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.     

The Synthesis of 5s RNA and Its Relationship to 18s and 28s Ribosomal RNA in the Bobbed Mutants of DROSOPHILA MELANOGASTER

Ribosomes contain one molecule each of 5S, 18S and 28S RNA. In Drosophila melanogaster although the genes for 18S+28S are physically separated from the 5S RNA genes, the multiplicity of various ribosomal RNA genes is roughly the same. Thus a coordinate synthesis of these three molecules might seem feasible. This problem has been approached by determining the molar ratios of various RNA's in ovaries and in adult flies. In ovaries there is a slight excess of 5S RNA molecules over other rRNA's, but in adult flies no such differences exist. Bobbed mutants also have the same molar ratios as wild-type flies. Results on 5S RNA synthesis in both in vitro and in vivo studies show that it is reduced in coordination with 18S+28S rRNA in the bobbed mutants of Drosophila melanogaster. Various possibilities are discussed in considering the implications of these results.     

Proportion of HeLa cell genome complementary to transfer RNA and 5 s RNA

The proportion of the HeLa cell genome complementary to tRNA and to 5 s RNA has been investigated by using the technique of RNA-DNA hybridization. The specificity of hybrids formed between HeLa DNA and these highly purified RNA classes has been assessed by analysis of the size distribution and nucleotide composition of the RNA recovered from the ribonuclease-treated hybrids.     

Launching of the yeast 20 s RNA narnavirus by expressing the genomic or antigenomic viral RNA in vivo

20 S RNA virus is a persistent positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome encodes only its RNA polymerase, p91, and resides in the cytoplasm in the form of a ribonucleoprotein complex with p91. We succeeded in generating 20 S RNA virus in vivo by expressing, from a vector, genomic strands fused at the 3'-ends to the hepatitis delta virus antigenomic ribozyme. Using this launching system, we analyzed 3'-cis-signals present in the genomic strand for replication. The viral genome has five-nucleotide inverted repeats at both termini (5'-GGGGC... GCCCC-OH). The fifth G from the 3'-end was dispensable for replication, whereas the third and fourth Cs were essential. The 3'-terminal and penultimate Cs could be eliminated or modified to other nucleotides; however, the generated viruses recovered these terminal Cs. Furthermore, extra nucleotides added at the viral 3'-end were eliminated in the launched viruses. Therefore, 20 S RNA virus has a mechanism(s) to maintain the correct size and sequence of the viral 3'-end. This may contribute to its persistent infection in yeast. We also succeeded in generating 20 S RNA virus similarly from antigenomic strands provided active p91 was supplied from a second vector in trans. Again, a cluster of four Cs at the 3'-end in the antigenomic strand was essential for replication. In this work, we also present the first conclusive evidence that 20 S and 23 S RNA viruses are independent replicons.     

Genetic Analysis of the 5s RNA Genes in DROSOPHILA MELANOGASTER

The 5S RNA genes of Drosophila melanogaster in either an isogenic wild-type or a multiply inverted (SM1) chromosome 2 increase their multiplicity when opposite a deficiency for the 5S gene site. This is analogous to the compensation phenomenon previously described for the 18S and 28S ribosomal RNA genes of the X chromosome nucleolus organizer region. Molecular hybridization of 5S RNA to DNA containing various doses of the 56F1-9 region of chromosome 2 demonstrates that most, if not all, of the 5S genes reside in or near this region. Also, a deficiency missing approximately one-half of the wild-type number of 5S genes was isolated and genetically localized. This mutant has a phenotype like that of bobbed, a mutant known to be partially deficient in 18S and 28S ribosomal RNA genes. Finally, we report the existence of a chromosomal rearrangement which splits the second chromosome into two segments, each containing 5S DNA.     

Isolation of total RNA from baker’s yeast

A simple method of production of total RNA from baker’s yeast was developed. Total RNA was isolated from yeast (Saccharomyces cerevisiae) biomass using lysis with sodium dodecyl sulfate at 100°C for 40–60 min and subsequent precipitation of the target product with 3 M NaCl. The preparation obtained was characterized in detail: yield of total RNA from 1 kg of pressed yeast, 9.25 g; optical density at 260 nm of 1 mg of RNA dissolved in 1 ml of water, 20.2 U; content of the acid-soluble fraction, 2.02%; and protein content, 1.8%. Total tRNA was isolated from total RNA by fractional precipitation with ethanol followed by gel filtration.