Abstract: The cell-free synthesis of three major proteins from virion RNA of nondefective Rous sarcoma virus (RSV), but not from RNA of transformation-defective deletion mutants, has been observed. The apparent molecular weights of these transformation-specific proteins are approximately 60,000 (60K), 25K, and 17K. Tryptic maps of methionine-containing peptides revealed the 17K, 25K, and 60K proteins to be overlapping in sequence. However, only partial homology was observed between the 17K, 25K and 60K proteins synthesized from Schmidt-Ruppin strain, subgroup D, RSV RNA and those synthesized from Prague strain, subgroup B, RSV, RNA. About half of the methionine peptides in the Schmidt-Ruppin strain, subgroup D, 60K protein were shared with the Prague strain, subgroup D, 60K protein, and the rest were distinct to each. The virion RNAs coding for the 60K, 25K, and 17K proteins were found to be polyadenylated and to sediment with maximal mRNA activity at about 23, 19 to 20, and 18S, respectively. In addition, transformation-specific proteins with molecular weights of 39K and 33K were observed by in vitro synthesis. These proteins are also related to the 60K, 25K, and 17K proteins and were synthesized from polyadenylated RSV RNA of approximately 21 to 22S. RNase T1-resistant oligonucleotides were analyzed in parallel, and the src-specific oligonucleotides were found to be first present in equimolar amounts in those gradient fractions sedimenting at 21 to 22S. Our data suggest that synthesis of the 60K protein is initiated near the 5' terminus of the src gene, whereas the 39K, 33K, 25K, and 17K proteins are initiated internally in the src gene. All of these proteins appear to be initiated independently, but they may have a common termination site.

Abstract: A rapid chemical procedure has been developed and used for the synthesis of 29 oligodeoxyribonucleotides to build synthetic genes for human insulin. The gene for insulin B chain, 104 base pairs, and the one for A chain, 77 base pairs, were designed from the amino acid sequence of human polypeptides. They bear single-stranded cohesive termini for the EcoRI and BamHI restriction endonucleases and are designed to be inserted separately into a pBR322 plasmid. The synthetic fragments, deca- to pentadecanucleotides, were synthesized by a block phosphotriester method with trinucleotides as building blocks. Final purification was by high-performance liquid chromatography. All 29 oligonucleotides were pure and had the correct sequences.

Abstract: Previous studies by Guntaka et al. have shown that the unintegrated DNA intermediates of avian RNA tumor virus replication can be readily isolated from cultures of the quail tumor line QT-6 at 1 day after infection. The intermediates include double-stranded linear and covalently closed circular DNA species. Using the analysis procedure of Southern together with previously obtained information regarding the sites of action of certain restriction endonucleases on avian sarcoma virus DNA, we have further characterized the viral DNA intermediates. Evidence is presented that, relative to the RNA genome, most of the linear species possess a direct terminal sequence redundancy equivalent to 0.5 X 10(6) +/- 0.3 X 10(6) daltons of double-stranded DNA. Some of the circular forms also possess a sequence redundancy of 0.21 X 10(6) +/- 0.03 X 10(6) daltons.

Abstract: Reverse phase high-pressure liquid chromatography (HPLC) using columns containing microparticulate materials with bonded octadecyl groups has been developed as a rapid and efficient method for the separation of nucleosides, nucleotides, and, in particular, of protected oligonucleotides which are standard intermediates in the stepwise synthesis of deoxyribopolynucleotides. Reported are extensive studies of the influence of the different purine and pyrimidine bases, of protecting groups, of the phosphate groups, and of the chain lengths of oligonucleotides on their retention on such columns. Further, the application of HPLC in the stepwise synthesis of an oligonucleotide, d(G-G-A-A-G-C-T-T-A-A-C), has been described. The methods, which are herein described, lend themselves to separations on a preparative scale and effect a marked reduction (up to 50%) in the time required for the synthesis of oligonucleotides.

Abstract: A SERIES of 2–5 linked oligoadenylic acid triphosphate (2–5A) inhibitors of cell-free protein synthesis are formed from ATP by an enzyme (2–5A synthetase) activated in interferon-treated cell extracts or rabbit reticulocyte lysates by double-stranded RNA1–4. The major active species is the trimer, pppA2′p5′A2′p5′A. Cell-free protein synthesis is inhibited by subnanomolar concentrations of 2–5A and this inhibition seems to be mediated at least in part, by a nuclease which degrades mRNA5. However, 2–5A, the nuclease and the inhibition of protein synthesis are all unstable in cell-free systems. 2–5A is rapidly degraded in such systems prepared from control (or interferon-treated) cells, nuclease activity is transient and in the absence of a 2–5A regenerating system protein synthesis resumes if fresh mRNA is added6,7. Extracts derived from cells that have never been treated with interferon, therefore, have mechanisms for the synthesis of 2–5A (rabbit reticulocytes) and for responding to and degrading 2–5A. Thus, the 2–5A system, in addition to any role it has in the antiviral action of interferon, may also be involved in the regulation of normal cell growth and development. The extraordinary potency of 2–5A and its instability in cell-free systems makes the task of detecting 2–5A in intact cells very difficult. As an alternative we have studied the effect of exogenous 2–5A on protein synthesis in intact cells, using a recently described method for making animal cells reversibly permeable to small molecules8. Here we report that pppA2′p5′A2′p5′A and related 2′–5′ linked oligonucleotides inhibit protein synthesis in hypertonically treated BHK–21 cells.

Abstract: Evidence is presented for a previously undiscovered protein that is associated with the virion of cowpea mosaic virus (CPMV). It co-purified with the viral RNA. The bond between this genomeassociated protein and the viral RNA is stable to treatments that generally disrupt non-covalent bonds, including heating in sodium dodecylsulfate solution and extraction with phenol. The protein has been characterized by peptide mapping and gel electrophoresis; its molecular weight was estimated to be ∼5000 from mobility in gels. One of the zones seen on autoradiographs of gel electrophoresis profiles of ribonuclease T1-digested 32P-labeled CPMV RNAs was designated oligonucleotide-proteinT1. Only this zone was found when the digestion was repeated using iodinated RNA. This zone was proteasesensitive, while other T1-generated oligonucleotides were not. The oligonucleotide-proteinT1 was not a substrate for polynucleotide kinase, though other ribonuclease-T1-genearted oligonucleotides did accept a 5′-terminal phosphoryl group under the same conditions. Since the intact CPMV RNAs also were not substrates for polynucleotide kinase and lack any of the usual 5′-ends [Klootwijk, J., Klein, I., Zabel, P. & van Kammen, A., Cell, 11, 73–82 (1977)], this substance is probably (an apparently covalent) protein-oligonucleotide complex derived from the 5′-end of CPMV RNAs.

Abstract: Current studies were undertaken to compare the genomes of Kirsten murine sarcoma virus (Ki-MuSV), Harvey murine sarcoma virus (Ha-MuSV), and the replication-defective endogenous rat virus to understand the function of these viral RNAs. Genome organization and sequence homology were studied by fingerprinting large RNase T1-resistant oligonucleotides and by cross-protecting homologous oligonucleotides against RNase A and T1 digestion with complementary DNA prepared from each of the other viral RNA. Ki-MuSV and Ha-MuSV were found to share an extensive series of rat-derived oligonucleotides begining ca. 1 kilobase (kb) from the 3' end and extending to within 1.5 kb of the 5'end of Ki-MuSV RNA. The total map distance covered in ca. 5.5 kb. The eight oligonucleotides covering the 1.5 kb at the 5' end of Ki-MuSV RNA were not found in Ha-MuSV RNA. Five out of these eight oligonucleotides, however, could be designated with certainty to be of rat virus origin. Since Ha-MuSV is 6.5 kb in size and Ki-MuSV is 8 kb in size, the major difference between them is the 1.5 kb from the replication-defective endogenous rat virus sequences at the 5' end of Ki-MuSV not present in Ha-MuSV. Consistent with the difference in the genome structure, these two sarcoma viral RNA'S yielded distinct major translation products in cell-free systems, I.E., A 50,000-dalton polypeptide (P50) from Ki-MuSV and a 22,000-dalton polypeptide (p22) from Ha-MuSV. These polypeptides may provide the necessary protein makers for identifying in vivo virus-coded proteins.

Abstract: A simple procedure is described to prepare nucleoside 3'(2'),5'-bisphosphates from the corresponding nucleosides with the use of pyrophosphoryl chloride. This method is rapid, gives nearly quantitative yields and, most importantly, can be used for a variety of nucleosides with base and sugar modifications. Since 3',5'-bisphosphates are donors in the T4 RNA ligase reaction, a single residue can be enzymatically attached to the 3' end of oligoribonucleotides. By these procedures, five different ring-modified nucleosides and one sugar-modified nucleoside were incorporated onto the 3' end of (Ap)3C. In two cases, an additional step of synthesis with RNA ligase resulted in the modified nucleotide being located in an internal position in the oligonucleotide. Thus, a general method for the synthesis of oligoribonucleotides containing modified nucleosides is outlined. Since many of the modified nucleosides are fluorescent, oligomers containing them should be useful in a variety of physical and biochemical studies.

Abstract: The sequence of the 5′ noncoding region of tobacco mosaic virus RNA has been determined. The noncoding region is 68 nucleotides long and is unusual in that it contains no internal guanosine residues. The long T1 oligonucleotide containing the guanosine-free tract was isolated from a T1 ribonuclease digest of tobacco mosaic virus RNA and sequenced by labelling techniques in vitro using polynucleotide kinase. The guanosine-free tract is terminated by the first potential initiation codon in the RNA molecule and several lines of evidence suggest that this AUG triplet is operational in initiating viral protein synthesis (see following paper). The 5′-noncoding region cannot base-pair extensively with the 3′-terminal sequence of 18-S ribosomal RNA from rabbit reticulocytes.

Abstract: Adenovirus type 2-infected HeLa cells were labeled with 32PO4 during the period 14 to 17 h postinfection. Viral mRNA's with polyadenylic acid were isolated by polyuridylic acid Sepharose chromatography and fractionated according to size by electrophoresis through an acrylamide-agarose slab gel. Messenger bands were eluted and partially degraded with alkali. RNA fragments from each band that contain polyadenylic acid were isolated by polyuridylic acid Sepharose chromatography and fingerprinted two-dimensionally after T1 RNase digestion. Three bands, with mobilities of approximately 26S, 21S, and 18S, shared two large characteristic T1 oligonucleotides in common in the fingerprints of their 3'-terminal sequences. These oligonucleotides were mapped with a Hpa II restriction fragment of adenovirus type 2 DNA with coordinates 49-50.2. We conclude that the three mRNA's are coterminal in sequence at their 3' ends and overlap at internal positions. Implications for the protein-coding potential of these mRNA's and the mechanisms of adenovirus tyep 2 late RNA processing are discussed.

Abstract: The polycytidylic acid [poly(C)] tract in foot and mouth disease virus RNA has been located about 400 nucleotides from the 5' end of the RNA by analysis of the products from the digestion of the RNA with RNase H in the presence of oligodeoxyguanylic acid [oligo(dG)]. This treatment produces a small fragment (S) containing the small protein covalently linked to the RNA and a large fragment (L) that migrates faster than untreated RNA on low-percentage polyacrylamide gels, lacks the poly(C) tract as shown by RNase T1 digestion and oligo(dG)-cellulose binding, and is no longer infective. Polyacrylamide gel electrophoresis of fragment S suggests that it is about 400 nucleotides long, in agreement with the size estimated from the proportion of radioactivity in the fragment. Analysis of the RNase T1 digestion products of S shows that it contains only those oligonucleotides mapping close to the poly(C) tract that is situated near the 5' end of the virus RNA.

Abstract: The principal RNA species isolated from labeled preparations of the arenavirus Pichinde usually include a large viral RNA species L (apparent molecular weight = 3.2 X 10(6)), and a smaller viral RNA species S (apparent molecular weight = 1.6 X 10(6)). In addition, either little or considerable quantities of 28S rRNA as well as 18S rRNA can also be obtained in virus extracts, depending on the virus stock and growth conditions used to generate virus preparations. Similar RNA species have been identified in RNA extracted from Tacaribe and Tamiami arenavirus preparations. Oligonucleotide fingerprint analyses have confirmed the host ribosomal origin of the 28S and 18S species. Such analyses have also indicated that the Pichinde viral L and S RNA species each contain unique nucleotide sequences. Viral RNA preparations isolated by conventional phenol-sodium dodecyl sulfate extraction often have much of their L and S RNA species in the form of aggregates as visualized by either electron microscopy or oligonucleotide fingerprinting of material recovered from the top of gels (run by using undenatured RNA preparations). Circular and linear RNA forms have also been seen in electron micrographs of undenatured RNA preparations, although denatured viral RNA preparations have yielded mostly linear RNA species with few RNA aggregates or circular forms.

Abstract: A comparison has been made by oligonucleotide analysis of three fractions of HeLa cell hnRNA: (1) the "snap-back" fraction (ds-hnRNA, 5% of the total); (2) the fraction that self-anneals during prolonged incubation (25% of total); and (3) the fraction that hybridizes most rapidly to an excess of HeLa cell DNA (rep-hnRNA, 10% of the total). T1 fingerprints of each of these hnRNA fractions were similar to one another and featured the largest T1 oligonucleotides of known sequence previously isolated from ds-hnRNA (Robertson, H.D., et al. (1977) J. Mol. Biol. 115, 571--590; Jelinek, W. (1977 J. Mol. Biol. 115, 591--602). When hybridized to DNA either in solution or immobilized on filters, the isolated ds-hnRNA and the rep-hnRNA fractions showed similar hybridization kinetics in the COt range of "intermediate" repetitive DNA sequences; the ds-hnRNA and the rep-hnRNA also self-annealed to equal extents in the absence of any DNA. DNA of all buoyant density classes contained the T1 oligonucleotides diagnostic of the ds-hnRNA and the rep-hnRNA. While hnRNA is rich in inverted repeated sequences, cytoplasmic mRNA contains far fewer such sequences.

Abstract: The avian retrovirus RNA-directed DNA polymerase contains an activity that is capable of removing hydrogen bonds from duplex nucleic acid molecules. This "unwinding-like" activity appears to be specific in its action, affecting RNA.DNA and DNA.DNA duplex molecules but not RNA.RNA duplexes. Studies with defined RNA.DNA hybrid molecules (e.g., Rous sarcoma virus RNA and complementary DNAs representing specific regions of the Rous sarcoma virus genome) and DNA.DNA duplexes indicate that, although this activity can remove a portion of the hydrogen bonds from these double-stranded structures, complete separation of complementary strands is not accomplished. The unwinding-like activity exhibits sensitivities to temperature and monovalent and divalent cation concentrations. It can also remove a specific large oligonucleotide from the 5' end of the viral genome subsequent to RNase H hydrolysis of viral RNA complexed to DNA present at that terminus. This reverse transcriptase-associated unwinding-like activity is discussed with respect to recently proposed models of retrovirus proviral DNA synthesis.

Abstract: Termination of RNA synthesis with 3'-O-Methylnucleoside 5'-triphosphates have been studied using E. coli RNA polymerase holoenzyme and poly [d(A-T)] as well as unfractionated T7 D delta III DNA as templates. It was shown that the termination can be used for DNA sequencing. A sequence of a part of RNA synthesized from AI promoter of the DNA have been determined. Syntheses of four 3'-O-Methylnucleoside 5'-triphosphates are described.

Abstract: We have studied the topography of 16S RNA in the inactive form of the 30S ribosomal subunit (Ginsburg, I., et al. (1973) J. Mol. Biol. 79, 481), using the guanine-specific reagent kethoxal. Oligonucleotides surrounding reactive guanine residues were isolated and quantitated by means of diagonal electrophoresis and sequenced. Comparison of these results with experiments on active or reactivated subunits reveals the following: (1) Most of the sites which are reactive in active 30S subunits are much more reactive (average 13-fold) in inactive subunits. Upon reactivation, these sites return to a less reactive state. Thus, a reversible increase in accessibility of specific 16S RNA sites parallels the reversible loss of protein synthesis activity of 30S subunits. (2) The number of kethoxal-reactive sites in inactive subunits is about twice that of active subunits. The nucleotide sequences and locations of the additional accessible sites in inactive subunits have been determined. (3) Sites that can be located in the 16S RNA sequence are distributed throughout the RNA chain in inactive subunits, in contrast to the clustering observed in active subunits. (4) The sites of kethoxal substitution are single stranded. Yet, of the 30 sites that can be located, 23 were predicted to be base paired in the proposed secondary structure model for 16S RNA (Ehresmann, C., et al. (1975), Nucleic Acids Res. 2, 265).