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PubMed1998

Regulation of MHC class I gene expression is at transcriptional and post-transcriptional level in bovine placenta.

A previous study of MHC in cattle trophoblast demonstrated low or absent class I expression, using a broad specificity monoclonal antibody. The study reported here uses MHC-defined cattle and embryo transfer to ensure MHC incompatibility between dam and calf. Transcription and expression of defined class I genes was examined in placentomes taken at term, using monoclonal antibodies to bovine class I, a gene-specific DNA-based typing system, and in situ hybridisation. Results demonstrate intermediate levels of fetal MHC class I mRNA in trophoblast, but no detectable fetal class I protein. This suggests a level of transcriptional down-regulation, and a post-transcriptional block which might involve other gene products, such as beta2-microglobulin (beta 2m), or proteins involved in generation/transport of peptides.

Animals↗
PubMed1997

The polyhomeotic locus of Drosophila melanogaster is transcriptionally and post-transcriptionally regulated during embryogenesis.

The polyhomeotic (ph) locus of Drosophila is a complex locus essential for the maintenance of segmental identity. Genetic analysis suggested that two independent units contribute to ph function. Comparison of genomic sequence shows that the ph locus has been duplicated, and that it contains proximal and distal transcription units. The proximal transcription unit encodes two embryonic mRNAs of 6.4 and 6.1 kb, and the distal unit encodes a 6.4-kb embryonic mRNA. The proximal and distal transcription units are differentially regulated at the mRNA level during development as shown by developmental Northern analysis. The distal protein is very similar to the proximal product, except for the absence of an amino terminal region, and a small region near the carboxy terminus. The long open reading frame in the distal cDNA does not begin with an ATG codon, and an internal ATG is used for a start codon. We show that the proximal protein occurs in two forms that are developmentally regulated, and that probably arise from use of two different initiator methionine codons. We find no evidence for differential binding of proximal and distal products to polytene chromosomes. Nevertheless, we show that mutations in the proximal and distal proteins have differing effects on regulation of a reporter under the control of a regulatory region from bithoraxoid, suggesting that ph proximal and distal proteins have different functions. These results show that the ph locus undergoes complex developmental regulation, and suggest that Polycomb group regulation may be more dynamic than anticipated.

Animals↗
PubMedDate not supplied

Transcriptional pathways for synapse-specific, neuregulin-induced and electrical activity-dependent transcription.

Innervation-dependent expression of acetylcholine receptor (AChR) genes in skeletal muscle is mediated by multiple transcriptional pathways. One pathway leads to activation of AChR genes selectively in synaptic nuclei and requires an Ets binding site that binds GABP. A second pathway leads to repression of AChR transcription in nuclei throughout the myofiber and requires inactivation of E-box-binding proteins, including myogenic bHLH proteins. Taken together, these studies indicate that separate pathways regulate innervation-dependent transcription.

Animals↗
PubMed2001

Differential induction of spermidine/spermine N1-acetyltransferase activity in cisplatin-sensitive and -resistant ovarian cancer cells in response to N1,N12-bis(ethyl)spermine involves transcriptional and post-transcriptional regulation.

The growth inhibition that occurs in cisplatin-sensitive 2008 human ovarian cancer cells in response to the spermine analogue, N1,N12-bis(ethyl)spermine (BESpm), is associated with a potent induction of spermidine/spermine N1-acetyltransferase (SSAT), the rate-limiting enzyme in polyamine catabolism. Conversely, in cisplatin-resistant C13* cells, which are less responsive to BESpm, enzyme induction does not occur at comparable levels after exposure to the bis(ethyl)-derivative. In this study, we investigated the molecular mechanisms underlying the differential induction of SSAT activity in cisplatin-sensitive and -resistant cells. Northern blot analysis revealed a difference in the level of SSAT mRNA expression in the two cell lines; in particular, 2008 cells treated with 10 microM BESpm for progressively increasing periods of time accumulated more heteronuclear (3.5 kb) and mature (1.3/1.5 kb) SSAT mRNAs than its resistant variant. SSAT mRNA accumulation paralleled enzyme activity and both were almost completely prevented in the two lines by co-treatment with 5 microg/ml actinomycin-D (Act-D), suggesting that transcription plays a major role in the analogue-mediated induction of SSAT. Moreover, when Act-D was added 48 h after BESpm exposure, SSAT mRNA and enzyme activity were stabilised in both cell lines. Therefore, the marked difference in the induction of SSAT activity seems to be related to increased enzyme synthesis, particularly in sensitive cells, whose SSAT protein turnover was also greatly reduced (half-life >12 h in 2008 cells versus 5 h in C13* cells) in the presence of BESpm. These findings suggest that cisplatin-resistance modulates the SSAT response to BESpm at transcriptional and post-transcriptional levels.

Acetyltransferases↗
PubMed2003

The first strand transfer reaction of HIV-1 reverse transcription is more efficient in infected cells than in cell-free natural endogenous reverse transcription reactions.

BACKGROUND: In the presence of dNTPs, intact HIV-1 virions are capable of reverse transcribing at least part of their genome, a process known as natural endogenous reverse transcription (NERT). PCR analysis of virion DNA produced by NERT revealed that the first strand transfer reaction (1stST) was inefficient in intact virions, with minus strand (-) strong stop DNA (ssDNA) copy numbers up to 200 times higher than post-1stST products measured using primers in U3 and U5. This was in marked contrast to the efficiency of 1stST observed in single-round cell infection assays, in which (-) ssDNA and U3-U5 copy numbers were indistinguishable. OBJECTIVES: To investigate the reasons for the discrepancy in first strand transfer efficiency between intact cell-free virus and the infection process. STUDY DESIGN: Alterations of both NERT reactions and the conditions of cell infection were used to test whether uncoating and/or entry play a role in the discrepancy in first strand transfer efficiency. RESULTS AND CONCLUSIONS: The difference in 1stST efficiency could not be attributed simply to viral uncoating, since addition of very low concentrations of detergent to NERT reactions removed the viral envelope without disrupting the reverse transcription complex, and these conditions resulted in no improvement in 1stST efficiency. Virus pseudotyped with surface glycoproteins from either vesicular stomatitis virus or amphotrophic murine leukaemia virus also showed low levels of 1stST in low detergent NERT assays and equivalent levels of (-) ssDNA and 1stST in single-round infections of cells, demonstrating that the gp120-mediated infection process did not select for virions capable of carrying out 1stST. These data indicate that a post-entry event or factor may be involved in efficient HIV-1 reverse transcription in vivo.

Cell Line↗
PubMed1989

Characterization of primary transcripts and identification of transcription initiation sites on the heavy and light strands of mouse mitochondrial DNA.

Total RNA from Ehrlich ascites mitochondria pretreated with RNase-free DNase was capped in vitro with [alpha-32P]GTP and guanylyl transferase. The cappable RNAs representing the primary transcripts show a heterogeneous size distribution with four major species of 46, 63, 94, and 152 nucleotides and four minor species of 19, 24, 104, and 790 nucleotides in size. Hybridization with the D-loop DNA probes shows that the 19-nucleotide-long capped RNA is coded by the H-strand of mitochondrial DNA while the rest are coded by the L-strand. S1 nuclease mapping and primer extension analyses suggest the occurrence of a transcription initiation of H-strand at about 19 nucleotides upstream from the start of the tRNA(Phe) gene. All of the L-strand cappable RNAs have a common 5' end mapping to nucleotide 16,183 +/- 5 of the genome. The 3' ends of four major cappable RNA species line up to the conserved sequence boxes, putative start sites of DH-DNA; and in fact about 2% of these cappable species are found to exist as DNA-linked RNA under steady-state conditions. The 3' end of the 790-nucleotide cappable RNA lies close to the start of the tRNA(Pro) gene, suggesting that it may be the true precursor of L-strand transcript endonucleolytically processed at the 3' end. The level of L-strand-coded cappable RNAs varies markedly under different growth conditions. Treatment with cycloheximide results in a reduction while chloramphenicol caused over 3-fold induction, suggesting that these "primer" RNAs may have an additional regulatory function.

Animals↗
PubMed2002

Transcription-mediated amplification and hybridisation protection assay to determine BCR-ABL transcript levels in patients with chronic myeloid leukaemia.

Detection of BCR-ABL transcripts in chronic myeloid leukaemia (CML) is used to confirm the diagnosis and to monitor residual disease. Quantitative techniques are required to predict response to therapy or early relapse. We have evaluated an assay in which transcription-mediated amplification (TMA) of BCR-ABL and ABL transcripts is achieved using reverse transcriptase and RNA polymerase. The products are quantified in the hybridisation protection assay (HPA) using acridinium ester-labelled DNA probes and chemiluminescence. The method is a single tube procedure which uses small amounts of RNA (<500 ng/triplicate analysis), is technically simple (requiring just two waterbaths and a luminometer), rapid (total assay time <4 h) and sensitive (capable of detecting one BCR-ABL-positive K562 cell in the presence of 10(4)-10(5) BCR-ABL-negative cells). BCR-ABL signals from patient RNA samples were quantified relative to known amounts of K562 RNA and normalised to levels of ABL. BCR-ABL/ABL ratios ranged from 0.15 to 1.59 (median 0.65) in RNA from diagnostic blood or bone marrow of 18 CML patients and were < or =0.0001 in 20 normal controls. Sequential samples analysed from six CML patients post-allogeneic bone marrow transplantation who relapsed and received donor lymphocyte infusions showed BCR-ABL/ABL ratios which reflected patient status or treatment. A BCR-ABL/ABL ratio of 0.01 served as a useful arbitrary indicator value, with results above and below this value generally correlating with relapse or remission, respectively.

Acridines↗
PubMed1988

RNA transcription in myocardial-cell nuclei during postnatal development. A study establishing an assay system for transcription in vitro.

A system for RNA transcription in vitro was established in order to determine the relative rate of RNA synthesis in neonatal and adult rat myocardial cells. This assay system optimizes the incorporation of [3H]UMP into RNA by using 3.5 x 10(7) myocardial-cell nuclei, and minimizes RNA degradation for at least 1 h in transcription in vitro, by the addition of human placental RNAase inhibitor. A 100% increase in the incorporation of [3H]UMP into myocardial-cell RNA was found on addition of this inhibitor. Myocardial-cell nuclei derived from 5-, 10-, 15-, 20-, and greater than 100-day-old rat hearts indicated that there is a progressive decrease in RNA synthesis with age. A 3-fold increase in RNA synthesis in 5-day-old myocardial cell nuclei as compared with 20-day-old rat heart was found. RNA synthesis in the adult myocardial cell nuclei decreased more than 10-fold in comparison with the 5-day-old newborn. The incorporation of [3H]UMP into rat liver nuclear RNA was 3-fold greater than in the myocardial-cell nuclear RNA, even when compared with the highly active transcription of 12-day-old heart nuclei. In order to determine the relationship between total RNA synthesis and the extent of specific gene expression in myocardial-cell nuclei during development, two distinct cDNA probes were used for Northern-blot analysis. Our results indicate that myosin-heavy-chain gene expression is remarkably decreased with age, whereas the 'housekeeping' gene is continually expressed independently of age.

Animals↗
PubMed2000

Transcriptional and post-transcriptional regulation of monocyte chemoattractant protein-3 gene expression in human endothelial cells by phorbol ester and cAMP signalling.

Monocyte chemoattractant protein-3 (MCP-3) is one of the most broadly active chemokines, potentially inducing chemotaxis of all leucocytic cells. In the present study, we examined the regulation of MCP-3 mRNA and protein production in endothelial cells by protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA) and cAMP signalling. On stimulation of endothelial cells with 10 nM PMA, MCP-3 mRNA increased to 300-fold the basal level at 3 hr and rapidly declined to 0.2-fold the basal level at 24 hr. PMA-induced MCP-3 mRNA and protein production of human endothelial cells were partially inhibited by pretreatment with the adenylate cyclase activator, forskolin, or membrane-permeable cAMP derivative. The PMA-induced MCP-3 mRNA increase was almost abrogated when cells were pretreated with cycloheximide (CHX). Forskolin inhibited the transcription of PMA-induced MCP-3 gene expression. Following PMA stimulation for 3 hr, subsequent addition of actinomycin D suppressed the rapid decay of PMA-induced MCP-3 mRNA. These results suggest that PMA induces the transcriptional activation of the MCP-3 gene through de novo protein synthesis and the rapid decay of PMA-induced MCP-3 mRNA through de novo synthesis of adenosine/uridine (AU)-rich element binding proteins and cAMP signalling inhibits the PMA-induced transcriptional activation of the MCP-3 gene expression.

Adenylyl Cyclases↗
PubMed1999

The function of CreA, the carbon catabolite repressor of Aspergillus nidulans, is regulated at the transcriptional and post-transcriptional level.

The creA gene of A. nidulans encodes a wide-domain regulatory protein mediating carbon catabolite repression. Northern blot analysis of creA mRNA revealed a complex expression profile: the addition of monosaccharides to a carbon-starved culture of A. nidulans provoked a strong transient stimulation of creA transcript formation within a few minutes. In the case of repressing carbon sources, creA mRNA levels were subsequently downregulated, whereas the high creA mRNA levels were maintained in a creA mutant strain and in the presence of derepressing monosaccharides. A high creA transcript level is essential to achieve carbon catabolite repression and is dependent on glucose transport and, at least partially, on the creB gene product. Subsequent downregulation of creA mRNA levels, on the other hand, is typical of carbon catabolite repression and requires a functional CreA recognition site in the creA promoter (and thus involves autoregulation) and formation of glucose-6-phosphate. Despite the presence of continuing high transcript levels of creA in the presence of derepressing carbohydrates, EMSA demonstrated the presence of only low levels of a CreA-DNA complex in respective cell-free extracts. Upon transfer of carbon catabolite derepressed mycelia to catabolite-repressing conditions, a CreA-DNA complex is formed, and this process is dependent on de novo protein synthesis.

Aspergillus nidulans↗
PubMed1983

Transcription termination in vitro at the tryptophan operon attenuator is controlled by secondary structures in the leader transcript.

The role of alternative RNA secondary structures in regulating transcription termination at the attenuator of the tryptophan (trp) operon of Serratia marcescens was examined in vitro by transcribing mutant DNA templates having deletions of different segments of the trp leader region. Deletions that removed sequences corresponding to successive segments of postulated RNA secondary structures either increased or decreased transcription termination at the attenuator. The results obtained are consistent with the hypothesis that transcription termination results from RNA polymerase recognition of a particular RNA secondary structure, the terminator. This structure forms only in the absence of an alternative, preceding, RNA secondary structure, the antiterminator.

Chromosome Deletion↗
PubMed1986

Mechanism for the autogenous control of the crp operon: transcriptional inhibition by a divergent RNA transcript.

Expression of the crp gene is negatively autoregulated by the complex of cyclic AMP and its receptor protein (cAMP-CRP). We find a second promoter in this region that is strongly activated in vitro and in vivo by cAMP-CRP. Transcription from this promoter is initiated 3 nucleotides upstream and on the opposite strand from the start of crp mRNA. The addition of the purified 5' segment of the divergent RNA specifically inhibits crp transcription in vitro. cAMP-CRP does not block crp expression if the new promoter is altered so that divergent RNA cannot be made. The initial nucleotides of the divergent RNA are complementary to 10 of the first 11 nucleotides of the crp mRNA. Since the next 11 nucleotides of crp mRNA are A + U-rich, and RNA hybrid between the divergent RNA and the 5' end of crp mRNA could produce a structure similar to a rho-independent terminator, leading to inhibition of crp transcription.

Bacterial Proteins↗
PubMed1988

Enhanced B2 transcription in simian virus 40-transformed cells is mediated through the formation of RNA polymerase III transcription complexes on previously inactive genes.

RNA polymerase III (pol III) transcribes the highly repeated murine B2 elements. We showed previously that the B2 RNAs are induced 4-fold in normal growing cells and 20-fold in simian virus 40-transformed cells relative to the levels in normal confluent cells. By employing chromatin as a template in a partially purified pol III transcription system, we now demonstrate that the augmented expression results from the formation of pol III transcription complexes on previously inactive B2 genes. Extracts prepared from normal growing cells and transformed cells transcribed cloned pol III templates 5-fold more efficiently than extracts from normal confluent cells. This increase was attributed to 5-fold greater levels of factor IIIC; the levels of pol III and factor IIIB were the same in all extracts. We discuss how the levels of IIIC and differing accessibility of this factor to repressed B2 genes mediate the formation of pol III transcription complexes in normal growing and transformed cells.

Animals↗
PubMed1996

Amplification of the full-length hepatitis A virus genome by long reverse transcription-PCR and transcription of infectious RNA directly from the amplicon.

The genetic study of RNA viruses is greatly facilitated by the availability of infectious cDNA clones. However, their construction has often been difficult. While exploring ways to simplify the construction of infectious clones, we have successfully modified and applied the newly described technique of "long PCR" to the synthesis of a full-length DNA amplicon from the RNA of a cytopathogenic mutant (HM 175/24a) of the hepatitis A virus (HAV). Primers were synthesized to match the two extremities of the HAV genome. The antisense primer, homologous to the 3' end, was used in both the reverse transcription (RT) and the PCR steps. With these primers we reproducibly obtained a full-length amplicon of approximately 7.5 kb. Further, since we engineered a T7 promoter in the sense primer, RNA could be transcribed directly from the amplicon with T7 RNA polymerase. Following transfection of cultured fetal rhesus kidney cells with the transcription mixture containing both the HAV cDNA and the transcribed RNA, replicating HAV was detected by immunofluorescence microscopy and, following passage to other cell cultures, by focus formation. The recovered virus displayed the cytopathic effect and large plaque phenotype typical of the original virus; this result highlights the fidelity of the modified long reverse transcription-PCR procedure and demonstrates the potential of this method for providing cDNAs of viral genomes and simplifying the construction of infectious clones.

Animals↗
PubMed1996

Inhibition of macrophage scavenger receptor activity by tumor necrosis factor-alpha is transcriptionally and post-transcriptionally regulated.

Regulation of expression of the scavenger receptor is thought to play a critical role in the accumulation of lipid by macrophages in atherosclerosis. Tumor necrosis factor-alpha (TNF-alpha) has been shown to suppress macrophage scavenger receptor function (van Lenten, B.J., and Fogelman, A.M. (1992) J. Immunol. 148, 112-6). However, the mechanism by which it does so is unknown. We evaluated the mechanism by which TNF-alpha inhibited macrophage scavenger receptor surface expression and binding of acetylated low density lipoprotein (aLDL). Binding of aLDL to phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophages was suppressed by TNF-alpha in a dose-dependent manner. Inhibition of aLDL binding was paralleled by a reduction of macrophage scavenger receptor protein as detected by the Western blot. TNF-alpha partially decreased macrophage scavenger receptor mRNA steady state levels in PMA-differentiated THP-1 macrophages, a result that was confirmed by reverse transcription-polymerase chain reaction. PMA increased the luciferase activity driven by the macrophage scavenger receptor promoter in the transfected cells, whereas TNF-alpha partially reduced luciferase activity. However, macrophage scavenger receptor mRNA half-life was dramatically reduced in cells treated with TNF-alpha relative to untreated cells. Reduction in macrophage scavenger receptor message in response to TNF-alpha was dependent on new protein synthesis because it was blocked by cycloheximide. These results indicate that TNF-alpha regulates macrophage scavenger receptor expression in PMA-differentiated THP-1 macrophages by transcriptional and post-transcriptional mechanisms but principally by destabilization of macrophage scavenger receptor mRNA.

Blotting, Northern↗
PubMed1996

Synergistic activation of interleukin-8 gene transcription by all-trans-retinoic acid and tumor necrosis factor-alpha involves the transcription factor NF-kappaB.

Induction of interleukin-8 (IL-8) by IL-1 or tumor necrosis factor (TNF), and repression by interferons or glucocorticoids have been shown to involve sequences between nucleotides -94 and -71 of the 5'-flanking region, and the transcription factors NF-IL-6 and NF-kappaB. The A3 cell line was derived from the human melanoma cell line G-361 by stable transfection with part of the IL-8 promoter (nucleotides -101 to +40 from transcription start) fused to the luciferase coding region. These regulatory sequences were sufficient for transcriptional activation by all-trans-retinoic acid (ATRA), 9-cis-retinoic acid, IL-1beta, or TNF-alpha. Simultaneous treatment of A3 cells with ATRA and TNF-alpha resulted in a dose- and time-dependent synergistic increase in luciferase expression and IL-8 mRNA levels. Transient transfections of the parental cell line demonstrated that the NF-kappaB binding site is essential for this synergistic transactivation. Electrophoretic mobility shift assays with nuclear extracts of A3 cells showed that stimulation with ATRA and TNF-alpha for more than 16 h resulted in enhanced NF-kappaB binding compared to that induced by TNF-alpha alone. The simultaneous treatment with ATRA and TNF-alpha also resulted in changes in the composition of NF-kappaB complexes bound to the IL-8 NF-kappaB site, preventing the formation of two TNF-alpha-inducible binding activities. We suggest that these complexes consist of repressive factors which, when removed, allow enhanced binding of NF-kappaB to its cognate site.

Drug Synergism↗
PubMed1996

An ATPase component of the transcription elongation complex is required for factor-dependent transcription termination by vaccinia RNA polymerase.

Vaccinia virus RNA polymerase terminates transcription in response to a specific signal UUUUUNU in the nascent transcript. Transduction of this signal to the elongating polymerase requires a virus-encoded termination factor, VTF. The existence of a second termination factor was suggested by the finding that transient exposure of purified elongation complexes to heparin rendered them refractory to VTF-induced termination. Loss of termination competence correlated with the removal of several polypeptide components of the elongation complex. We present the identification of factor X, an activity that restored VTF responsiveness to heparin-stripped ternary complexes. We propose that factor X, which has an associated DNA-dependent ATPase activity, mediates the requirement for ATP hydrolysis during transcription termination.

Adenosine Triphosphatases↗
PubMed1996

Transcription of the transforming growth factor-beta2 gene is dependent on an E-box located between an essential cAMP response element/activating transcription factor motif and the TATA box of the gene.

Transforming growth factor-beta2 (TGF-beta2) is an important regulator of cell proliferation and differentiation; however, its transcriptional regulation is not well understood. Here we report characterization of an essential E-box motif, positioned at -50/-45 between a previously described functional cAMP response element/activating transcription factor site and the TATA box of the human TGF-beta2 promoter. By site-directed mutagenesis, we demonstrate that this E-box motif is necessary for the promoter activity, not only in differentiated cells derived from embryonal carcinoma cells, but also in choriocarcinoma cells and in MCF-7 breast carcinoma cells. We also demonstrate that the transcription factors USF1 and USF2 bind to this E-box motif in vitro when nuclear extracts from each of these cell lines are examined by gel retardation assays. Moreover, using a dominant-negative USF2 protein, we show that USF proteins are critical for TGF-beta2 promoter activity in vivo. The importance of the E-box motif described in this study is supported by the presence of an E-box motif in the same position in the chicken TGF-beta2 gene promoter.

Cell Line↗