) polymerase ofthe oncornaviruses and indicated that the inhibitory polynucleotides blocked the association of enzyme and active nucleic acid template (7). Although the extension of these enzyme analyses to cell culture (16,19,21) and in vivo virus inhibition studies (20) did demonstrate significant antiviral activity, the actual mechanism of inhibition remains unclear. The replication of the oncornaviruses is a complex process that is strongly dependent on host cell metabolism (18), and mechanistic studies involving these viruses may remain difficult and equivocal.Efforts to find a less complex virus-host cell system in which to study the direct inhibitory -activity of the single-stranded polymers demonstrated that the replication ofvesicular stomatitis (VS) virus in mouse L-cell culture was sensitive to poly(2'-fluoro-2'-deoxyuridylic acid)[(dUfl)J], a potent inhibitor of the avian myeloblastosis virus RNA-dependent DNA polymerase (8). This system appeared to be amenable to our studies since VS virus carries a viral-specific, RNA-dependent RNA polymerase, viral replication is relatively independent of host cell DNA and RNA synthesis, and (dUfl), was found to be nontoxic to the host cells. The present study demonstrates that the VS virus RNA polymerase is sensitive to (dUfl)!,, inhibition and the polynucleotide is active when administered postadsorption and presents data which suggest that the inhibition is not mediated by interferon.(A preliminary report on these investigations was presented at the Fourteenth Interscience

Bacillus amyloliquefaciens BaM-2 produces large amounts of extracellular enzymes, and the synthesis of these proteins appears to be dependent upon abnormal ribonucleic acid metabolism. A polynucleotide phosphorylase (nucleoside diphosphate:polynucleotide nucleotidyl transferase) was identified, purified, and characterized from this strain. The purification scheme involved cell disruption, phase partitioning, differential (NH4)2SO4 solubilities, agarose gel filtration, and diethylaminoethyl-Sephadex chromatography. The purified enzyme demonstrated the reactions characteristic of polynucleotide phosphorylase: polymerization, phosphorolysis, and inorganic phosphate exchange with the 3phosphate of a nucleotide diphosphate. The enzyme was apparently primer independent and required a divalent cation. The reactions for the synthesis of the homopolyribonucleotides, (A),, and (G),, were optimized with respect to pH and divalent cation concentration. The enzyme is sensitive to inhibition by phosphate ion and heparin and is partially inhibited by rifamycin SV and synthetic polynucleotides.