A collection of randomly generated temperature-sensitive (ts) vaccinia virus (strain IHD-W) mutants were reported by S. Dales et al., (1978, Virology, 84, 403-428) in 1978 and characterized by electron microscopy. We have performed further genetic analysis on the Dales collection of mutants to make the mutants more useful to the scientific community. We obtained the entire Dales collection, 97 mutants, from the American Type Culture Center (ATCC). All 97 mutants were grown and reassessed for temperature sensitivity. Of these, 16 mutants were either very leaky or showed unacceptably high reversion indices even after plaque purification and therefore were not used for further analysis. The remaining 81 ts mutants were used to perform a complete complementation analysis with each other and the existing Condit collection of ts vaccinia virus (strain WR) mutants. Twenty-two of these 81 Dales mutants were dropped during complementation analysis due to erratic or weak behavior in the complementation test. Of the 59 mutants that were fit for further investigation, 30 fall into 13 of Condit's existing complementation groups, 5 comprise 3 previously identified complementation groups independent of the Condit collection, and 24 comprise 18 new complementation groups. The 59 mutants which were successfully characterized by complementation will be accessioned by and made available to the scientific community through the ATCC.

Prior phenotypic analysis of a vaccinia virus gene A18R mutant, Cts23, showed the synthesis of longer than wild type (Wt) length viral transcripts during the intermediate stage of infection, indicating that the A18R protein may act as a negative transcription elongation factor. The purpose of the work described here was to determine a biochemical activity for the A18R protein.Pulse-labeled transcription complexes established from intermediate virus promoters on bead-bound DNA templates were assayed for transcript release during an elongation step that contained nucleotides and various proteins. Pulse-labeled transcription complexes elongated in the presence of only nucleotides were unable to release nascent RNA. The addition of Wt extract during the elongation phase resulted in release of the nascent transcript, indicating that additional factors present in the Wt extract are capable of inducing transcript release. Extract from Cts23 or mock-infected cells was unable to induce release. The lack of release upon addition of Cts23 extract suggests that A18R is involved in release of nascent RNA. By itself, purified polyhistidine-tagged A18R protein (His-A18R) was unable to induce release; however, release did occur in the presence of purified His-A18R protein plus extract from either Cts23 or mock-infected cells. These data taken together indicate that A18R is necessary but not sufficient for release of nascent transcripts. We have also demonstrated that the combination of A18R protein and mock extract induces transcript release in an ATP-dependent manner, consistent with the fact that the A18R protein is an ATP-dependent helicase. Further analysis revealed that the release activity is not restricted to a vaccinia intermediate promoter but is observed using pulse-labeled transcription complexes initiated from all three viral gene class promoters. Therefore, we conclude that A18R and an as yet unidentified cellular factor(s) are required for the in vitro release of nascent RNA from a vaccinia virus transcription elongation complex.Elongation and termination are key control points in both prokaryotic and eukaryotic transcription (1). Transcriptional events such as pausing, arrest, and termination are regulated by cis-and trans-acting factors that decide the fate of a given transcript, either elongation or termination. A paused complex, in which the 3Ј end of the nascent RNA is retained in the polymerase catalytic site, can be induced by a DNA sequencespecific element, such as a T-rich sequence, or blockage of the RNA polymerase by so-called negative elongation factors. Elongation of a paused complex may resume either spontaneously or in response to positive elongation factors. An arrested complex, in which the catalytic site of the polymerase has slipped backwards and out of context with the 3Ј end of the nascent transcript, must cleave the nascent RNA to return the 3Ј end to the catalytic site in order to relieve arrest. Cleavage is an endogenous activity of the RNA polymerase but is activated in response to trans-a...