Showing papers on "Transcription (biology) published in 1986"
TL;DR: A gene expression system based on bacteriophage T7 RNA polymerase has been developed and high levels of accumulation suggest that the RNAs are relatively stable, perhaps in part because their great length and/or stem-and-loop structures at their 3' ends help to protect them against exonucleolytic degradation.
6,415 citations
TL;DR: Isolation and renaturation of proteins purified from sodium dodecyl sulfate polyacrylamide gels allowed the identification of two polypeptides as those responsible for recognizing and interacting specifically with the GC-box promoter elements characteristic of Sp1 binding sites.
Abstract: The biochemical analysis of cellular trans-activators involved in promoter recognition provides an important step toward understanding the mechanisms of gene expression in animal cells. The promoter selective transcription factor, Sp1, has been purified from human cells to more than 95 percent homogeneity by sequence-specific DNA affinity chromatography. Isolation and renaturation of proteins purified from sodium dodecyl sulfate polyacrylamide gels allowed the identification of two polypeptides (105 and 95 kilodaltons) as those responsible for recognizing and interacting specifically with the GC-box promoter elements characteristic of Sp1 binding sites.
1,284 citations
TL;DR: Transcriptionally active nuclear extracts have been prepared from rat liver, brain, and spleen and the adenovirus-2 major late promoter directs efficient transcription by RNA polymerase II in all of these extracts, whereas the promoter of the mouse albumin gene is significantly used only in the liver extract.
1,149 citations
TL;DR: The RNA polymerase II transcription factor Sp1 is a protein that binds to specific DNA sequences and activates RNA synthesis from a select group of promoters and appears to be important for modulation of gene expression in higher organisms.
1,022 citations
TL;DR: In this paper, the authors describe a method to clone a functional gene for bacteriophage T7 RNA polymerase, which is useful for synthesizing large amounts of RNA in vivo or in vitro, and can produce a single RNA selectively from a complex mixture of DNAs.
Abstract: This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells.
971 citations
TL;DR: Characterization of 27 insertional mutants of the hGR allowed the location of at least four functional domains, two of which correspond to the predicted DNA- and steroid-binding domains, raising the possibility that other regions in the receptor are necessary for full transcriptional activation but are not specifically involved in steroid or DNA binding.
919 citations
TL;DR: It is concluded that four extra, reading frame-restoring nucleotides are added during or after transcription of the frameshift gene by an RNA-editing process.
785 citations
TL;DR: A DNA-binding gel electrophoresis assay was used to detect a protein(s) in HeLa cell nuclear extracts that specifically binds to the 5' activating element.
764 citations
TL;DR: During HL60 differentiation, a DNase I hypersensitive site in the chromatin about 300 bases downstream of the 5′ end of of intron 1 increases in intensity relative to other sites, possibly reflecting events associated with the termination of transcription.
Abstract: The c-myc gene product is a nuclear protein1,2 expressed in a wide variety of cell types3. It has been implicated in the control of normal cell growth as well as transformation4–7, but its exact function is unknown. When the human promyelocytic leukaemia cell line HL60 is treated with retinoic acid, the cells differentiate into granulocytes, and there is a reduction in steady state c-myc RNA of more than 10-fold8. Nuclear runoff assays show that this reduction is caused by a corresponding decrease in the transcription of exon 2. However, only a minor decrease in exon 1 transcription is observed upon differentiation. In undifferentiated HL60 cells there is an approximately 3-fold molar excess of exon 1 transcription over exon 2, and this excess increases to about 15-fold in differentiated cells. This observation suggests that a major component of c-myc transcriptional down-regulation in HL60 cells is at the level of elongation rather than at the level of initiation. The position of the elongation block was mapped to the region of the boundary between exon 1 and intron 1. During HL60 differentiation, a DNase I hypersensitive site in the chromatin about 300 bases downstream of the 5′ end of of intron 1 increases in intensity relative to other sites, possibly reflecting events associated with the termination of transcription. Our runoff analysis also revealed transcription of both strands immediately upstream of exon 1 in HL60 cells. The sense strand transcription of this region produces a novel c-myc RNA which initiates several hundred bases upstream of the previously defined promoters and is found in a variety of cell types.
716 citations
TL;DR: Findings suggest that one important component of the AIDS virus transcriptional control region interacts with a cellular transcription factor, Sp1, and that this factor must function in conjunction with transcriptional elements located downstream of the RNA cap site to mediate the response of the LTR to viral trans-activation.
Abstract: The nature and position of transcriptional control elements responsible for the expression of genes encoded by the retrovirus associated with acquired immune deficiency syndrome (AIDS) have not been precisely defined. In this study it is shown that the mammalian Sp1 transcription factor binds to promoter sequences within the AIDS retrovirus long terminal repeat (LTR) and activates RNA synthesis five- to eightfold in reconstituted reactions in vitro. Experiments in which regions of DNA were protected from added reagents by specifically bound proteins (footprinting) indicated that the upstream promoter region of the AIDS virus LTR lies between -45 and -77 (relative to the RNA start site, +1) and contains three tandem, closely spaced SP1 binding sites of variable affinity. Base-substitution mutations targeted to one or all three Sp1 binding sites were found both to eliminate the binding of Sp1 and to cause up to a tenfold reduction in transcriptional efficiency in vitro. These findings suggest that one important component of the AIDS virus transcriptional control region interacts with a cellular transcription factor, Sp1, and that this factor must function in conjunction with transcriptional elements located downstream of the RNA cap site to mediate the response of the LTR to viral trans-activation.
683 citations
TL;DR: The E7 protein has been identified in CaSki cells and the prevalence of its mRNA suggests a possible function in progression to, or long-term maintenance of, the malignant state.
Abstract: Human papillomavirus type 16 DNA and RNA were characterized in the cervical cancer-derived CaSki cell line, which contains only integrated DNA, and in a cervical cancer, which contains predominantly plasmid DNA In both, a major RNA can code for the early open reading frame E7 and a minor one can code for E6 The cervical cancer, but not the CaSki cell line, contains a minor RNA that can code for an intact E2 protein, and this may relate to the continued presence of plasmid DNA The RNA mapping data suggest that the poly(A)+ RNA is transcribed from a minor fraction of the several hundred gene copies present, and in the cervical cancer these genomes appear to be integrated The E7 protein has been identified in CaSki cells and the prevalence of its mRNA suggests a possible function in progression to, or long-term maintenance of, the malignant state
TL;DR: The results suggest that in PC12 cells c-fos transcription is activated by a protein-synthesis-independent mechanism, whereas c-myc stimulation requires new protein synthesis.
Abstract: Stimulation of quiescent 3T3 cells with purified growth factors or of the pheochromocytoma cell line PC12 with nerve growth factor results in the rapid transient induction of c-fos, c-myc, and actin gene transcription (M.E. Greenberg and E.B. Ziff, Nature [London] 312:711-716; M.E. Greenberg, L.A. Greene, and E.B. Ziff, J. Biol. Chem. 26:14101-14110). We used protein synthesis inhibitors to investigate whether synthesis of new proteins plays a role in the rapid induction and subsequent repression of the transcription of these genes. Pretreatment of quiescent 3T3 cells with the inhibitor anisomycin before growth factor stimulation caused a superinduction of c-fos and c-myc mRNA levels upon growth factor addition. Nuclear runoff transcription analyses of 3T3 cells indicated that anisomycin potentiated c-fos, c-myc, and also actin expression at the transcriptional level, possibly by inhibiting transcriptional repression. Somewhat different results were obtained when PC12 cells were incubated with either anisomycin or cycloheximide. In PC12 cells protein synthesis inhibitors superinduced nerve growth factor activation of c-fos mRNA production but completely abolished the activation of c-myc. The results suggest that in PC12 cells c-fos transcription is activated by a protein-synthesis-independent mechanism, whereas c-myc stimulation requires new protein synthesis. The difference in the effect of anisomycin on growth factor activation of c-myc expression in 3T3 versus PC12 cells may be due to differential stringency of protein synthesis inhibition in the two cells or could reflect cell type differences in c-myc regulation.
TL;DR: Treating HOS cells in vitro with MNNG resulted in fusion of two chromosomally disparate loci, met and tpr, generating the active met oncogene, related to the tyrosine kinase gene family.
TL;DR: Transcription of these three genes is also rapidly and transiently induced by cycloheximide, an inhibitor of protein synthesis, indicating that they are under the control of short-lived repressors.
Abstract: Exposure of mouse resident and thioglycollate-elicited peritoneal macrophages to IFN-gamma leads to a marked increase in the TNF-alpha (tumor necrosis factor/cachectin), IL-1 and u-PA (urokinase-type plasminogen activator) mRNA levels. Nuclear run-on experiments show that IFN-gamma acts by enhancing the transcription of these three genes. Transcription of these three genes is also rapidly and transiently induced by cycloheximide, an inhibitor of protein synthesis, indicating that they are under the control of short-lived repressors.
TL;DR: The shortened form of the self-splicing ribosomal RNA intervening sequence of Tetrahymena thermophila acts as an enzyme in vitro that can act as an RNA polymerase, differing from the protein enzyme in that it uses an internal rather than an external template.
Abstract: A shortened form of the self-splicing ribosomal RNA (rRNA) intervening sequence of Tetrahymena thermophila acts as an enzyme in vitro. The enzyme catalyzes the cleavage and rejoining of oligonucleotide substrates in a sequence-dependent manner with Km = 42 microM and kcat = 2 min-1. The reaction mechanism resembles that of rRNA precursor self-splicing. With pentacytidylic acid as the substrate, successive cleavage and rejoining reactions lead to the synthesis of polycytidylic acid. Thus, the RNA molecule can act as an RNA polymerase, differing from the protein enzyme in that it uses an internal rather than an external template. At pH 9, the same RNA enzyme has activity as a sequence-specific ribonuclease.
TL;DR: The results suggest that the E2 binding activity is a cellular transcriptional factor, the concentration or binding activity of which increases as a result of the action of the E1A gene product.
TL;DR: Assays in microinjected frog oocytes and transfected mouse L cells indicate that equivalent point mutations, introduced at each residue of the CAT pentanucleotide of each promoter, lead to similar changes in promoter activity and alter binding of the nuclear protein fraction.
TL;DR: Results indicate that the strategy of generating antisense RNA to inhibit gene expression may be useful in delineating the function of protooncogenes.
Abstract: Antisense RNA complementary to c-fos mRNA was produced in mouse 3T3 cells by gene transfer techniques. Transcriptional units were constructed consisting of a steroid-inducible mouse mammary tumor virus (MMTV) promoter, mouse or human 5' c-fos gene fragments in either the sense (5' to 3') or antisense (3' to 5') orientation, and splice and poly(A) signals from the human beta-globin gene. A gene that confers neomycin resistance was included in the vectors to allow isolation of stable transformants. Dexamethasone caused a marked induction of hybrid MMTV-fos-globin RNA. Induction of the hybrid transcript containing antisense c-fos RNA decreased colony formation following DNA transfer and inhibited the proliferation of cells into which the antisense transcriptional unit had been integrated. In contrast, colony formation and cell proliferation were not inhibited by induction of hybrid RNA containing c-fos RNA sequences in the sense orientation. These results indicate that the strategy of generating antisense RNA to inhibit gene expression may be useful in delineating the function of protooncogenes. The c-fos gene product appears to have a required role in normal cell division.
TL;DR: The Notch locus is essential for proper differentiation of the ectoderm in Drosophila melanogaster and appears to have evolved by repeated tandem duplications of the DNA coding for the 38-amino-acid-long protein segments, followed by insertion of intervening sequences.
Abstract: The Notch locus is essential for proper differentiation of the ectoderm in Drosophila melanogaster. Notch corresponds to a 37-kilobase transcription unit that codes for a major 10.4-kilobase polyadenylated RNA. The DNA sequence of this transcription unit is presented, except for portions of the two largest intervening sequences. DNA sequences also were obtained from three Notch cDNA clones, allowing the 5' and 3' ends of the gene to be mapped, and the structures and locations of nine RNA coding regions to be determined. The major Notch transcript encodes a protein of 2,703 amino acids. The protein is probably associated with cell surfaces and carries an extracellular domain composed of 36 cysteine-rich repeating units, each of about 38 amino acids. The gene appears to have evolved by repeated tandem duplications of the DNA coding for the 38-amino-acid-long protein segments, followed by insertion of intervening sequences. These repeating protein segments are quite homologous to portions of mammalian clotting factors IX and X and to the product of the Caenorhabditis elegans developmental gene lin-12. They are also similar to mammalian growth hormones, typified by epidermal growth factor.
TL;DR: In this paper, sequences flanking the mouse c-fos gene were tested for the ability to form specific DNA-protein complexes with factors present in crude nuclear extracts prepared from mammalian cells.
Abstract: We tested sequences flanking the mouse c-fos gene for the ability to form specific DNA-protein complexes with factors present in crude nuclear extracts prepared from mammalian cells. Three such complexes were detected. One complex formed in a region necessary for the induction of c-fos expression by serum growth factors. Two additional complexes formed at sequences that contribute to basal c-fos promoter activity in vivo. These complexes represent three novel sequence-specific DNA-binding activities which appear to participate in the regulation of c-fos transcription.
TL;DR: The mapping of the 5′‐ends of the virus‐cell fusion transcripts indicates that transcription is initiated at a viral promoter, suggesting a functional role of HPV18 early genes for the malignant phenotype of these cells.
Abstract: Transcription of human papillomavirus type 18 (HPV18) DNA in the human cervical carcinoma cell lines HeLa, C4-1 and SW756 was studied by nucleotide sequence analysis of HPV18-positive cDNA clones isolated from a HeLa, C4-1 and SW756 cDNA library, respectively, and the cDNA sequences were used to predict the potential encoded proteins. The cDNA clones from all three cell lines were found to be derived from virus-cell fusion transcripts in which 3'-terminal host cell sequences (different for each cell line) were spliced to 5'-terminal exon sequences from the HPV18 E6-E7-E1 region. Three different types of cDNA clones can be distinguished according to the splicing patterns observed in the 5' terminal HPV18 sequences. They carry as potential protein-coding regions the HPV18 specific open reading frames E6 and E6* (generated by splicing and identical with E6 up to the E6* splice junction), E7 and E1 (only in HeLa). Translation of specific cellular genes from the chimeric viral-cellular transcripts seems to be unlikely. The mapping of the 5'-ends of the virus-cell fusion transcripts indicates that transcription is initiated at a viral promoter. The similar patterns of HPV18 transcription in the three different cervical carcinoma cell lines suggest a functional role of HPV18 early genes for the malignant phenotype of these cells.
TL;DR: It is shown that the ability of this promoter to be activated by a low intracellular concentration of NRI depends on two binding sites for NRI located approximately 110 and 140 bp, respectively, upstream of the start of transcription.
TL;DR: The antibody and an alkaline phosphatase-labeled second antibody were used in an immunodetection method for measurement of hybrids formed between immobilized DNA probes of various lengths and 23 S ribosomal RNA, the colorimetric response of this assay increased linearly with the amount of hybrid formed.
TL;DR: Downstream of the transcription start site in the Drosophila heat-shock gene hsp22, a region that is necessary for efficient transcription, and also for selective translation during heat shock is identified, which coincides with a region of sequence homology between the heat- shock genes.
TL;DR: It is concluded that all classes of genes become competent for activation during cycle 10, and that subsequent activation is differentially suppressed by functions associated with nuclear division.
TL;DR: Results show that the class II intron products are similar to those of nuclear pre-mRNA splicing, particularly the spliced exons and broken form of the lariat.
TL;DR: Hormonal regulatory elements within the phosphoenolpyruvate carboxykinase (GTP) (PEPCK) promoter region were mapped using a series of 5' deletions linked to the amino-3'-glycosyl phosphotransferase structural gene, confirming the existence of the hormone regulatory domains within the PEPCK promoter.
TL;DR: In this paper, a simple approach has been developed for the unambiguous identification and purification of sequence-specific DNA-binding proteins solely on the basis of their ability to bind selectively to their target sequences.
Abstract: A simple approach has been developed for the unambiguous identification and purification of sequence-specific DNA-binding proteins solely on the basis of their ability to bind selectively to their target sequences. Four independent methods were used to identify the promoter-specific RNA polymerase II transcription factor MLTF as a 46-kilodalton (kDa) polypeptide. First, a 46-kDa protein was specifically cross-linked by UV irradiation to a body-labeled DNA fragment containing the MLTF binding site. Second, MLTF sedimented through glycerol gradients at a rate corresponding to a protein of native molecular weight 45,000 to 50,000. Third, a 46-kDa protein was specifically retained on a biotin-streptavidin matrix only when the DNA fragment coupled to the matrix contained the MLTF binding site. Finally, proteins from the most highly purified fraction which were eluted and renatured from the 44- to 48-kDa region of a sodium dodecyl sulfate-polyacrylamide gel exhibited both binding and transcription-stimulatory activities. The DNA-binding activity was purified 100,000-fold by chromatography through three conventional columns plus a DNA affinity column. Purified MLTF was characterized with respect to the kinetic and thermodynamic properties of DNA binding. These parameters indicate a high degree of occupancy of MLTF binding sites in vivo.