Showing papers on "Transcription (biology) published in 1983"

Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei

Abstract: We have developed a procedure for preparing extracts from nuclei of human tissue culture cells that directs accurate transcription initiation in vitro from class II promoters. Conditions of extraction and assay have been optimized for maximum activity using the major late promoter of adenovirus 2. The extract also directs accurate transcription initiation from other adenovirus promoters and cellular promoters. The extract also directs accurate transcription initiation from class III promoters (tRNA and Ad 2 VA).

Yeast RNA polymerase II genes: isolation with antibody probes.

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.

Beta-galactosidase gene fusions for analyzing gene expression in Escherichia coli and yeast

Abstract: Publisher Summary Gene fusions can be constructed either in vivo , using spontaneous nonhomologous recombination or semi-site specific transposon recombination, or in vitro , with recombinant DNA technology. This chapter describes in vitro methods and lists some recently developed β -galactosidase gene fusion vectors. With these methods, gene-controlling elements from any source can be fused to the β -galactosidase structural gene and examined in the prokaryote bacterium Escherichia coli or the lower eukaryote yeast Saccharomyces cerevisiae. β -galactosidase gene fusions can be constructed both with transcription initiation control signals and with transcription plus translation initiation control signals. The β -galactosidase gene is convenient for making translational fusions because it is possible to remove its translation initiation region along with up to at least the first 27 amino acid codons without affecting β -galactosidase enzymic activity. The focus here is on these transcription-translation fusions because they provide all the gene initiation signals from the other gene. β -Galactosidase expression from a gene fusion can be used not only to measure gene expression and regulation, but also to isolate mutations and additional gene fusions.

Specific transcription and RNA splicing defects in five cloned beta-thalassaemia genes.

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.

The glucocorticoid receptor binds to defined nucleotide sequences near the promoter of mouse mammary tumour virus.

Abstract: Glucocorticoids are known to induce the transcription of integrated proviral mouse mammary tumour virus (MMTV) genes in a variety of cell lines derived from mouse mammary tumours Chimaeric genes in which selectable markers are linked to the long terminal repeat (LTR) region of MMTV can be induced by the synthetic glucocorticoid dexamethasone after introduction into mouse fibroblasts This suggests that the regulatory elements required for hormonal induction are located within the cloned LTR fragments The idea is supported by the observation that glucocorticoid receptors bind to certain cloned fragments of MMTV DNA in vitro Using filter binding studies and monoclonal antibodies to the glucocorticoid receptor we have now delimited the receptor binding region to a DNA segment of 152 base pairs (bp) that has been shown to be relevant for hormonal induction In nuclease protection experiments we have identified partially homologous receptor binding sequences located in this region, all of which share the hexanucleotide 5'-TGTTCT-3'

Expression and regulation of immunoglobulin heavy chain gene transfected into lymphoid cells.

Abstract: A plasmid including a mouse immunoglobulin mu gene was transfected into the IgG-secreting human lymphoid line HMy2 and mouse B- and pre-B-cell lines WEHI 231 and 18-81; stably transfected cells were selected. Transfected HMy2 cells synthesized mouse immunoglobulin mu chains as a major secreted protein but the WEHI 231 and 18-81 transfectants transcribed the introduced mu gene at lower levels. In HMy2 transfectants, most of the transcription of the introduced heavy chain gene initiated 40 and 62 bp upstream of the beginning of the VH exon translation start, although a small proportion of transcripts initiating further upstream was detected. WEHI 231 and 18-81 transfectants gave a much higher proportion of upstream initiation. Transient expression of the VH exon was monitored following transfection of mouse myeloma with the VH gene DNA in various plasmid constructs. VH transcription was only observed if the plasmids contained a segment derived from the large VH-CH intron of the immunoglobulin heavy chain locus. This segment, located between JH and switch regions, functioned both downstream of the VH exon and upstream in either orientation. The existence of a transcription enhancer element in this region is therefore proposed.

The gene structure and replication of influenza virus.

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

Structure and transcription of the nopaline synthase gene region of T-DNA.

Abstract: We present the DNA sequence and plant-tumor transcription pattern of some 2400 base pairs from the right border region of pTi T37 DNA from the virulent Agrobacterium tumefaciens strain T37. This region includes the entire transcription unit encompassing the nopaline synthase gene, together with parts of other transcription units. The strategy used to determine the sequence also produced two opposing series of defined, asymmetric deletions across the target DNA region, some of which may serve future purposes in the exploitation of this sequence, which is known to be expressed in a wide variety of host plant tissues.

Actively transcribed genes are associated with the nuclear matrix

Abstract: In the chicken oviduct, it has been well documented that steroid hormones stimulate the transcription of specific genes such as the ovalbumin gene. In addition to the presence of specific hormone receptors in the tissue, gene expression seems to require that target genes exist in large DNase I sensitive chromosomal domains. This structure appears necessary but not sufficient for transcriptional activation. In search of still other levels of control, we have investigated the interactions of genes with the nuclear matrix, a structure which has been implicated in DNA synthesis, transcription and RNA processing. Here we have isolated nuclear matrix and used a nondegradative method to fractionate nuclear DNA based on its preferential association with the matrix. The preparation was digested with a restriction enzyme and both matrix-bound and released DNAs were recovered. We found that only actively expressed genes were associated with the matrix. Furthermore, within a 100-kilobase (kb) DNase I sensitive chromosomal domain, only the transcribed regions were associated with the matrix. This association was shown to be reversible when hormone was withdrawn. Our results suggest that the nuclear matrix is the site of nuclear transcription and may represent another potential level of control for regulation of gene expression in the eukaryotic cell.

Expression of recombinant plasmids in mammalian cells Is enhanced by sodium butyrate

Abstract: We have studied the effects of sodium butyrate on DNA-mediated gene transfer in an effort to investigate interrelationships between chromatin structure and expression of recombinant plasmids. Our results demonstrate that butyrate affects the early stages of gene activity following DNA uptake at least two levels. First, the number of cells able to express foreign DNA increases from 10% to up to 40%. Second, there is an increase in enhancer-dependent transcription, approximately 30 fold in HeLa cells, involving the SV40 early promoter. Stable transformation efficiencies increase to 4% and 10% in HeLa S3 and monkey kidney CV-1 cells, respectively. Finally, expression of integrated recombinant plasmid genes is reinducible by a second treatment five weeks after initial exposure to this agent.

Rifampin: Mechanisms of Action and Resistance

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.

RNA from the yeast transposable element Ty1 has both ends in the direct repeats, a structure similar to retrovirus RNA.

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?

Changes in liver-specific compared to common gene transcription during primary culture 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.

Organization and expression of the immediate early genes of human cytomegalovirus.

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

Expression of a cellular oncogene during liver regeneration.

Abstract: The number of transcripts of the cellular oncogene ras, which is homologous to the transforming gene of Harvey sarcoma virus, increases during liver regeneration in rats. The increase in these transcripts in liver polysomal polyadenylated RNA occurs at the time of activation of DNA synthesis during the regenerative process induced by partial hepatectomy or carbon tetrachloride injury. The number of ras transcripts returns to basal levels within 72 hours. These observations show that transcription of a cellular oncogene increases in a regulated way in a nonneoplastic growth process.

In vitro transcription: whole-cell extract.

Abstract: Publisher Summary This chapter focuses on three classes of nuclear DNA-dependent RNA polymerases that have been identified in eukaryotic cells. Synthesis of mature RNA molecules requires additional enzymes and factors other than those needed to bring about accurate transcriptional initiation. For analysis by hybridization and SI nuclease digestion, it is important first to remove the template DNA. Titrations both of DNA and of extract yield nonlinear responses. At a constant extract concentration, measuring runoff transcription as a function of DNA concentration yields a threshold DNA concentration below which no transcription occurs and an inhibitory effect of high DNA concentration. For a given promoter, short runoff transcripts have a higher optimum DNA concentration than longer runoff transcripts. A number of inhibitory activities can be removed by fractionation on phosphocellulose, yielding a more efficient transcription extract.

Differential gene expression in the gastrula of Xenopus laevis

Abstract: A modified cloning method designed to produce differential complementary DNA libraries permits the isolation of sequences that are present in the RNA population of any developmental stage or tissue, but are not present or are much less abundant in another stage or tissue. Selective complementary DNA cloning is especially useful when the differentially expressed RNA's are of low to moderate abundance in the cells in which they occur. A class of cytoplasmic polyadenylated RNA's differentially expressed in gastrula embryos of Xenopus laevis (DG RNA's) has been isolated. These DG RNA's occur very rarely or not at all in unfertilized eggs and blastulae, accumulate as the result of transcription before and during gastrulation, and, with some exceptions, decline in abundance as development proceeds. Many of these RNA molecules appear to be translated at the gastrula stage. Thus, DG RNA's may encode proteins that are important in the process of gastrulation.