Showing papers on "RNA published in 1987"

Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates

Abstract: A method is described to synthesize small RNAs of defined length and sequence using T7 RNA polymerase and templates of synthetic DNA which contain the T7 promoter. Partially single stranded templates which are base paired only in the -17 to +1 promoter region are just as active in transcription as linear plasmid DNA. Runoff transcripts initiate at a unique, predictable position, but may have one nucleotide more or less on the 3' terminus. In addition to the full length products, the reactions also yield a large amount of smaller oligoribonucleotides in the range from 2 to 6 nucleotides which appear to be the result of abortive initiation events. Variants in the +1 to +6 region of the promoter are transcribed with reduced efficiency but increase the variety of RNAs which can be made. Transcription reaction conditions have been optimized to allow the synthesis of milligram amounts of virtually any RNA from 12 to 35 nucleotides in length.

Expression of a multidrug-resistance gene in human tumors and tissues

Abstract: The identification and cloning of a segment of a human multidrug resistance gene (mdr1) was reported recently. To examine the molecular basis of one type of multidrug resistance, we have prepared RNA from human tumors and normal tissues and measured their content of mdr1 RNA. We find that the mdr1 gene is expressed at a very high level in the adrenal gland; at a high level in the kidney; at intermediate levels in the lung, liver, lower jejunum, colon, and rectum; and at low levels in many other tissues. The mdr1 gene is also expressed in several human tumors, including many but not all tumors derived from the adrenal gland and the colon. In addition, increased expression was detected in a few tumors at the time of relapse following initial chemotherapy. Although controlled clinical studies will be required, our results suggest that measurement of mdr1 RNA may prove to be a valuable tool in the design of chemotherapy protocols.

Characterization and chromosomal localization of a cDNA encoding brain amyloid of Alzheimer's disease

Abstract: Four clones were isolated from an adult human brain complementary DNA library with an oligonucleotide probe corresponding to the first 20 amino acids of the beta peptide of brain amyloid from Alzheimer's disease. The open reading frame of the sequenced clone coded for 97 amino acids, including the known amino acid sequence of this polypeptide. The 3.5-kilobase messenger RNA was detected in mammalian brains and human thymus. The gene is highly conserved in evolution and has been mapped to human chromosome 21.

Neural cell adhesion molecule: structure, immunoglobulin-like domains, cell surface modulation, and alternative RNA splicing.

Abstract: The neural cell adhesion molecule, N-CAM, appears on early embryonic cells and is important in the formation of cell collectives and their boundaries at sites of morphogenesis. Later in development it is found on various differentiated tissues and is a major CAM mediating adhesion among neurons and between neurons and muscle. To provide a molecular basis for understanding N-CAM function, the complete amino acid sequences of the three major polypeptides of N-CAM and most of the noncoding sequences of their messenger RNA's were determined from the analysis of complementary DNA clones and were verified by amino acid sequences of selected CNBr fragments and proteolytic fragments. The extracellular region of each N-CAM polypeptide includes five contiguous segments that are homologous in sequence to each other and to members of the immunoglobulin superfamily, suggesting that interactions among immunoglobulin-like domains form the basis for N-CAM homophilic binding. Although different in their membrane-associated and cytoplasmic domains, the amino acid sequences of the three polypeptides appear to be identical throughout this extracellular region (682 amino acids) where the binding site is located. Variations in N-CAM activity thus do not occur by changes in the amino acid sequence that alter the specificity of binding. Instead, regulation is achieved by cell surface modulation events that alter N-CAM affinity, prevalence, mobility, and distribution on the surface. A major mechanism for modulation is alternative RNA splicing resulting in N-CAM's with different cytoplasmic domains that differentially interact with the cell membrane. Such regulatory mechanisms may link N-CAM binding function with other primary cellular processes during the embryonic development of pattern.

Interaction of antibiotics with functional sites in 16S ribosomal RNA

Abstract: Chemical footprinting shows that several classes of antibiotics (streptomycin, tetracycline, spectinomycin, edeine, hygromycin and the neomycins) protect concise sets of highly conserved nucleotides in 16S ribosomal RNA when bound to ribosomes These findings have strong implications for the mechanism of action of these antibiotics and for the assignment of functions to specific structural features of 16S rRNA

Rna ribozyme polymerases, dephosphorylases, restriction endoribonucleases and methods.

Abstract: RNA enzymes or ribozymes can act as endoribonucleases, catalyzing the cleavage of RNA molecules with a sequence specificity of cleavage greater than that of known ribonucleases and approaching that of the DNA restriction endonucleases, thus serving as RNA sequence specific endoribonucleases. An example is a shortened form of the self-splicing ribosomal RNA intervening sequence of Tetrahymena (L-19 IVS RNA). Site-specific mutagenesis of the enzyme active site of the L-19 IVS RNA alters the substrate sequence specificity in a predictable manner, allowing a set of sequence-specific endoribonucleases to be synthesized. Varying conditions allow the ribozyme to act as a polymerase (nucleotidyltransferase), a dephosphorylase (acid phosphatase or phosphotransferase) or a sequence-specific endoribonuclease.

RNA complementary to a herpesvirus alpha gene mRNA is prominent in latently infected neurons.

Abstract: In initial attempts to define the molecular events responsible for the latent state of herpes simplex virus, in situ hybridization was utilized to search for virally encoded RNA transcripts in latently infected sensory neurons. The use of cloned probes representing the entire viral genome indicated that transcripts encoded within terminal repeats were present. When the alpha genes encoding ICP-0, ICP-4, and ICP-27 and the gamma 1 gene encoding VP-5 were employed, only RNA transcripts hybridizing to the ICP-0 probe were detected. In latently infected cells, the ICP-0--related transcripts were localized principally in the nucleus; this was not the case in acutely (productively) infected neurons or in neurons probed for RNA transcripts coding for actin. In Northern blotting experiments, an RNA of 2.6 kilobases was detected with the ICP-0 probe. When single-stranded DNAs from the ICP-0 region were used as probes, RNA from the strand complementary to that encoding ICP-0 messenger RNA (mRNA) was the major species detected. This RNA species may play a significant role in maintaining the latent infection.

A small catalytic oligoribonucleotide

Abstract: A 19-nucleotide RNA fragment can cause rapid, highly specific cleavage of a 24-nucleotide RNA fragment under physiological conditions. Because each 19-mer can participate in many cleavage reactions, this molecule has all the properties associated with an RNA enzyme.

Anti-termination of transcription within the long terminal repeat of HIV-1 by tat gene product.

Abstract: Human immunodeficiency virus-1 (HIV-1) gene expression is controlled by cellular transcription factors and by virally encoded trans-activation proteins of the HIV-1 tat and art/trs genes, which are essential for viral replication1,9–11. Tat trans-activates HIV-1 gene expression by interacting with the trans-acting response element (TAR) located within the HIV-1 long terminal repeat (LTR) (ref. 2). In transient expression assays, tat mediates its effects largely by increasing the steady-state levels of messenger RNA species that contain the TAR sequence at or near their 5′ ends2–4, suggesting a function for tat either in transcription or in subsequent RNA processing. The tat gene could also facilitate translation of mRNA containing the TAR sequence5–8. To determine the mechanism of trans-activation by tat, we analysed the structure and rate of synthesis of RNA species directed by the HIV-1 LTR in transient expression assays both in the presence and absence of tat. Although the rate of HIV-1 transcription initiation was not affected by tat, transcriptional elongation beyond position +59 was seen only in the presence of tat. Thus, tat trans-activates HIV-1 transcription by relieving a specific block to transcriptional elongation within the TAR sequence.

Apolipoprotein B-48 is the product of a messenger RNA with an organ-specific in-frame stop codon.

Abstract: The primary structure of human apolipoprotein (apo) B-48 has been deduced and shown by a combination of DNA excess hybridization, sequencing of tryptic peptides, cloned complementary DNAs, and intestinal messenger RNAs (mRNAs) to be the product of an intestinal mRNA with an in-frame UAA stop codon resulting from a C to U change in the codon CAA encoding Gln2153 in apoB-100 mRNA. The carboxyl-terminal Ile2152 of apoB-48 purified from chylous ascites fluid has apparently been cleaved from the initial translation product, leaving Met2151 as the new carboxyl-terminus. These data indicate that approximately 85% of the intestinal mRNAs terminate within approximately 0.1 to 1.0 kilobase downstream from the stop codon. The other approximately 15% have lengths similar to hepatic apoB-100 mRNA even though they have the same in-frame stop codon. The organ-specific introduction of a stop codon to a mRNA appears unprecedented and might have implications for cryptic polyadenylation signal recognition and RNA processing.

Probing the structure of RNAs in solution

Abstract: During these last years, a powerful methodology has been developed to study the secondary and tertiary structure of RNA molecules either free or engaged in complex with proteins. This method allows to test the reactivity of every nucleotide towards chemical or enzymatic probes. The detection of the modified nucleotides and RNase cleavages can be conducted by two different paths which are oriented both by the length of the studied RNA and by the nature of the probes used. The first one uses end-labeled RNA molecule and allows to detect only scissions in the RNA chain. The second approach is based on primer extension by reverse transcriptase and detects stops of transcription at modified or cleaved nucleotides. The synthesized cDNA fragments are then sized by electrophoresis on polyacrylamide:urea gels. In this paper, the various structure probes used so far are described, and their utilization is discussed.

Antisense RNA inactivation of myosin heavy chain gene expression in Dictyostelium discoideum

Abstract: The role of myosin in the contraction of striated muscle cells is well known, but its importance in nonmuscle cells is not yet clear. The function of myosin in Dictyostelium discoideum has been investigated by isolating cells which specifically lack myosin heavy chain (MHC A) protein. Cells were transformed with a vector encoding RNA complementary to mhcA messenger RNA (antisense RNA). Stable transformants have a dramatic reduction in the amount of MHC A protein, grow slowly, and generate giant multinucleated progeny, indicating an impairment in cytokinesis. Surprisingly, the cells adhere to surfaces, extend pseudopods and are capable of ameboid locomotion. The developmental sequence that is initiated by starving cells is severely impaired by the lack of myosin. The cells are unable to form multicellular aggregates normally and do not undergo subsequent morphogenesis. By changing the food source from liquid medium to bacteria, expression of the endogenous mhcA messenger RNA can be increased relative to expression of antisense RNA. When grown in this way, the transformed cells accumulate MHC A protein, remain mononucleate, and proceed through development normally.

Three rat preprotachykinin mRNAs encode the neuropeptides substance P and neurokinin A.

Abstract: Synthetic oligonucleotides were used to screen a rat striatal cDNA library for sequences corresponding to the tachykinin peptides substance P and neurokinin A. The cDNA library was constructed from RNA isolated from the rostral portion of the rat corpus striatum, the site of striatonigral cell bodies. Two types of cDNAs were isolated and defined by restriction enzyme analysis and DNA sequencing to encode both substance P and neurokinin A. The two predicted preprotachykinin protein precursors (130 and 115 amino acids in length) differ from each other by a pentadecapeptide sequence between the two tachykinin sequences, and both precursors possess appropriate processing signals for substance P and neurokinin A production. The presence of a third preprotachykinin mRNA of minor abundance in rat striatum was established by S1 nuclease protection experiments. This mRNA encodes a preprotachykinin of 112 amino acids containing substance P but not neurokinin A. These three mRNAs are derived from one rat gene as a result of differential RNA processing; thus, this RNA processing pattern further increases the diversity of products that can be generated from the preprotachykinin gene.

Isolation and characterization of the c-fos(rat) cDNA and analysis of post-translational modification in vitro.

Abstract: c-fos mRNA accumulates to a level of 0.2% of cellular poly(A)-containing RNA 45 min after treatment of rat pheochromocytoma (PC12) cells with 1 mM barium chloride. Several clones of the c-fos(rat) cDNA were isolated from a cDNA library constructed from this RNA population. Nucleotide sequence analysis of a full-length cDNA clone reveals striking conservation among the c-fos genes isolated from rat, mouse and human cells, and confirms the c-fos gene structure predicted from an analysis of c-fos genomic clones. Translation of an SP6-derived transcript of the c-fos(rat) cDNA in a messenger-dependent rabbit reticulocyte lysate yields the complete c-fos protein. It undergoes extensive post-translational modification in the lysate, particularly in the presence of additional cAMP. The c-fos protein synthesized in vitro appears to be phosphorylated by the cAMP-dependent protein kinase.

Rapid Evolution of RNA Viruses

Abstract: The high error rate inherent in all RNA synthesis provides RNA virus genomes with extremely high mutation rates. Thus nearly all large RNA virus clonal populations are quasispecies collections of differing, related genomes (14, 49). These rapidly mutating populations can remain remarkably stable under certain conditions of replication. Under other conditions, virus-population equilibria become disturbed, and extremely rapid evolution can result. This extreme variability and rapid evolution can cause severe problems with previously unknown virus diseases (such as AIDS). It also presents daunting challenges for the design of effective vaccines for the control of diseases caused by rapidly evolving RNA virus populations.

Calretinin: a gene for a novel calcium-binding protein expressed principally in neurons.

Abstract: A novel gene of the calmodulin superfamily, encoding a 29-kD neuronal protein here named "calretinin," has been isolated as a cDNA clone from chick retina. The encoded sequence includes four putative calcium-binding sites and a fusion protein binds calcium. The most similar protein known is the 28-kD intestinal calcium-binding protein, calbindin (58% homology). Both genes date from before the divergence of chicks from mammals. The distribution of calretinin and calbindin mRNAs in chick tissues has been mapped using RNA gel blots and in situ hybridization. RNAs from both genes are abundant in the retina and in many areas of the brain, but calretinin RNA is absent from intestine and other nonneural tissues. Calretinin and calbindin are expressed in different sets of neurons throughout the brain. Calretinin RNA is particularly abundant in auditory neurons with precisely timed discharges.

Ribosomal RNA sequence suggests microsporidia are extremely ancient eukaryotes

Abstract: A comparative sequence analysis of the 18S small subunit ribosomal RNA (rRNA) of the microsporidium Vairimorpha necatrix is presented. The results show that this rRNA sequence is more unlike those of other eukaryotes than any known eukaryote rRNA sequence. It is concluded that the lineage leading to microsporidia branched very early from that leading to other eukaryotes.

Identification of the iron-responsive element for the translational regulation of human ferritin mRNA

Abstract: Regulated translation of messenger RNA offers an important mechanism for the control of gene expression. The biosynthesis of the intracellular iron storage protein ferritin is translationally regulated by iron. A cis-acting element that is both necessary and sufficient for this translational regulation is present within the 5' nontranslated leader region of the human ferritin H-chain messenger RNA. In this report the iron-responsive element (IRE) was identified by deletional analysis. Moreover, a synthetic oligodeoxynucleotide was shown to be able to transfer iron regulation to a construct that would otherwise not be able to respond to iron. The IRE has been highly conserved and predates the evolutionary segregation between amphibians, birds, and man. The IRE may prove to be useful for the design of translationally regulated expression systems.

Production of infectious RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants.

Abstract: We constructed full-length cDNA clones of Sindbis virus that can be transcribed in vitro by SP6 RNA polymerase to produce infectious genome-length transcripts. Viruses produced from in vitro transcripts are identical to Sindbis virus and show strain-specific phenotypes reflecting the source of RNA used for cDNA synthesis. The cDNA clones were used to confirm the mapping of the causal mutation of ts2 to the capsid protein. A general strategy for mapping Sindbis virus mutations is described and was used to identify two lethal mutations in an original full-length construct which did not produce infectious transcripts. An XbaI linker was inserted in the cDNA clone near the transcriptional start of the subgenomic mRNA; the resulting virus retains the XbaI recognition sequence, thus providing formal evidence that viruses are derived from in vitro transcripts of cDNA clones. The potential applications of the cDNA clones are discussed.

The chemistry of self-splicing RNA and RNA enzymes.

Abstract: Proteins are not the only catalysts of cellular reactions; there is a growing list of RNA molecules that catalyze RNA cleavage and joining reactions. The chemical mechanisms of RNA-catalyzed reactions are discussed with emphasis on the self-splicing ribosomal RNA precursor of Tetrahymena and the enzymatic activities of its intervening sequence RNA. Wherever appropriate, catalysis by RNA is compared to catalysis by protein enzymes.

Molecular cloning of complementary DNA encoding the avian receptor for vitamin D.

Abstract: Vitamin D3 receptors are intracellular proteins that mediate the nuclear action of the active metabolite 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Two receptor-specific monoclonal antibodies were used to recover the complementary DNA (cDNA) of this regulatory protein from a chicken intestinal lambda gt11 cDNA expression library. The amino acid sequences that were deduced from this cDNA revealed a highly conserved cysteine-rich region that displayed homology with a domain characteristic of other steroid receptors and with the gag-erbA oncogene product of avian erythroblastosis virus. RNA selected via hybridization with this DNA sequence directed the cell-free synthesis of immunoprecipitable vitamin D3 receptor. Northern blot analysis of polyadenylated RNA with these cDNA probes revealed two vitamin D receptor messenger RNAs (mRNAs) of 2.6 and 3.2 kilobases in receptor-containing chicken tissues and a major cross-hybridizing receptor mRNA species of 4.2 kilobases in mouse 3T6 fibroblasts. The 4.2-kilobase species was substantially increased by prior exposure of 3T6 cells to 1,25(OH)2D3. This cDNA represents perhaps the rarest mRNA cloned to date in eukaryotes, as well as the first receptor sequence described for an authentic vitamin.

Anti-sense regulation of gene expression in plant cells

Abstract: Regulation of expression of genes encoded for in plant cell genomes is achieved by integration of a gene under the transcriptional control of a promoter which is functional in the host and in which the trans­cribed strand of DNA is complementary to the strand of DNA that is transcribed from the endogenous gene(s) one wishes to regulate. The integrated gene, referred to as anti-sense, provides an RNA sequence capable of binding to naturally existing RNAs, exemplified by polygalacturonase, and inhibiting their expression, where the anti-sense sequence may bind to the coding, non-coding, or both, portions of the RNA. The anti-­sense construction may be introduced into the plant cells in a variety of ways and be integrated into the plant genome for inducible or constitutive transcrip­tion of the anti-sense sequence. A wide variety of plant cell properties may be modified by employing this technique.