Showing papers on "Transcription (biology) published in 1985"
TL;DR: A coupled system that permits the exclusive expression of genes under the control of a T7 RNA polymerase promoter and its use to express high levels of phage T7 gene 5 protein, a subunit of T7 DNA polymerase is described.
Abstract: The RNA polymerase gene of bacteriophage T7 has been cloned into the plasmid pBR322 under the inducible control of the lambda PL promoter. After induction, T7 RNA polymerase constitutes 20% of the soluble protein of Escherichia coli, a 200-fold increase over levels found in T7-infected cells. The overproduced enzyme has been purified to homogeneity. During extraction the enzyme is sensitive to a specific proteolysis, a reaction that can be prevented by a modification of lysis conditions. The specificity of T7 RNA polymerase for its own promoters, combined with the ability to inhibit selectively the host RNA polymerase with rifampicin, permits the exclusive expression of genes under the control of a T7 RNA polymerase promoter. We describe such a coupled system and its use to express high levels of phage T7 gene 5 protein, a subunit of T7 DNA polymerase.
3,214 citations
TL;DR: Analysis of the amino acid sequence reveals nine tandem similar units, each consisting of approximately 30 residues and containing two invariant pairs of cysteines and histidines, the most common ligands for zinc in the 7S particle of Xenopus laevis oocytes, which suggests that the protein contains repetitive zinc‐binding domains.
Abstract: The 7S particle of Xenopus laevis oocytes contains 5S RNA and a 40-K protein which is required for 5S RNA transcription in vitro. Proteolytic digestion of the protein in the particle yields periodic intermediates spaced at 3-K intervals and a limit digest containing 3-K fragments. The native particle is shown to contain 7-11 zinc atoms. These data suggest that the protein contains repetitive zinc-binding domains. Analysis of the amino acid sequence reveals nine tandem similar units, each consisting of approximately 30 residues and containing two invariant pairs of cysteines and histidines, the most common ligands for zinc. The linear arrangement of these repeated, independently folding domains, each centred on a zinc ion, comprises the major part of the protein. Such a structure explains how this small protein can bind to the long internal control region of the 5S RNA gene, and stay bound during the passage of an RNA polymerase molecule.
2,194 citations
TL;DR: The effects of 5′ deletions in a plant viral promoter in tobacco callus as well as in regenerated plants, includ ing different plant tissues, are analysed to allow a more direct assessment of deletion effects.
Abstract: Although promoter regions for many plant nuclear genes have been sequenced, identification of the active promoter sequence has been carried out only for the octopine synthase promoter. That analysis was of callus tissue and made use of an enzyme assay. We have analysed the effects of 5' deletions in a plant viral promoter in tobacco callus as well as in regenerated plants, including different plant tissues. We assayed the RNA transcription product which allows a more direct assessment of deletion effects. The cauliflower mosaic virus (CaMV) 35S promoter provides a model plant nuclear promoter system, as its double-strand DNA genome is transcribed by host nuclear RNA polymerase II from a CaMV minichromosome. Sequences extending to -46 were sufficient for accurate transcription initiation whereas the region between -46 and -105 increased greatly the level of transcription. The 35S promoter showed no tissue-specificity of expression.
1,674 citations
TL;DR: It is found that the HPV 18 DNA is integrated into the cellular genome and is amplified in HeLa and 756 cells, and some of the transcripts are composed of HPV 18 and cellular sequences.
Abstract: DNA of human papillomavirus (HPV) types 16 and 18 has been found closely associated with human genital cancer, supporting the concept that members of this virus group are key factors in the aetiology of genital cancer. HPV 18 DNA sequences were also detected in cell lines derived from cervical cancer. We have now analysed these cell lines, HeLa, C4-1 and 756, for the structural organization and transcription of the HPV 18 genome and we find that the HPV 18 DNA is integrated into the cellular genome and is amplified in HeLa and 756 cells. Almost the complete HPV 18 genome seems to be present in 756 cells, with the early region being disrupted into two portions in each integrated copy. In HeLa and C4-1 cells, a 2-3 kilobase (kb) segment of HPV 18-specific sequences is missing from the E2 to L2 region. HPV 18 sequences are specifically transcribed from the E6-E7-E1 region into poly(A)+ RNAs of 1.5-6.5 kb. Hybridization analysis of cDNA clones indicated that some of the transcripts are composed of HPV 18 and cellular sequences. In addition, poly(A)+ RNA hybridizing with HPV 16 DNA was found in two out of three cervical carcinoma biopsies.
1,458 citations
TL;DR: The HCMV enhancer, which seems to have little cell type or species preference, is severalfold more active than the SV40 enhancers, a property that makes it a useful component of eukaryotic expression vectors.
1,336 citations
TL;DR: The enzyme that carries out RNA synthesis provides the cell with the means to adjust the patterns of transcription in response to environmental and developmental signals, and the presence of promoter-specific transcription factors enables other components of the transcriptional machinery, including the RNA polymerase, to initiate transcription with selectivity and accuracy.
Abstract: The enzymatic machinery that carries out RNA synthesis provides the cell with the means to adjust the patterns of transcription in response to environmental and developmental signals. In eukaryotes, this regulation is mediated in part by promoter-specific transcription factors, which are DNA-binding proteins with the ability to discriminate between distinctive DNA sequence elements found in the promoter regions of different genes. The presence of these factors bound to DNA enables other components of the transcriptional machinery, including the RNA polymerase, to initiate transcription with selectivity and accuracy.
1,048 citations
983 citations
TL;DR: Human T-lymphotropic virus type III (HTLV-III) encodes a trans-acting factor that activates the expression of genes linked to the HTLV- III long terminal repeat by functional mapping of complementary DNA transcripts of viral messenger RNA's.
Abstract: Human T-lymphotropic virus type III (HTLV-III) encodes a trans-acting factor that activates the expression of genes linked to the HTLV-III long terminal repeat. By functional mapping of complementary DNA transcripts of viral messenger RNA's the major functional domain of the gene encoding this factor was localized to a region immediately before the env gene of the virus, a region previously thought to be noncoding. This newly identified gene consists of three exons, and its transcription into messenger RNA involves two splicing events bringing together sequences from the 5' part (287 base pairs), middle (268 base pairs), and 3'part (1258 base pairs) of the HTLV-III genome. A similar messenger RNA with a truncated second exon (70 base pairs) does not encode a trans-acting function. It is proposed that this second messenger RNA is the transcript of a gene (3'-orf) located after the env gene. Messenger RNA's were also identified for the env and gag-pol genes of HTLV-III.
846 citations
TL;DR: The results suggest that c-fos, c-myc, and actin induction may be general nuclear responses to growth or differentiation factors in a variety of different cell types.
811 citations
TL;DR: The study of fusion genes showed that in addition to the 5' activating element, transient accumulation of c-fosH RNA following serum stimulation requires sequences at the 3' end of the c- fosH gene.
753 citations
TL;DR: It is shown that the yeast positive regulatory protein GAL4 binds to four sites in the upstream activating sequence UASG to activate transcription of the adjacent GAL1 and GAL10 genes, consistent with the idea that GAL2 protein binds to three related 17 bp sequences, each of which displays approximate 2-fold rotational symmetry.
TL;DR: Analysis of this region by replacement of specific portions with nondiscriminatory control elements from viral systems shows that a transcriptional enhancer is located in the distal portion of the 5' flanking DNA; its position has been mapped by deletion analysis.
Abstract: The 59 flanking DNA of the rat insulin I gene contains sequences controlling cell-specific expression. Analysis of this region by replacement of specific portions with nondiscriminatory control elements from viral systems shows that a transcriptional enhancer is located in the distal portion of the 59 flanking DNA; its position has been mapped by deletion analysis. Additional experiments suggest that another distinct regulatory element is located more proximal to the transcription start site. The activity of both elements is restricted to pancreatic B cells. The combinatorial effect of multiple control elements could explain the cell-specific expression of insulin genes.
TL;DR: It is suggested that all the transcripts that gave rise to these pseudogenes were actually produced in the germ line cell, and that at least one processed pseudogene, the human DHFR psi 1, has been formed so recently that it is polymorphic.
Abstract: The processed pseudogenes reported to date fall into three categories: those that are a complete copy of the mRNA transcribed from the functional gene, those that are only a partial copy of the corresponding mRNA, and those that contain sequences in addition to those expected to be present in the mRNA. The general structural characteristics of these processed pseudogenes include the complete lack of intervening sequences found in the functional counterparts, a poly A tract at the 3' end, and direct repeats flanking the pseudogene sequence. In all the cases studied, these pseudogenes have been found to be on a different chromosome from their functional counterpart. These characteristics have led investigators to suggest that an RNA intermediate, in many cases the mRNA of the functional gene, is involved in the production of these pseudogenes. The mechanism by which processed pseudogenes arose involves the integration of the mRNA, or its cDNA copy, into a staggered chromosome break, followed by DNA synthesis and repair. I suggest that all the transcripts that gave rise to these pseudogenes were actually produced in the germ line cell. The transcripts that gave rise to the processed pseudogenes that are direct copies of the corresponding mRNA resulted from RNA polymerase II transcription of the functional counterpart. Pseudogenes that are not a direct copy of the corresponding mRNA may have resulted from RNA polymerase III transcription. If this is indeed the case, one need not postulate the involvement of retroviruses to explain the presence of processed pseudogenes corresponding to genes that are not expressed in the germ line. Following the integration event, processed pseudogenes can no longer be transcribed to produce the functional mRNA from which they arose. This inability to be transcribed by RNA polymerase II is not surprising considering that processed pseudogenes seem to be randomly integrated into the genome. Therefore, integration of a processed pseudogene such that RNA polymerase II transcriptional promoters are correctly positioned 5' to the resultant pseudogene is an unlikely event. The presence of processed pseudogenes seems peculiar to mammals. In fact, evolutionary studies indicate that processed pseudogenes are of relatively recent origin. In fact, at least one processed pseudogene, the human DHFR psi 1, has been formed so recently that it is polymorphic.
TL;DR: An in vitro transcription system derived from uninfected HeLa cells that accurately initiates RNA synthesis at the herpes virus thymidine kinase (TK) promoter is characterized and three distinctive protein binding sites are identified.
TL;DR: Results suggest that direct binding of MLTF to an upstream element activates transcription from the major late promoter.
TL;DR: The CSF-1 appears to be encoded by a single-copy gene, but its expression results in the synthesis of several messenger RNA species, ranging in size from about 1.5 to 4.5 kilobases.
Abstract: Complementary DNA (cDNA) clones encoding human macrophage-specific specific colony-stimulating factor (CSF-1) were isolated. One cDNA clone codes for a mature polypeptide of 224 amino acids and a putative leader of 32 amino acids. This cDNA, which was cloned in the Okayama-Berg expression vector, specifies the synthesis of biologically active CSF-1 in COS cells, as determined by a specific radioreceptor assay, macrophage bone marrow colony formation, and antibody neutralization. Most of the cDNA isolates contain part of an intron sequence that changes the reading frame, resulting in an abrupt termination of translation; these cDNA's were inactive in COS cells. The CSF-1 appears to be encoded by a single-copy gene, but its expression results in the synthesis of several messenger RNA species, ranging in size from about 1.5 to 4.5 kilobases.
TL;DR: The mechanism of action of actinomycin binds to a premelted DNA conformation present within the transcriptional complex, interfering with the elongation of growing RNA chains.
Abstract: Recent advances in understanding how actinomycin binds to DNA have suggested its mechanism of action. Actinomycin binds to a premelted DNA conformation present within the transcriptional complex. This immobilizes the complex, interfering with the elongation of growing RNA chains. The model has a number of implications for understanding RNA synthesis.
TL;DR: In the most actively expressing transformants the amount of mRNA from the introduced chimaeric genes was half that of the endogenous wild‐type gene, and in general transcriptional fusions in which a linker sequence interrupted the 5′‐untranslated region gave rise to less chimaic mRNA accumulation than a translational fusion.
Abstract: Promoter DNA sequences from a petunia chlorophyll a/b binding protein gene were fused to octopine synthase DNA sequences and the resulting chimaeric genes were introduced into petunia and tobacco cells. Populations of transformed regenerated petunia plants containing the chimaeric genes were examined so that the expression of any particular construction could be compared between independent transformants. Substantial variation was observed between transformants in the level of chimaeric gene expression. In general, transcriptional fusions in which a linker sequence interrupted the 5'-untranslated region gave rise to less chimaeric mRNA accumulation than a translational fusion. In the most actively expressing transformants the amount of mRNA from the introduced chimaeric genes was half that of the endogenous wild-type gene. Transcription initiated at the same place in the chimaeric and endogenous genes. Construction of the translational cab/ocs fusion caused three amino acid changes in the octopine synthase protein and functional octopine synthase enzyme was absent from plants in which mRNA for the chimaeric gene was abundantly expressed.
01 Jan 1985
TL;DR: This review will attempt to cover the present information on the multiple forms of eukaryotic DNA-dependent RNA polymerases, both at the structural and functional level.
Abstract: This review will attempt to cover the present information on the multiple forms of eukaryotic DNA-dependent RNA polymerases, both at the structural and functional level. Nuclear RNA polymerases constitute a group of three large multimeric enzymes, each with a different and complex subunit structure and distinct specificity. The review will include a detailed description of their molecular structure. The current approaches to elucidate subunit function via chemical modification, phosphorylation, enzyme reconstitution, immunological studies, and mutant analysis will be described. In vitro reconstituted systems are available for the accurate transcription of cloned genes coding for rRNA, tRNA, 5 SRNA, and mRNA. These systems will be described with special attention to the cellular factors required for specific transcription. A section on future prospects will address questions concerning the significance of the complex subunit structure of the nuclear enzymes; the organization and regulation of the gene coding for RNA polymerase subunits; the obtention of mutants affected at the level of factors, or RNA polymerases; the mechanism of template recognition by factors and RNA polymerase.
TL;DR: It appears that longer stretches of poly(dA-dT) are more effective upstream promoter elements for constitutive expression, and these transcriptional effects may be due to exclusion of nucleosomes from poly( dA- dT) regions.
Abstract: pet56, his3, and ded1 are adjacent but unrelated genes located on chromosome XV of the yeast Saccharomyces cerevisiae. his3 and pet56 are transcribed in opposite directions from initiation sites separated by approximately equal to 200 base pairs. Under normal growth conditions, both genes are transcribed at a similar basal level. Deletion analysis of the his3 gene indicates that the upstream promoter element for constitutive expression is defined by a 17-base-pair region that contains 15 thymidine residues in the coding strand. Sequential deletions of the pet56 gene indicate that this same region is required for wild-type transcription levels. Thus, this poly(dA-dT) sequence acts bidirectionally to activate transcription of two unrelated genes. Transcription of the ded1 gene is initiated approximately equal to 300 base pairs downstream from the his3 gene, and it occurs at a 5-fold higher level. This gene contains a 34-base-pair region containing 28 thymidine residues in the coding strand located upstream from the ded1 TATA box. Deletion of this dA-dT stretch significantly reduces transcription below the wild-type level. Thus, for at least three different yeast genes, naturally occurring stretches of poly(dA-dT) serve as upstream promoter elements for constitutive expression. In addition, it appears that longer stretches of poly(dA-dT) are more effective upstream promoter elements. These transcriptional effects may be due to exclusion of nucleosomes from poly(dA-dT) regions.
TL;DR: It is shown that the rate of transcription of MT2A is the same on treatment with interferon or dexamethasone, but that the mRNA accumulates much faster with dexam ethon, indicating that post-transcriptional events are important in the latter case.
Abstract: Complementary DNAs corresponding to the interferon (IFN)-induced messenger RNAs for histocompatibility locus antigens (HLA), metallothionein-II (MT2), 2',5'-oligoadenylate synthetase and about eight other proteins of unknown sequence have been isolated recently, and by interferon regulation of transcription has been demonstrated for several of the eight mRNAs, with a significant increase apparent in as little as 5 min. We now show that IFN-alpha treatment results in a three- to fivefold increase in the transcription of MT2 and HLA class I genes in human T98G neuroblastoma cells. Furthermore, comparison of regions upstream of the MT2A gene, two HLA genes and one HLA class II gene reveals a homologous sequence of approximately 30 base pairs (bp) which may be involved in regulating transcription of interferon-induced genes. Transcription of the mRNA for human MT2A is induced by glucocorticoids or metal ions and the regulatory elements have been mapped by promoter-fusion experiments. We now show that the rate of transcription of MT2A is the same on treatment with interferon or dexamethasone, but that the mRNA accumulates much faster with dexamethasone, indicating that post-transcriptional events are important in the latter case.
TL;DR: The results suggest that a labile, cell type-specific protein may interact with the retroviral LTR and regulate transcription of genes under LTR control.
Abstract: In several bursal lymphoma cell lines in which c-myc transcription is regulated by avian leukosis virus (ALV) long terminal repeat (LTR) sequences, protein synthesis inhibition decreases the transcriptional activity of c-myc as well as other LTR driven viral genes. This decrease in transcription is associated with a change in the chromatin structure of c-myc, as measured by deoxyribonuclease I (DNase I) hypersensitivity, and a shift of transcription from the LTR to the normal c-myc promoter. In contrast, cycloheximide had little or no effect on the transcription of LTR driven genes in infected chicken embryo fibroblasts treated with the drug. These results suggest that a labile, cell type-specific protein may interact with the retroviral LTR and regulate transcription of genes under LTR control. Further, the results demonstrate that the increase in intracellular concentration of c-myc RNA induced by cycloheximide treatment of normal cells is the result of stabilization of this message.
TL;DR: It is demonstrated that the expression of this trans-activating factor maps to the 3' open reading frames of the viral transforming region of the bovine papilloma virus, and a premature termination codon engineered into the E2 ORF eliminates expression ofThis diffusible transactivation function establishing the E1 gene product as the diffusibles trans-Activating factor.
TL;DR: That the evolution of viroids has involved RNA rearrangements of domains is supported by the partial duplication of coconut cadang cadang viroid, which arises de novo during each infection.
Abstract: On the basis of sequence homology a model is proposed for five structural and functional domains in viroids. These domains include (i) a conserved central region capable of forming two alternative structures that may regulate two phases of the viroid replication cycle, (ii) a region associated with pathogenicity, (iii) a domain with high sequence variability, (iv and v) two terminal domains that are interchangeable between viroids. That the evolution of viroids has involved RNA rearrangements of domains is supported by the partial duplication of coconut cadang cadang viroid, which arises de novo during each infection. Similar RNA rearrangements have been established for animal viral defective interfering RNAs, which arise by some form of discontinuous transcription. This mechanism could account for the origin of viroids and also RNA viruses, whereby modules of genetic information may have undergone repeated exchange between RNA pathogens and the RNA of their hosts.
TL;DR: Observations suggest that anti-sense RNA may be generally useful for suppressing the expression of specific genes in vivo and may be a potential molecular alternative to classical genetic analysis.
Abstract: Plasmid DNA directing transcription of the noncoding (anti-sense) DNA strand can specifically inhibit the expression of several test genes as well as normal, endogenous genes. The anti-sense plasmid constructions can be introduced into eukaryotic cells by transfection or microinjection and function in both transient and stable transformation assays. Anti-sense transcripts complementary to as little as 52 bases of 5' untranslated target gene mRNA specifically suppress gene activity as well as, or more efficiently than, anti-sense transcripts directed against the protein coding domain alone. Conditional anti-sense inhibition is accomplished with the use of hormone-inducible promoter sequences. Suppression of endogenous actin gene activity by anti-sense RNA is detected as a decrease in growth rate and as a reduction in the number of actin microfilament cables. These observations suggest that anti-sense RNA may be generally useful for suppressing the expression of specific genes in vivo and may be a potential molecular alternative to classical genetic analysis.
TL;DR: It is suggested that these sites represent a class of vegetatively expressed Streptomyces promoter that is utilised by a form of RNA polymerase holoenzyme that also recognizes typical promoters of other bacterial genera.
TL;DR: Evidence is presented indicating that the Notch repeat is a member of a novel family of repetitive elements, which is term the opa family, and the data suggest that some of these elements may be not only transcribed but also translated.
TL;DR: The 21-base pair repeat elements of the SV40 promoter contain six tandem copies of the GGGCGG hexanucleotide (GC-box), each of which can bind, with varying affinity, to the cellular transcription factor, Sp1.
Abstract: The 21-base pair repeat elements of the SV40 promoter contain six tandem copies of the GGGCGG hexanucleotide (GC-box), each of which can bind, with varying affinity, to the cellular transcription factor, Sp1. In vitro SV40 early RNA synthesis is mediated by interaction of Sp1 with GC-boxes I, II, and III, whereas transcription in the late direction is mediated by binding to GC-boxes III, V, and VI.
TL;DR: Evidence is consistent with the view that gpntrA is a sigma subunit for RNA polymerase, which binds to five sites in the glnA promoter-regulatory region and appears to activate transcription initiation.
Abstract: In enteric bacteria the products of two nitrogen regulatory genes, ntrA and ntrC, activate transcription of glnA, the structural gene encoding glutamine synthetase, both in vivo and in vitro. The ntrC product (gpntrC) is a DNA-binding protein, which binds to five sites in the glnA promoter-regulatory region and appears to activate transcription initiation. Using as an assay the stimulation of glnA transcription in a coupled in vitro transcription-translation system, we have partially purified the ntrA gene product (gpntrA). The following evidence is consistent with the view that gpntrA is a sigma subunit for RNA polymerase: (i) The gpntrA activity copurifies with the sigma 70 holoenzyme (E sigma 70) and core (E) forms of RNA polymerase through several steps but can be separated from them by chromatography on heparin agarose. (ii) After further purification by molecular sieve chromatography, the partially purified gpntrA fraction allows transcription of glnA from the same startpoint used in vivo; transcription is dependent on gpntrC and on added E. The gpntrA fraction does not allow transcription from promoters that we have used as controls, including lacUV5. E sigma 70 has the reverse specificity.