Construction and characterization of a plasmid containing a nearly full-size DNA copy of bacteriophage MS2 RNA.

An apparently full-length complementary DNA copy of in vitro polyadenylated MS2 RNA was synthesized with avian myeloblastosis virus RNA-dependent DNA polymerase. After the MS2 RNA template was removed from the complementary DNA strand with T₁ and pancreatic RNase digestion, the complementary DNA became a good template for the synthesis of double-stranded MS2 DNA with Escherichia coli DNA polymerase I. We then constructed molecular chimeras by inserting the double-stranded MS2 DNA into the PstI restriction endonuclease cleavage site of the E. coli plasmid pBR322 by means of the poly(dA)· poly(dT) tailing procedure. An E. coli transformant carrying a plasmid with a nearly full-length MS2 DNA insertion, called pMS2-7, was chosen for further study. Correlation between the restriction cleavage site map of pMS2-7 DNA and the cleavage map predicted from the primary structure of MS2 RNA, and nucleotide sequence analysis of the 5′ and 3′ end regions of the MS2 DNA insertion, showed that the entire MS2 RNA had been faithfully copied, and that, except for 14 nucleotides corresponding to the 5′-terminal sequence of MS2 RNA, the fulllength DNA copy of the viral genetic information had been inserted into the plasmid. Restriction endonuclease analysis of the chimera plasmid DNA also revealed the presence of an extra DNA insertion which was identified as the translocatable element IS1³ (see following paper).     

Plasmid rolling circle replication: identification of the RNA polymerase-directed primer RNA and requirement for DNA polymerase I for lagging strand synthesis.

Plasmid rolling circle replication involves generation of single-stranded DNA (ssDNA) intermediates. ssDNA released after leading strand synthesis is converted to a double-stranded form using solely host proteins. Most plasmids that replicate by the rolling circle mode contain palindromic sequences that act as the single strand origin, sso. We have investigated the host requirements for the functionality of one such sequence, ssoA, from the streptococcal plasmid pLS1. We used a new cell-free replication system from Streptococcus pneumoniae to investigate whether host DNA polymerase I was required for lagging strand synthesis. Extracts from DNA polymerase I-deficient cells failed to replicate, but this was corrected by adding purified DNA polymerase I. Efficient DNA synthesis from the pLS1-ssoA required the entire DNA polymerase I (polymerase and 5'-3' exonuclease activities). ssDNA containing the pLS1-ssoA was a substrate for specific RNA polymerase binding and a template for RNA polymerase-directed synthesis of a 20 nucleotide RNA primer. We constructed mutations in two highly conserved regions within the ssoA: a six nucleotide conserved sequence and the recombination site B. Our results show that the former seemed to function as a terminator for primer RNA synthesis, while the latter may be a binding site for RNA polymerase.     

Replication of RNA viruses: structure of a 6 s RNA synthesized by the Q RNA polymerase

The in vitro synthesis of RNA catalyzed by the Qβ RNA polymerase has been studied using a single-stranded 6 s RNA template. Whereas Qβ RNA replication results in the synthesis predominantly of single-stranded Qβ RNA, the predominant reaction product of 6 s RNA replication was found to be double stranded. When treated with formaldehyde to dissociate complementary base pairs, 6 s RNA exhibited a decrease in molecular weight as indicated by its slower sedimentation rate and faster electrophoretic mobility. 6 s RNA also exhibited a hyperchromic thermal transition indicative of double-stranded RNA and differing markedly from that of single-stranded RNA. The T_m of this transition increased linearly with the logarithm of ionic strength. Renaturation of 6 s RNA below the T_m occurred slowly and was also dependent upon ionic strength.