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At least 1,783 records · Page 99Linked to original sources
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↗
PubMed1995

Presence and instability of repetitive elements in sequences the altered expression of which characterizes risk for colonic cancer.

50C10 and 52H10 are two DNA clones previously reported by us to be overexpressed in human colonic mucosa at high risk for development of colonic tumors. This report presents sequencing data that reveal that these clones contain repetitive Alu elements. Each Alu sequence is associated with a 3'-oligoadenylate [oligo(A)] sequence, which is demonstrated to exhibit instability in human colonic tumors. The oligo(A) sequences only decrease in length, unlike microsatellites, which can either increase or decrease. Rigorous quantitative analysis of the length of the oligo(A) sequence in colonic tumors demonstrates that the standard deviation of the length of the sequence in tumors is a function of the mean length; i.e., as the oligo(A) sequence becomes progressively shorter, the variance increases. Both measurements, therefore, provide a quantitative index of the extent of instability in a tissue. Comparison of instability at the oligo(A) loci defined by 50C10 and 52H10 to instability at a CA microsatellite upstream of the apoD gene, and comparison of an oligo(A) and a CA microsatellite both in the 3' untranslated region of the cyclin D1 mRNA demonstrate that instability in a tumor, when present, is more prominent for the oligo(A) sequences than for the microsatellite (P < 0.0001). This suggests either that the mechanisms that generate oligo(A) instability are more penetrant than those that generate microsatellite instability, or that the instability at oligo(A) sequences takes place earlier in the development of the tumor and is selected for, thus becoming more prominent. These features of oligo(A) instability suggest that they may be uniquely useful in detecting and quantifying instability in tissues. Further, the presence of repetitive sequence elements in loci overexpressed in colonic mucosa at risk may be related to an extensive literature that demonstrates that a variety of repetitive sequences accumulate in the cellular RNA population during carcinogenesis and in tumors. Such RNA sequences could play a mechanistic role in tumor development.

Amino Acid Sequence↗
PubMed1995

Double-stranded RNA: the variables controlling its degradation by RNases.

The kinetics of single-stranded (SS) and double-stranded (ds) polyribonucleotides cleavage by three mammalian pancreatic type ribonucleases have been studied under low and high salt conditions. The values kcat, Km, and kcat/Km for depolymerization of poly(U), poly(A).poly(U), poly(I) and poly(I).poly(C) by bovine RNase A, bovine seminal RNase, and human seminal RNase have been determined and compared to each other. The Km values of bovine RNase A for (ss) or (ds) substrates were of the same order of magnitude under low and high ionic strength conditions, while their kcat values were found to differ considerably. Qualitatively similar results were obtained with bovine and human seminal RNases, i.e., the activity ratios (ssRNA/dsRNA) were mostly determined by the ratio of kcat values. It was shown that the modest levels of activity toward dsRNAs shown by single-strand-preferring RNases may occur by a mechanism consisting in the binding of the RNase to single nucleotides which are wound off the double helix because of thermal fluctuations. A higher activity and its enhancement as a function of number and location of the positive charges present on the RNase surface (human seminal RNase > bovine seminal RNase > bovine RNase A), as well as its increase under low ionic strength conditions, could instead be explained by the increased occurence of the splitting mechanism based on the binding of the RNase to single-stranded RNA sequences transiently exposed from the RNA double-helix.

Animals↗
PubMed1995

Using in vitro selection to direct the covalent attachment of human immunodeficiency virus type 1 Rev protein to high-affinity RNA ligands.

We have used an in vitro selection procedure called crosslinking SELEX (SELEX = systematic evolution of ligands by exponential enrichment) to identify RNA sequences that bind with high affinity and crosslink to the Rev protein from human immunodeficiency virus type 1 (HIV-1). A randomized RNA library substituted with the photoreactive chromophore 5-iodouracil was irradiated with monochromatic UV light in the presence of Rev. Those sequences with the ability to photocrosslink to Rev were partitioned from the rest of the RNA pool, amplified, and used for the next round of selection. Rounds of photocrosslinking selection were alternated with rounds of selection for RNA sequences with high affinity to Rev. This iterative, dual-selection method yielded RNA molecules with subnanomolar dissociation constants and high efficiency photocrosslinking to Rev. Some of the RNA molecules isolated by this procedure form a stable complex with Rev that is resistant to denaturing gel electrophoresis in the absence of UV irradiation. In vitro selection of nucleic acids by using modified nucleotides allows the isolation of nucleic acid molecules with potentially limitless chemical capacities to covalently attack a target molecule.

Base Sequence↗
PubMed1992

Specificity of Rous sarcoma virus nucleocapsid protein in genomic RNA packaging.

Site-directed mutagenesis has shown that the nucleocapsid (NC) protein of Rous sarcoma virus (RSV) is required for packaging and dimerization of viral RNA. However, it has not been possible to demonstrate, in vivo or in vitro, specific binding of viral RNA sequences by NC. To determine whether specific packaging of viral RNA is mediated by NC in vivo, we have constructed RSV mutants carrying sequences of Moloney murine leukemia virus (MoMuLV). Either the NC coding region alone, the psi RNA packaging sequence, or both the NC and psi sequences of MoMuLV were substituted for the corresponding regions of a full-length RSV clone to yield chimeric plasmid pAPrcMNC, pAPrc psi M, or pAPrcM psi M, respectively. In addition, a mutant of RSV in which the NC is completely deleted was tested as a control. Upon transfection, each of the chimeric mutants produced viral particles containing processed core proteins but were noninfectious. Thus, MoMuLV NC can replace RSV NC functionally in the assembly and release of mature virions but not in infectivity. Surprisingly, the full-deletion mutant showed a strong block in virus release, suggesting that NC is involved in virus assembly. Mutant PrcMNC packaged 50- to 100-fold less RSV RNA than did the wild type; in cotransfection experiments, MoMuLV RNA was preferentially packaged. This result suggests that the specific recognition of viral RNA during virus assembly involves, at least in part, the NC protein.

Amino Acid Sequence↗
PubMed1997

The Streptomyces galP1 promoter has a novel RNA polymerase recognition sequence and is transcribed by a new form of RNA polymerase in vitro.

We report the identification of DNA sequences that determine the activity of the Streptomyces galP1 promoter and a new form of RNA polymerase holoenzyme that recognizes these sequences in vitro. Base substitutions were introduced throughout the galP1 promoter region, and bases at positions -34, -36, and -11 with respect to the transcription start site were shown to be required for promoter function. These bases correspond in their positions to regions known to be important for RNA polymerase binding in several classes of eubacterial promoters, but the sequences themselves are not similar to those previously described. The -35 region of the galP1 promoter consists of six G residues, and base changes in this G hexamer had a dramatic effect on promoter activity. By using galP1-containing DNA template, a new RNA polymerase activity was purified from Streptomyces. Holoenzyme reconstitution experiments identified a new sigma factor that directs galP1 transcription in vitro. DNase I protection experiments identified a binding site for this new holoenzyme immediately upstream of the galP1 transcription start site.

Base Sequence↗
PubMed1988

The 5'-terminal sequence of VSV(NJ) (Ogden): is the interaction of the NS protein with the NS binding site responsible for heterotypic interference activity?

The 5'-terminal sequence of VSV(NJ) (Ogden) and VSV(NJ) (Hazelhurst) was compared in an attempt to understand why the defective interfering particle, DI-LT, heterotypically interferes with VSV(NJ) (Ogden) but not with VSV(NJ) (Hazelhurst). The 5'-terminal sequence of VSV(NJ) (Ogden) genomic RNA was determined by direct RNA sequencing and by DNA sequencing of cDNA clones of the 3'-terminal sequence of VSV(NJ) (Ogden) DI particle genome. Primer extension analysis of the 5'-terminus of VSV(NJ) (Ogden) standard genomic RNA confirmed these data. Within the last 47 nucleotides, equivalent to the negative-strand leader RNA, the only nucleotide changes between VSV(NJ) (Ogden) and VSV(NJ) (Hazelhurst) occur between nucleotides 19 and 26, representing part of the putative NS binding region described by Isaac and Keene (J. Virol. 43, 241-249 (1982] for VSV(IND) DI particles. The spacer (S) region, located between the polyadenylation signal of the L gene and the 47th nucleotide of the leader RNA, contains more differences. The polyadenylation signal of the L gene is fully conserved, but the remainder of the L gene region (177 nucleotides) has highly diverged between VSV(NJ) (Ogden) and VSV(NJ) (Hazelhurst). The changes in the NS binding region of the negative-strand leader RNA provide further evidence for the divergent evolution of VSV(NJ) (Ogden) and VSV(NJ) (Hazelhurst). The NS binding region has been implicated as a crucial site for the initiation of RNA transcription and replication. The interaction of the NS protein with this site may determine the ability of DI particles to interfere heterotypically.

Base Sequence↗
PubMed1989

A functional analysis of the repeated methionine initiator tRNA genes (IMT) in yeast.

Standard laboratory yeast strains have from four to five genes encoding the methionine initiator tRNA (IMT). Strain S288C has four IMT genes with identical coding sequences that are colinear with the RNA sequence of tRNA(IMet). Each of the four IMT genes from strain S288C is located on a different chromosome. A fifth IMT gene with the same coding sequence is present in strain A364A but not in S288C. By making combinations of null alleles in strain S288C, we show that each of the four IMT genes is functional and that tRNA(IMet) is not limiting in yeast strains with three or more intact genes. Strains containing a single IMT2, 3 or 4 gene grow only after amplification of the remaining IMT gene. Strains with only the IMT1 gene intact are viable but grow extremely slow; normal growth is restored by the addition of another IMT gene by transformation, providing a direct test for IMT function.

Base Sequence↗
PubMed1988

Alternative use of a polyadenylation signal and of a downstream 3' splice site. Effect of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole.

A minor pathway for the processing of transcripts from the early region 3 transcription unit of adenovirus 2 is described. It results from the selection of a promoter-proximal polyadenylation site P1 instead of the major promotor-distal sites P2 and P4. 5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) considerably reduces the use of the major pathways and enhances the minor one. The characterization of three novel nuclear RNAs whose amount increases in DRB-treated cells leads us to propose the following steps for the selection of P1. The nascent transcript is first cleaved in the branch point-3' splice site region of the first E3 IVS (intervening sequence), generating a promoter-proximal RNA with a heterogeneous 3' end (F-RNA) and a promoter-distal RNA with a heterogeneous 5' end (G-RNA). This cleavage prevents the formation of the premessenger RNAs ending at P2 and P4. The 3' end of F-RNA is about 130 nucleotides downstream from P1 and F-RNA contains the signals required for cleavage-polyadenylation. It may thus generate CP1, which is the transcript ending at P1. The cleavage of the nascent transcript in a region crucial for spliceosome assembly suggests that defective assembly may render RNA sequences at the 3' end of IVS accessible to intracellular nucleases and trigger the use of an upstream polyadenylation site. Such a mechanism may explain the alternative use of neighbouring, mutually exclusive, splice and polyadenylation sites.

Adenoviridae↗
PubMed1991

Cryptomonad algae are evolutionary chimaeras of two phylogenetically distinct unicellular eukaryotes.

Although it is widely accepted that the plastids of plants and algae originated as endosymbionts, the details of this evolutionary process are unclear. It has been proposed that in organisms whose plastids are surrounded by more than two membranes, the endosymbiont was a eukaryotic alga rather than a photosynthetic prokaryote. The DNA-containing nucleomorph of cryptomonad algae appears to be the vestigial nucleus of such an algal endosymbiont. Eukaryotic-type ribosomal RNA sequences have been localized to a nucleolus-like structure in the nucleomorph. In support of the hypothesis that cryptomonads are evolutionary chimaeras of two distinct eukaryotic cells, we show here that Cryptomonas phi contains two phylogenetically separate, nuclear-type small-subunit rRNA genes, both of which are transcriptionally active. We incorporate our rRNA sequence data into phylogenetic trees, from which we infer the evolutionary ancestry of the host and symbiont components of Cryptomonas phi. Such trees do not support the thesis that chromophyte algae evolved directly from a cryptomonad-like ancestor.

Animals↗
PubMed1998

Human Tra2 proteins are sequence-specific activators of pre-mRNA splicing.

The RNA-binding protein Tra2 is an important regulator of sex determination in Drosophila. Recently, two mammalian Tra2 homologs of unknown function have been described. Here, we show that human Tra2 proteins are present in HeLa cell nuclear extracts and that they bind efficiently and specifically to a previously characterized pre-mRNA splicing enhancer element. Indeed, both purified proteins bound preferentially to RNA sequences containing GAA repeats, characteristic of many enhancer elements. Neither Tra2 protein functioned in constitutive splicing in vitro, but both activated enhancer-dependent splicing in a sequence-specific manner and restored it after inhibition with competitor RNA. Our findings indicate that mammalian Tra2 proteins are sequence-specific splicing activators that likely participate in the control of cell-specific splicing patterns.

Animals↗
PubMed2000

Identification of a cis-acting replication element within the poliovirus coding region.

The replication of poliovirus, a positive-stranded RNA virus, requires translation of the infecting genome followed by virus-encoded VPg and 3D polymerase-primed synthesis of a negative-stranded template. RNA sequences involved in the latter process are poorly defined. Since many sequences involved in picornavirus replication form RNA structures, we searched the genome, other than the untranslated regions, for predicted local secondary structural elements and identified a 61-nucleotide (nt) stem-loop in the region encoding the 2C protein. Covariance analysis suggested the structure was well conserved in the Enterovirus genus of the Picornaviridae. Site-directed mutagenesis, disrupting the structure without affecting the 2C product, destroyed genome viability and suggested that the structure was required in the positive sense for function. Recovery of revertant viruses suggested that integrity of the structure was critical for function, and analysis of replication demonstrated that nonviable mutants did not synthesize negative strands. Our conclusion, that this RNA secondary structure constitutes a novel poliovirus cis-acting replication element (CRE), is supported by the demonstration that subgenomic replicons bearing lethal mutations in the native structure can be restored to replication competence by the addition of a second copy of the 61-nt wild-type sequence at another location within the genome. This poliovirus CRE functionally resembles an element identified in rhinovirus type 14 (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998) and the cardioviruses (P. E. Lobert, N. Escriou, J. Ruelle, and T. Michiels, Proc. Natl. Acad. Sci. USA 96:11560-11565, 1999) but differs in sequence, structure, and location. The functional role and evolutionary significance of CREs in the replication of positive-sense RNA viruses is discussed.

Analysis of Variance↗
PubMed1981

Nucleotide sequence of Xenopus laevis 18S ribosomal RNA inferred from gene sequence.

18S ribosomal RNA in Xenopus laevis is 1,825 nucleotides long, as inferred from sequence analysis of an 18S gene. All the 40 rRNA methyl groups can be located in the sequence. Comparison with the yeast (Saccharomyces cerevisiae) 18S sequence reveals extensive regions of high homology interspersed with tracts having little or no homology. Regions of high homology contain almost all the RNa methyl groups. Major regions of low homology area considerably richer in C + G in Xenopus than in yeast.

Animals↗
PubMed1975

Comparison of an avian osteopetrosis virus with an avian lymphomatosis virus by RNA-DNA hybridization.

Myeloblastosis-associated virus (MAV)-2(0), a virus which was derived from avian myeloblastosis virus and induced a high incidence of osteopetrosis, was compared with avian lymphomatosis virus 5938, a recent field isolate which induced a high incidence of lymphomatosis. The following information was obtained. (i) MAV-2(0) induced osteopetrosis, nephroblastoma, and a very low incidence of hepatocellular carcinoma. No difference was seen in the oncogenic spectrum of end point and plaque-purified MAV-2(0). (ii) 125I-labeled RNA sequences from MAV-2(0) formed hybrids with DNA extracted from osteopetrotic bone at a rate suggesting five proviral copies per haploid cell genome. The extent of hybridization of MAV-2(0) RNA with DNA from osteopetrotic tissue was more extensive (87%) than was observed in reactions with DNA from uninfected chicken embryos (52%). (iii) Competition of unlabeled viral RNA in hybridization reactions between the radioactive RNA from the two viruses and their respective proviral sequences present in tumor tissues showed that 15 to 20% of the viral sequences detected in these reactions were unshared. In contrast, no differences were detected in competition analyses of RNA sequences from the two viruses detected in DNA of normal chicken cells. (iv) MAV-2(0) 35S RNA was indistinguishable in size from avian lymphomatosis virus 5938 35S RNA by polyacrylamide gel electrophoresis.

Animals↗
PubMed1989

Identification of cellular proteins that bind to the human immunodeficiency virus type 1 trans-activation-responsive TAR element RNA.

Human immunodeficiency virus type 1 (HIV-1) trans-activator protein Tat activates the expression of its viral long terminal repeat (LTR) through a target transactivation-responsive element termed TAR. We have constructed cell lines that constitutively express the HIV-1 Tat protein. Analyses of nuclear proteins from these cells and from matched control cells that do not express Tat have identified three proteins that bind to a radiolabeled HIV-1 TAR RNA probe. These polypeptides are 100 kDa, 62 kDa, and 46 kDa in size. Competition experiments using a wild-type TAR RNA sequence, a biologically inactive mutant sequence of TAR, and an unrelated RNA species demonstrated that these proteins show higher binding affinity to wild-type TAR than to the other two non-trans-activatable sequences. We hypothesize that these cellular proteins may mediate a function necessary in Tat-dependent activation of the LTR. The fact that no differences were seen in the binding profiles of nuclear proteins to TAR RNA in Tat-producing and Tat-nonproducing cells suggests that Tat does not directly interact with TAR.

Base Sequence↗
PubMed2005

A complex adaptive systems approach to the kinetic folding of RNA.

The kinetic folding of RNA sequences into secondary structures is modeled as a complex adaptive system, the components of which are possible RNA structural rearrangements (SRs) and their associated bases and base pairs. RNA bases and base pairs engage in local stacking interactions that determine the probabilities (or fitnesses) of possible SRs. Meanwhile, selection operates at the level of SRs; an autonomous stochastic process periodically (i.e., from one time step to another) selects a subset of possible SRs for realization based on the fitnesses of the SRs. Using examples based on selected natural and synthetic RNAs, the model is shown to reproduce characteristic (nonlinear) RNA folding dynamics such as the attainment by RNAs of alternative stable states. Possible applications of the model to the analysis of properties of fitness landscapes, and of the RNA sequence-to-structure mapping are discussed.

Genes, Bacterial↗
PubMed1996

Identification of nuclear and cytoplasmic proteins that interact specifically with an AU-rich, cis-acting inhibitory sequence in the 3' untranslated region of human papillomavirus type 1 late mRNAs.

Expression of human papillomavirus late genes encoding L1 and L2 capsid proteins is restricted to terminally differentiated epithelial cells. We have previously identified and characterized an AU-rich, cis-acting negative regulatory element in the 3' untranslated region of human papillomavirus type 1 late mRNAs. This element acts posttranscriptionally to reduce mRNA levels and the translation efficiency of mRNAs. The experiments reported here are a continuation of our previous work. We have used RNA gel shifts and UV cross-linking assays to identify cellular proteins that interact with the inhibitory RNA sequence of human papillomavirus type 1. RNA gel shift assays established that cellular proteins interact with the AU-rich sequence. The binding of nuclear proteins was inhibited by competition with poly(U), whereas the binding of cytoplasmic proteins was inhibited by competition with poly(U) and also by competition with poly(A) and poly(G). Two nuclear proteins and two cytoplasmic proteins that bind specifically to the AU-rich RNA sequence were identified by UV cross-linking. These proteins did not bind to the 3' untranslated region of human papillomavirus type 1 early mRNAs, which does not show inhibitory activity. The cellular proteins identified in our experiments may therefore be involved in the inhibition of human papillomavirus type 1 late gene expression in nondifferentiated epithelial cells.

Base Sequence↗
PubMed2004

The first complete monogenean ribosomal RNA gene operon: sequence and secondary structure of the Gyrodactylus salaris Malmberg, 1957, large subunit ribosomal RNA gene.

The sequence of the Gyrodactylus salaris Malmberg, 1957, large subunit, or 28S, ribosomal RNA (rRNA) gene has been determined. This gene is the final portion of the Gyrodactylus rRNA gene operon to be sequenced and results in the first complete sequence of all rRNA genes and spacers from a monogenean. The nucleotide sequence was used to predict the secondary structure of the large subunit rRNA, and regions of conserved and variable sequence and structure were identified. The site where the 5' terminus of the 5.8S rRNA binds to a region within the large subunit rRNA was predicted and complements the anticipated interaction of the 3' terminus of the 5.8S with the 5' terminus of the large subunit rRNA. The large subunit gene may be useful in phylogenetic analysis of the Monogenea or Platyhelminthes and comparisons with other eukaryotes. The variable domains C and H may be most suitable for this purpose.

Animals↗