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At least 757 records · Page 42Linked to original sources
PubMed1999

Consistent detection of CALM-AF10 chimaeric transcripts in haematological malignancies with t(10;11)(p13;q14) and identification of novel transcripts.

The t(10;11)(p13-14;q14-21) is a rare but recurring translocation associated with acute lymphoblastic leukaemia (ALL) and acute myeloid leukaemia (AML). Recently the CALM gene was cloned from the t(10;11) breakpoint of U937 and fused to AF10, a putative transcription factor, which had been identified as one of the fusion partners of the MLL gene. In order to define the involvement of these genes in primary leukaemias and cell lines with t(10;11), we analysed the expression of fusion transcripts by reverse transcriptase-polymerase chain reaction (RT-PCR) in five patient samples including ALL, AML and lymphoblastic lymphoma, and three monocytic cell lines (P31/Fujioka, KP-Mo-TS and U937). The CALM-AF10 fusion transcript was detected in all samples; however, the AF10-CALM fusion was not detected in two patient samples and one cell line. In RT-PCR analysis there were six isoforms of the CALM-AF10 fusion transcripts and five of AF10-CALM fusion transcripts. We also detected novel transcripts in U937. Sequence analysis revealed that all these isoforms had in-frame junctions and that some of them resulted from alternative splicing at different exons of CALM and others from different breakpoints at CALM and/or AF10. There were at least two different breakpoints of CALM and three of AF10 gene. Our results suggest that the CALM-AF10 fusion gene is a constant feature and is involved in the pathogenesis of haematological malignancies with t(10;11)(p13-14;q14-21), showing various and often multilineage phenotypes. Thus, t(10;11) needs to be investigated by RT-PCR for identification of the genes involved.

Adult↗
PubMed2003

Transcriptional control for initiation of chromosomal replication in Escherichia coli: fluctuation of the level of origin transcription ensures timely initiation.

BACKGROUND: During the cell cycle, the initiation of chromosomal replication is strictly controlled. In Escherichia coli, the initiator DnaA and the replication origin oriC are major targets for this regulation. Here, we assessed the role of transcription of the mioC gene, which reads through the adjacent oriC region. This mioC-oriC transcription is regulated in coordination with the replication cycle so that it is activated after initiation and repressed before initiation. RESULTS: We isolated a strain bearing a mioC promoter mutation that causes constitutive mioC-oriC transcription from the chromosome. A quantitative S1 nuclease assay indicated that in this mutant, the level of transcription does not fluctuate. Introduction of this mutation suppressed the growth defect of an overinitiation-type dnaAcos mutant, and severely inhibited the growth of initiation-defective dnaA mutants at semipermissive temperatures in a dnaA allele-specific manner. These results suggest that mioC-oriC transcription inhibits initiation at oriC. Indeed, flow cytometry analysis and quantification of DNA replication in synchronized cultures revealed that the mioC promoter mutation alters the control of the initiation of chromosomal replication, for instance by delaying replication within the cell cycle. CONCLUSIONS: These results suggest that the transcriptional regulation of the mioC gene is required for cell cycle-coordinated initiation of chromosomal replication.

Bacterial Outer Membrane Proteins↗
PubMed1993

Light-regulated and endogenous fluctuations of chloroplast transcript levels in Chlamydomonas. Regulation by transcription and RNA degradation.

Changes in the relative sizes of pools of transcripts of organelle genes during plastid development are common in flowering plants, but technical difficulties have prevented direct determinations of the effects of changes in rates of transcription and degradation on such fluctuations. It has been possible to follow both rates in Chlamydomonas reinhardtii. In synchronous or asynchronous cultures of cells grown in 12 h light/12 h dark periods, sizes of pools of transcripts of the chloroplast genes atpA, atpB, tufA, and psaB fluctuate. Differences in chloroplast transcript abundances in light/dark cycles were found to be cell cycle-independent but controlled by either an endogenous rhythm (atpA, atpB, and tufA) or by light (psaB). In vivo labeling experiments showed that transcriptional regulation and light/dark-regulated degradation both contribute, in gene-specific manners, to the level of transcripts of individual C. reinhardtii chloroplast genes in cells grown in alternating light/dark cycles.

Animals↗
PubMed1999

Plasminogen activator inhibitor type-2 (PAI-2) gene transcription requires a novel NF-kappaB-like transcriptional regulatory motif.

Induction of human plasminogen activator inhibitor type-2 (PAI-2) gene transcription is the response of macrophages to inflammatory stimuli, such as the pleiotropic cytokine, tumour necrosis factor-alpha (TNFalpha). Here we have examined whether PAI-2 gene transcription in response to TNFalpha may be mediated through a regulatory pathway involving the transcription factor, NF-kappaB. We have tested the function of two potential NF-kappaB-like sites present in the PAI-2 proximal promoter for responsiveness to TNFalpha using chloramphenicol acetyl transferase reporter gene deletion and mutation analyses. While no evidence was found for TNFalpha regulation of the PAI-2 gene through either of these two sites, one of the NF-kappaB-like motifs, transcriptional regulatory motif (TRM), present at position -400 was found to be essential for constitutive PAI-2 transcription, as mutation of this motif abolished basal PAI-2 promoter activity in both monocyte-like U937 cells and HT1080 fibrosarcoma cells. Competition electrophoretic mobility shift assays identified four TRM-binding proteins present in U937, HT1080 and HeLa cell extracts, which bound to this motif but were not components of the NF-kappaB regulatory complex. Expression screening of a HeLa cell cDNA library using the -400 TRM as a probe identified two cDNAs encoding partial peptides which specifically bound the TRM motif. DNA sequence analysis revealed that one cDNA was novel, and the second cDNA encoded exon 5 of the nephroblastoma overexpressed (novH) proto-oncogene, suggesting a new role for this peptide in gene regulation. Taken together, these findings identify a new regulatory element required for constitutive PAI-2 transcription, and identify potential DNA-binding proteins associated with this element that may play a role in PAI-2 gene regulation.

Base Sequence↗
PubMed1979

Electron microscopic analysis of transcription: mapping of initiation sites and direction of transcription.

An electron microscope technique is described that allows rapid characterization of transcription in vitro. DNA is transcribed with Escherichia coli RNA polymerase in vitro, and the RNA is hybridized to its template. Measurement of the resulting transcription R-loop molecules allows accurate mapping of transcription initiation sites (promoter sites) and analysis of the direction and rate of transcription and the level of transcription from each initiation site. The two major early promoters pR and pL of bacteriophage lambda have been mapped within 0.1-0.3 map units of the known positions and three additional sites have been confirmed. Six transcription initiation sites have been preliminarily mapped on plasmid pSF2124 DNA.

Bacteriophage lambda↗
PubMed1983

Topography of transcription: path of the leading end of nascent RNA through the Escherichia coli transcription complex.

A cleavable dinucleotide photoaffinity reagent was prepared and used to map the path of the leading end of the RNA transcript across the surface of Escherichia coli RNA polymerase/T7 DNA transcription complexes. By using 5'-(4-azidophenacylthio)phosphoryladenylyl(3'-5')uridine, transcription was specifically initiated at the A1 promoter of bacteriophage T7 D111 or D123 DNA. Transcription complexes containing radiolabeled RNA chains of various lengths (4-116 nucleotides) were prepared, and the 5' end of the RNA transcript was then covalently attached to the nearby polymerase subunits or DNA by irradiation with UV light. The photoaffinity-labeled enzyme subunits and DNA were separated, the radiolabeled RNAs were cleaved from each, and the lengths and sequences of RNA attached to each component were determined. The leading end of RNA chains up to 12 bases long was found to label the DNA and the beta and beta' subunits of RNA polymerase, with more than 90% of the label going to the DNA. When the RNA transcript reached 12 bases in length, the 5' end diverged from the DNA and only the beta and beta' enzyme subunits were labeled thereafter. These two subunits were heavily labeled by RNA chains 12 to as many as 94 bases long. No significant labeling of the alpha subunit occurred. The sigma subunit was not labeled by RNAs longer than the trinucleotide.

Base Sequence↗
PubMed1985

Initiation of transcription from each of the two human mitochondrial promoters requires unique nucleotides at the transcriptional start sites.

Promoters for transcriptional initiation on each strand of human mtDNA lie in close proximity in the displacement-loop region. Transcription initiates within these promoter elements, providing an opportunity to analyze the effects of nucleotide sequence changes on the efficiency and fidelity of this event within two delimited regions. Each promoter was individually altered by a site-directed mutagenesis approach and assayed in vitro for the ability to support transcription. The data clearly confirm the previous assignment of promoter functions for these elements and show that single nucleotide substitutions immediately upstream of the transcriptional start sites can abolish transcription. In all cases of decreased transcription, the correct site of initiation is maintained. Although the heavy-strand promoter and light-strand promoter are similar in primary sequence, they exhibit remarkably different sensitivities to base substitutions.

DNA, Mitochondrial↗
PubMed1992

Molecular cloning of human CREB-2: an ATF/CREB transcription factor that can negatively regulate transcription from the cAMP response element.

The cAMP response element (CRE) is an octanucleotide motif (TGACGTCA) that mediates diverse transcriptional regulatory effects. In this report we describe the isolation and characterization of a full-length cDNA that encodes a CRE binding protein called CREB-2. Like other ATF/CREB transcription factors, the 351-amino acid CREB-2 protein contains a COOH-terminal leucine-zipper motif and an adjacent basic domain. CREB-2 mRNA is expressed ubiquitously in human tumor cell lines and mouse organs suggesting that it is involved in regulating transcription in a wide variety of cell types. Overexpression of CREB-2 resulted in a consistent and significant repression of CRE-dependent transcription in CV-1 cells. Deletional analyses localized the transcriptional repressor activity of CREB-2 to a 102-amino acid COOH-terminal region (amino acids 249-351) that contains the leucine-zipper and basic domains of the molecule. These results demonstrate that CRE-dependent transcription can be both positively and negatively regulated by structurally related members of the ATF/CREB family.

Amino Acid Sequence↗
PubMed1998

Stimulation of bacteriophage T4 middle transcription by the T4 proteins MotA and AsiA occurs at two distinct steps in the transcription cycle.

The bacteriophage T4 encodes proteins that are responsible for tightly regulating mRNA synthesis throughout phage development in Escherichia coli. The three classes of T4 promoters (early, middle, and late) are utilized sequentially by the host RNA polymerase as a result of phage-induced modifications. One such modification is the tight binding of the T4 AsiA protein to the sigma70 subunit of the RNA polymerase. This interaction is pivotal for the transition between T4 early and middle transcription, since it both inhibits recognition of host and T4 early promoters and stimulates T4 middle mode synthesis. The activation of T4 middle transcription also requires the T4 MotA protein, bound specifically to its recognition sequence, the "Mot box," which is centered at position -30 of these promoters. Accordingly, the two T4 proteins working in concert are sufficient to effectively switch the transcription specificity of the RNA polymerase holoenzyme. Herein, we investigate the mechanism of transcription activation and report that, while the presence of MotA and AsiA increases the initial recruitment of RNA polymerase to a T4 middle promoter, it does not alter the intrinsic stability of the discrete complexes formed. In addition, we have characterized the RNA polymerase-promoter species by UV laser footprinting and followed their evolution from open into initiating complexes. These data, combined with in vitro transcription assays, indicate that AsiA and MotA facilitate promoter escape, thereby stimulating the production of full-length transcripts.

Adenosine Triphosphate↗
PubMed1999

Arterivirus discontinuous mRNA transcription is guided by base pairing between sense and antisense transcription-regulating sequences.

To generate an extensive set of subgenomic (sg) mRNAs, nidoviruses (arteriviruses and coronaviruses) use a mechanism of discontinuous transcription. During this process, mRNAs are generated that represent the genomic 5' sequence, the so-called leader RNA, fused at specific positions to different 3' regions of the genome. The fusion of the leader to the mRNA bodies occurs at a short, conserved sequence element, the transcription-regulating sequence (TRS), which precedes every transcription unit in the genome and is also present at the 3' end of the leader sequence. Here, we have used site-directed mutagenesis of the infectious cDNA clone of the arterivirus equine arteritis virus to show that sg mRNA synthesis requires a base-pairing interaction between the leader TRS and the complement of a body TRS in the viral negative strand. Mutagenesis of the body TRS of equine arteritis virus RNA7 reduced sg RNA7 transcription severely or abolished it completely. Mutations in the leader TRS dramatically influenced the synthesis of all sg mRNAs. The construction of double mutants in which a mutant leader TRS was combined with the corresponding mutant RNA7 body TRS resulted in the specific restoration of mRNA7 synthesis. The analysis of the mRNA leader-body junctions of a number of mutants with partial transcriptional activity provided support for a mechanism of discontinuous minus-strand transcription that resembles similarity-assisted, copy-choice RNA recombination.

5' Untranslated Regions↗
PubMed1995

The translation product of the presumptive Thermococcus celer TATA-binding protein sequence is a transcription factor related in structure and function to Methanococcus transcription factor B.

A gene for a putative homolog of TATA-binding protein (TBP) from Thermococcus celer has been expressed in Escherichia coli, and the function of the purified recombinant protein was studied in a Methanococcus-derived cell-free transcription system. Thermococcus TBP can replace archaeal transcription factor B (aTFB) in cell-free transcription reactions. This transcriptional activation is TATA box-dependent and occurs both on tRNA(Val) and protein-encoding genes as templates indicating that Thermococcus TBP is a general transcription factor. Antibodies raised against Thermococcus TBP bind to Methanococcus aTFB and inhibit a TFB activity. These findings demonstrate that Thermococcus TBP (like eucaryal TBPs) can direct specific transcription from TATA boxes.

Archaea↗
PubMed1996

Translocation and transcriptional arrest during transcript elongation by RNA polymerase II.

RNA polymerase II may stop transcription, or arrest, while transcribing certain DNA sequences. The molecular basis for arrest is not well understood, but a connection has been suggested between arrest and a transient failure of the polymerase to translocate along the template. We have investigated this question by monitoring the movement of RNA polymerase II along a number of templates, using exonuclease III protection as our assay. We found that normal transcription is accompanied by essentially coordinate movement of the active site and both the leading and trailing edges of the polymerase. However, as polymerase approaches an arrest site, translocation of the body of the polymerase stops while transcription continues, leading to an arrested complex in which the 3' end of the transcript is located much closer than normal to the front edge of the polymerase. Surprisingly, mutated arrest sites that no longer block transcription continue to direct the transient failure of polymerase translocation. As transcription proceeds through these sequences, the initially stationary polymerase moves forward 10-15 bases along the template in response to the addition of only 3 bases to the nascent RNA. Mutagenesis studies indicate that the sequences responsible for the transient block to polymerase movement are located downstream of the T-rich motif required for arrest. Our results indicate that blocking translocation is not sufficient to cause arrest.

Base Composition↗
PubMed1999

Induction of coproporphyrinogen oxidase in Chlamydomonas chloroplasts occurs via transcriptional regulation of Cpx1 mediated by copper response elements and increased translation from a copper deficiency-specific form of the transcript.

Coproporphyrinogen III oxidase, encoded by a single nuclear gene in Chlamydomonas reinhardtii, produces three distinct transcripts. One of these transcripts is greatly induced in copper-deficient cells by transcriptional activation, whereas the other forms are copper-insensitive. The induced form of the transcript was expressed coordinately with the cytochrome c6-encoding (Cyc6) gene, which is known to be transcriptionally regulated in copper-deficient cells. The sequence GTAC, which forms the core of a copper response element associated with the Cyc6 gene, is also essential for induction of the Cpx1 gene, suggesting that both are targets of the same signal transduction pathway. The constitutive and induced Cpx1 transcripts have the same half-lives in vivo, and all encode the same polypeptide with a chloroplast-targeting transit sequence, but the shortest one representing the induced form is a 2-4-fold better template for translation than are either of the constitutive forms. The enzyme remains localized to a soluble compartment in the chloroplast even in induced cells, and its abundance is not affected when the tetrapyrrole pathway is manipulated either genetically or by gabaculine treatment.

Amino Acid Sequence↗
PubMed1999

Identification of conserved cis-elements and transcription factors required for sterol-regulated transcription of stearoyl-CoA desaturase 1 and 2.

We previously identified stearoyl-CoA desaturase 2 (SCD2) as a new member of the family of genes that are transcriptionally regulated in response to changing levels of nuclear sterol regulatory element binding proteins (SREBPs) or adipocyte determination and differentiation factor 1 (ADD1). A novel sterol regulatory element (SRE) (5'-AGCAGATTGTG-3') identified in the proximal promoter of the mouse SCD2 gene is required for induction of SCD2 promoter-reporter genes in response to cellular sterol depletion (Tabor, D. E., Kim, J. B., Spiegelman, B. M., and Edwards, P. A. (1998) J. Biol. Chem. 273, 22052-22058). In this report, we demonstrate that this novel SRE is both present in the promoter of the SCD1 gene and is critical for the sterol-dependent transcription of SCD1 promoter-reporter genes. Two conserved cis elements (5'-CCAAT-3') lie 5 and 48 base pairs 3' of the novel SREs in the promoters of both the SCD1 and SCD2 murine genes. Mutation of either of these putative NF-Y binding sites attenuates the transcriptional activation of SCD1 or SCD2 promoter-reporter genes in response to cellular sterol deprivation. Induction of both reporter genes is also attenuated when cells are cotransfected with dominant-negative forms of either NF-Y or SREBP. In addition, we demonstrate that the induction of SCD1 and SCD2 mRNAs that occurs during the differentiation of 3T3-L1 preadipocytes to adipocytes is paralleled by an increase in the levels of ADD1/SREBP-1c and that the SCD1 and SCD2 mRNAs are induced to even higher levels in response to ectopic expression of ADD1/SREBP-1c. We conclude that transcription of both SCD1 and SCD2 genes is responsive to cellular sterol levels and to the levels of nuclear SREBP/ADD1 and that transcriptional induction requires three spatially conserved cis elements, that bind SREBP and NF-Y. Additional studies demonstrate that maximal transcriptional repression of SCD2 reporter genes in response to an exogenous polyunsaturated fatty acid is dependent upon the SRE and the adjacent CCAAT motif.

3T3 Cells↗
PubMed1999

Estrogen inducibility of c-Ha-ras transcription in breast cancer cells. Identification of functional estrogen-responsive transcriptional regulatory elements in exon 1/intron 1 of the c-Ha-ras gene.

Although mutation of ras gene is rare in human breast cancer, overexpression of normal c-Ha-ras gene is frequently observed. Using a mouse mammary metastasis model consisting of genetically related mammary tumor sublines with variant metastatic potential, we have previously (i) demonstrated a direct correlation between c-Ha-ras mRNA and protein levels and metastatic potential and (ii) identified a novel hormone-responsive transcriptional regulatory element in intron 1 of the mouse c-Ha-ras gene that contains the consensus half-site of a glucocorticoid response element and flanking consensus half-sites for estrogen response element. Here, we have examined the functionality of intron 1 sequence in context of upstream sequences by using transient transfection assays with plasmids expressing chloramphenicol acetyltransferase. Intron 1 sequence and sequences similar to intron 1 element located in exon 1 function as transcriptional regulatory elements that confer hormonal inducibility to chloramphenicol acetyltransferase gene expression both independently and in context of 5'-flanking sequences. Measurement of c-Ha-ras transcription rates and protein expression by nuclear run-on and metabolic labeling assays showed a 5-12-fold enhancement, respectively, following treatment with 17beta-estradiol that was blunted by ICI 182,780 in the nonmetastatic variant. In contrast, constitutive overexpression of c-Ha-ras transcripts and protein in the metastatic subline was unaffected by estrogen and ICI 182,780. Gel shift assays demonstrated specific interaction of c-Ha-ras exon 1 sequence with nuclear proteins of human breast cancer MCF-7 cells with formation of two complexes, one of which contains estrogen receptor. Our data demonstrate a direct (i) interaction of c-Ha-ras sequence with estrogen receptor and (ii) stimulatory effect of estrogen on c-Ha-ras gene transcription and suggest that alteration in transcriptional regulation of c-Ha-ras gene by estrogen may play an important role in progression of breast cancer.

Animals↗
PubMed1999

The homeodomain transcription factor NK-4 acts as either a transcriptional activator or repressor and interacts with the p300 coactivator and the Groucho corepressor.

NK-4 (tinman) encodes an NK-2 class homeodomain transcription factor that is required for development of the Drosophila dorsal mesoderm, including heart. Genetic evidence suggests its important role in mesoderm subdivision, yet the properties of NK-4 as a transcriptional regulator and the mechanism of gene transcription by NK-4 are not completely understood. Here, we describe its properties as a transcription factor and its interaction with the p300 coactivator and the Groucho corepressor. We demonstrate that NK-4 can activate or repress target genes in cultured cells, depending on functional domains that are conserved between Drosophila melanogaster and Drosophila virilis NK-4 genes. Using GAL4-NK-4 fusion constructs, we have mapped a transcriptional activation domain (amino acids 1-110) and repression domains (amino acids 111-188 and the homeodomain) and found an inhibitory function for the homeodomain in transactivation by NK-4. Furthermore, we demonstrate that NK-4-dependent transactivation is augmented by the p300 coactivator and show that NK-4 physically interacts with p300 via the activation domain. In addition, cotransfection experiments indicate that the repressor activity of NK-4 is strongly enhanced by the Groucho corepressor. Using immunoprecipitation and in vitro pull-down assays, we show that NK-4 directly interacts with the Groucho corepressor, for which the homeodomain is required. Together, our results indicate that NK-4 can act as either a transcriptional activator or repressor and provide the first evidence of NK-4 interactions with the p300 coactivator and the Groucho corepressor.

Amino Acid Sequence↗
PubMed2000

CRTR-1, a developmentally regulated transcriptional repressor related to the CP2 family of transcription factors.

CP2-related proteins comprise a family of DNA-binding transcription factors that are generally activators of transcription and expressed ubiquitously. We reported a differential display polymerase chain reaction fragment, Psc2, which was expressed in a regulated fashion in mouse pluripotent cells in vitro and in vivo. Here, we report further characterization of the Psc2 cDNA and function. The Psc2 cDNA contained an open reading frame homologous to CP2 family proteins. Regions implicated in DNA binding and oligomeric complex formation, but not transcription activation, were conserved. Psc2 expression in vivo during embryogenesis and in the adult mouse demonstrated tight spatial and temporal regulation, with the highest levels of expression in the epithelial lining of distal convoluted tubules in embryonic and adult kidneys. Functional analysis demonstrated that PSC2 repressed transcription 2.5-15-fold when bound to a heterologous promoter in ES, 293T, and COS-1 cells. The N-terminal 52 amino acids of PSC2 were shown to be necessary and sufficient for this activity and did not share obvious homology with reported repressor motifs. These results represent the first report of a CP2 family member that is expressed in a developmentally regulated fashion in vivo and that acts as a direct repressor of transcription. Accordingly, the protein has been named CP2-Related Transcriptional Repressor-1 (CRTR-1).

Amino Acid Sequence↗
PubMed2001

Huntingtin is present in the nucleus, interacts with the transcriptional corepressor C-terminal binding protein, and represses transcription.

Huntingtin is a protein of unknown function that contains a polyglutamine tract, which is expanded in patients with Huntington's disease (HD). We investigated the localization and a potential function for huntingtin in the nucleus. In human fibroblasts from normal and HD patients, huntingtin localized diffusely in the nucleus and in subnuclear compartments identified as speckles, promyelocytic leukemia protein bodies, and nucleoli. Huntingtin-positive nuclear bodies redistributed after treatment with sodium butyrate. By Western blot, purified nuclei had low levels of full-length huntingtin compared with the cytoplasm but contained high levels of N- and C-terminal huntingtin fragments, which tightly bound the nuclear matrix. Full-length huntingtin co-immunoprecipitated with the transcriptional corepressor C-terminal binding protein, and polyglutamine expansion in huntingtin reduced this interaction. Full-length wild-type and mutant huntingtin repressed transcription when targeted to DNA. Truncated N-terminal mutant huntingtin repressed transcription, whereas the corresponding wild-type fragment did not repress transcription. We speculate that wild-type huntingtin may function in the nucleus in the assembly of nuclear matrix-bound protein complexes involved with transcriptional repression and RNA processing. Proteolysis of mutant huntingtin may alter nuclear functions by disrupting protein complexes and inappropriately repressing transcription in HD.

Alcohol Oxidoreductases↗