Direct evidence that ribosome bound RNA-dependent RNA polymerase does not play a role in globin messenger RNA replication.

The radioactively labelled product of RNA-dependent RNA polymerase+ from ribosomes of immature chicken erythrocytes was tested for the presence of newly replicated globin mRNA using unlabelled globin complementary DNA. No radioactively labelled globin mRNA sequences were found in the product, providing direct confirmation that this RNA-dependent RNA polymerase is not involved in globin mRNA amplification.     

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.     

A characterization of globin mRNA sequences in the nucleus of duck immature red blood cells

Studies were performed with duck immature red blood cells to identify and characterize the globin mRNA sequences in nuclear RNA. Annealing of ³H-globin cDNA to unlabeled nuclear RNA has identified three distinct size classes of nuclear RNA molecules containing globin mRNA sequences. The largest size class contained 1–2% of total nuclear globin mRNA sequences and sedimented through 85% formamide-sucrose gradients at the same rate as 28S ribosomal RNA. Chromatography on oligo(dT)-cellulose indicated that most of these molecules are not polyadenylated. The bulk of nuclear globin mRNA sequences (70%) was contained in polyadenylated RNA molecules which sedimented at 16.5S. The remainder of nuclear globin mRNA sequences (～30%) was detected in molecules sedimenting at 10S (the position of cytoplasmic globin mRNA).To determine whether a precursor-product relationship exists between these nuclear molecules and cytoplasmic globin mRNA, pulse-label and chase experiments were performed. Labeled globin mRNA sequences were assayed by annealing to globin cDNA-cellulose. Labeled 28S nuclear globin RNA sequences could not be detected, perhaps due to technical reasons. 16.5S nuclear globin RNA was labeled and chased into cytoplasmic globin mRNA sequences. The half-life of 16.5S nuclear globin RNA was estimated to be less than 30 min. These results demonstrate that in duck immature red blood cells, globin mRNA is transcribed as a larger precursor. Furthermore, size characterization of this precursor during pulse-label and chase periods suggests that it is processed within the nucleus to 10S globin RNA.     

Globin mRNA contains a sequence complementary to double-stranded region of nuclear pre-mRNA.

Melted ds RNA isolated from rabbit bone marrow pre-mRNA was hybridized with excess of globin mRNA which was prepared from rabbit reticulocytes. 7-9% of ds sequences became RNAase-stable and about 30% of the sequences could be bound to poly(U)-Sepharose through poly (A) of mRNA. The size of RNAase-stable hybrid is about 30 nucleotides, that is one fourth of the length of one strand of the ds RNA.