[Ribosomal RNA sequencing with reverse transcriptase: its application to the phylogenetic study of cells].
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Base Sequence↗
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Assessment of phylogenetic relationships among ciliated protists using partial ribosomal RNA sequences derived from reverse transcripts.
Partial sequences were derived for the 16S-like ribosomal RNA of Halteria grandinella, Glaucoma chattoni, Colpidium campylum, Opisthonecta henneguyi and Colpoda inflata. Using isolated bulk nucleic acid preparations, partial rRNA sequences were determined using a series of oligonucleotide primers complementary to conserved sequence elements of the molecule and a dideoxynucleotide sequencing reaction using reverse transcriptase. Sequences were aligned and an evolutionary tree was generated by a distance-matrix method, comparing these partial sequences to the complete sequences of species of Tetrahymena, Paramecium, Euplotes, Oxytricha and Stylonychia. Halteria shows greater similarity to the stichotrichs Oxytricha and Stylonychia than to the hypotrich Euplotes, while these four genera share the same branch. Glaucoma and Colpidium appear closely related to Tetrahymena, with all three being more closely related to Opisthonecta than to Paramecium. Colpoda diverges near the base of the branch shared by Paramecium and the four oligohymenophorean genera. These results are discussed with reference to published revisions of the systematics of the phylum Ciliophora.
Animals↗
Linkage of a 7S RNA sequence and kappa light chain genes in the mouse.
A mouse 7S RNA cDNA plasmid clone was employed to identify and map DNA restriction fragment variants using recombinant inbred (RI) and congenic mouse strains. More than a dozen such restriction variants were identified and mapped to different regions of the mouse genome. One such variant, designated Rn7s-6, showed close linkage to the Ly-2,3-Igk-V (T lymphocyte antigens 2 and 3, kappa immunoglobulin variable region) cluster of markers on chromosome 6. No recombinants were detected among three of these markers in 59 RI strains. On the basis of these data, the Rn7s-6 sequence may be placed within 1.3 centimorgans of Ly-3 and one of the Igk-V-region markers, Igk-Ef1. Two mouse stocks with previously identified crossovers within the Ly-2,3-Igk-V region were used to sublocalize Rn7s-6. The results are consistent with the gene order (Ly-2, Ly-3)-(Rn7s-6, Igk-Ef1)-Igk-Ef2. Several mouse plasmacytomas, known to have various parts of the kappa chain complex deleted, retain the Rn7s-6 sequence. The Rn7s-6 variant is a plus/minus variant; no sequence allelic to Rn7s-6 is found in inbred strains that share the Ly-3a-Igk-Ef1a haplotype.
Animals↗
RNA sequence- and shape-dependent recognition by proteins in the ribonucleoprotein particle.
At all stages of its life (from transcription to translation), an RNA transcript interacts with many different RNA-binding proteins. The composition of this supramolecular assembly, known as a ribonucleoprotein particle, is diverse and highly dynamic. RNA-binding proteins control the generation, maturation and lifespan of the RNA transcript and thus regulate and influence the cellular function of the encoded gene. Here, we review our current understanding of protein-RNA recognition mediated by the two most abundant RNA-binding domains (the RNA-recognition motif and the double-stranded RNA-binding motif) plus the zinc-finger motif, the most abundant nucleic-acid-binding domain. In addition, we discuss how not only the sequence but also the shape of the RNA are recognized by these three classes of RNA-binding protein.
Animals↗
Lentiviral siRNAs targeting multiple highly conserved RNA sequences of human immunodeficiency virus type 1.
The high mutation rate of the human immunodeficiency virus (HIV) makes it difficult for any therapy employing a single anti-HIV targeting mechanism to sustain prolonged effect. In an attempt to explore novel therapy for AIDS, we developed and tested lentiviral small interfering RNA (siRNA) vectors targeting multiple highly conserved regions in the HIV type 1 (HIV-1) genome. The siRNA expression cassette was cloned into an extensively deleted HIV-1-derived lentiviral self-inactivating insulator (SIN) insulator [corrected] vector. Although some of the siRNAs targeting sites were also present in the helper construct of the vector system, the production of these lentiviral siRNA vectors were not significantly affected. When tested against different HIV-1 strains including pNL4-3 (subtype B), p89.6 (subtype B) and p90CF402.1.8 (subtype A/E recombinant), the siRNAs targeting conserved gag, pol, int and vpu, but not U3, nef or U5 regions, efficiently inhibited replication of all three viral strains. These lentiviral siRNA vectors also protected host cells from syncytium-forming macrophage- and T-cell-tropic HIV-1-induced cytotoxicity. Transduction of a long-term chronically infected human lymphoma cell line with lentiviral siRNAs resulted in stable inhibition of HIV-1 replication. Northern analysis showed that both genomic and subgenomic viral RNA species were downregulated. In addition, the viral RNA was inhibited in both the nuclear and cytoplasmic compartments of [corrected] chronically infected cells after prolonged passage, suggesting that [corrected] lentiviral siRNAs have a nuclear effect [corrected] Using these lentiviral siRNA [corrected] vectors, we further demonstrated reduced replication kinetics of HIV-1 in primary human peripheral blood lymphocytes. These results suggest that lentiviral siRNAs targeting multiple conserved HIV-1 sequences holds significant promise for the treatment of HIV-1 infections.
Base Sequence↗
Detection and cellular localization of enterovirus RNA sequences in spinal cord of patients with ALS.
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Amyotrophic Lateral Sclerosis↗
Small RNA sequences are readily stabilized by inclusion in a carrier rRNA.
This laboratory previously showed that an RNA derived from 5S ribosomal RNA could be used as a carrier to harbor a nucleic acid "tag" for monitoring genetically engineered or naturally occurring bacteria. The prototype system expressed a specific tagged RNA that was stable and accumulated to high levels. For such a system to be useful there should, however, be little limitation on the sequence composition and length of the insert. To test these limitations, a collection of insertion sequences were created and introduced into the artificial 5S rRNA cassette. This library consisted of random 13- and 50-base oligonucleotides that were inserted into the carrier RNA. We report here that essentially all of the insert-containing RNAs are stable and accumulate to detectable levels. Tagged RNAs were produced by both plasmid-borne and chromosomally integrated expression systems in E. coli and several Pseudomonas strains without obvious effect on the host cell. It is anticipated that in addition to its intended use in environmental monitoring, this system can be used for in vivo selection of useful artificial RNAs. Because the carrier lends stability to the RNAs, the system may also be useful in RNA production.
Base Sequence↗
Expression of xenotropic-like env RNA sequences in normal DBA/2 and NZB mouse tissues.
Using a DNA probe prepared from cloned env gene sequences of Friend spleen focus-forming viruses, we detected the differential expression of multiple RNA species in uninfected DBA/2 fibroblasts and in various tissues from adult DBA/2 and NZB mice. The size of the major RNA species detected was estimated to be 24S . The 24S RNA species was enriched in polyadenylate-selected preparations and thus may represent a message for endogenous viral envelope glycoproteins. The viral origin of the 24S RNA was further characterized by its hybridization to DNA probes containing the long terminal repeats of Harvey murine sarcoma virus, mouse mammary tumor virus, or the U3 region of an endogenous xenotropic virus. Although the env-related 24S RNA failed to react with either Harvey murine sarcoma virus or mouse mammary tumor virus long terminal repeat probes, it hybridized well with the xenotropic virus long terminal repeat probe. Therefore, it is likely that the RNA detected with the Friend spleen focus-forming virus env probe reflects transcription of xenotropic envelope sequences in uninfected tissues. Our finding that the level of 24S RNA varied in different organs indicated some tissue specificity in the expression of these xenotropic-like env proteins.
Animals↗
Assessing the odd secondary structural properties of nuclear small subunit ribosomal RNA sequences (18S) of the twisted-wing parasites (Insecta: Strepsiptera).
We report the entire sequence (2864 nts) and secondary structure of the nuclear small subunit ribosomal RNA (SSU rRNA) gene (18S) from the twisted-wing parasite Caenocholax fenyesi texensis Kathirithamby & Johnston (Strepsiptera: Myrmecolacidae). The majority of the base pairings in this structural model map on to the SSU rRNA secondary and tertiary helices that were previously predicted with comparative analysis. These regions of the core rRNA were unambiguously aligned across all Arthropoda. In contrast, many of the variable regions, as previously characterized in other insect taxa, had very large insertions in C. f. texensis. The helical base pairs in these regions were predicted with a comparative analysis of a multiple sequence alignment (that contains C. f. texensis and 174 published arthropod 18S rRNA sequences, including eleven strepsipterans) and thermodynamic-based algorithms. Analysis of our structural alignment revealed four unusual insertions in the core rRNA structure that are unique to animal 18S rRNA and in general agreement with previously proposed insertion sites for strepsipterans. One curious result is the presence of a large insertion within a hairpin loop of a highly conserved pseudoknot helix in variable region 4. Despite the extraordinary variability in sequence length and composition, this insertion contains the conserved sequences 5'-AUUGGCUUAAA-3' and 5'-GAC-3' that immediately flank a putative helix at the 5'- and 3'-ends, respectively. The longer sequence has the potential to form a nine base pair helix with a sequence in the variable region 2, consistent with a recent study proposing this tertiary interaction. Our analysis of a larger set of arthropod 18S rRNA sequences has revealed possible errors in some of the previously published strepsipteran 18S rRNA sequences. Thus we find no support for the previously recovered heterogeneity in the 18S molecules of strepsipterans. Our findings lend insight to the evolution of RNA structure and function and the impact large insertions pose on genome size. We also provide a novel alignment template that will improve the phylogenetic placement of the Strepsiptera among other insect taxa.
Animals↗
Self-splicing and enzymatic activity of an intervening sequence RNA from Tetrahymena.
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Animals↗
Compilation of small ribosomal subunit RNA sequences.
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Animals↗
Analysis of ribosomal RNA sequences by combinatorial clustering.
We present an analysis of multi-aligned eukaryotic and procaryotic small subunit rRNA sequences using a novel segmentation and clustering procedure capable of extracting subsets of sequences that share common sequence features. This procedure consists of: i) segmentation of aligned sequences using a dynamic programming procedure, and subsequent identification of likely conserved segments; ii) for each putative conserved segment, extraction of a locall homogeneous cluster using a novel polynomial procedure; and iii) intersection of clusters associated with each conserved segment. Aside from their utilit in processing large gap-filled multi-alignments, these algorithms can be applied to a broad spectrum of rRNA analysis functions such as subalignment, phylogenetic subtree extraction and construction, and organism tree-placement, and can serve as a framework to organize sequence data in an efficient and easily searchable manner. The sequence classification we obtained using the method presented here shows a remarkable consistency with the independently constructed eukaryotic phylogenetic tree.
Algorithms↗
Comparison of phylogenetic relationships based on phospholipid fatty acid profiles and ribosomal RNA sequence similarities among dissimilatory sulfate-reducing bacteria.
Twenty-five isolates of dissimilatory sulfate-reducing bacteria were clustered based on similarity analysis of their phospholipid ester-linked fatty acids (PLFA). Of these, 22 showed that phylogenetic relationships based on the sequence similarity of their 16S rRNA directly paralleled the PLFA relationships. Desulfobacter latus and Desulfobacter curvatus grouped with the other Desulfobacter spp. by 16S rRNA comparison but not with the PLFA analysis as they contained significantly more monoenoic PLFA than the others. Similarly, Desulfovibrio africanus clustered with the Desulfovibrio spp. by 16S rRNA but not with them when analyzed by PLFA patterns because of higher monoenoic PLFA content. Otherwise, clustering obtained with either analysis was essentially congruent. The relationships defined by PLFA patterns appeared robust to shifts in nutrients and terminal electron acceptors. Additional analyses utilizing the lipopolysaccharide-lipid A hydroxy fatty acid patterns appeared not to shift the relationships based on PLFA significantly except when completely absent, as in Gram-positive bacteria. Phylogenetic relationships between isolates defined by 16S rRNA sequence divergence represent a selection clearly different from the multi-enzyme activities responsible for the PLFA patterns. Determination of bacterial relationships based on different selective pressures for various cellular components provides more clues to evolutionary history leading to a more rational nomenclature.
Desulfovibrio↗
Adenovirus transcription. II. RNA sequences complementary to simian virus 40 and adenovirus 2DNA in AD2+ND1- and AD2+ND3-infected cells.
The genomes of the two nondefective adenovirus 2/simian virus 40 (Ad2/SV 40) hybrid viruses, nondefective Ad2/SV 40 hybrid virus 1 (Ad2+ND1) and nondefective hybrid virus 3 (Ad2+ND3), WERE FORMED BY A DELETION OF ABOUT 5% OF Ad2 DNA and insertion of part of the SV40 genome. We have compared the cytoplasmic RNA synthesized during both the early and late stages of lytic infection of human cells by these hybrid viruses to that expressed in Ad2-infected and SV40-infected cells. Separated strands of the six fragments of 32P-labeled Ad2 DNA produced by cleavage with the restriction endonuclease EcoRI (isolated from Escherichia coli) and the four fragments of 32P-labeled SV40 DNA produced by cleavage with both a restriction nuclease isolated from Haemophilus parainfluenzae, Hpa1, and EcoRI were prepared by electrophoresis of denatured DNA in agarose gels. The fraction of each fragment strand expressed as cytoplasmic RNA was determined by annealing fragmented 32P-labeled strands to an excess of cellular RNA extracted from infected cells. The segment of Ad2 DNA deleted from both hybrid virus genomes is transcribed into cytoplasmic mRNA during the early phase of Ad2 infection. Hence, we suggest that Ad2 codes for at least one "early" gene product which is nonessential for virus growth in cell culture. In both early Ad2+ND1 and Ad2+ND3-infected cells, 1,000 bases of Ad2 DNA adjacent to the integrated SV40 sequences are expressed as cytoplasmic RNA but are not similarly expressed in early Ad2-infected cells. The 3' termini of this early hybrid virus RNA maps in the vicinity of 0.18 on the conventional SV40 map and probably terminates at the same position as early lytic SV40 cytoplasmic RNA. Therefore, the base sequence in this region of SV40 DNA specifies the 3' termini of early messenger RNA present in both hybrid virus and SV40-infected cells.
Adenoviridae↗
In vitro release of alpha 1-acid glycoprotein RNA sequences shows fidelity with the acute phase response in vivo.
The acute phase reaction of rat liver to subcutaneous turpentine challenge results in a 20- to 100-fold increase in alpha 1-acid glycoprotein (alpha AGP) mRNA. We utilized this response to establish conditions appropriate for study of RNA transport in vitro using hybridization with 32P-labeled exon and intron alpha AGP sequences. Contamination of nuclear preparations by membrane-absorbed cytoplasmic RNA was eliminated by detergent-rinsing. The in vitro incubation conditions that most reflected the in vivo state required RNase inhibitor (purified from placenta), polyvinylpyrrolidone to prevent nuclear swelling, and addition of ATP. Under these circumstances, alpha AGP sequences were transported only from turpentine-stimulated preparations, were found only in poly(A)+ RNA, and were the same size as authentic cytoplasmic mRNA. Omission of polyvinylpyrrolidone resulted in release of some alpha AGP sequences in smaller, more heterogeneous poly(A)- RNA, and leakage of some alpha AGP sequences was observed from control preparations. Omission of ATP resulted in restriction of mature alpha AGP mRNA to the nucleus. In contrast to alpha AGP mRNA, transport of albumin mRNA was decreased 3-4X in turpentine-treated preparations. The largest alpha AGP intron was not found in RNA transported from treated nuclei in complete medium. The intron-containing fragments remained in the nucleus, largely in poly(A)- RNA of a size consistent with free intron. Some hybridization of intron sequences was observed with cytoplasmic and nuclear membrane-associated poly(A)+ RNA preparations which may represent 3'-processing catabolites; leakage of these sequences was considerably greater in the absence of PVP. On the basis of densitometric estimates, a 5-fold increase in the amount of alpha AGP exon sequences was observed in nuclear RNA, comparing treated with control animals, but transport of alpha AGP exon sequences was detectable only from treated nuclei, indicating at least a 50-fold increase in abundance of alpha AGP sequences. This suggests that a selective gating mechanism may be operative at the level of post-transcriptional nucleocytoplasmic transport during induction of alpha AGP in the acute phase response.
Animals↗
Detection by RNA blot hybridization of RNA sequences homologous to the BglII-N fragment of herpes simplex virus type 2 DNA.
RNA species, extracted at the time of peak synthesis of the alpha, beta, and gamma classes of herpes simplex virus polypeptides from lytically infected Vero cells, were examined for homology to the BglII-N fragment (map units 0.58 to 0.63) of herpes simplex virus type 2 DNA. By using northern blot analysis, two major and several minor polyadenylated RNA species showed homology to the BglII-N fragment at times corresponding to the maximum synthesis of the beta (7 h postinfection) and gamma (12 h postinfection) herpes simplex virus polypeptides. No alpha RNA homologous to the BglII-N fragment was detected.
Animals↗
A reproducible microanalytical method for the detection of specific RNA sequences by dot-blot hybridization.
A rapid, microanalytical procedure for the reproducible isolation of RNA from small cultured cell samples and application to dot-blot hybridization is described. The procedure employs guanidine hydrochloride solubilization of whole cells, disruption by syringing, and selective precipitation of RNA with ethanol. The method can be performed in a single tissue culture tube and obviates the need for removal of nuclei or for organic solvent extractions. Recovery of RNA from small cell samples (10(6) cells) is 51%, while 97% of the DNA and 99% of the protein are eliminated by the procedure. Detection of specific RNA by dot-blot hybridization using a labeled probe demonstrates high reproducibility of recovered RNA and lack of "masking" with up to a 10-fold excess of starting cell material. Applicability of the procedure to detection of virus-specific RNA in cells persistently infected with mouse hepatitis virus is described.
Animals↗
Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information.
This paper presents a new computer method for folding an RNA molecule that finds a conformation of minimum free energy using published values of stacking and destabilizing energies. It is based on a dynamic programming algorithm from applied mathematics, and is much more efficient, faster, and can fold larger molecules than procedures which have appeared up to now in the biological literature. Its power is demonstrated in the folding of a 459 nucleotide immunoglobulin gamma 1 heavy chain messenger RNA fragment. We go beyond the basic method to show how to incorporate additional information into the algorithm. This includes data on chemical reactivity and enzyme susceptibility. We illustrate this with the folding of two large fragments from the 16S ribosomal RNA of Escherichia coli.
Base Composition↗