New procedure for isolation of Rous sarcoma virus-specific RNA from infected cells.

The use of mercurated "strong stop" complementary DNA (complementary to the 5'-terminal 101 nucleotides of Rous sarcoma virus RNA) in the isolation of virus-specific RNA from infected chicken embryo fibroblasts is described. Strong stop Rous sarcoma virus complementary DNA was mercurated chemically, and, as a result of the low complexity of this DNA, short hybridization times (up to 15 min) and heating in the absence of formamide were found to be adequate conditions for the isolation of virus-specific RNA. The purity of the isolated RNA was demonstrated by analysis of labeled RNase T1-resistant oligonucleotides by two-dimensional polyacrylamide gel electrophoresis. The isolated RNA could be translated in the in vitro protein synthesis system derived from rabbit reticulocytes, and an analysis of polypeptides programmed by isolated RNA before and after immunoprecipitation further demonstrated both the purity of the isolated mRNA and the quantitative nature of the isolation procedure.     

Nuclear steady-state RNA from chicken immature red blood cells. Distribution of globin-coding and poly (A) sequences

Nuclear steady-state RNA and polysomal RNA of chicken immature red blood cells were isolated and separated on formamide sucrose gradients. For comparison the distribution of 9 S globin mRNA was investigated by gradient centrifugation of 125I-labelled mRNA. The material was either pooled into two fractions (less than 20 S; greater than 20 S) and translated in an Ehrlich ascites cell-free system or each gradient fraction was analyzed by hybridization with [3H]-poly (U) or [3H]-labelled DNA complementary to purified 9 S globin mRNA (globin cDNA). In neither case could evidence be obtained for the existence of a high molecular weight RNA as a probable globin mRNA precursor. Further analysis was performed by electrophoresis of RNA on exponential polyacrylamide gels in formamide and subsequent hybridization with cDNA. The results are consistent with those of gradient centrifugation and demonstrate that the distribution of globin-coding sequences in nuclear steady state RNA corresponds to that of cytoplasmic 9 S globin mRNA.     

In vitro RNA synthesis and expression of vitellogenin gene in isolated chicken liver nuclei.

Optimal conditions for prolonged in vitro synthesis of RNA in isolated chicken liver nuclei have been described. It is shown by incorporation of gamma32P-GTP into RNA, analysis of the product on sucrose density gradient, and digestion with alkaline phosphatase and ribonuclease A that there is reinitiation of RNA synthesis. Polynucleotide kinase activity has been ruled out as explanation for the incorporation of gamma32P-GTP. alpha-Amanitin inhibits RNA synthesis by about 50%. Nuclei prepared from estradiol-treated chicks have twice the RNA synthesis activity as the controls. RNA is synthesized in the presence of Hg-UTP and the mercurated product separated by affinity chromatography on sulfhydryl-Sepharose column under stringent conditions. Vitellogenin mRNA sequences are measured by hybridization with DNA complementary to vitellogenin mRNA. Estradiol treatment leads to a 10-fold increase in vitellogenin mRNA sequences.     

Globin-coding sequences in the nuclear 28S pre-mRNA and in the cytoplasmic RNA of pigeon erythroid cells

The steady-state content of globin-coding sequences in nuclear and cytoplasmic RNA of pigeon erythroid cells was estimated by hybridization in the excess of nuclear 28S RNA and cytoplasmic poly(A) + RNA with [3H]DNA, synthesized on globin mRNA. Sequences of 9S globin mRNA are found in 0.06% of molecules of non-ribosomal 28S nuclear RNA (pre-mRNA) of erythroblasts and in 0.5% of molecules of non-ribosomal 28S nuclear RNA of reticulocytes. The content of globin mRNA in erythroblast cytoplasm is, respectively lower than in that of reticulocytes.     

Initiation of simian virus 40 DNA replication in vitro: identification of RNA-primed nascent DNA chains.

Cell-free extracts of simian virus 40 (SV40)-infected CV-1 cells can initiate large tumor antigen dependent bidirectional replication in circular DNA molecules containing a functional SV40 origin of replication (ori). To determine whether or not DNA replication under these conditions involves RNA-primed DNA synthesis, replication was carried out in the presence of 5-mercuri-deoxycytidine triphosphate to label nascent DNA chains. Newly synthesized mercurated DNA was isolated by its affinity for thiol-agarose, and the 5'-ends of the isolated chains were radiolabeled to allow identification of RNA primers. At least 50% of the isolated chains contained 4 to 7 ribonucleotides covalently linked to their 5'-end; 80% of the oligoribonucleotides began with adenosine and 19% began with guanosine. About 60% of the nascent DNA chains annealed to the SV40 ori region, and about 80% of these chains were synthesized in the same direction as early mRNA. These results are consistent with the properties of SV40 DNA replication in vivo and support a model for initiation of SV40 DNA replication in which DNA primase initiates DNA synthesis on that strand of ori that encodes early mRNA.     

Inhibition of pre-mRNA splicing by antisense RNA in vitro: effect of RNA containing sequences complementary to exons.

The objective of the experiments described in this paper was to determine the feasibility of inhibition of pre-mRNA splicing by antisense RNA in vitro. Three different types of antisense RNA were utilized: antisense RNA complementary to the spliced RNA molecule; antisense RNA complementary to the unprocessed mRNA precursor molecule; and antisense RNA complementary to the 5' and 3' splice junctions. Whereas antisense RNA complementary to mRNA had little effect on splicing, antisense RNAs complementary to mRNA precursor or to splice junctions strongly inhibited splicing of pre-mRNA molecule. The results obtained indicate that the inhibitory effect is most likely due to hybrid formation between pre-mRNA and antisense RNA molecules and that antisense RNA complementary to the exon portion but not to the intron portion of splice junction exhibit an inhibitory effect. This inhibition can be overcome by bringing together 5' and 3' splice junctions via hybrid formation with antisense RNA complementary to the spliced RNA molecule.     

Purification of globin messenger RNA from dimethylsulfoxide-induced friend cells and detection of a putative globin messenger RNA precursor

Procedures are described that permit the detection and isolation of a specific messenger RNA as well as its precursor from total cell extracts. DNA complementary to the mRNA was elongated by the addition of dCMP residues and annealed with labeled cell RNA. The elongated DNA with RNA hybridized to it was isolated by chromatography on a poly(I)-Sephadex column. The method was used to isolate ³²P-labeled globin mRNA from labeled Friend cells, a mouse erythroleukaemic cell line, induced with dimethylsulfoxide to synthesize hemoglobin. ³²P-labeled globin mRNA isolated by this procedure was estimated to be 80% pure by hybridization analysis and sedimented as a single peak at 10 S. Partial sequences were determined for 16 oligonucleotides derived from the purified ³²P-labeled globin mRNA by RNAase T₁ digestion. The partial sequences for nine oligonucleotides corresponded to those predicted from the amino acid sequences of α and β globin; the other oligonucleotides were presumably derived from non-translated regions.In order to detect a possible precursor to globin mRNA, RNA from induced Friend cells pulse-labeled with [³²P]phosphate for 20 minutes was centrifuged through a sucrose gradient and the resulting fractions were analyzed for globinspecific sequences. Two peaks of globin-specific RNA were detected, a larger one at 10 S, the position of mature globin mRNA, and a smaller one at 15 S.