Infectious positive- and negative-strand transcript rnas from bacteriophage Qβ cDNA clones

Ribosomal protein S1 functions as a termination factor in RNA synthesis by Qβ phage replicase

“…Qb( þ ) RNA was isolated from purified wild-type phage particles lysed with SDS, followed by centrifugation through a 15% sucrose cushion and phenol extraction 25 . Qb( À ) RNA was prepared by transcription of SmaI-digested plasmid pQb8 30 .…”

“…It is commonly adopted that, at replication of the Qb phage RNA, S1 is only needed for copying the plus strand, and not the minus strand 7,[11][12][13][14] . To check this issue, we explored effects of S1 on copying the transcript synthesized from a plasmid carrying the Qb( À ) cDNA under the T7 promoter 30 . Figure 5a demonstrates that the transcript is indeed the minus strand as, in contrast to the plus strand, it is copied by Qb replicase independently of the host factor (protein Hfq) 31 ; yet the effects of S1 on copying the two templates is the same: in each case, S1 promotes accumulation of the single-stranded RNA and reduces the proportion of double-stranded RNA in the reaction products.…”

Synthesis of biologically active influenza virus core proteins using a vaccinia virus-T7 RNA polymerase expression system

“…There have been a number of reports showing rescue of ssRNA viruses from cDNA-derived RNAs containing non-viral nucleotides at the 3' and 5' ends (van der Werf et al, 1986;Shaklee, et al, 1988;Dzianott & Bujarski, 1989;Pattnaik et al, 1992). In all cases analysed, these extra nucleotides were not retained in the progeny virus, showing that the viral replicase initiated RNA synthesis at the correct site to remove the extra nucleotides.…”

Infectious cucumber mosaic virus RNA transcribed in vitro from clones obtained from cDNA amplified using the polymerase chain reaction

“…Nevertheless amplification of eDNA by the PCR may be worthy of investigation as a method of generating virus mutants. The finding that infectivity was reduced by addition of one G residue, and to a greater extent by two G residues, to the 5' end of the transcripts (Table 1) confirms the importance of the Y-terminal structure of transcripts for infectivity (Dawson et al, 1986;Van der Werf et al, 1987;Janda et al, 1987;Rice et al, 1987;Shaklee et al, 1988;Eggen et al, 1989a;Heaton et al, 1989) but, in contrast, infectivity appears to be relatively insensitive to T-terminal extensions (Janda et al, 1987;Eggen et al, 1989b). BamHI-cleaved pCMVI-(A to E), pCMV2-(A to E) and pCMV3-(A to E) contained four additional nucleotides beyond the 3' terminus of the RNAs; however, there was little difference between the infectivity of the transcripts and that of CMV-Q RNA.…”

“…A limitation on the production of infectious transcripts using E. coli RNA polymerase has been the variable efficiency of different commercial batches of the enzyme Janda et al, 1987). This has led to the use of more efficient and reliable RNA polymerases from bacteriophages T3, T7 and SP6 for the synthesis of infectious transcripts from cDNA clones of a range of plant viruses (Janda et al, 1987;Vos et al, 1988;Domier et al, 1989;Eggen et al, 1989a, b;Heaton et al, 1989;Petty et al, 1989;Quillet et al, 1989;Weiland & Dreher, 1989), animal viruses (van der Werf et al, 1986;Rice et al, 1987) and bacteriophage Qfl (Shaklee et al, 1988).…”