ABSTRACT Synthesis of single-stranded DNA ofbacteriophageM13 is blockedbyrifampicin. This inhibi-tionis notobservedinanEscherichiacoli mutant possess-ing an RNA polymerase resistant to rifampicin. Sincerifampicin stops single-strand synthesis faster thanchloramphenicol, inhibition by rifampicin does not seemtoresultfromatranscriptionalblockofproteinsynthesis,particularly in viewofthelonghalf-life ofthemessengerRNAfor M13. It is, therefore, concluded that the inter-action ofrifampicin withRNApolymerasedirectly affectssingle-strand DNA synthesis. Possible mechanisms arediscussed. TheDNAof the filamentous phage M13 replicates in threestages (1, 2). First, the infecting single-stranded DNA is converted by host enzymes into a double-stranded form (SS -- RF).Early in the infection the replicative formsmulti- ply to establish a pool of RFmolecules (RF -- RF). Later, double-strand synthesis ceasesalmost completely, andtheRF molecules serve as precursors for single-strand synthesis (RF SS).Single-stranded
During the replication of the minicircular DNA of Escherichia coli 15 an open circular form II has been identified as a precursor of superhelical form I. Pulse‐labeled form II DNA contains a newly synthesized linear strand which is non‐covalently associated with a circular template strand. Equilibrium centrifugation of isolated linear strands in alkaline CsCI shows that the incorporation of label occurs with equal frequency in either of the complementary strands indicating a symmetrical mechanism of replication. Labeling of replicative intermediates is observed in short pulses at 25 °C. Upon alkali denaturation these molecules yield small labeled DNA fragments (4–6 S) suggesting a discontinuous mode of strand synthesis.
Recently, it was reported that 3' truncation of an integrated surface gene (pre-S2/S) cloned from a hepatitis B virus (HBV)-associated hepatoma gave rise to the generation of a C-terminally truncated middle surface protein (MHBst), which surprisingly exerted a transcriptional transactivator function. To define the molecular requirements for the generation of transactivating MHBs(t) proteins, a 3' deletion analysis of the HBV pre-S2/S gene was performed. In cotransfection experiments with reporter plasmid pSV2CAT, full-length pre-S2/S genes or pre-S2/S genes with minor 3'-terminal deletions did not exhibit transactivator activity. In contrast, deletion of C-terminal hydrophobic region III of the S domain generated the transactivator function. Further stepwise 3' deletions, removing hydrophobic region II and both hydrophilic regions of the S domain, did not interfere with the transactivator activity; it was completely abolished, however, after additional deletion of hydrophobic region I. The results of this study define a range within the S open reading frame (between HBV nucleotides 221 and 573), termed the trans-activity-on region, in which 3' deletions give rise to the generation of transactivating MHBs(t) proteins. Within this region, not only 3' deletions but also the introduction of a stop codon activated the transactivator function, indicating that point mutations of integrated HBV DNA also may give rise to the synthesis of transactivating MHBs(t) proteins in vivo.
We have developed an in situ hybridization assay capable of detecting enteroviral RNA in myocardial cells, using molecularly cloned coxsackievirus B3 cDNA as a diagnostic probe. Because of the high degree of nucleic acid sequence homology among the numerous enteroviral serotypes, including the group A and B coxsackieviruses and the echoviruses, detection of these various agents commonly implicated in human viral heart disease is possible in a single hybridization assay. We demonstrate the considerable potential of this method for an unequivocal diagnosis of enteroviral heart disease as well as for pathogenicity studies. Using athymic mice persistently infected with coxsackievirus B3 as a model system, we show that the myocardium is affected in a disseminated, multifocal manner.
