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PubMed2000

Studies on anti-human immunodeficiency virus oligonucleotides that have alternating methylphosphonate/phosphodiester linkages.

Preliminary investigations of the physical properties of oligonucleotide analogs that contain alternating methylphosphonate/phosphodiester linkages are described. An alternating oligo-2'-O-methylribonucleoside methylphosphonate, oligomer 1676, whose sequence is complementary to the upper hairpin region of human immunodeficiency virus TAR RNA, has been synthesized. This 15-mer forms a very stable duplex with its complementary RNA target, whose melting temperature is 71 degrees C. Introduction of two mismatched bases reduces the melting temperature by 16 degrees C. Similar results were obtained with the all-phosphodiester version of oligomer 1676, which demonstrates that introduction of the methylphosphonate linkages does not significantly perturb the ability of the oligo-2'-O-methylribonucleoside methylphosphonate to bind to RNA. Unlike the phosphodiester oligomer, however, oligomer 1676 is completely resistant to hydrolysis by the 3'-exonuclease activity found in mammalian serum. The interactions between nuclease-resistant, 5'-psoralen-derivatized, alternating oligo-2'-deoxypyrimidine methylphosphonates and double-stranded DNA were also studied. A 15-mer that contains thymine, 5-methylcytosine, and 5-propynyl-uracil forms a triplex with a polypurine tract found in the env gene of human immunodeficiency virus proviral DNA with an apparent dissociation constant of 400 nM at 22 degrees C. Maximal triplex formation by these oligomers is observed at approximately 2.5 mM magnesium, whereas maximal triplex formation by the corresponding all-phosphodiester oligomers occurs between 10 and 20 mM magnesium. This reduced magnesium dependence most likely results from reduced charge repulsion between the backbones of the methylphosphonate oligomer and purine strand of the target. The nuclease stability and ability of the methylphosphonate oligomers to form stable complexes with their target nucleic acids suggest that these oligomers are potential candidates for use as antisense or antigene agents in cell culture.

Binding Sites↗
PubMed1973

Enzymatic synthesis of DNA complementary to purified 14S messenger RNA of immunoglobulin light chain.

The 14S messenger RNA, which contains poly(adenylic acid), of MOPC 41 (mouse plasmocytoma) immunoglobulin light chain, purified to a single peak as shown by polyacrylamide gel electrophoresis, was used to synthesize complementary DNA with the RNA-dependent DNA polymerase of avian myeloblastosis virus. DNA synthesis is entirely dependent on added RNA template and oligo(dT) primer. Both the size and the concentration of the primer affect the reaction. The product behaves similarly to DNA during centrifugation in cesium sulfate density gradients. It is shown by hybridization that the DNA made is complementary to the purified template, light-chain mRNA. The high specific activity of the complementary DNA should make it suitable for gene-dosage experiments. According to alkaline sucrose gradient analyses, some complete complementary DNA transcripts of the 14S mRNA seem to be made. Oligo(dG) can also function as a primer for DNA synthesis, possibly by annealing to an internal cluster of cytidines in the mRNA, that correspond to the bases coding for amino-acids 119 and 120 of the MOPC 41 light chain.

Animals↗
PubMed1991

Molecular characterization of the Prospect Hill virus M RNA segment: a comparison with the M RNA segments of other hantaviruses.

Complementary DNA representing the genomic M RNA segment of the Prospect Hill (PH) Hantavirus was cloned and its nucleotide sequence determined. The PH virus M RNA segment is 3707 nucleotides in length and has a long open reading frame in the viral complementary-sense RNA with a coding capacity of 1142 amino acids. The predicted gene product of the PH virus M segment was compared with the corresponding gene products of Hantaan virus strain 76-118 (Hantaan), Sapporo rat virus strain SR-11 (SR) and Puumala virus strain Hällnäs B1 (Hällnäs). The amino acid sequence identities between the G1 and G2 proteins of PH virus and Hällnäs virus are respectively 74% and 79%. In contrast, the amino acid sequence similarities between the G1 proteins of PH virus and SR virus or Hantaan virus are only 50%. However the G2 proteins of SR and Hantaan viruses were more closely related to the G2 protein of PH virus with amino acid sequence similarities of approximately 62%. The G1 proteins of all four viruses had three potential asparagine-linked glycosylation sites conserved and there was one conserved site in the G2 proteins. Hydrophilicity plots of the four virus glycoproteins were very similar. The region of greatest hydrophilicity was conserved in the Hällnäs, SR and Hantaan viruses, and was located near the C terminus of the G1 protein, whereas the region of greatest hydrophilicity in the PH virus glycoprotein precursor is located closer to the N terminus of the G1 protein. Our data demonstrate that despite differences in the serotypic profiles and virulence of PH and Hällnäs viruses, their G1 and G2 proteins are closely related. We conclude that PH and Hällnäs viruses may have evolved along a separate evolutionary pathway in the Hantavirus genus from that of SR and Hantaan viruses.

Amino Acid Sequence↗
PubMed1995

Inhibition of phage phi 29 assembly by antisense oligonucleotides targeting viral pRNA essential for DNA packaging.

A sensitive and efficient system for the functional assay of antisense oligonucleotides (oligos) was developed based on an in vitro viral assembly system. A 120-base RNA (pRNA), which indispensably participates in bacteriophage phi 29 DNA packaging, was the target for antisense action. Antisense oligos bound to pRNA, as revealed by a slower electrophoretic mobility of pRNA/oligo complexes in comparison with native pRNA. Infectious viruses were assembled in vitro with synthetic pRNA and DNA, as well as with viral proteins produced from cloned genes. Up to 10(7) plaque-forming units per milliliter were obtained in the absence of antisense oligos, while as few as zero plaques were detected in the presence of certain antisense oligos. A 1-base mismatch greatly influenced the inhibitory effect of the antisense oligos, but this 1-based mismatch was not important when the mismatch was placed at the end of the oligo. Five oligos did not bind pRNA or inhibit the assembly of the virion, suggesting that the RNA sequences complementary to these oligos are nonessential or buried internally in the RNA. Viral assembly was strongly inhibited by antisense oligos P15 and P10, targeting either the 5'- or the 3'-end of the pRNA, respectively. Viral assembly was also strongly inhibited by oligo P6, targeting an internal region, residues 75-91, of pRNA. Oligo P6 inhibited DNA packaging activity by blocking the binding of pRNA to the procapsid, while P10 and P15 inhibited DNA packaging activity but did not block the binding of pRNA to the procapsid, suggesting that in addition to the reported internal domain for procapsid binding, pRNA contains another domain at the paired 5'/3'-ends with a yet to be defined role in DNA translocation.

Bacillus Phages↗
PubMed1978

Estrogen withdrawal in chick oviduct. Characterization of RNA synthesized in isolated nuclei using a mercurated precursor.

1. As a prerequisite for analyzing the effect of estrogen on transcription in chick oviduct, we describe suitable methods for the synthesis (under conditions restricting reinitiation), and isolation of RNA transcripts from oviduct nuclei in vitro, utilizing mercurated UTP (Hg-UTP) as an RNA precursor and chromatography on sulphydryl-Sepharose (SH-Sepharose) to recover mercurated RNA (Hg-RNA). The techniques described include treatment of Hg-RNA with p-hydroxymercuribenzoate, to improve the efficiency of binding to SH-Sepharose, and elution of Hg-RNA from SH-Sepharose after treatment with 60% formamide at 90 degrees C, to eliminate contamination by aggregated nucleic acid. 2. RNA synthesized by endogenous form B RNA polymerase (using either UTP or Hg-UTP as precursor) was recovered in nuclear lysates in the form of 30--85-S heterogeneous RNA . protein complexes, and after removal of protein, was 10--12 S in size. 3. The nature of RNA transcripts synthesized in vitro was examined by hybridization. More than 90% of the RNA was complementary to "unique" DNA sequences, and 50--60% of the hybridized RNA could be competed with homologous, steady-state nuclear RNA, indicating a significant degree with homologous, steady-state nuclear RNA, indicating a significant degree of homology between in vitro transcripts and in vivo RNA. The level of homology was similar whether RNA synthesis was performed in low salt, or in high salt in the presence of heparin. Possible reasons for only partial competition in these experiments are discussed. 4. Withdrawal of estrogen from chicks leads to a 50% reduction in endogenous RNA polymerase activities in nuclei within 48 h. Similar levels of competition with Hg-RNA transcripts for "unique" DNA were obtained using oviduct nuclear RNAs isolated before or after estrogen withdrawal, and even with liver nuclear RNA. Thus, in oviduct, those sequences present in primary transcripts, and analyzed under our experimental conditions, are present in different hormonal states and also in other chick tissues.

Amanitins↗
PubMed1983

Androgen regulation of prostatic binding protein messenger RNA studied by using cloned complementary DNA.

The construction of a double-stranded cDNA library using rat prostatic poly(A)RNA and pBR322/kappa 1776 system and the isolation of three prostatic binding protein (PBP) cDNA clones are described. These cDNA clones were characterized and identified by in situ hybridization, mRNA selection-translation and immuno-precipitation as coding for the three subunit components, C1, C2, and C3, of PBP. These clones were used in hybridization experiments with prostatic poly(A)RNA to determine the effect of testosterone on the levels of PBP-mRNA. The results showed that synthesis of these mRNAs varied in response to either androgen withdrawal or replacement. Accumulation of PBP-mRNAs coding for C2 and C3 components occurred 1 hr after androgen administration to castrated rat, whereas the mRNA coding for the C1 component did not appear until 4 hr after androgen replacement. Quantitation of PBP-mRNA sequences in nuclear and polysomal poly(A)RNAs showed that they did not vary coordinately in response to androgen withdrawal. These results indicate differential regulation of PBP genes and suggest possible multiple levels of androgen control of PBP synthesis.

Animals↗
PubMed2000

Antisense RNA regulation of the pAD1 par post-segregational killing system requires interaction at the 5' and 3' ends of the RNAs.

The par stability determinant of the Enterococcus faecalis plasmid pAD1 is the first antisense RNA-regulated post-segregational killing system (PSK) identified in a Gram-positive organism. Par encodes two small, convergently transcribed RNAs, designated RNA I and RNA II, which are the toxin and antidote of the par PSK system respectively. RNA I encodes an open reading frame for a 33-amino-acid toxin called Fst. Expression of fst is regulated post-transcriptionally by RNA II. In this paper, RNA II is shown to interact with RNA I by a unique antisense RNA mechanism. RNA I and RNA II contain complementary direct repeats at their 5' ends and a complementary transcriptional terminator stem-loop at their 3' ends. Deletion of the terminator or mutations within the terminator loop of RNA II severely reduced the rate of interaction in vitro. Mutations in the 5' direct repeats of RNA II prevented the RNAs from interacting in vitro. For these mutations in RNA II, complementary mutations in RNA I were shown to restore interaction. The reduced binding efficiency of the RNA II mutants was paralleled by the failure of these mutants to suppress par-mediated killing in vivo. These results indicate that regions at both the 5' and the 3' ends of the par transcripts are important for RNA I-RNA II interaction.

3' Untranslated Regions↗
PubMed1977

Synthesis of RNA from cellulose-bound complementary DNA.

Radioactively labeled RNAs were synthesized from cellulose-bound cDNA templates using Escherichia coli RNA polymerase. Hybridization of this RNA to excess unlabeled cDNA approached 100%, indicating the complementarity of product and template. The average length of the RNA product, as determined by formamide gels, was approximately 40% of the template length. Hybridization of unlabeled globin RNA produced by this technique to labeled globin cDNA indicated the population of RNA sequences represented at least 80% of the template sequences. Approximately 30% of the RNA product by mass contains poly(A) tails as determined by binding to oligo(dT)-cellulose. The template can be reused for several cycles of synthesis with little loss of synthetic capability and therefore, can amplify the amount of mRNA initially used to produce the template.

Cellulose↗
PubMed1981

Azocarcinogen-induced release of chromatin-associated RNA into liver cytoplasm: the possible role of reiterated RNA sequences in the control of RNA processing.

In the liver of rats fed the azocarcinogen 3'-methyl-4-dimethylaminoazobenzene (3'MeDAB) reiterated RNA sequence transcribed from middle repetitive DNA are released into the cytoplasm. The same repetitive nucleotide sequences can be isolated from the chromatin of the liver of control animals in the form of metabolically highly active, 13 000 daltons RNA. This small, chromatin-associated RNA originates from nuclear RNA larger than 10 S. The discontinuation of the feeding of the azocarcinogen will not stop the release of the nuclear reiterated RNA sequences into the cytoplasm. The repetitive sequences of nuclear RNA which are released into the cytoplasm in animals fed the azocarcinogen can no longer be found in the chromatin in the form of small RNA molecules. The results can be explained by the assumption that the reiterated RNA sequences are involved in the upholding of RNA processing. A cell-specific processing of RNA will be maintained by the interaction of reiterated RNA fragments from already processed RNA with the reiterated complementary sequences on RNA yet to be processed. Existence of such a feed-back circuit would make it possible to explain how a temporary interference of the azocarcinogen with RNA processing will result in the disappearance of specific reiterated RNA sequences from the chromatin. It could also explain the continuation of the release of the same repeated RNA sequences into the cytoplasm as part of larger RNA molecules even after the removal of the carcinogen.

Animals↗
PubMed1987

Hantaan virus M RNA: coding strategy, nucleotide sequence, and gene order.

The M genome segment of Hantaan virus was molecularly cloned and the nucleotide sequence of cDNA was determined. The virion RNA is 3616 bases long with 3'- and 5'-terminal nucleotide sequences complementary for 18 bases. A single long open reading frame in the viral complementary-sense RNA had the potential to encode 1135 amino acids or a polypeptide of 126,000 Da. Amino-terminal sequences of isolated G1 and G2 envelope glycoproteins were determined, revealing a gene order with respect to message sense RNA of 5'-G1-G2-3'. Mature G1 begins 18 amino acids beyond the first AUG of the open reading frame, preceded by a short, hydrophobic leader sequence. G2 begins at the 649th amino acid of the open reading frame and also follows a hydrophobic sequence. Carboxy termini of G1 and G2 were localized and gene order was verified by immune precipitation of Hantaan proteins with antisera to synthetic peptides generated by using amino acid sequences derived from the cDNA sequence. The antipeptide sera were also reactive by immunoblotting with SDS-denatured G1 and G2. Molecular weights of 64,000 and 53,700 were calculated for the G1 and G2 glycoproteins, respectively, from their predicted amino acid sequences. Five potential asparagine-linked glycosylation sites were contained within the G1 amino acid sequence and two within the G2 sequence. These data are consistent with our previous estimates of the molecular weights and extent of glycosylation of the Hantaan envelope glycoproteins.

Amino Acid Sequence↗
PubMed1987

Molecular characterization of the genomic S RNA segment from lymphocytic choriomeningitis virus.

We have used cDNA clones derived from the genomic S RNA segment of lymphocytic choriomeningitis virus (LCMV), Armstrong strain, as hybridization probes to monitor virus gene expression during acute infections. Our results with strand-specific probes confirm the ambisense character of the LCMV S RNA segment and document the presence of both genomic sense and genomic complementary sense RNA species over the time course of infection. We have used nucleotide sequence information to predict primary amino acid sequences for the major viral structural proteins, nucleoprotein (NP) and glycoprotein (GP-C). Antibodies raised against synthetic peptides derived from these predicted protein sequences have indicated that the gene order for the S segment is 3' NP----5' GP-C and provided direct demonstration that the GP-1 portion of the GP-C precursor is encoded nearest the 5' end of the S segment. Comparison of the predicted amino acid sequences for NP and GP-C between the Armstrong CA-1371 strain and the WE strain shows over 90% amino acid identity. This suggests that significant differences described for the pathogenic potential of the Arm and WE strains in C3H mice reside in one or a very few critical amino acid changes.

Amino Acid Sequence↗
PubMed1989

Determination of the coding capacity of the M genome segment of nephropathia epidemica virus strain Hällnäs B1 by molecular cloning and nucleotide sequence analysis.

The M genome RNA segment of nephropathia epidemica virus (NEV) strain Hällnäs B1 was characterized by molecular cloning and DNA nucleotide sequencing of the corresponding cDNA clones. The size of the M RNA segment is 3682 nucleotides. The 3' and 5' terminal sequences are complementary for 21 bases and their predicted secondary structure is very stable. The viral complementary messenger RNA possesses a single long open reading frame with a coding capacity of 1148 amino acids (polypeptide of 126 kDa). A comparison of the NEV M segment to that of Hantaan virus strain 76-118 reveals 61% sequence homology at the nucleotide level and 53% at the deduced amino acid level. Four out of five potential asparagine-linked glycosylation sites of the encoded glycoproteins have been conserved between NEV and Hantaan M. The isoelectric points (IEP) are nearly identical. Furthermore it was found that 90% of all cysteine residues have been conserved. Putative NEV G1 and G2 are preceded by a short hydrophobic sequence as shown for G1 and G2 of Hantaan virus. Hydrophilicity profiles of the two segments are of striking similarity. These data indicate that NEV- and Hantaan virus M-encoded polypeptides seem to be very similar in structure and function despite the relatively low amino acid sequence homology.

Amino Acid Sequence↗
PubMed1975

Messenger RNA species synthesized in vitro by the virion-associated RNA polymerase of vesicular stomatitis virus.

The virion-associated RNA-dependent RNA polymerase of vesicular stomatitis virus (VSV) synthesizes in vitro two size classes of RNA products similar to those observed in VSV-infected cells. One RNA product sediments at 31S with an approximate molecular weight of 2.1 X 106. The smaller products consist of at least three classes of RNA sedimenting at 17S, 14.5S, and 12S with molecular weights of 0.7 X 106, 0.52 X 106, and 0.37 X 106, respectively. Hybridization experiments show that both the 31S and 12-18S RNA products are complementary to the genome RNA, and that each class is transcribed from different nucleotide sequences. From the molecular weights of the RNA species and the hybridization experiments, it seems that almost the entire VSV genome RNA is transcribed in vitro.

Animals↗
PubMed1983

A simple procedure for the extraction of double-stranded RNA from virus-infected plants.

A simple procedure for the isolation of double-stranded (ds) RNA from virus-infected plants is described. The method is based on grinding plant tissue in 4% p-aminosalicylic acid and recovery of ds RNA by phenol extraction and precipitation with 30% ethanol. The presence of both negative and positive virus RNA strands in RNA fractionated in agarose gels was verified by Northern blot hybridization with polynucleotide kinase labelled genomic RNA or complementary DNA (cDNA) probes. The procedure enabled detection of three major ds RNA species (MWs 4.2, 1.05 and 0.48 X 10(6)) and at least 4 minor bands with estimated MWs of 3.5, 2.5, 2.2 and 2.0 X 10(6) in Nicotiana tabacum plants systemically infected with tobacco mosaic virus (TMV). Cucumber mosaic virus (CMV)-infected Pachystachys coccinea plants contained 2 minor bands of MWs 0.49 and 0.35 X 10(6) in addition to the previously described 4 major ds RNAs and ds CARNA 5 (MW 0.22 X 10(6)). The patterns of ds RNA are useful for diagnosing natural infections of CMV and TMV in N. glauca plants and of citrus tristeza virus in Citrus spp.

Mosaic Viruses↗
PubMed1994

In situ detection of Pbs21 mRNA during sexual development of Plasmodium berghei.

The patterns of expression of ribosomal RNA and mRNA encoding the parasite surface antigen Pbs21 have been investigated during the sexual stages of development of the malaria parasite, Plasmodium berghei, using the technique of non-radioactive in situ RNA hybridisation. An RNA probe complementary to a region of the small subunit of P. berghei ribosomal RNA hybridised to parasites at all stages of development in a smear of blood taken from mice infected with P. berghei. Messenger RNA encoding Pbs21, in contrast, was detected only within parasites committed to sexual development within the vertebrate host and, furthermore, was shown to be expressed in a sex-specific manner, exclusively within female gametocytes. At later stages of sexual development, Pbs21 mRNA was detected at high levels in female gametes and ookinetes. We have previously shown that Pbs21 protein is first detectable only after the initiation of gametogenesis which occurs following transmission to the insect vector. These results suggest, therefore, that post-transcriptional mechanisms operate to regulate the translation of Pbs21 mRNA as it accumulates during female gametocytogenesis.

Animals↗
PubMed1988

RNA mediated formation of a phosphorothioate diester bond.

Previous results showed that multimeric, tandemly sequence-repeated forms of satellite tobacco ringspot virus RNA of the encapsidated polarity (STobRV (+)RNA) autolytically process at a specific phosphodiester bond, the junction. Substituting a phosphorothioate diester bond for the STobRV (+)RNA junction drastically slowed autolytic processing. Here we show that for the complementary STobRV (-)RNA, in contrast, replacing sets of phosphodiester bonds with phosphorothioate diester bonds, even at the junction, did not greatly slow autolytic processing or spontaneous ligation, the usual reactions of the unmodified RNA. In the ligation reaction STobRV (-)RNA directed the formation of an ApG phosphorothioate diester bond.

Base Sequence↗
PubMed1996

Crystal structures of an A-form duplex with single-adenosine bulges and a conformational basis for site-specific RNA self-cleavage.

BACKGROUND: Bulged nucleotides are common secondary structural motifs in RNA molecules and are often involved in RNA-RNA and RNA-protein interactions. RNA is selectively cleaved at bulge sites (when compared to other sites within stems) in the presence of divalent metal cations. The effects of bulge nucleotides on duplex stability and topology have been extensively investigated, but no detailed X-ray structures of bulge-containing RNA fragments have been available. RESULTS: We have crystallized a self-complementary RNA-DNA chimeric 11-nucleotide sequence containing single-adenosine bulges under two different conditions, giving two distinct crystal forms. In both lattices the adenosines are looped out, leaving the stacking interactions in the duplex virtually unaffected. The bulges cause the duplex to kink in both cases. In one of the structures, the conformation of the bulged nucleotide places its modeled 2'-oxygen in line with the adjacent phosphate on the 3' side, where it is poised for nucleophilic attack. CONCLUSIONS: Single adenosine bulges cause a marked opening of the normally narrow RNA major groove in both crystal structures, rendering the bases more accessible to interacting molecules compared with an intact stem. The geometries around the looped-out adenosines are different in the two crystal forms, indicating that bulges can confer considerable local plasticity on the usually rigid RNA double helix. The results provide a conformational basis for the preferential, metal-assisted self-cleavage of RNA at bulged sites.

Crystallography, X-Ray↗