SummaryRNAIII is a 514 nt regulatory RNA that is the effector molecule of the staphylococcal agr quorum-sensing system, regulating a large set of virulence and other accessory genes at the level of transcription. RNAIII was discovered nearly 20 years ago and we long ago hypothesized that it would function by regulating the synthesis or activity of one or more intermediary transcription factors. We have finally confirmed this hypothesis, showing that Staphylococcus aureus RNAIII regulates the synthesis of a major pleiotropic transcription factor, Rot, by blocking its translation. RNAIII has a complex secondary structure with several stable hairpins that have highly C-rich end loops, unusual in an AT-rich organism. We noted that these loops are complementary to two G-rich stem loops of the rot mRNA translation initiation region (TIR). Pairing of the complementary RNAs would be predicted to occlude the rot Shine-Dalgarno (SD) site and to block rot translation. Through a combination of transcriptional and translational fusions and Northern and Western blot hybridization analyses, we show that RNAIII does, indeed, block rot translation. Through alterations in the C-rich loops of RNAIII and the G-rich loops of rot, we show that the sequences of these loops are critical for inhibition of rot translation and suggest that this inhibition is affected by pairing between the complementary stem loops, followed by the cleavage of rot mRNA. We propose that the RNAIII-rot mRNA interaction plays a key role in agr regulation of staphylococcal virulence.

Successful cancer gene therapy requires a vector that systemically and specifically targets tumor cells throughout the body. Although several vectors have been developed to express cytotoxic genes via tumor-specific promoters or to selectively replicate in tumor cells, most are taken up and expressed by just a few targeted tumor cells. By contrast, we show here that blood-borne Sindbis viral vectors systemically and specifically infect tumor cells. A single intraperitoneal treatment allows the vectors to target most tumor cells, as demonstrated by immunohistochemistry, without infecting normal cells. Further, Sindbis infection is sufficient to induce complete tumor regression. We demonstrate systemic vector targeting of tumors growing subcutaneously, intrapancreatically, intraperitoneally and in the lungs. The vectors can also target syngeneic and spontaneous tumors in immune-competent mice. We document the anti-tumor specificity of a vector that systemically targets and eradicates tumor cells throughout the body without adverse effects.

curious accident of sequence organization but has a Medical School, New York, NY 10016, USA substantive biological rationale that is derived from the 1 Corresponding author plasmid's need to prevent recycling of the initiator because recycling would obviate control of plasmid copy number. pT181 and other closely related rolling circle plasmidsRecycling is prevented by a modification of the initiator, have the nicking site for initiation of replication consisting of the attachment to one subunit of a short between the arms of a GC-rich inverted repeat oligonucleotide representing sequences 3Ј to the nick site sequence adjacent to the binding site for the dimeric in the double strand origin (DSO) (Rasooly and Novick, initiator protein. Replication is initiated by the initi-1993). The resulting heterodimer, RepC/C*, is present in ator-induced extrusion of this sequence as a cruciform, great excess, is metabolically stable, can bind to the pT181 creating a single-stranded region for nicking by the DSO and can inhibit but not initiate replication (Jin et al., protein. Nicking is followed by assembly of the repli-1997). We have proposed that this oligonucleotide is some without relaxation of the secondary structure. generated by a short 3Ј extension of the leading strand at Following termination, the initiator protein is released the end of the replication cycle and becomes attached to with a short oligonucleotide attached to one subunit, the protein via the normal termination mechanism (Rasooly which prevents it from being recycled, a necessary and Novick, 1993). feature of the plasmid's replication control system. TheSince RepC/C* is isolated from pT181-containing cells, modified initiator can cleave single-stranded substrates there must always be at least a small fraction of RepC/C, and can nick and relax supercoiled plasmid DNA corresponding to material that has not yet been used for weakly. Although it can bind to its recognition sequence replication, and accounting for the in vitro replication in the leading strand origin, the modified protein activity originally observed in extracts of pT181-concannot induce cruciform extrusion, and it is proposed taining staphylococci by Khan et al. (1981). In our initial that this inability is the key to understanding the studies of RepC/C* (Rasooly and Novick, 1993; Rasooly biological rationale for having the nicking site at the et al., 1994a,b), we attributed the weak nicking, relaxing tip of a cruciform: the need to provide the functional and replication activity seen in these preparations to initiator with a catalytic advantage over the modified this small fraction of RepC/C, which was estimated on one sufficient to offset the numerical advantage and theoretical grounds to be 5-10%. The more recent results metabolic stability of the latter.reported here and elsewhere (Jin et al., 1997) suggest that Keywords: cruciform/initiation nicking site/recycling/ the level of RepC/C present in these preparations is RepC/rolling circle considerably lower than 5% and although it can a...

A phosphoenolpyruvate : dihydroxyacetone phosphotransferase was induced in Escherichia coli grown on dihydroxyacetone as sole carbon source or in its presence. This is the first example of a triose which can be acted upon by the membrane complex to provide a central intermediate in glycolysis. The presence of this system explains the ability of a mutant, in which the ATPdependent glycerol kinase is genetically replaced by a glycerol : NAD 2-oxidoreductase, to grow on glycerol.

Staphylococcus aureus plasmid pT181 replicates via a rolling circle mechanism. The synthesis of the pT181 initiator protein (RepC) is regulated by antisense RNAs, and RepC is inactivated after usage by the attachment of an oligonucleotide to one of its subunits. The inactivated heterodimeric RepC/C* has been shown be unable to initiate replication in vitro (Rasooly, A., and Novick, R. P. (1993) Science 262, 1048 -1050). The inactive RepC/C* has been found to be very stable and constitute about 90 -95% of the total RepC antigen inside the cell. We studied the specific interaction of the RepC/C and RepC/C* complex with the pT181 double strand origin. The results indicated that RepC/C and RepC/C* footprint supercoiled DNA differently although their footprints on linear DNA are similar; we also find that RepC/C is able to enhance cruciform extrusion while RepC/C* cannot. RepC/C* binds and bends the double strand origin much more weakly than does RepC/C. These results suggest that the attached oligonucleotide induces a conformational change in the RepC/C* molecule that is responsible for its lack of activity.All known bacterial plasmids encode a specific initiator and control their replication primarily by negatively regulating its synthesis. Often, the linkage between replication and the control of initiator synthesis is indirect in that the regulatory factors act in trans and respond to the copy number of the plasmid. This type of replication control requires that the initiator be utilized stoichiometrically rather than catalytically since the latter would obviate such control. Plasmids such as pT181 and its relatives, which replicate by the rolling circle mechanism, inactivate the initiator protein automatically as a part of the termination process (1). ColE1 and its relatives, which replicate by the mechanism, utilize a long RNA primer as initiator and automatically degrade this primer as replication proceeds. Plasmids such as R1, which also replicate by the mechanism, require new protein synthesis for each round of replication (2), suggesting that the initiator is inactivated after use.The inactivation mechanism for pT181 is unique; the initiator protein, RepC, is a dimer, referred to as RepC/C, and following the completion of a round of replication, an oligonucleotide, representing sequences immediately 3Ј to the initiation nicking site is attached to the active site tyrosine of one of its subunits (1). Our initial experiments suggested that the heterodimeric derivative, RepC/C*, lacked the nicking, relaxation, and replication activities of the native protein. This was puzzling. Since only one of the two subunits was modified, the unmodified subunit would be expected, a priori, to have nicking activity, the reversal of which would result in relaxation, and to be able to initiate but not terminate replication. Testing of these possibilities was complicated by the fact that preparations of total RepC antigen from pT181-containing cells generally consist of 90 -95%