Influenza defective interfering viral RNA is formed by internal deletion of genomic RNA.

The 3'- and 5'-terminal nucleotide sequences of the defective interfering (DI) RNAs present in a preparation of DI influenza virus were determined. It was found that all DI RNAs possessed identical terminal sequences for at least the first 13 nucleotides at the 5' end and at least the last 12 nucleotides at the 3' end. The sequence of the DI RNAs is (5')A-G-U-A-G-A-A-A-C-A-A-G-G-...-C-C-U-G-C-U-U-U-C-G-C-U-OH(3'). In addition, the same sequences were present at the 3' and 5' termini of the viral polymerase genes (P1, P2, and P3) from which these DI RNAs originate. These results indicate that DI RNAs of influenzing virus are formed by an internal deletion of the genomic RNA.     

DNA-directed in vitro synthesis of proteins involved in bacterial transcription and translation.

The in vitro synthesis of elongation factor (EF)-Tu (tufB), the beta beta' subunits of RNA polymerase, ribosomal proteins L10 and L12 directed by DNA from the transducing phage lambda rifd 18, EF-Tu (tufA), EF-G, and the alpha subunit of RNA polymerase directed by DNA from the transducing phage lambda fus3 has been investigated in a crude and a partially defined protein-synthesizing system. Proteins L10 and L12 are synthesized in the partially defined system almost as well as in the crude system. However, the synthesis of EF-Tu, EF-G, and the alpha and beta beta' subunits of RNA polymerase is far less efficient in the partially defined system. An active fraction that stimulates the synthesis of these latter proteins has been obtained by fractionation of a high-speed supernatant on DEAE-cellulose. Because previous studies showed that this fraction (1 M DEAE salt eluate) contains a protein, called L factor, that stimulates beta-galactosidase synthesis in vitro, L factor was tested for activity. Although L factor stimulates the synthesis of the beta beta' subunits, it has little or no effect on the in vitro synthesis of the other products studied. In the present experiments, the ratio of L12/L10 and of EF-Tu (tufA)/EF-G formed is 4-6. These values are consistent with in vivo results.     

Initiator RNA in Discontinuous Polyoma DNA Synthesis

During replication of polyoma DNA in isolated nuclei, RNA was found attached to the 5' ends of growing progeny strands. This RNA starts with either ATP or GTP and can be labeled at its 5' end with (32)P from beta-labeled nucleotides. Digestion of progeny strands with pancreatic DNase released (32)P-labeled RNA that, on gel electrophoresis, gave a distinct peak in the position expected for a decanucleotide. We believe that this short RNA is involved in the initiation of the discontinuous synthesis of DNA and propose the name "initiator RNA" for it. The covalent linkage of initiator RNA to 5' ends of growing DNA chains was substantiated by the finding that (32)P was transferred to ribonucleotides by alkaline hydrolysis of purified initiator RNA obtained by DNase digestion of polyoma progeny strands synthesized from [alpha-(32)P]dTTP. While initiator RNA was quite homogeneous in size, it had no unique base sequence since digestion with pancreatic RNase of initiator RNA labeled at its 5' end with (32)P released a variety of different [(32)P]oligonucleotides. The switch from RNA to DNA synthesis during strand elongation may thus depend on the size of initiator RNA rather than on a specific base sequence.     

In vitro regulation of DNA-dependent synthesis of Escherichia coli ribosomal protein L12.

The DNA of the transducing phage lambdarifd18 contains, among others, the genes for the ribosomal proteins L11, L1, L10, and L12 and the beta and beta' subunits of RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). In a coupled in vitro protein-synthesis system, lambdarifd18 DNA directs the synthesis of about four to five molecules of L12 per molecule of L10. This is consistent with the finding that there are four copies of L12 per ribosome. The ratio of L12/L10 was also examined from an EcoRI fragment of lambdarifd18 that contains the L10 gene and about 50% of the L12 gene. A significantly lower ratio of truncated L12/L10 was observed compared to the intact phage. The binding of RNA polymerase to various lambdarifd18 DNA restriction fragments was used to locate possible promoter sites. These binding experiments suggest that the beta and beta' subunits of RNA polymerase are cotranscribed with at least ribosomal protein L12 and, also, that there may be an additional promoter site for the L12 gene within the structural gene for L10.     

Nucleotide sequence homology at the 3'' termini of RNA from vesicular stomatitis virus and its defective interfering particles.

Vesicular stomatitis virus (VSV) and defective interfering (DI) particle RNAs were labeled at their 3' ends by using RNA ligase and cytidine 3',5'-bis[32P]phosphate. The RNAs were subjected to partial digestion with alkali and analyzed by oligonucleotide fingerprinting in two dimensions. VSV and DI particle RNAs have complete sequence homology for the first eight bases from the 3' end. The following four positions contain three mismatched nucleotides in which guanosine residues in one strand are replaced by uridine residues in the other. There is again complete homology for the next five bases (positions 13-17). The locations of purine residues within the sequence were confirmed by partial digestion with RNase T1 and RNase U2 and separation by size on 20% acrylamide gels. The latter method also indicated that sequences of VSV and DI particle RNAs diverge beyond the 18th nucleotide from the 3' termini.     

X-ray structure of a dinucleoside monophosphate A2''p5''C that contains a 2''-5'' link found in (2''-5'')oligo(A)s induced by interferons: single-stranded helical conformation of 2''-5''-linked oligonucleotides.

In order to understand why DNA and RNA have the 3'-5' and not the 2'-5' link and to delineate the stereochemistry of the 2'-5' phosphodiester links, we crystallized and carried out a very accurate x-ray diffraction analysis of A2 p5'C, an analog of A2' p5'A. Contrary to numerous reports in the literature that conclude that the tendency for 2'-5' nucleotides to stack intramolecularly is stronger than for 3'-5' counterparts, we find hardly any intramolecular base stacking for this molecule but find an intramolecular "stacking" of the ribose oxygen-4' of cytidine on top of the adenine ring. Although A2' p5'C shows the standard conformational features usually found for 3'-5' nucleotides, the overall stereochemistry of 2'-5' nucleotides is quite different because the 2' link orients the backbone inwards to the bases unlike the 3' and 5' links that orient it away from the bases. With the conformational features found for A2' p5'C, it is possible to build a very compact right-handed single-stranded helix but not a double helix. Such a preference for single-stranded helices may be the reason for the absence of 2'-5' bonds in DNA and RNA even though the 2'-5' bonds are formed more readily then 3'-5' bonds.     

Epstein-Barr virus RNA VII: size and direction of transcription of virus-specified cytoplasmic RNAs in a transformed cell line

At least three separate regions of the Epstein-Barr virus (EBV) genome encode RNA in a cell line that is growth transformed and nonpermissively infected with EBV. Six polyadenylylated cytoplasmic RNAs have been identified from these three regions. An abundant RNA 3.0-3.1 kilobases (kb) long is encoded by DNA of the internal reiteration, IR, and DNA that maps at 25.7-30 megadaltons. A second, abundant, 2.9-kb RNA is primarily encoded by DNA at 110-03 megadaltons but probably has a 3' end to the left of 110 megadaltons. A third, abundant, 3.7-kb RNA is largely encoded by DNA at 63-66 megadaltons and has a 5' end to the left of 63 megadaltons. A less-abundant 1.5-kb RNA is also encoded by IR. The least-abundant polyadenylylated RNAs identified are 2.3 and 2.0 kb. These RNAs have 3' ends mapping of 5-7 megadaltons and 5' ends mapping to the right of 7 megadaltons. The data suggest that there may be two additional polyadenylylated cytoplasmic RNAs, a 3-kb RNA mapping at 26.2-30 megadaltons and a minor RNA mapping at 102-110 megadaltons. An abundant 0.16-kb nonpolyadenylylated RNA is also present in the cytoplasm of IB-4 cells. This RNA precipitates from the cytoplasm in the presence of high concentrations of magnesium, indicating that it is complexed with protein or polyribosomes.