Showing papers on "RNA published in 1983"
TL;DR: The RNA moieties of ribonuclease P purified from both E. coli and B. subtilis can cleave tRNA precursor molecules in buffers containing either 60 mM Mg2+ or 10 mM MG2+ plus 1 mM spermidine, and in vitro, the RNA and protein subunits from one species can complement sub units from the other species in reconstitution experiments.
2,524 citations
TL;DR: The approach described here permits the application of recombinant DNA technology to analyses of complex neurobiological systems in the absence of prior structural or biological information.
Abstract: Alternative processing of the RNA transcribed from the calcitonin gene appears to result in the production of a messenger RNA in neural tissue distinct from that in thyroidal 'C' cells The thyroid mRNA encodes a precursor to the hormone calcitonin whereas that in neural tissues generates a novel neuropeptide, referred to as calcitonin gene-related peptide (CGRP) The distribution of CGRP-producing cells and pathways in the brain and other tissues suggests functions for the peptide in nociception, ingestive behaviour and modulation of the autonomic and endocrine systems The approach described here permits the application of recombinant DNA technology to analyses of complex neurobiological systems in the absence of prior structural or biological information
2,243 citations
TL;DR: PMOV-psi- has a defect in the packaging of genomic RNA into virions but can provide in trans the products necessary for virion production, and can be used to produce helper-free stocks of natural or synthetic defective retroviruses.
1,810 citations
01 Jan 1983
TL;DR: A method for isolation of large, translationally active RNA species is presented and yields large mRNA precursors up to 10 kb and mRNA species which translate very well, however, small RNA species are recovered with a poor yield.
Abstract: A method for isolation of large, translationally active RNA species is presented. The procedure involves homogenization of cells or tissues in 5 m guanidine monothiocyanate followed by direct precipitation of RNA from the guanidinium by 4 m LiCl. Modifications are described for use with tissue culture cells, yeast, tissues, or isolated nuclei. The advantages of the procedure include speed, simplicity, avoidance of an ultracentrifugation, and its applicability to large numbers of small samples. The procedure yields large mRNA precursors up to 10 kb and mRNA species which translate very well. However, small (<300 nucleotides) RNA species are recovered with a poor yield.
1,348 citations
TL;DR: The Shine-Dalgarno sequence, appropriately positioned upstream from an exposed initiator codon, is sufficient to define a bacterial ribosome bnding site?
1,199 citations
TL;DR: All the advantages that nitrocellulose blotting previously afforded for DNA analysis are now available for RNA analysis as well.
Abstract: Publisher Summary The technique of E.M. Southern for transferring electrophoretically separated DNA fragments to nitrocellulose paper for subsequent hybridization with radioactive RNA or DNA probes has proved to be a powerful tool for the analysis of DNA. Until recently, the only comparable procedure for RNA has been to transfer and covalently couple it to activated cellulose paper (diazobenzyloxymethyl paper, DBM paper) according to the method of Alwine et al . Although DBM paper does offer the advantage of covalent attachment, it has several drawbacks as discussed in this chapter. The chapter also describes a related method for dotting RNA onto nitrocellulose pretreated with high salt. Thus, all the advantages that nitrocellulose blotting previously afforded for DNA analysis are now available for RNA analysis as well.
1,000 citations
TL;DR: The identity of genes that specify the largest RNA polymerase II subunits, the 220-000- and 150,000-dalton polypeptides, was confirmed by competitive radioimmune assay.
Abstract: Genes encoding yeast RNA polymerase II subunits were cloned. Efficient isolation of these genes was accomplished by probing a phage lambda gt11 recombinant DNA expression library with polyvalent antibodies directed against purified yeast RNA polymerase II. The identity of genes that specify the largest RNA polymerase II subunits, the 220,000- and 150,000-dalton polypeptides, was confirmed by competitive radioimmune assay. Both of these genes exist in single copy in the yeast Saccharomyces cerevisiae.
952 citations
TL;DR: An assay was developed whereby transcription of a cloned, rearranged kappa gene could be detected following its transfection into antibody-secreting mouse myeloma cells, suggesting that after rearrangement the kappa variable region promoter is activated by sequences more than 2.6 kb downstream of J kappa.
724 citations
TL;DR: Transcriptional analysis of five different cloned β-thalassaemia genes introduced into cultured mammalian cells revealed specific defects in transcription and RNA splicing that results in the formation of abnormal β-globin RNA that contains an extra exon.
Abstract: Transcriptional analysis of five different cloned beta-thalassaemia genes introduced into cultured mammalian cells revealed specific defects in transcription and RNA splicing. A single base change 87 base pairs to the 5' side of the mRNA cap site significantly lowers the level of transcription and therefore appears to represent a promoter mutation. Three genes contain different single base changes in the first intervening sequence (IVS) 5' splice site. One mutation, at IVS1 position 1, inactivates the splice site completely; the other two, at IVS1 positions 5 and 6, reduce its activity. Each mutation activates the same three cryptic splice sites. The fifth gene contains a single base change within IVS2 at position 745, which results in the formation of abnormal beta-globin RNA that contains an extra exon.
557 citations
TL;DR: The ability of purified U1 small nuclear RNA-protein complexes (U1 snRNPs) to bind in vitro to two RNAs transcribed from recombinant DNA clones by bacteriophage T7 RNA polymerase is studied.
498 citations
TL;DR: Direct sequence analysis of c-fos (mouse) RNA by primer extension demonstrates that the fourth discontinuity is due to a 104 bp deletion in the v- fos gene, and as a consequence of the deletion, the predicted v-fOS and c-Fos gene products differ at their C termini.
TL;DR: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purified from the breast muscles of 3-week-old chickens and used to raise a specific antiserum in rabbits coupled to an in vitro translation assay to monitor the purification of GAPDH mRNA.
Abstract: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purified from the breast muscles of 3-week-old chickens and used to raise a specific antiserum in rabbits. This antiserum was coupled to an in vitro translation assay to monitor the purification of GAPDH mRNA. RNA was isolated from identical breast muscles and consecutively fractionated with several techniques to yield a preparation of GAPDH mRNA which was at least 50% pure. Double-stranded cDNA was made against this purified RNA, inserted into pBR322, and used to transform Escherichia coli. Recombinants were screened by colony filter hybridization with a cDNA probe made against the purified RNA. The hybridization-positive clone with the largest insert, pGAD-28, was then characterized by using pGAD-28-cellulose to select complementary RNA from total poly(A) RNA and then translating the hybridization-selected RNA in vitro. The single translation product was shown to be GAPDH by (1) comigration with pure GAPDH on sodium dodecyl sulfate-polyacrylamide gels, (2) precipitation with specific anti-GAPDH antiserum, (3) cyanylation fingerprinting, and (4) AMP-agarose affinity chromatography. pGAD-28 was mapped with several restriction enzymes and then sequenced by the method of Maxam and Gilbert [Maxam, A. M., & Gilbert, W. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 560]. The 1261-nucleotide insert was found to contain 29 nucleotides of noncoding sequence at the 5' end, the entire coding region, and 230 nucleotides of the 3'-noncoding region including a poly(A) addition signal (AATAAA) and the first five residues of the poly(A) tail.
TL;DR: The murine immune interferon (IFN-gamma) gene was cloned and expressed under control of the simian virus 40 early promoter in the monkey COS-1 cell line and expressed in Escherichia coli under trp promoter control.
Abstract: The murine immune interferon (IFN-gamma) gene was cloned and expressed under control of the simian virus 40 early promoter in the monkey COS-1 cell line. A protein is secreted from these cells having the biological, antigenic, and biochemical characteristics of natural murine IFN-gamma. Cloned murine IFN-gamma cDNAs were obtained by using RNA from both mitogen-induced murine spleens and the transfected COS cells, and both code for identical proteins. The mature murine IFN-gamma encoded is 136 amino acids long, 10 amino acids shorter than human IFN-gamma. The nucleotide homology between the murine and human IFN-gamma genes is 60-65%, whereas the encoded proteins are only 40% homologous. Murine IFN-gamma cDNA was expressed in Escherichia coli under trp promoter control.
TL;DR: Two families of fungal mitochondrial introns that include all known sequences have been recognized are extended to incorporate a plant mitochondrial intron and several introns in chloroplast‐ and nuclear‐encoded rRNA and tRNA precursors.
Abstract: Two families of fungal mitochondrial introns that include all known sequences have been recognized. These families are now extended to incorporate a plant mitochondrial intron and several introns in chloroplast- and nuclear-encoded rRNA and tRNA precursors. Members of the same family share distinctive sequence stretches and a number of potential RNA secondary structures that would bring these stretches and the intron-exon junctions into relatively close proximity. Using several of these introns which have been extensively studied by either biochemical or genetic means, an attempt is made to integrate the available data into a common picture.
TL;DR: The existence of amino acid sequence homology between retroviral reverse transcriptase and the putative polymerases of HBV and CaMV is reported, suggesting the replication cycle of a plant virus, cauliflower mosaic virus, includes a reverse transcription step.
Abstract: In infected cells, the RNA genomes of RNA tumour viruses are copied into DNA by a virus-encoded reverse transcriptase enzyme1–3 This transfer of information from RNA into DNA was thought to be a unique feature of RNA tumour viruses3, but recent results suggest it may be a more general strategy Hepatitis B virus (HBV) has a double-stranded DNA genome, and it has recently been shown that the minus DNA strand of the HBV genome is copied from a plus-strand RNA template, leading to the suggestion that reverse transcription is central to the life cycle of HBV4–6 More recently it has been suggested that the replication cycle of a plant virus, cauliflower mosaic virus (CaMV), includes a reverse transcription step7–9 We report here the existence of amino acid sequence homology between retroviral reverse transcriptase and the putative polymerases of HBV and CaMV
TL;DR: The structure of the messenger RNA encoding the precursor to mouse submaxillary epidermal growth factor (EGF) was determined from the sequence of a set of overlapping complementary DNA's (cDNA).
Abstract: The structure of the messenger RNA (mRNA) encoding the precursor to mouse submaxillary epidermal growth factor (EGF) was determined from the sequence of a set of overlapping complementary DNA's (cDNA). The mRNA is unexpectedly large, about 4750 nucleotide bases, and predicts the sequence of preproEGF, a protein of 1217 amino acids (133,000 molecular weight). The EGF moiety (53 amino acids) is flanked by polypeptide segments of 976 and 188 amino acids at its amino and carboyxl termini, respectively. The amino terminal segment of the precursor contains seven peptides with sequences that are similar but not identical to EGF.
TL;DR: When an in vitro synthesized beta-globin pre-mRNA containing a splice junction mutation is microinjected, the affected junction is not spliced, indicating that sequences necessary for accurate splicing in human cells are also necessary for spliced in oocytes.
TL;DR: Evidence for a Host-Cell Nuclear Requirement for primary Transcription for Primary Transcription is found and evidence for overlapping Coding Regions using Diff erent Reading Frames in Viruses is found.
Abstract: PERSPECTIVES AND SUMMARy 468 MORPHOLOGY OF THE INFLUENZA VIRUS PARTICLE (VIRION) 469 GENOME STRUCTURE 471 The Segmented Genome 471 ASSigning Gene Functions to RNA Segments 472 Sequences Common to all RNA Segments 474 THE SEQUENCES OF INFLUENZA VIRUS RNA SEGMENTS 474 RNA Segments 1,2, and 3: Transcriptase-Associated Proteins PBI, PB2, and PA ... 476 RNA Segment 4: the Hemagglutinin (HA) 476 RNA Segment 5: the N uc/eocapsid Protein (N P) 481 RNA Segment 6: the Neuraminidase (N A) 482 RNA Segment 7: the Membrane Protein (M 1) and Nonstructural Protein (M2) ......... 484 RN A Segment 8: Nonstructural Proteins NS1 and NS2 487 Overlapping Coding Regions Using Diff erent Reading Frames in Viruses 489 INFLUENZA VIRUS TRANSCRIPTION AND REPLICATION 490 Evidence for a Host-Cell Nuclear Requirement for Primary Transcription 490 In Vitro Transcription of Influenza Virus mRN As 491 In Vivo Transcription of mRN As 494 Template A( )cRNA Synthesis 495 Virion RN A Synthesis 496 The Control of RNA SyntheSis 496 Spliced mRNAs and Their Controlled Synthesis 497 Subgenomic RN As: A Replication Error Produces Defective Interfering Particles .... 498 The Packaging of Eight RNA Segments 498 CONCLUDING REMARKS 499
TL;DR: In vitro translation shows that the p135 gene of E26 is a genetic hybrid of three distinct elements, ∼1.2kb derived from the 5′ region of the retroviral gag gene, mybE and the ets sequence, linked in the order 5′-Δgag–mybE–ets-3′.
Abstract: Only two avian oncogenic viruses specifically cause acute leukaemias yet do not transform chicken fibroblasts in culture: E26, which causes erythroblastosis and a low level of concomitant myeloblastosis in chickens, and avian myeloblastosis virus (AMV), which causes myeloblastosis exclusively. Both viruses are replication-defective and share a sequence termed myb (also known as amv) which is unrelated to essential virion genes and is therefore thought to be part of the transforming onc genes of these viruses. However, the genetic structure of the two viruses differs. E26 has a genomic RNA of 5.7 kilobases (kb) and encodes a 135,000 molecular weight gag-related protein (p135) with probable transforming function. We show here by in vitro translation that the 5.7-kb E26 RNA directs the synthesis of p135. Oligonucleotide analysis indicates that E26 RNA contains an internal 0.8-kb subset of the 1.2-kb AMV-related sequence (mybA), termed mybE. A 2.46-kb molecular clone prepared from cDNA transcribed in vitro from E26 RNA contained an E26 transformation-specific (ets) sequence flanked by mybE and an env-related sequence. A complete DNA sequence of this clone indicates that the 1.5-kb ets sequence extends the open reading frame of mybE for 491 amino acids. Thus, the p135 gene of E26 is a genetic hybrid of three distinct elements, approximately 1.2 kb derived from the 5' region of the retroviral gag gene, mybE and the ets sequence, linked in the order 5'-delta gag-mybE-ets-3'. The myeloid leukaemogenicity shared by E26 and AMV correlates with the common myb sequence, while the distinct erythroid leukaemogenicity of E26 correlates with ets and the E26-specific linkage of myb to delta gag.
TL;DR: Direct evidence was obtained for 3'-to-5' directionality in the decay of the long-lived mRNA encoded by the ompA gene, and no preferential stability was observed for translated versus untranslated mRNA segments.
Abstract: An assay was developed to investigate the fate of specific segments of beta-lactamase (bla) and ompA gene transcripts in Escherichia coli. DNA probes cloned in bacteriophage M13 were treated with an endonuclease capable of cleaving single-stranded DNA, the fragments produced were annealed with total cellular RNA, and the resulting RNA . DNA hybrids were subjected to S1 nuclease treatment and gel fractionation. By using this assay, direct evidence was obtained for 3'-to-5' directionality in the decay of the long-lived mRNA encoded by the ompA gene, and no preferential stability was observed for translated versus untranslated mRNA segments. In the case of bla mRNA, initial cleavage of the full-length transcript was rate limiting, and no decay intermediates were detected. No difference in degradation rate was seen for bla transcripts having variant 3' or 5' termini.
TL;DR: No strict correlation exists between enzyme sensitivity and MIC values, since inhibition of RNA synthesis does not always show up to the same extent in the two different test systems used for the determination of these values.
Abstract: Rifampin specifically inhibits bacterial RNA polymerase, the enzyme responsible for DNA transcription, by forming a stable drug-enzyme complex with a binding constant of 10(-9) M at 37 C. The corresponding mammalian enzymes are not affected by rifampin. Bacterial resistance to rifampin is caused by mutations leading to a change in the structure of the beta subunit of RNA polymerase. Such resistance is not an all-or-nothing phenomenon; rather, a large number of RNA polymerases with various degrees of sensitivity to rifampin have been found. No strict correlation exists between enzyme sensitivity and MIC values, since inhibition of RNA synthesis does not always show up to the same extent in the two different test systems used for the determination of these values.
TL;DR: The RNA homologous to the yeast transposable element Ty1 is one of the more abundant poly(A)+ RNAs in many strains of the yeast Saccharomyces cerevisiae and the 5' and 3' ends of Ty1 RNA have been determined from analysis of cDNA.
Abstract: The RNA homologous to the yeast transposable element Ty1 is one of the more abundant poly(A)+ RNAs in many strains of the yeast Saccharomyces cerevisiae. The 5' and 3' ends of Ty1 RNA have been determined from analysis of cDNA. The 5' end is 245 bases into the left delta sequence measured from the left side of the Ty1 element. The delta sequence is a direct repeat of about 340 base pairs present at each end of the Ty1 element. The Ty1 transcription includes 93-97 bases of the left delta sequence and continues through the entire internal portion of the element and through about 295 bases of the right delta sequence before reaching the 3' end located 38-46 bases from the right side of the right delta sequence. Because the delta sequences present at each end of a single Ty1 element have identical or very similar DNA sequences, these end points for Ty1 RNA raise several questions about the expression of Ty1 elements. First, what are the initiation and termination signals, because the Ty1 transcript must read through a DNA sequence that is identical to the 3' end at about 50 bases from the 5' end? Second, why is the direction of transcription of the Ty1 element opposite to that of genes that are overexpressed after the insertion of a Ty1 element? Third, because the Ty1 RNA itself has direct repeats of about 45 bases, a structure analogous to retrovirus RNAs, is the Ty1 RNA an intermediate in the transposition of Ty1?
TL;DR: A prompt differential transcriptional effect seems to underlie the gradual loss of tissue specificity of the primary cultures of mouse hepatocytes.
Abstract: Liver-specific mRNA sequences were examined in primary cultures of mouse hepatocytes. After cell disaggregation by collagenase treatment and for at least 24 h in culture, little change in liver-specific mRNA concentrations was noted. Gradually over a period of 140 h, liver-specific mRNAs declined. In contrast, transcriptional assays in which liver cell nuclei were used to produce 32P-labeled nuclear RNA showed that liver-specific gene transcription was greatly diminished within 24 h, while polymerase II transcription of "common" genes and transcription of tRNA and rRNA did not decline. Thus, a prompt differential transcriptional effect seems to underlie the gradual loss of tissue specificity of the primary cultures.
TL;DR: 1,10-Phenanthroline also inhibits binding of factor A to the intragenic control region of the 5 S RNA gene, and this reagent specifically inhibits factor A-dependent synthesis of 5 SRNA but notfactor A-independent tRNA synthesis in a HeLa cell in vitro transcription system.
TL;DR: Immunoprecipitation of viral proteins synthesized in vitro as well as in vivo demonstrated that the predominant immediate early protein is synthesized as a protein of 75,000 daltons, but is presumably modified in vivo, resulting in a broad banding pattern ranging from75,000 to 68,000Daltons.
Abstract: The immediate early genes of human cytomegalovirus were characterized according to map location, RNA transcripts, and translation products. Three regions in the large unique component (0.709 to 0.751 map units) were transcribed in the presence of an inhibitor of protein synthesis (anisomycin). A single size class of polyadenylated mRNA, 1.95 kilobases (kb), was transcribed abundantly relative to the other size classes. The predominant 1.95-kb viral RNA was transcribed from right to left on the prototype arrangement of the viral genome and spanned a region of approximately 2.8 kb (0.739 to 0.751 map units). This mRNA codes for a 75,000-dalton protein that represents the predominant immediate early protein detected in infected cells. Immunoprecipitation of viral proteins synthesized in vitro as well as in vivo demonstrated that the predominant immediate early protein is synthesized as a protein of 75,000 daltons, but is presumably modified in vivo, resulting in a broad banding pattern ranging from 75,000 to 68,000 daltons. A different immediate early viral gene (0.732 to 0.739 map units) is transcribed from left to right at relatively low levels. The 39 ends of the above viral RNAs terminate at approximately 230 base pairs apart in the region of approximately 0.739 map units. Five RNA size classes ranging from 2.25 to 1.10 kb were detected, but the 1.75-kb and 1.40-kb RNA size classes were more abundant from this region. At least four minor proteins are coded by these mRNAs, with apparent molecular weights ranging from 56,000 to 16,500. Last, a 1.95-kb mRNA was transcribed from a third region (0.709 to 0.728 map units). This viral mRNA was present at relatively low concentration and coded for a minor protein of 68,000 daltons. Since immediate early gene expression of human cytomegalovirus is dominated by the synthesis of an mRNA from the region of 0.739 to 0.751 map units that codes for the predominant immediate early protein found in the infected cell, we hypothesize that this protein is the major regulatory protein influencing the switch from restricted to extensive transcription. Images
TL;DR: It is suggested that U1 RNP is essential for the splicing of mRNA precursors, and sera directed against the Sm class of small nuclear RNPs, including a mouse monoclonal antibody, specifically inhibited splicing.
TL;DR: Methods that are used to study the in vitro synthesis of proteins, each comprise several percent of total cellular protein, such as globin, prolactin, the caseins, and to the synthesis of the lysosomal proteins cathepsin D, β-glucuronidase, and glucocerebrosidase are described.
Abstract: Publisher Summary Cell-free extracts of wheat germ embryos, containing ribosomes and soluble factors, support the in vitro translation of a wide variety of mRNAs into protein. This chapter describes methods that are used to study the in vitro synthesis of proteins, each comprise several percent of total cellular protein, such as globin, prolactin, the caseins, and to the synthesis of the lysosomal proteins cathepsin D, β-glucuronidase, and glucocerebrosidase. The wheat germ system is used widely because, it is relatively easy to prepare and contains a relatively low amount of endogenous mRNA. The low concentration of endogenous mRNA present in the wheat germ extract competes with the added RNA for ribosomes and factors required for translation; therefore, it is advantageous to reduce the concentration of endogenous RNA by treating with nuclease. The scale of the translation reaction required for detection of the protein under study depends upon the amount of its mRNA in the total RNA preparation.
TL;DR: The genetic heterogeneity generated upon passage of foot-and-mouth disease virus (FMDV) in cell culture has been evaluated by T1-oligonucleotide fingerprinting of genomic RNA, with some mutations present in only one of the cloned genomes analyzed.
TL;DR: Oligonucleotide map analysis of the large (L), medium (M), and small (S) genome segments of Hantaan virus demonstrated that each RNA species was unique with respect to each other and was different from host cell ribosomal RNA.
TL;DR: The coronavirus system may be a useful model for the study of synthesis, glycosylation, and transport of O-linked cellular glycoproteins.
Abstract: Publisher Summary Coronaviruses have recently emerged as an important group of animal and human pathogens that share a distinctive replicative cycle. Some of the unique characteristics in the replication of coronaviruses include generation of a 3' coterminal-nested set of five or six subgenomic mRNAs, each of which appears to direct the synthesis of one protein. Two virus-specific RNA polymerase activities have been identified. Many of the distinctive features of coronavirus infection and coronavirus-induced diseases may result from the properties of the two coronavirus glycoproteins. The intracellular budding site, which may be important in the establishment and maintenance of persistent infections, appears to be due to the restricted intracytoplasmic migration of the E1 glycoprotein, which acts as a matrix-like transmembrane glycoprotein. E1 also exhibits distinctive behavior by self-aggregating on heating at 100°C in sodium dodecyl sulfate (SDS) and by its interaction with RNA in the viral nucleocapsid. The E1 of mouse hepatitis virus (MHV) is an O-linked glycoprotein, unlike most other viral glycoproteins. Thus, the coronavirus system may be a useful model for the study of synthesis, glycosylation, and transport of O-linked cellular glycoproteins.