Showing papers on "Transcription (biology) published in 1989"

Regulation of lymphokine messenger RNA stability by a surface-mediated T cell activation pathway

Abstract: Quiescent T cells can be induced to express many genes by mitogen or antigen stimulation. The messenger RNAs of some of these genes undergo relatively rapid degradation compared to messenger RNAs from constitutively expressed genes. A T cell activation pathway that specifically regulates the stability of messenger RNAs for the lymphokines interleukin-2, interferon-gamma, tumor necrosis factor-alpha, and granulocyte-macrophage colony-stimulating factor is induced by stimulation of the CD28 surface molecule. This pathway does not directly affect the steady-state messenger RNA level, transcription, or messenger RNA half-life of other T cell activation genes, including c-myc, c-fos, IL-2 receptor, and the 4F2HC surface antigen. These data show that stimuli received at the cell surface can alter gene expression by inducing specific changes in messenger RNA degradation.

Cloning of cDNA for the major DNA-binding protein of the erythroid lineage through expression in mammalian cells

Abstract: Genes expressed in erythroid cells contain binding sites for a cell-specific factor believed to be an important regulator for this haematopoietic lineage. Using high-level transient expression in mammalian cells, we have identified complementary DNA encoding the murine protein. The factor, a new member of the zinc-finger family of DNA-binding proteins, is restricted to erythroid cells at the level of RNA expression and is closely homologous between mouse and man.

Tumor necrosis factor alpha activates human immunodeficiency virus type 1 through induction of nuclear factor binding to the NF-kappa B sites in the long terminal repeat.

Abstract: Expression of human immunodeficiency virus type 1 (HIV-1) can be activated in a chronically infected T-cell line (ACH2 cells) by a cytokine, human tumor necrosis factor alpha (TNF-alpha). TNF-alpha treatment of ACH2 cells resulted in an increase in steady-state levels of HIV RNA and HIV transcription. Gel mobility shift assays demonstrated that the transcriptional activation of the HIV long terminal repeat (LTR) by TNF-alpha was associated with the induction of a nuclear factor(s) binding to the NF-kappa B sites in the LTR. Deletion of the NF-kappa B sites from the LTR eliminated activation by TNF-alpha in T cells transfected with plasmids in which the HIV LTR directed the expression of the bacterial chloramphenicol acetyltransferase gene. Thus, TNF-alpha appears to activate HIV RNA and virus production by ACH2 cells through the induction of transcription-activating factors that bind to the NF-kappa B sequences in the HIV LTR.

Transcription-based amplification system and detection of amplified human immunodeficiency virus type 1 with a bead-based sandwich hybridization format

Abstract: The in vitro amplification of biologically important nucleic acids has proceeded principally by a strategy of DNA replication. Polymerase chain reaction was the first such protocol to achieve this goal. In this report, a transcription-based amplification system (TAS) is described. Each cycle of the TAS is composed of two steps. The first is a cDNA synthesis step that produces one copy of a double-stranded DNA template for each copy of RNA or DNA target nucleic acid. During the course of this cDNA synthesis step, a sequence recognized by a DNA-dependent RNA polymerase is inserted into the cDNA copy of the target sequence to be amplified. The second step is the amplification of the target sequence by the transcription of the cDNA template into multiple copies of RNA. This procedure has been applied to the detection of human immunodeficiency virus type 1 (HIV-1)-infected cells. After four cycles of TAS, the amplification of the vif region of the HIV-1 RNA genome was measured to be, on the average, 38- to 47-fold per cycle, resulting in a 2-5 x 10(6)-fold increase in the copy number of the original target sequence. This amplification by the TAS protocol allows the detection of fewer than one HIV-1-infected CEM cell in a population of 10(6) uninfected CEM cells. Detection of the TAS-generated RNA from HIV-1-infected cells can easily be accomplished by means of a bead-based sandwich hybridization protocol, which provides additional specificity for the identification of the amplified HIV-1-specific sequence.

Cationic liposome-mediated RNA transfection

Abstract: We have developed an efficient and reproducible method for RNA transfection, using a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), incorporated into a liposome (lipofectin). Transfection of 10 ng to 5 micrograms of Photinus pyralis luciferase mRNA synthesized in vitro into NIH 3T3 mouse cells yields a linear response of luciferase activity. The procedure can be used to efficiently transfect RNA into human, rat, mouse, Xenopus, and Drosophila cells. Using the RNA/lipofectin transfection procedure, we have analyzed the role of capping and beta-globin 5' and 3' untranslated sequences on the translation efficiency of luciferase RNA synthesized in vitro. Following transfection of NIH 3T3 cells, capped mRNAs with beta-globin untranslated sequences produced at least 1000-fold more luciferase protein than mRNAs lacking these elements.

Absolute mRNA quantification using the polymerase chain reaction (PCR). A novel approach by a PCR aided transcript titration assay (PATTY).

Abstract: The polymerase chain reaction (PCR) is used as part of a new approach to the absolute quantification of mRNA. We describe a PCR aided transcript titration assay (PATTY) which is based on the co-amplification of an in vitro generated transcript differing by a single base exchange from the target mRNA. Identical portions of a total RNA sample are "spiked" with different amounts of this mutated standard RNA, converted to cDNA and amplified by PCR. Because the base exchange creates a novel restriction endonuclease site, the ratio of co-amplified DNA derived from target mRNA to amplified DNA derived from standard RNA can be determined after restriction endonuclease digestion and separation by gel electrophoresis. This method gives accurate results within 24 hours and is useful especially for the quantification of either low-abundance mRNA or more abundant mRNA present in very small amounts of total RNA. The low-abundance mRNA encoding 4-coumarate:CoA ligase (4CL) in cultured potato cells (Solanum tuberosum L.) was measured in a case study. About 100 molecules per assay could be accurately detected by the new method.

A third human retinoic acid receptor, hRAR-gamma

Abstract: Retinoic acid receptors (RARs) are retinoic acid (RA)-inducible enhancer factors belonging to the superfamily of steroid/thyroid nuclear receptors. We have previously characterized two human RAR (hRAR-alpha and hRAR-beta) cDNAs and have recently cloned their murine cognates (mRAR-alpha and mRAR-beta) together with a third RAR (mRAR-gamma) whose RNA was detected predominantly in skin, a well-known target for RA. mRAR-gamma cDNA was used here to clone its human counterpart (hRAR-gamma) from a T47D breast cancer cell cDNA library. Using a transient transfection assay in HeLa cells and a reporter gene harboring a synthetic RA responsive element, we demonstrate that hRAR-gamma cDNA indeed encodes a RA-inducible transcriptional trans-activator. Interestingly, comparisons of the amino acid sequences of all six human and mouse RARs indicate that the interspecies conservation of a given member of the RAR subfamily (either alpha, beta, or gamma) is much higher than the conservation of all three receptors within a given species. These observations indicate that RAR-alpha, -beta, and -gamma may perform specific functions. We show also that hRAR-gamma RNA is the predominant RAR RNA species in human skin, which suggests that hRAR-gamma mediates some of the retinoid effects in this tissue.

Illegitimate transcription: transcription of any gene in any cell type.

Abstract: Using in vitro amplification of cDNA by the polymerase chain reaction, we have detected spliced transcripts of various tissue-specific genes (genes for anti-Mullerian hormone, beta-globin, aldolase A, and factor VIIIc) in human nonspecific cells, such as fibroblasts, hepatoma cells, and lymphoblasts. In rats, erythroid- and liver-type pyruvate kinase transcripts were also detected in brain, lung, and muscle. The abundance of these "illegitimate" transcripts is very low; yet, their existence and the possibility of amplifying them by the cDNA polymerase chain reaction provide a powerful tool to analyze pathological transcripts of any tissue-specific gene by using any accessible cell.

Transcription activation by the adenovirus E1a protein

Abstract: The adenovirus Ela protein stimulates transcription of a wide variety of viral and cellular genes. It is shown here that Ela has the two functions characteristic of a typical cellular activator: one direct Ela to the promoter, perhaps by interacting with a DMA-bound protein, and the other, an activating region, enables the bound activator to stimulate transcription.

The c-fos transcript is targeted for rapid decay by two distinct mRNA degradation pathways.

Abstract: Rapid degradation of c-fos proto-oncogene mRNA is crucial for transient c-fos gene expression. Experiments were performed to investigate the cellular mechanisms responsible for the extremely short half-life of human c-fos mRNA in growth-factor-stimulated fibroblasts. These experiments demonstrate the existence of two distinct cellular pathways for rapid c-fos mRNA degradation. Each of these pathways recognizes a different, functionally independent instability determinant within the c-fos transcript. One instability determinant, which is located within the c-fos 3'-untranslated region, is a 75-nucleotide AU-rich segment. Insertion of this element into beta-globin mRNA markedly reduces the half-life of that normally long-lived message. Nevertheless, specific deletion of the AU-rich element from c-fos mRNA has little effect on the transcript's cytoplasmic half-life due to the presence of the other c-fos instability determinant, which is located in the protein-coding segment of the c-fos message. Examination of mRNA decay in cells treated with transcription inhibitors indicates that one c-fos mRNA degradation pathway is dependent on RNA synthesis, whereas the other is not.

Regulation of Proenkephalin by Fos and Jun

Abstract: Fos and Jun form a heterodimeric complex that associates with the nucleotide sequence motif known as the AP-1 binding site. Although this complex has been proposed to function as a transcriptional regulator in neurons, no specific target gene has yet been identified. Proenkephalin mRNA increased in the hippocampus during seizure just after an increase in c-fos and c-jun expression was detected. Fos-Jun complexes bound specifically to a regulatory sequence in the 5' control region of the proenkephalin gene. Furthermore, c-fos and c-jun stimulated transcription from this control region synergistically in transactivation assays. These data suggest that the proenkephalin gene may be a physiological target for Fos and Jun in the hippocampus and indicate that these proto-oncogene transcription factors may play a role in neuronal responses to stimulation.

Abscisic acid-responsive sequences from the em gene of wheat.

Abstract: We demonstrate that a chimeric gene containing the beta-glucuronidase (GUS) reporter gene linked to a 646-base pair 5' fragment (-554 to +92) from the abscisic acid (ABA)-regulated Em gene from wheat is correctly expressed in transgenic tobacco. We observe high activity only in embryos of mature seeds, and immature seeds cultured on ABA show enhanced expression. Using a rice transient assay, we identify a 260-base pair fragment (-168 to +92) that accounts for the ABA-specific 15-fold to 20-fold increase in GUS expression. A 50-base pair sequence (-152 to -103) fused 5' in either orientation to a truncated cauliflower mosaic virus promoter (35S) increases GUS activity threefold in the presence of ABA. Insertion of the Em 5'-untranslated region (+6 to +86) between the 35S promoter and the ATG of GUS results in a 10-fold increase in GUS activity in the absence of ABA. These results suggest the following two functional fragments of the Em 5' region: an ABA response element from -152 to -103 and an element between +6 and +86 that quantitatively increases the ABA response.

Three amino acids of the oestrogen receptor are essential to its ability to distinguish an oestrogen from a glucocorticoid-responsive element.

Abstract: Steroid hormone receptors activate specific gene transcription by binding as hormone-receptor complexes to DNA enhancer elements termed hormone responsive elements. A highly conserved 66-amino-acid region of the oestrogen and glucocorticoid receptors which corresponds to part of the receptor DNA-binding domain (region C) determines the specificity of target gene recognition. This region contains two subregions (CI and CII), encoded in two separate exons, that are analogous to the 'zinc fingers' of the transcription factor TFIIIA. The N-terminal CI finger determines the recognition specificity of the hormone responsive element. A chimaeric oestrogen receptor, in which the CI finger is replaced with the corresponding glucocorticoid receptor CI finger region, activates transcription from a reporter gene containing a glucocorticoid-responsive element, but not from a reporter gene containing an oestrogen-responsive element. We report here that three amino acids located at the C-terminal side of the oestrogen receptor CI finger play a key part in this specificity.

COUP transcription factor is a member of the steroid receptor superfamily.

Abstract: THE COUP (chicken ovalbumin upstream promoter) transcription factor (COUP-TF) exists in a number of different tissues and is essential for expression of the chicken ovalbumin gene1-3. It binds to the ovalbumin promoter and, in conjunction with a second protein (S300-II), stimulates initiation of transcription in vitro. COUP-TF also binds specifically to the rat insulin promoter element4,5, although the two binding sites share little sequence similarity. Here we report the isolation of a human complementary DNA clone encoding COUP-TF. Comparison of the amino-acid sequence of COUP-TF with known sequences reveals that it is a member of the steriod/thyroid hormone/vitamin receptor super-family6. Consequently, it is the first member of this family that has been shown to function in a cell–free transcription system7,8. We conclude that this superfamily of gene regulators contains proteins which bind and activate distal promoter elements of eukaryotic genes.

Human immunodeficiency virus 1 tat protein binds trans-activation-responsive region (TAR) RNA in vitro

Abstract: tat, the trans-activator protein for human immunodeficiency virus 1 (HIV-1), has been expressed in Escherichia coli from synthetic genes. Purified tat binds specifically to HIV-1 trans-activation-responsive region (TAR) RNA in gel-retardation, filter-binding, and immunoprecipitation assays. tat does not bind detectably to antisense TAR RNA sequences, cellular mRNA sequences, variant TAR RNA sequences with altered stem-loop structures, or TAR DNA.

Characterization of junD: a new member of the jun proto-oncogene family.

Abstract: In an extensive screen of a cDNA library prepared from serum-stimulated mouse NIH 3T3 cells, we identified three distinct jun-related clones. Two of them were carrying c-jun and junB sequences respectively, whereas the sequence of the third group of clones (junD) was distinct from these two and from v-jun. The amino acid sequences derived from these jun-related clones are very well conserved in five distinct regions including the putative DNA binding domain. Truncated c-Jun and JunD proteins containing the C-terminus recognize the same DNA sequences which were defined as the PEA1/AP1 binding sequence or TPA response element (TRE). Furthermore, both can trans-activate a promoter including the TRE, and this activation is further enhanced by c-fos. Contrary to c-jun and junB transcription, which are strongly stimulated by serum or TPA treatment of quiescent 3T3 cells, junD transcription is not significantly stimulated in these conditions. The tissue distribution and levels of expression of junD mRNA differ from that of c-jun and junB mRNA. These observations suggest that each of these Jun-related gene products has a distinct role in the control of gene activity and growth in the organism.

Oligodeoxynucleotides: Antisense Inhibitors of Gene Expression

Abstract: Publisher Summary A new type of drug molecule synthesized with a specific base sequence designed to interact with a target nucleic acid containing the complementary sequence are being developed. In the earliest and most developed example of this approach, oligodeoxynucleotides and their analogs have been applied as antisense inhibitors of gene expression to bring about translation arrest. This chapter discusses the use of antisense oligodeoxynucleotides as a therapeutic approach. Once this approach is recognized in principle, other examples can be seen to fit a pattern, such as the use of oligodeoxynucleotides to form a triple-strand helix with duplex DNA. However, with a different base triplet code to arrest transcription, the opening of duplex DNA is not considered facile enough to allow the access of an oligo to interact with one of the strands. The development of synthetic oligoribonucleotides as ribozymes is another example in which the information for the selectivity is encoded in the portions of the oligomer flanking the catalytic site. Another example is the use of oligodeoxynucleotides to bind to protein sites that recognize specific sequences of bases, thus, inhibiting polymerase action or bringing about transcription arrest by sequestering specific transcription cofactors. Thus, a new area of pharmacology is developing that is differentiated by the purposeful inclusion of information in the form of nucleic-acid bases in a putative oligonucleotide drug molecule.

Function of a Bacterial Activator Protein that Binds to Transcriptional Enhancers

Abstract: The nitrogen regulatory (NtrC) protein of enteric bacteria, which binds to sites that have the properties of transcriptional enhancers, is known to activate transcription by a form of RNA polymerase that contains the NtrA protein (sigma 54) as sigma factor (referred to as sigma 54-holoenzyme). In the presence of adenosine triphosphate, the NtrC protein catalyzes isomerization of closed recognition complexes between sigma 54-holoenzyme and the glnA promoter to open complexes in which DNA in the region of the transcription start site is locally denatured. NtrC is not required subsequently for maintenance of open complexes or initiation of transcription.

Viral proteins containing the purine NTP-binding sequence pattern

Abstract: A compilation is presented of viral proteins containing the NTP-binding sequence pattern, and criteria are suggested for assessment of the functional significance of the occurrence of this pattern in protein sequences. It is shown that the distribution of NTP-binding pattern-containing proteins through the viral kingdom is strongly non-random. Sequence comparisons led to delineation of several families of these proteins, some of which could be brought together into superfamilies including also cellular proteins. The available biochemical evidence is compatible with the proposal that viral proteins in which the NTP-binding pattern is evolutionarily conserved might all be NTPases involved in: i) duplex unwinding during DNA and RNA replication, transcription, recombination and repair, and possibly mRNA translation; ii) DNA packaging, and iii) dNTP generation.

jun-D: a third member of the jun gene family.

Abstract: The protooncogene c-jun encodes a component of the transcription factor AP-1. Both murine c-jun and a related gene (jun-B) are rapidly activated in BALB/c3T3 cells by serum growth factors. We report here the cloning and analysis of a cDNA encoding a third member of the murine jun family, jun-D. The amino acid sequence encoded by jun-D has two extensive regions of homology with the other Jun proteins. One homology region includes the DNA-binding domain and sequences required for dimer formation and interaction with the Fos oncoprotein; the other includes the acidic sequence thought to be involved in gene activation. All three jun mRNAs are present in a variety of murine tissues and cell lines. In resting 3T3 cells, jun-D is expressed at a higher level compared to c-jun and jun-B, and its transcription is stimulated only slightly by serum growth factors. Thus, jun-D appears to be regulated differently than c-jun and jun-B.

Identification of the sigma E subunit of Escherichia coli RNA polymerase: a second alternate sigma factor involved in high-temperature gene expression.

Abstract: The rpoH gene of Escherichia coli encodes sigma 32, the 32-kD sigma-factor responsible for the heat-inducible transcription of the heat shock genes. rpoH is transcribed from at least three promoters. Two of these promoters are recognized by RNA polymerase containing sigma 70, the predominant sigma-factor. We purified the factor responsible for recognizing the third rpoH promoter (rpoH P3) and identified it as RNA polymerase containing a novel sigma-factor with an apparent Mr of 24,000. This new sigma, which we call sigma E, is distinct from the known sigma factors in molecular weight and promoter specificity. sigma E holoenzyme will not recognize the sigma 70- or sigma 32-controlled promoters we tested, but it does transcribe the htrA gene, which is required for viability at temperatures greater than 42 degrees C. The in vivo role of sigma E is not known. The transcripts from the sigma E-controlled rpoH P3 and htrA promoters are most abundant at very high temperature, suggesting the sigma E holoenzyme may transcribe a second set of heat-inducible genes that are involved in growth at high temperature or in thermotolerance.

IL-4 down-regulates IL-1 and TNF gene expression in human monocytes.

Abstract: The effects of IL-4 on IL-1 and TNF gene expression in human peripheral monocytes (PBM) were examined. Highly purified PBM cultured for 24 to 96 h in the presence of IL-4 produce neither IL-1 nor TNF protein. RNA hybridization studies demonstrated that IL-4 does not induce transcription of IL-1 or TNF. Preincubation of PBM with IL-4 did, however, inhibit LPS-induced IL-1 and TNF production in a dose- and time-related fashion. Maximal inhibition occurred after 96 h of incubation with 200 U/ml IL-4. RNA hybridization studies demonstrated that IL-4 suppressed the expression of IL-1 alpha, IL-1 beta, and TNF mRNA. These results suggest IL-4 modulates monocyte production of TNF and IL-1 by down-regulation of gene expression. This unique property of IL-4 may be important in the regulation of the immune response.

Novel method for studying mRNA phenotypes in single or small numbers of cells

Abstract: Biological processes, such as growth control, are often governed by biochemical steps involving mRNA transcripts that are short-lived and have a low copy number. Furthermore, the cells involved in these processes are often available in low numbers from in vivo sources. We now report a method that is superior to in situ hybridization, RNA blot analysis, and the nuclease protection assay for the study of short-lived, low-copy-number mRNA transcripts. The method consists of a microprocedure for isolating RNA from one to a few thousand cells and two coupled enzymatic steps: reverse transcription of whole cellular RNA, followed by amplification of the cDNA by a specifically primed polymerase chain reaction to give specific cDNA fragments that can be visualized on agarose gels by ethidium bromide staining. With this method we have detected actin mRNA from a single cell, or less than 100 cRNA molecules, and have quantified differences in RNA concentrations of less than threefold. The reverse transcription reaction products can be divided for the polymerase chain reaction, and several mRNA species can be assayed simultaneously. Therefore, we call the method single-cell mRNA phenotyping. This technique is applicable to the analysis of low-copy-number growth factor transcripts in cells in culture and in vivo.