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.     

Structure of the genome of Moloney murine leukemia virus: a terminally redundant sequence

The genome of the Moloney strain of murine leukemia virus (Mo-MuLV) has been analyzed by digestion with ribonuclease T1 and separation of the digestion products by two-dimensional gel electrophoresis. Thirty large oligonucleotides isolated from such a fingerprint have been characterized. One of these oligonucleotides (number 21) was found to be present in twice the molar yield of the rest. The 30 oligonucleotides were mapped on the genome by determining their yields in various size classes of 3' terminal fragments of Mo-MuLV RNA. The physical map obtained in this way suggested that oligonucletoide 21 was present very near the 3' end of the geome as well as in another location near or at the 5' end. The genome structure suggested by these results was confirmed by analyzing oligonucleotides in Mo-Mulv RNA complementary to strong stop DNA, which is shown to be a copy of the 5' terminal 134 nucleotides of the MoMuLV genome. Some of the oligonucleotides in the RNA protected from RNAase digestion by hybridization to this DNA, including oligonucleotide 21, were present near both the 3' and 5' ends. Comparison of these with the nucleotide sequence of strong stop DNA shows that there is a terminal redundancy of 49-60 nucleotides in the Mo-MuLV genome RNA.     

RNA synthesis of vesicular stomatitis virus. VIII. Oligonucleotides of the structural genes and mRNA.

The single-stranded RNA genome of vesicular stomatitis virus (VSV, Indiana serotype, San Juan strain) yields approx. 75 RNase T1-resistant oligonucleotides ranging in size from 10 to 50 bases. Each of the five structural genes, isolated as duplex RNA molecules hybridized to complementary mRNA, contains two or more of these large oligonucleotides. One of the oligonucleotides is identified as part of the non-coding region near the 3' end of the genome. Comparison of these results with others indicate that the RNA sequence of VSV is apparently stable in the laboratory but not in the wild. RNase T1-resistant oligonucleotides are also shown for all five VSV mRN species. Whether the mRNA for these digestions are are isolated from duplex RNA molecules or as single-stranded RNA species, the oligonucleotide patterns for each mRNA are virtually identical, indicating that each mRNA is transcribed from contiguous sequences on the genome. Comparison with published oligonucleotide patterns obtained from other isolates of VSV or from VSV deletion mutants indicate that identity and changes in their genome structure can be correlated with specific structural genes.     

A fluorimetric assay for available lysine in proteins

A two-dimensional fingerprinting gel system that provides sensitive analyses with high resolution of T1-resistant oligonucleotides of large RNA molecules is described. Unique oligonucleotides less than 30 bases in length are recovered quantitatively while longer oligonucleotides are recovered in very large (～90%) yields by active transfer of the fingerprint to DEAE paper. After elution of the oligonucleotides from DEAE paper, secondary analysis is performed by digestion of oligonucleotides with pancreatic RNase and separation of the products by high-voltage electrophoresis on polyethyleneimine cellulose. The complete analysis of up to 40 oligonucleotides can be accomplished within 4 days.     

Nucleotide sequence study of mouse 5.8S ribosomal RNA.

The primary structure of 5.8S mouse ribosomal RNA has been studied and compared to the structures previously established for other animal species. The results obtained show that mouse 5.8S ribosomal RNA yields pancreatic oligonucleotides with the same nucleotide sequence as the homologous oligonucleotides from rat cells. Furthermore T1 oligonucleotides of 5.8S ribosomal RNA from rat, mouse and human cells behave identically on fingerprinting fractionation and have the same composition as judged by pancreatic digestion. These results strongly suggest that the primary structures of 5.8S ribosomal RNA from rat, mouse and human cells are identical. This identity of structure is also found when the presence of several modified bases (psi and methylated bases) is considered. The findings emphasize the remarkable evolutionary stability of ribosomal gene structure. Comparison of the terminal regional of 5.8S RNA with those of 18S RNA reveals differences which imply a more complex mechanism underlying the maturation of 45S precursor RNA than the finding of identical structure would have suggested.     

Synthesis,purification and crystallization of guanine-rich RNA oligonucleotides

Guanine-rich RNA oligonucleotides display many novel structural motifs in recent crystal structures. Here we describe the procedures of the chemical synthesis and the purification of such RNA molecules that are suitable for X-ray crystallographic studies. Modifications of the previous purification methods allow us to obtain better yields in shorter time. We also provide 24 screening conditions that are very effective in crystallization of the guanine-rich RNA oligonucleotides. Optimal crystallization conditions are usually achieved by adjustment of the concentration of the metal ions and pH of the buffer. Crystals obtained by this method usually diffract to high resolution. Published: November 22, 2004.     

RNA oligonucleotide synthesis via 5'-silyl-2'-orthoester chemistry

The chemical synthesis of RNA oligonucleotides is a valuable resource for biological research. A new approach for RNA synthesis that is now as reliable and efficient as DNA synthesis methods is described in this report. A 5'-O-silyl ether is used in conjunction with acid-labile orthoester protecting groups on the 2'-hydroxyls. RNA synthesis proceeds efficiently on commercial synthesizers in high yields. Analysis by anion-exchange HPLC shows that the quality and yields of RNA synthesized with this chemistry are unprecedented. Furthermore, this chemistry enables analysis and purification of stable 2'-O-protected RNA. This property serves to minimize possibilities for degradation of the RNA. In addition, it now possible to analyze troublesome sequences, which, when fully 2'-O-deprotected, do not easily resolve into one major conformation due to strong secondary structure. When ready for use, the RNA is easily 2'-O-deprotected in mild-acidic aqueous buffers in 30 min. This new RNA chemistry has enabled the routine high-quality synthesis of RNA oligonucleotides up to 50 bases in length regardless of sequence or secondary structure.     

Predicting oligonucleotide affinity to nucleic acid targets

A computer program, OligoWalk, is reported that predicts the equilibrium affinity of complementary DNA or RNA oligonucleotides to an RNA target. This program considers the predicted stability of the oligonucleotide-target helix and the competition with predicted secondary structure of both the target and the oligonucleotide. Both unimolecular and bimolecular oligonucleotide self structure are considered with a user-defined concentration. The application of OligoWalk is illustrated with three comparisons to experimental results drawn from the literature.     

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.     

Structural and functional characterization of mouse U7 small nuclear RNA active in 3'' processing of histone pre-mRNA.

Oligonucleotides derived from the spacer element of the histone RNA 3' processing signal were used to characterize mouse U7 small nuclear RNA (snRNA), i.e., the snRNA component active in 3' processing of histone pre-mRNA. Under RNase H conditions, such oligonucleotides inhibited the processing reaction, indicating the formation of a DNA-RNA hybrid with a functional ribonucleoprotein component. Moreover, these oligonucleotides hybridized to a single nuclear RNA species of approximately 65 nucleotides. The sequence of this RNA was determined by primer extension experiments and was found to bear several structural similarities with sea urchin U7 snRNA. The comparison of mouse and sea urchin U7 snRNA structures yields some further insight into the mechanism of histone RNA 3' processing.     

Chromatographic separation of mononucleotides derived from transfer ribonucleic acids

1. A method is described for fractionating alkaline and enzymic hydrolysates of ribonucleic acids and oligonucleotides on DEAE-cellulose columns by using a constant-composition eluent. 2. The relative partition coefficients of the four major and several minor nucleotides present in nucleic acid digests are given. 3. The influence of pH and of molarity of the buffer on the separation is described. 4. Relations for the quantitative determination of the major nucleotides from their absorption zone areas on the chromatographic record are derived. 5. Hydrolysis of oligonucleotides with T2 ribonuclease yields solely the nucleoside 3'-monophosphates, thereby simplifying the subsequent chromatographic separation. 6. Examples of the qualitative and quantitative analysis of oligonucleotides from enzymic hydrolysates of RNA are given.     

Formation of specific T3-RNA-polymerase complexes with oligoribonucleotides and inhibition of DNA-dependent RNA synthesis

It was shown previously that E. coli RNA polymerase and T7 RNA polymerase being incubated with oligonucleotides of different length derived from RNA endonuclease hydrolysate bind selectively to certain oligonucleotides with the length larger than or equal to 5. The data presented demonstrate that T3 RNA polymerase also binds selectively from the isoplith mixtures certain oligonucleotides starting from pentanucleotides. Adding of excess of T3 RNA polymerase it was possible to exhaustively extract the recognizable oligonucleotides from the isoplith mixture. However, the exhausted by T3 RNA polymerase mixture of pentanucleotides still contained those which are bound selectively by T7 and E. coli RNA polymerases. The data suggest that various RNA-polymerases recognize different oligoribonucleotides. It was shown that T3 DNA inhibits the selective binding of penta-or heptaribonucleotides to T3 RNA polymerase competing obviously for the enzyme. The T3 RNA polymerase bound penta- or heptanucleotides inhibit DNA-dependent RNA synthesis carried out by the enzyme; the isoplith mixtures which do not contain T3 RNA polymerase bound oligonucleotides are deprived of the inhibitory properties. Only those isoplith mixtures contain T3 RNA polymerase bound oligonucleotides which were derived from symmetrically transcribed RNA which have obviously promoter simulating sequences. The data provide evidence that T2 RNA polymerase binds selectively the oligonucleotides mimicking the promotor recognition sites.     

"Fingerprinting" high molecular weight RNA by two-dimensional gel electrophoresis: application to poliovirus RNA.

Conditions are described under which complete RNase T1 digests of high molecular weight RNA can be separated into numerous components by two-dimensional gel electrophoresis. Small and large oligonucleotides (n = 1 - 2c0) can be resolved without losses. The procedure yields fingerprints which are diagnostic for a particular species of RNA and an index of its purity as will be shown for the genomes of poliovirus type 1 and 2.     

A shell program for the design of PCR primers using genetics computer group (GCG) software (7.1) on VAX/VMS systems.

A number of software analysis packages for the design of PCR primers are available for PCs; however, software for users that depend on VAX/VMS operating systems is not available. By treating oligonucleotides as RNA molecules, I have designed an alternative means toward studying oligonucleotide interactions using software that is currently available from The Genetics Computer Group (GCG, Madison, WI). The oligonucleotide interactions with self and non-self are analyzed by the GCG FOLD program, a program which finds a secondary structure of minimum free energy for an RNA molecule. This approach allows the identification of self-priming primer pairs, and the interaction energies provide a guideline for the prediction of optimal PCR primers.     

Perylene attached to 2'-amino-LNA: synthesis, incorporation into oligonucleotides, and remarkable fluorescence properties in vitro and in cell culture

During recent years, fluorescently labeled oligonucleotides have been extensively investigated within diagnostic approaches. Among a large variety of available fluorochromes, the polyaromatic hydrocarbon perylene is an object of increasing interest due to its high fluorescence quantum yield, long-wave emission compared to widely used pyrene, and photostability. These properties make perylene an attractive label for fluorescence-based detection in vitro and in vivo. Herein, the synthesis of 2'- N-(perylen-3-yl)carbonyl-2'-amino-LNA monomer X and its incorporation into oligonucleotides is described. Modification X induces high thermal stability of DNA:DNA and DNA:RNA duplexes, high Watson-Crick mismatch selectivity, red-shifted fluorescence emission compared to pyrene, and high fluorescence quantum yields. The thermal denaturation temperatures of duplexes involving two modified strands are remarkably higher than those for double-stranded DNAs containing modification X in only one strand, suggesting interstrand communication between perylene moieties in the studied 'zipper' motifs. Fluorescence of single-stranded oligonucleotides having three monomers X is quenched compared to modified monomer (quantum yields Phi F = 0.03-0.04 and 0.67, respectively). However, hybridization to DNA/RNA complements leads to Phi F increase of up to 0.20-0.25. We explain it by orientation of the fluorochrome attached to the 2'-position of 2'-amino-LNA in the minor groove of the nucleic acid duplexes, thus protecting perylene fluorescence from quenching with nucleobases or from the environment. At the same time, the presence of a single mismatch in DNA or RNA targets results in up to 8-fold decreased fluorescence intensity of the duplex. Thus, distortion of the duplex geometry caused by even one mismatched nucleotide induces remarkable quenching of fluorescence. Additionally, a perylene-LNA probe is successfully applied for detection of mRNA in vivo providing excitation wavelength, which completely eliminates cell autofluorescence.     

Assignment of the large oligonucleotides of vesicular stomatitis virus to the N, NS, M, G, and L genes and oligonucleotide gene ordering within the L gene.

Analyses of prototype vesicular stomatitis (VSV, Indiana serotype) mRNA-32P-labeled viral RNA duplexes have established the assignments of 65 of the 72 large oligonucleotides that are recovered by two-dimensional electrophoresis of RNase T1 digests of the viral RNA. Fifty of the oligonucleotides are recovered in the L RNA duplex, four each in the N, M, and NS duplexes, and three in the G RNA duplex. Studies of three small defective-particle RNA species indicate that they have only L gene oligonucleotides in addition to three of the seven unassigned oligonucleotides. Some L gene ordering of oligonucleotides can be postulated from the defective-particle RNA sequence analyses. Analyses of naturally occurring alternate isolates of VSV Indiana have established that by comparison to the prototype virus strain, the alternate isolates minimally have genome sequence differences in L, G, N, NS and/or unassigned regions of the genome. Changes in the genome have also been induced by vitro high-level mutagenesis of the prototype virus.     

Genomic RNA of mengovirus V. Recognition of common features by ribosomes and eucaryotic initiation factor 2.

Binding of ribosomes to the 32P-labeled genomic RNA of mengovirus was studied in lysates of mouse L929 and Krebs ascites cells under conditions for initiation of translation. Upon total digestion with RNase T1, the 32P-labeled RNA protected in either 40S or 80S initiation complexes yielded four unique, large oligonucleotides. Each of these oligonucleotides occurred once in the viral RNA molecule. The same four oligonucleotides were recovered from 80S initiation complexes formed in lysates in which unlabeled mengovirus RNA had been translated extensively, indicating that recognition by ribosomes was not modulated detectably by a viral translation product. The recognition of intact, 32P-labeled mengovirus RNA by eucaryotic initiation factor 2 (eIF-2) was examined by direct complex formation. Fingerprint analysis of the RNA protected by eIF-2 against RNase T1 digestion yielded three T1 oligonucleotides that were identical to three of the four oligonucleotides protected in either 40S or 80S initiation complexes. A physical map of the large T1 oligonucleotides of the mengovirus RNA molecule was constructed, and the four protected oligonucleotides were found to map internally, within the region between the polycytidylate tract and the 3' end. For either ribosomes or eIF-2, the protected oligonucleotides could not be arranged in a continuous sequence, suggesting that they constitute at least two widely separated domains. These results show that ribosomes recognize and blind to more than a single sequence in mengovirus RNA, located internally in regions that are far removed from the 5' end of the molecule. eIF-2 itself binds with high specificity to mengovirus RNA, recognizing apparently three of the four sequences recognized by ribosomes.