Genetic structure, transforming sequence, and gene product of avian sarcoma virus UR1

We analyzed the genetic structure and gene products of the newly isolated avian sarcoma virus UR1, which recently has been shown to be replication defective and to contain no sequences homologous to the src gene of Rous sarcoma virus. The sizes of the genomic RNAs of UR1 and its associated helper virus, UR1AV, were determined to be 29S and 35S (5.9 and 8.5 kilobases), respectively, by gel electrophoresis and sucrose gradient sedimentation. RNase T1 oligonucleotide mapping of purified viral RNAs indicated that UR1 RNA contains eight unique oligonucleotides in the middle of the genome and shares four 5'-terminal and three 3'-terminal oligonucleotides with UR1AV RNA. The unique sequences of UR1 and Fujinami sarcoma virus were found to be closely related to each other by molecular hybridization of UR1 RNA with DNA complementary to the unique sequence of Fujinami sarcoma virus RNA, but minor differences were found by oligonucleotides fingerprinting. In the regions flanking the unique sequences, UR1 and Fujinami sarcoma viral RNAs contain distinct oligonucleotides, which are shared with oligonucleotides of the respective helper viral RNAs. Cell transformed with UR1 produce a single 29S RNA species which contains a UR1 unique sequence; this species is most likely the mRNA coding for the transforming protein. In UR1-transformed cells, a phosphoprotein fo 150,000 daltons (p150) was detected by immunoprecipitation with antiserum against gag proteins. p150 was associated with a protein kinase activity that was capable of phosphorylating p150 itself, immunoglobulin G of antiserum, and a soluble substrate, alpha-casein. This enzyme transferred phosphate exclusively to tyrosine residues of substrates in vitro, but p 150 labeled in vivo with 32P contained both phosphoserine and phosphotyrosine. The in vitro kinase reaction was not affected by the presence of cyclic AMP or cyclic GMP and strongly preferred Mn2+ over Mg2+. Thus, the properties of UR1 protein are almost identical to those of Fujinami sarcoma virus protein.     

Nucleoside triphosphate phosphohydrolase associated with cytoplasmic polyhedrosis virus.

Nucleoside triphosphate phosphohydrolase [EC 3.6.1.15] activity was found to be included in silkworm cytoplasmic polyhedrosis (CP) virus, which synthesizes mRNA carrying the 5'-terminal modification. This enzyme releases orthophosphate from the gamma-position in a nucleoside triphosphate, leaving nucleoside diphosphate. The rate of hydrolysis of ATP is faster than that of any other ribonucleoside triphosphate. Deoxy ATP is hydrolyzed rather faster than ATP. However, polynucleotides carrying triphosphate at the 5'-terminus, that is, 4S RNA which was synthesized by E. coli RNA polymerase [EC 2.7.7.6] using calf thymus DNA as a template, and the phage Q beta RNA (30S), are not effective substrates for this enzyme. Although the CP virion loses the viral genome and one kind of protein component on proteolytic treatment with pronase, the partially degraded virion still retains phosphohydrolase activity. The phosphohydrolase must therefore be associated firmly with the virion. This enzyme does not require the presence of nucleic acid for its function. Phosphohydrolysis of ATP by this enzyme activity represents a first step in the synthesis of the 5'-terminal modified mRNA of CP virus.     

Origin and degradation of the RNA primers at the 5' termini of nascent DNA chains in Bacillus subtilis

We had earlier characterized the nascent DNA synthesized in permeable cells of Bacillus subtilis in the presence of 5-mercurideoxycytidine triphosphate and 2',3'-dideoxyATP as being substituted at its 5' end with a ribonucleotide moiety of the sequence pApG(pC)1-2 DNA. In this paper, we examine the origin and turnover of the DNA-linked ribonucleotide and its relationship to DNA replication. At least 50% of the RNA-linked nascent DNA chains served as guanylate acceptors when incubated with GTP and the eukaryotic capping enzyme, indicating the presence of 5'-terminal di- or triphosphate groups and suggesting that the RNA moiety is synthesized de novo and is not a degradation product. In nascent DNA produced without limitation of chain growth by dideoxyATP, the degree of terminal ribonucleotide substitution was reduced by 50%, consistent with a linkage between RNA primer removal and DNA chain growth. Such a relationship was demonstrated directly by examining the RNA primer content of nascent DNA synthesized in the absence of dideoxyATP as a function of DNA chain length. As the DNA size increased from 40 to 200 nucleotide residues, the extent of RNA substitution declined from 80% to nearly 0%. Endgroup analysis showed that the loss of RNA was accompanied by a gradual shift from predominantly adenylate residues to 5'-terminal guanylate, consistent with a stepwise removal of ribonucleotides from the 5' end. Evidence that the nascent mercurated DNA synthesized under our experimental conditions was indeed a replicative intermediate came from the study of the time course of DNA chain growth and pulse-chase experiments. In the presence of the DNA ligase inhibitor NMN, mercurated DNA accumulated in two size classes with average length of approximately 750 and 8000 nucleotide residues, presumably representing the mature size of intermediates in discontinuous DNA synthesis. Comparison with the DNA size range at which the loss of the 5'-terminal RNA moiety occurred (40 to 200 residues) indicated that the processing of RNA primers occurred at an early stage during DNA chain elongation, and that moderate size intermediates in discontinuous DNA replication (greater than 200 nucleotides) have already lost their RNA primers.     

Terminal nucleotide sequences of 17-S ribosomal RNA and its immediate precursor 18-S RNA in yeast.

The 5' and 3'-terminal nucleotide sequences of 17-S rRNA and its immediate precursor 18-S RNA from the yeast Saccharomyces carlsbergensis have been analysed. Identification of the terminal oligonucleotides, as present in Ti ribonuclease digests, was performed by diagonal procedures. The major (molar yield 0.9) 5'-terminal oligonucleotide (molar yield 0.15) with the overall composition pU (U2,C2)G was observed. 18-S precursor RNA was found to contain the same 5'-terminal sequences as 17-S rRNA. However, the 3'-terminal sequences of the two types of RNA appeared to be different. The 17-S rRNA yields the oligonucleotide A-U-C-A-U-U-AOH while at least half of the 18-S RNA molecules contain the sequence U-U-U-C-A-A-U-AOH. In addition 18-S RNA yields several minor 3'-terminal oligonucleotides which appear to be structurally related to the major 3'-terminal sequence. These results demonstrate that the extra nucleotides in 18-S RNA relative to 17-S RNA are located exclusively at the 3'-terminus of the 18-S RNA molecule. The possibility that the 3'-terminal nucleotide sequence of 18-S RNA plays a role in the maturation process is discussed.     

Selectivity of RNA chain initiation in vitro. 1. Analysis of RNA initiations by two-dimensional thin-layer chromatography of 5'-triphosphate-labeled oligonucleotides.

A method for the rapid and quantitative analysis of 5'-terminal oligonucleotides of RNAs made in vitro is described. The method involves synthesis of RNA in the presence of [gamma-32P]ATP or GTP, isolation of the RNA, and digestion with T1 or pancreatic ribonucleases to release labeled 5'-triphosphate termanated oligonucleotides. The oligonucleotides are then subjected to chromatography on a polyethyleniminecellulose thin-layer system using 2 M LiCl, 0.01 M EDTA (pH 6.5) in the first dimension and 1.5 M LiCl, 1.8 M formic acid, 0.005 M EDTA (pH 2.0) in the second. RNAs made with E. coli RNA polymerase and lambdacb2, T7, T4, and adenovirus 2 DNA yield characteristic fingerprint patterns. The utility of this method in studying selectivity of in vitro RNA chain initiation is discussed.     

Initiation of adenovirus DNA replication: detection of covalent complexes between nucleotide and the 80-kilodalton terminal protein

We have previously shown that the 5'-terminal deoxycytidine residue of each nascent adenovirus 5 DNA strand synthesized in vitro is covalently linked to the 80-kilodalton (kd) terminal protein precursor via a phosphodiester bond to a serine residue in the protein. When extracts prepared from adenovirus 5-infected cells are incubated with [alpha-33P]dCTP as the only added deoxynucleoside triphosphate, complexes consisting of nucleotide covalently linked to the 80-kd protein can be detected. The nucleotide moieties present in such complexes include d(pC) and d(pCpA), the 5'-terminal nucleotide and dinucleotide of adenovirus 5 DNA, respectively, as well as some longer oligonucleotides. The formation of these complexes requires the presence of adenovirus DNA containing the attached 55-kd terminal protein and ATP. Extracts from H5ts125-infected cells which are defective in DNA replication catalyze complex formation to the same extent as extracts prepared from wild-type infected cells; thus, the presence of the adenovirus-coded 72-kd DNA-binding protein is apparently not required. Most, if not all, of the 80-kd protein-nucleotide complexes that are formed are noncovalently bound to the input viral DNA. These observations are consistent with the protein-priming model for the initiation of adenovirus DNA replication.     

In vitro RNA transcription by the New Jersey serotype of vesicular stomatitis virus. II. Characterization of the leader RNA.

The New Jersey serotype of vesicular stomatitis virus (VSV) was able to synthesize a small RNA (leader RNA) approximately 70 bases in length similar to the leader RNA synthesized in vitro by the genetically distinct Indiana serotype of VSV. Also, the New Jersey leader RNA contained the same 5'-terminal sequence, ppA-C-G, as the Indiana leader RNA and had a very similar base composition, with 42% AMP, 16% CMP, 18.6% GMP, and 23.4% UMP. The 3'-terminal sequence of the VSV New Jersey genome RNA was detemined and found to contain the sequence- Py-G-UOH, again the same as that of the Indiana serotype of VSV. Evidence that the New Jersey leader RNA is transcribed from the 3' end of the genome RNA was obtained from the fact that it can protect the 3'-terminal base of [3H]borohydride-labeled New Jersey genome RNA from RNase digestion. Although the New Jersey and Indiana leader RNAs were similar in many respects, they were unable to form RNase-resistant hybrids when annealed to heterologous genome RNA.     

Sequence determination of 5'-terminal and 3'-terminal T1 oligonucleotides of 18-S ribosomal RNA of a mouse cell line (L 5178 Y).

The 5' and 3'-terminal oligonucleotides of 18-S ribosomal RNA of L 5178 Y (a mouse cell line) obtained after total T1 ribonuclease hydrolysis were isolated by a diagonal procedure. They were localized on the fingerprint of T1-ribonuclease-hydrolysed 18-S RNA. These two oligonucleotides were partially hydrolysed by snake venom and spleen phsophodiesterases and resulting products were fractionated bidimensionally. Their base compositions were determined by total hydrolysis with piperidine or snake venom phosphodiesterase. From these results the following sequences were deduced: pU-A-C-C-U-G for the 5'-terminal oligonucleotide and G-A-U-C-A-U-U-Aoh for the 3'-terminal oligonucleotide. Quantitative studies indicated that these sequences represent at least 70% for the 5' oligonucleotide and 85% for the 3' oligonucleotide of the terminal sequences of the 18-S RNA.     

Characterization and translation of methylated and unmethylated vesicular stomatitis virus mRNA synthesized in vitro by ribonucleoprotein particles from vesicular stomatitis virus-infected L cells.

Ribonucleoprotein particles isolated from extracts of vesicular stomatitis virus (VSV) -infected L cells synthesized in vitro four classes of polyadenylated RNA sedimenting at 29S, 19S, 17S, and 13S. When synthesized in vitro in the presence of the methyl donor S-adenosyl methionine, these RNA species contained the following 5'-terminal structures: (i) m7G5ppp5'AmpAp(70%) ; (ii) m7G5'ppp5'AmpAmpNp (20%) and (iii) pppAp (10%). In the presence of the methylation inhibitor S-adenosylhomocysteine, however, the mRNA contained the 5'-terminal structures G5'ppp5'Ap (80%) and pppAp (20%). The mRNA's synthesized in vitro were translated in the homologous ascites and the heterologous wheat embryo cell-free systems. In both, the products were shown by sodium dodecyl sulfate gel electrophoresis and by immunoprecipitation to contain all five viral proteins, L, G, N, NS, and M. The presumed precursor to the G protein (G*) was also identified by fingerprint analysis. Methylated VSV mRNA was more active in protein synthesis than unmethylated mRNA in both the ascites system and the wheat embryo systems. Addition of S-adenosylmethionine stimulated translation of unmethylated mRNA in the wheat embryo but not in the ascites extract. S-adenosylhomocysteine, however, by preventing mRNA methylation inhibited the translation of unmethylated VSV mRNA in both systems. The mRNA methylating activity present in wheat embryo S-30 extracts was recovered in the ribosome-free supernatant fraction (S-150) and was insensitive to the protein synthesis inhibitor pactamycin.     

In vitro RNA transcription by the New Jersey serotype of vesicular stomatitis virus. I. Characterization of the mRNA species.

The in vitro RNA synthesis by the virion-associated RNA polymerase of vesicular stomatitis virus (VSV), New Jersey serotype, was compared with that of the serologically distinct Indiana serotype of VSV. The New Jersey serotype of VSV synthesized five distinct mRNA species in vitro, three of which were smaller than the corresponding species synthesized by the Indiana serotype of VSV. These included the mRNA's coding for the G, M, and NS proteins. By hybridization experiments, virtually no sequence homology was detected between the mRNA's of the two serotypes. Despite this lack of overall homology, the 12 to 18S mRNA species of both serotype contained a common 5'-terminal hexanucleotide sequence, G(5')ppp(5')A-A-C-A-G. The signicance of this finding in light of specific interactions between the two serotypes of VSV in vivo is discussed.     

Synthesis of oligodeoxyribonucleotides containing oleylamine moieties]

A method for directional introduction of oleylamine residues to any position of oligodeoxyribonucleotides during their automated synthesis was developed. The presence of oleylamine residues in 3'- or 5'-terminal nucleotides was shown to have no effect on the thermodynamic stability of DNA duplexes formed by such oligonucleotides and the complementary sequences. The rate of the snake venom phosphodiesterase hydrolysis of oligonucleotides containing oleylamine residues in the 3'-terminal units was shown to be markedly lower than that of natural oligonucleotides.