Nucleotide sequence of U-2 ribonucleic acid. The sequence of the 5'-terminal oligonucleotide.

The nucleotide sequences were determined for the 5'-oligonucleotides obtained by complete pancreatic RNase digestion (P25) and complete T1 RNase digestion (T27) of U-2 RNA. Complete digestion of oligonucleotide P25 with snake venom phosphodiesterase produced pm3 2,2,7G, pAm, pUm, and pCp in approximately equimolar ratios. Partial digestion of these oligonucleotides with snake venom phosphodiesterase produced -Um-C-Gp and pAm-Um, indicating the sequence of the 3'-terminal portion of the 5'-oligonucleotide is pAm-Um-C-Gp. The 5'-terminal oligonucleotide did not contain a 5'-phosphate and no free nucleoside was released from the 5' end by venom phosphodiesterase digestion. Since free pm3 2,2,7G was released by digestion with nucleotide pyrophosphatase and limited digestion with snake venom phosphodiesterase, this nucleotide is apparently linked to pAm in a pyrophosphate linkage. Mass spectrometry and thin layer chromatography in borate systems showed the ribose of m3 2, 2, 7G contains no 2'O-methyl residue. Moreover, the finding that the ribose of m3 2, 2, 7G was oxidized by NaIO4 and reduced by KB3H4 in intact U-2 RNA rules out other linkages involving the 2' and 3' positions. Accordingly, it is concluded that the structure of the 5'-terminal pentanucleotide of U-2 RNA is(see article).     

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.     

5' -and 3'-terminal nucleotide sequences of Tetrahymena pyriformis 17S rRNA.

Ribonuclease T1 oligonucleotides arising from the 5' and 3' termini of the 17S rRNA of Tetrahymena pyriformis were isolated by the diagonal method of Dahlberg (Dahlberg, J. E. (1968), Nature (London) 220, 548), and their nucleotide sequences were determined. The base sequence of the 3'-terminal fragment is (G)AUCAUUAoh, which is identical to that found in other 17S-18S eucaryotic rRNA species. The nucleotide sequence of the 5'-terminal oligonucleotide is pAACCUGp, which is identical in length to that found in other eucaryotes and shows a partial but significant sequence homology to the 5' RNase TI oligonucleotides isolated from other eucaryotic species.     

Studies on the conformation of the 3' terminus of 18-S rRNA

We have studied the conformation of the 3' end of 18-S RNA from human, hamster and Xenopus laevis cells. The 3'-terminal oligonucleotide in a T1 ribonuclease digest of 18-S RNA from HeLa cells was identified, using a standard fingerprinting method. The sequence (G)-A-U-C-A-U-U-A, established by Eladari and Galibert for HeLa 18-S rRNA, was confirmed. An identical 3' terminus is present in hamster fibroblasts and Xenopus laevis cells. The ease of identification of this oligonucleotide has enabled us to quantify its molar yield relative to several other oligonucleotides, and hence to analyse the 3' terminus by several conformation probes. Its sensitivity to S1 nuclease, limited T1 ribonuclease digestion, bisulphite modification and carbodiimide modification was consistent with the terminal oligonucleotide being in a highly exposed conformation. The m6/2A-m6/2A-C-containing sequence of 18-S rRNA also appears to be in an exposed location on the basis of three of these probes.     

Homologous terminal sequences of the genome double-stranded RNAs of bluetongue virus.

The 3'-terminal sequences of the 10 double-stranded RNA genome segments of bluetongue virus (serotypes 10 and 11) were determined. The double-stranded RNAs were 3' labeled with [5'-32P]pCp and resolved into 10 segments by electrophoresis. After denaturation, the two complementary strands of segments 4 through 10 were resolved into fast- and slow-migrating species by polyacrylamide gel electrophoresis, and their 3' end sequences were determined. Complete RNase T1 digestion of the individual 3'-labeled double-stranded RNA segments yielded two labeled oligonucleotides, one of which migrated faster than the other on 20% polyacrylamide-7 M urea gels. Sequence analyses of the two oligonucleotides of segments 4 through 10 confirmed the corresponding RNA sequence data. For RNA segments 1 through 3 the oligonucleotide analyses gave comparable results. The 3'-terminal sequences of the fast-migrating RNA species were HOCAAUUU. . . ; those of the slow-migrating RNA species were HOCAUUCACA. . . . Similar results were obtained for double-stranded RNA from bluetongue virus serotypes 10 and 11. Beyond the common termini, the sequences for each segment varied considerably.     

Non-ribosomal nucleotide sequences in 7-S RNA, the immediate precursor of 5.8-S ribosomal RNA in yeast.

The topography and the length of the non-ribosomal sequences present in 7-S RNA, the immediate precursor of 5.8-S ribosomal RNA, from the yeast Saccharomyces carlsbergensis were determined by analyzing the nucleotide sequences of the products obtained after complete digestion of 7-S RNA with RNase T1. The results show that 7-S RNA contains approximately 150 non-ribosomal nucleotides. The majority (90%) of the 7-S RNA molecules was found to have the same 5'-terminal pentadecanucleotide sequence as mature 5.8-S rRNA. The remaining 10% exhibited 5'-terminal sequences identical to those of 5.9-S RNA, which has the same primary structure as 5.8-S rRNA except for a slight extension at the 5' end [Rubin, G.M. (1974) Eur. J. Biochem. 41, 197--202]. These data show that the non-ribosomal nucleotides present in 7-S RNA are all located 3'-distal to the mature 5.8-S rRNA sequence. Moreover, it can be concluded that 5.9-S RNA is a stable rRNA rather than a precursor of 5.8-S rRNA. The 3'-terminal sequence of 5.8-S rRNA (U-C-A-U-U-UOH) is recovered in a much longer oligonucleotide in the T1 RNase digest of 7-S RNA having the sequence U-C-A-U-U-U-(C-C-U-U-C-U-C)-A-A-A-C-A-(U-U-C-U)-Gp. The sequences enclosed in brackets are likely to be correct but could not be established with absolute certainty. The arrow indicates the bond cleaved during processing. The octanucleotide sequence -A-A-A-C-A-U-U-C- located near the cleavage site shows a remarkable similarity to the 5'-terminal octanucleotide sequence of 7-S RNA (-A-A-A-C-U-U-U-C-). We suggest that these sequences may be involved in determining the specificity of the cleavages resulting in the formation of the two termini of 5.8-S rRNA.     

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.     

Characterisation of RNA fragments obtained by mild nuclease digestion of 30-S ribosomal subunits from Escherichia coli.

When Escherichia coli 30-S ribosomal subunits are hydrolysed under mild conditions, two ribonucleoprotein fragments of unequal size are produced. Knowledge of the RNA sequences contained in these hydrolysis products was required for the experiments described in the preceding paper, and the RNA sub-fragments have therefore been examined by oligonucleotide analysis. Two well-defined small fragments of free RNA, produced concomitantly with the ribonucleoprotein fragments, were also analysed. The larger ribonucleoprotein fragment, containing predominantly proteins S4, S5, S8, S15, S16 (17) and S20, contains a complex mixture of RNA sub-fragments varying from about 100 to 800 nucleotides in length. All these fragments arose from the 5'-terminal 900 nucleotides of 16-S RNA, corresponding to the well-known 12-S fragment. No long-range interactions could be detected within this RNA region in these experiments. The RNA from the smaller ribonucleoprotein fragment (containing proteins S7, S9 S10, S14 and S19) has been described in detail previously, and consists of about 450 nucleotides near the 3' end of the 16-S RNA, but lacking the 3'-terminal 150 nucleotides. The two small free RNA fragments (above) partly account for these missing 150 nucleotides; both fragments arose from section A of the 16-S RNA, but section J (the 3'-terminal 50 nucleotides) was not found. This result suggests that the 3' region of 16-S RNA is not involved in stable interactions with protein.     

RNA mimetics: oligoribonucleotide N3'-->P5' phosphoramidates.

The synthesis and properties of novel RNA mimetics, oligoribonucleotide N3'-->P5' phosphoramidates, are described. These oligonucleotides contain 3'-aminoribonucleosides connected via N3'-->P5' phosphoramidate linkages, replacing the native RNA O3'-->P5' phosphodiester counterparts. The key monomers 2'-t-butyldimethylsilyl-3'-(monomethoxytrityl)-amino-5'-phospho ramidi tes were synthesized and used to prepare the oligonucleotide phosphoramidates using a solid phase methodology based on the phosphoramidite transfer reaction. Oligoribophosphoramidates are very resistant to enzymatic hydrolysis by snake venom phosphodiesterase. These compounds form stable duplexes with complementary natural phosphodiester DNA and RNA strands, as well as with 2'-deoxy N3'-->P5' phosphoramidates. The increase in melting temperature, Delta T m, was 5-14 degrees C relative to the 2'-deoxy phosphoramidates for decanucleotides. Also, the thermal stability of the ribophosphoramidatehomoduplex was noticeably higher (Delta T m +9.5 degrees C) than that for the isosequential 2'-deoxy phosphoramidate complex. Furthermore, the oligopyrimidine ribo N3'-->P5' phosphoramidate formed an extremely stable triplex with an oligopurine/oligopyrimidine DNA duplex with Delta T m +14.3 degrees C relative to the 2'-deoxy N3'-->P5' phosphoramidate counterpart. The properties of the oligoribonucleotide N3'-->P5' phosphoramidates indicate that these compounds can be used as hydrolytically stable structural and functional RNA mimetics.     

Primary structure of tRNA2Leu of bovine mammary gland. Oligonucleotides of T1-RNAse hydrolysate. Reconstruction of a total nucleotide sequence

The oligonucleotides obtained by digestion of tRNA2Leu from cow mammary gland with T1 RNase were separated by micro-column chromatography on DEAE-cellulose in 7 M urea at pH 7,5 and 3,7, and in addition on Dowex 1 x 2. The digest consisted of 18 individual components, the larger being a tridecanucleotide. Micro-column chromatography of nucleotides on anion-exchanger AG 1 x 8 and nucleosides on Aminex A-6 was used to determine the base composition of the oligonucleotides. The oligonucleotide structure was established using terminal analysis, hydrolysis by pancreatic and U2-RNases and incomplete hydrolysis by snake venom phosphodiesterases. The total primary structure of tRNA2Leu was derived from overlapping fragments isolated after its complete hydrolysis with pancreatic and T1 RNase and using data obtained on S1-nuclease digestion of tRNA. The methods of rapid gel-sequencing were also employed for checking the nucleotide sequence of tRNA2Leu from cow mammary gland.     

Genetic structure and transforming sequence of avian sarcoma virus UR2.

We have recently shown that a newly isolated avian sarcoma virus, UR2, is defective in replication and contains no sequences homologous to the src gene of Rous sarcoma virus. In this study, we analyzed the genetic structure and transforming sequence of UR2 by oligonucleotide fingerprinting. The sizes of the genomic RNAs of UR2 and its associated helper virus, UR2AV, were determined to be 24S and 35S, respectively, by sucrose gradient sedimentation. The molecular weight of the 24S UR2 genomic RNA was estimated to be 1.1 x 10(6), corresponding to 3,300 nucleotides, by gel electrophoresis under the native and denatured conditions. RNase T1 oligonucleotide mapping indicated that UR2 RNA contains seven unique oligonucleotides in the middle of the genome and shares eight 5'- and six 3'-terminal oligonucleotides with UR2AV RNA. From these data, we estimated that UR2 RNA contains a unique sequence of about 12 kilobases in the middle of the genome, and contains 1.4 and 0.7 kilobases of sequences shared with UR2AV RNA at the 5' and 3' ends, respectively. Partial sequence analysis of the UR2-specific oligonucleotides by RNase A digestion revealed that there are no homologous counterparts to these oligonucleotides in the RNAs of other avian sarcoma and acute leukemia viruses studied to date. UR2-transformed non-virus-producing cells contain a single 24S viral RNA which is most likely the message coding for the transforming protein of UR2. On the basis of the uniqueness of the transforming sequence, we concluded that UR2 is a new member of the defective avian sarcoma viruses.     

3'-terminal processing of ribosomal RNA precursors in mammalian cells.

The 3'-terminal structures of ribosomal 28S RNA and its precursors from rat and mouse were analyzed by means of periodate oxidation followed by reduction with 3H-borohydride. 3'-terminal labeled nucleoside derivatives produced by RNase T2 digestion were determined by thin-layer chromatography and oligonucleotides generated by RNase T1 digestion were analyzed by DEAE-Sephadex chromatography. In the rat, the major 3'-terminal sequences of ribosomal 28S RNA, nucleolar 28S, 32S, 41S, and 45S RNAs were YGUoh, GZ2Uoh, GZ12Uoh, GZ2Uoh, and GZ7Goh, respectively, whereas in the mouse corresponding sequences were YGUoh, GZ1,2, or 3Uoh, Goh, Uoh and GZ 13Uoh, respectively. (Y: pyrimidine nucleoside, Z: any nucleoside other than guanosine) These results suggest that a "transcribed spacer" sequence is present at the 3'-terminus of the 45S pre-ribosomal RNA, which is gradually removed during the steps of processing.     

Nuclease resistance and RNase H sensitivity of oligonucleotides bridged by oligomethylenediol and oligoethylene glycol linkers

The properties of new chimeric oligodeoxynucleotides made of short sequences (tetramers, pentamers, octamers, and decamers) bridged by hexamethylenediol and hexaethylene glycol linkers have been investigated. These chimeric oligonucleotides showed an improved resistance toward snake venom 3'-phosphodiesterase, with an increased stability when a terminal 3'-3'-internucleotide phosphodiester bond is present. It also has been demonstrated that the hybrid complexes formed by bridged oligonucleotides and a complementary 20-mer RNA are able to elicit the activity of ribonuclease H (RNase H) from Escherichia coli. The substrate properties of chimeric oligonucleotides depend on the length of the oligonucleotide fragments bridged by linkers. Introduction of a nonnucleotide spacer into the native oligonucleotide only slightly hampers the extent of the RNA hydrolysis in the hybrid complexes, whereas a modification of the site of reaction is observed as a possible consequence of the steric disturbance due to the aliphatic linkers. Hence, these new chimeric oligonucleotides, namely, short oligonucleotide fragments bridged by nonnucleotide linkers, demonstrate a favorable combination of exonuclease resistance and high substrate activity toward RNase H. As a consequence, these chimeric oligonucleotides could be proposed as new, promising analogs to be used in the antisense strategy.     

Studies on the sequence of the 3'-terminal region of turnip-yellow-mosaic-virus RNA.

A fragment representing the 3'-terminal 'tRNA-like' region of turnip yellow mosaic (TYM) virus RNA has been purified following incubation of intact TYM virus RNA with Escherichia coli 'RNase P'. This fragment, which is 112+3-nucleotides long has been completely digested with T1 RNase and pancreatic RNase and all the oligonucleotides present in such digests have been sequenced using 32P-end labelling techniques in vitro. The TYM virus RNA fragment is free of modified nucleosides and does not contain a G-U-U-C-R sequence. Using nuclease P1 from Penicillium citrinum, the sequence of 26 nucleotides from the 5' end and 16 nucleotides from the 3' end of this fragment has been deduced. The nucleotide sequence at the 5' end of the TYM virus RNA fragment indicates that this fragment includes the end of the TYM virus coat protein gene.     

Oligonucleotide N3'-->P5' phosphoramidates as antisense agents.

Uniformly modified oligonucleotide N3'-->P5' phosphoramidates, where every 3'-oxygen is replaced by a 3'-amino group, were synthesized. These compounds have very high affinity to single-stranded RNAs and thus have potential utility as antisense agents. As was shown in this study, the oligonucleotide phosphoramidates are resistant to digestion with snake venom phosphodiesterase, to nuclease activity in a HeLa cell nuclear extract, or to nuclease activity in 50% human plasma, where no significant hydrolysis was observed after 8 h. These compounds were used in various in vitro cellular systems as antisense compounds addressed to different targeted regions of c-myb, c-myc and bcr-abl mRNAs. C-myb antisense phosphoramidates at 5 microM caused sequence and dose-dependent inhibition of HL-60 cell proliferation and a 75% reduction in c-myb protein and RNA levels, as determined by Western blot and RT-PCR analysis. Analogous results were observed for anti-c-myc phosphoramidates, where a complete cytostatic effect for HL-60 cells was observed at 1 microM concentration for fully complementary, but not for mismatched compounds, which were indistinguishable from untreated controls. This was correlated with a 93% reduction in c-myc protein level. Moreover, colony formation by the primary CML cells was also inhibited 75-95% and up to 99% by anti-c-myc and anti-bcr-abl phosphoramidate oligonucleotides, respectively, in a sequence- and dose-dependent manner within a 0.5 nM-5 microM dose range. At these concentrations the colony-forming ability of normal bone marrow cells was not affected. The presented in vitro data indicate that oligonucleotide N3'-->P5' phosphoramidates could be used as specific and efficient antisense agents.     

Nucleotide sequence of the 5'-terminal region of rat 18S ribosomal DNA

The 5'-terminal 597 base-pairs (bp) of the Sprague-Dawley rat 18S ribosomal RNA gee and 10 bp of the adjoining transcribed spacer have been sequenced. Previously sequenced 10 large oligonucleotides of rat 18S RNA were located in this region. This mammalian sequence has been compared with the known sequences of yeast and frog 18S rDNA's. The analysis indicates that 534 bp of the 597 bp (89%) are conserved between rat and frog sequences but only 75% of the nucleotides are conserved between rat and yeast in this region. Two large and two small sections have been identified where insertions have been introduced during evolution. Of these 58 bp long inserted sections of the rat rDNA sequence, 50 bp (86%) were G-C base-pairs.     

Nucleotide sequence at the 5' extremity of tobacco-mosaic-virus RNA. 1. The noncoding region (nucleotides 1-68)

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.     

Sequence specificity of internal methylation in B77 avian sarcoma virus RNA subunits.

Following ribonuclease digestion of methyl-3H-labeled B77 avian sarcoma virus RNA subunits, methylated oligonucleotides were isolated by diethylaminoethylcellulose chromotogrpahy. Partial nucleotide sequences were deduced from the known enzymatic specificities of the ribonucleases. In addition to methylated nucleosides in the 5'-terminal cap structure, m7G(5')GmpCp, N6-methyladenosine(m6A) was found to be present in only two internal sequences of the RNA molecule, Gpm6ApC and Apm6ApC. The average numbers of methylated nucleosides per RNA subunit are about 12-13 in Gpm6ApC, 1-2 in Apm6ApC, and 2 in m7GpppGmpCp. The sequences containing m6A in B77 sarcoma virus RNA are identical to m6A-containing sequences previously reported for the bulk mRNA from HeLa cells (Wei, C.M., Gershowitz, A., and Moss, B. (1976), Biochemistry 15, 397-401). Analysis of the oligonucleotides produced by RNase A digestion indicated that the sequence of bases on the 5' side of these trinucleotides is not specific. The oligonucleotide profile, however, was highly reproducible in different virus preparations. This suggests that the methylations occur at specific positions on the RNA molecule. Some of the methylated oligonucleotides produced by RNase A digestion appear to be present in less than molar amounts. Several hypotheses are proposed to explain this result.     

Specific interaction of hepatitis C virus protease/helicase NS3 with the 3'-terminal sequences of viral positive- and negative-strand RNA

The hepatitis C virus (HCV)-encoded protease/helicase NS3 is likely to be involved in viral RNA replication. We have expressed and purified recombinant NS3 (protease and helicase domains) and Delta pNS3 (helicase domain only) and examined their abilities to interact with the 3'-terminal sequence of both positive and negative strands of HCV RNA. These regions of RNA were chosen because initiation of RNA synthesis is likely to occur at or near the 3' untranslated region (UTR). The results presented here demonstrate that NS3 (and Delta pNS3) interacts efficiently and specifically with the 3'-terminal sequences of both positive- and negative-strand RNA but not with the corresponding complementary 5'-terminal RNA sequences. The interaction of NS3 with the 3'-terminal negative strand [called 3'(-) UTR(127)] was specific in that only homologous (and not heterologous) RNA competed efficiently in the binding reaction. A predicted stem-loop structure present at the 3' terminus (nucleotides 5 to 20 from the 3' end) of the negative-strand RNA appears to be important for NS3 binding to the negative-strand UTR. Deletion of the stem-loop structure almost totally impaired NS3 (and Delta pNS3) binding. Additional mutagenesis showed that three G-C pairs within the stem were critical for helicase-RNA interaction. The data presented here also suggested that both a double-stranded structure and the 3'-proximal guanosine residues in the stem were important determinants of protein binding. In contrast to the relatively stringent requirement for 3'(-) UTR binding, specific interaction of NS3 (or Delta pNS3) with the 3'-terminal sequences of the positive-strand RNA [3'(+) UTR] appears to require the entire 3'(+) UTR of HCV. Deletion of either the 98-nucleotide 3'-terminal conserved region or the 5' half sequence containing the variable region and the poly(U) and/or poly(UC) stretch significantly impaired RNA-protein interaction. The implication of NS3 binding to the 3'-terminal sequences of viral positive- and negative-strand RNA in viral replication is discussed.