scholarly journals Delineation of DNA sequences that are important for in vitro transcription from the adenovirus EIIa late promoter.

1988 ◽  
Vol 8 (5) ◽  
pp. 1906-1914 ◽  
Author(s):  
D H Huang ◽  
R G Roeder
Keyword(s):  
Dna Sequences ◽  
Nuclear Extract ◽  
In Vitro Transcription ◽  
Initiation Site ◽  
Late Promoter ◽  
Fold Reduction ◽  
Sequence Elements ◽  
A Cell ◽  
Circular Template

Late in infection, transcription of the EIIa gene is initiated primarily at map unit 72 of the adenovirus genome. A cell-free nuclear extract system was used to determine sequence elements important for the function of this late promoter. In such a system, the transcriptional activity of a circular template was found to be much higher (5- to 10-fold) than that of a linear template. The effect of template topology appeared to be dependent on two distal upstream elements with 5' boundaries located near -265 to -223 and -147 to -133 (in relation to the initiation site), since deletions of these regions reduced transcription of the circular template, in a stepwise fashion, to a level similar to that observed with the linear template. Further deletions revealed an element in the -116 region that appeared to be more important for transcription of the circular template (10-fold reduction) than for transcription of the linear template (3-fold reduction). Lastly, deletion of the TACAAA sequence in the -29 region resulted in further reduction in transcription, indicating that this element functions as a promoter in vitro.

Delineation of DNA sequences that are important for in vitro transcription from the adenovirus EIIa late promoter

1988 ◽  
Vol 8 (5) ◽  
pp. 1906-1914
Author(s):  
D H Huang ◽  
R G Roeder
Keyword(s):  
Dna Sequences ◽  
Nuclear Extract ◽  
In Vitro Transcription ◽  
Initiation Site ◽  
Late Promoter ◽  
Fold Reduction ◽  
Sequence Elements ◽  
A Cell ◽  
Circular Template

Late in infection, transcription of the EIIa gene is initiated primarily at map unit 72 of the adenovirus genome. A cell-free nuclear extract system was used to determine sequence elements important for the function of this late promoter. In such a system, the transcriptional activity of a circular template was found to be much higher (5- to 10-fold) than that of a linear template. The effect of template topology appeared to be dependent on two distal upstream elements with 5' boundaries located near -265 to -223 and -147 to -133 (in relation to the initiation site), since deletions of these regions reduced transcription of the circular template, in a stepwise fashion, to a level similar to that observed with the linear template. Further deletions revealed an element in the -116 region that appeared to be more important for transcription of the circular template (10-fold reduction) than for transcription of the linear template (3-fold reduction). Lastly, deletion of the TACAAA sequence in the -29 region resulted in further reduction in transcription, indicating that this element functions as a promoter in vitro.

scholarly journals Multiple sequence elements are required for maximal in vitro transcription of a human histone H2B gene.

1986 ◽  
Vol 6 (10) ◽  
pp. 3329-3340 ◽  
Author(s):  
H L Sive ◽  
N Heintz ◽  
R G Roeder
Keyword(s):  
Transcription Initiation ◽  
In Vitro Transcription ◽  
Initiation Site ◽  
Multiple Sequence ◽  
Histone H2b ◽  
Cis Acting ◽  
Promoter Sequences ◽  
Nuclear Extracts ◽  
Sequence Elements

As part of our studies on the cell cycle regulation of human histone gene expression, we examined the elements governing transcription of a human histone H2B gene in nuclear extracts derived from human HeLa cells. Circular templates were transcribed at 5- to 10-fold higher levels than were linear templates. A series of deletion, linker-substitution, and point mutants defined cis-acting promoter sequences that were recognized in nuclear extracts. These sequences extended from 118 to 21 base pairs 5' to the transcription initiation site. Elements recognized included (from 5' to 3') a series of direct repeats, a CCAAT homology, a human histone-specific hexamer, an H2B consensus element, and a TATA box. Sequence elements 5' to the hexamer were required for its function. In contrast, the H2B consensus element could function independently of more-5' promoter elements and in turn was essential for the function of upstream elements. An interesting feature of this consensus is that its core octanucleotide (ATTTGCAT) is found in several nonhistone genes. By comparison with functional elements in an H4 promoter, we infer that a combinatorial interaction of general and gene-specific factors may contribute to the S-phase elevation of H2B transcription.

Multiple sequence elements are required for maximal in vitro transcription of a human histone H2B gene

1986 ◽  
Vol 6 (10) ◽  
pp. 3329-3340
Author(s):  
H L Sive ◽  
N Heintz ◽  
R G Roeder
Keyword(s):  
Transcription Initiation ◽  
In Vitro Transcription ◽  
Initiation Site ◽  
Multiple Sequence ◽  
Histone H2b ◽  
Cis Acting ◽  
Promoter Sequences ◽  
Nuclear Extracts ◽  
Sequence Elements

As part of our studies on the cell cycle regulation of human histone gene expression, we examined the elements governing transcription of a human histone H2B gene in nuclear extracts derived from human HeLa cells. Circular templates were transcribed at 5- to 10-fold higher levels than were linear templates. A series of deletion, linker-substitution, and point mutants defined cis-acting promoter sequences that were recognized in nuclear extracts. These sequences extended from 118 to 21 base pairs 5' to the transcription initiation site. Elements recognized included (from 5' to 3') a series of direct repeats, a CCAAT homology, a human histone-specific hexamer, an H2B consensus element, and a TATA box. Sequence elements 5' to the hexamer were required for its function. In contrast, the H2B consensus element could function independently of more-5' promoter elements and in turn was essential for the function of upstream elements. An interesting feature of this consensus is that its core octanucleotide (ATTTGCAT) is found in several nonhistone genes. By comparison with functional elements in an H4 promoter, we infer that a combinatorial interaction of general and gene-specific factors may contribute to the S-phase elevation of H2B transcription.

scholarly journals Cyclic AMP-dependent protein kinase regulates transcription of the phosphoenolpyruvate carboxykinase gene but not binding of nuclear factors to the cyclic AMP regulatory element.

1990 ◽  
Vol 10 (7) ◽  
pp. 3357-3364 ◽  
Author(s):  
P G Quinn ◽  
D K Granner
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Rat Liver ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Nuclear Extract ◽  
In Vitro Transcription ◽  
Catalytic Subunit ◽  
Nuclear Factors ◽  
Dependent Protein

We have examined the binding of factors in rat liver nuclear extracts to the phosphoenolpyruvate carboxykinase (PEPCK) gene cyclic AMP (cAMP) response element (CRE) and other CREs and have isolated a rat liver CRE-binding protein (CREBP) cDNA. In addition, we have examined the influence of altering the phosphorylation state of nuclear factors on both CRE binding and in vitro transcription. Specific binding to the PEPCK CRE was measured in a mobility shift assay. CRE sequences of the PEPCK, somatostatin, and glycoprotein hormone alpha subunit genes competed equally for binding of rat liver nuclear factors to the PEPCK CRE, whereas mutant PEPCK CRE sequences did not compete for binding. Oligonucleotides complementary to rat pheochromocytoma CREBP (Gonzalez et al., Nature [London] 337:749-752, 1989) were used to prime rat liver and brain cDNA in the polymerase chain reaction. The predominant CREBP molecule obtained was identical to the rat pheochromocytoma CREBP except for a 14-amino-acid deletion in the N-terminal half that was also present in a human placental cDNA (Hoeffler et al., Science 242:1430-1433, 1988). The regulation of transcription by cAMP was examined by coincubation of rat liver nuclear extract with the purified catalytic subunit of cAMP-dependent protein kinase (protein kinase A). Although binding to the CRE was unaffected, in vitro transcription directed by the PEPCK promoter was stimulated by catalytic subunit, and this effect was blocked by protein kinase inhibitor peptide. In contrast, when nuclear extract was coincubated with phosphatase, there was substantial inhibition of in vitro transcription directed by the PEPCK promoter, but there was no effect on binding to the CRE. The major effects of catalytic subunit were exerted through the CRE, but residual stimulation was evident in promoter fragments containing only the TATA element. These data suggest that factors are bound to the CRE at constitutively high levels and that their capacity for transcriptional activation is regulated by phosphorylation.

Sequence requirements for premature transcription arrest within the first intron of the mouse c-fos gene

1991 ◽  
Vol 11 (5) ◽  
pp. 2832-2841
Author(s):  
N Mechti ◽  
M Piechaczyk ◽  
J M Blanchard ◽  
P Jeanteur ◽  
B Lebleu
Keyword(s):  
Rna Polymerase Ii ◽  
In Vitro Transcription ◽  
Rna Transcripts ◽  
First Intron ◽  
Nuclear Extracts ◽  
Intron 1 ◽  
A Cell ◽  
Sequence Requirements

A strong block to the elongation of nascent RNA transcripts by RNA polymerase II occurs in the 5' part of the mammalian c-fos proto-oncogene. In addition to the control of initiation, this mechanism contributes to transcriptional regulation of the gene. In vitro transcription experiments using nuclear extracts and purified transcription templates allowed us to map a unique arrest site within the mouse first intron 385 nucleotides downstream from the promoter. This position is in keeping with that estimated from nuclear run-on assays performed with short DNA probes and thus suggests that it corresponds to the actual block in vivo. Moreover, we have shown that neither the c-fos promoter nor upstream sequences are absolute requirements for an efficient transcription arrest both in vivo and in vitro. Finally, we have characterized a 103-nucleotide-long intron 1 motif comprising the arrest site and sufficient for obtaining the block in a cell-free transcription assay.

Transcriptional and posttranscriptional regulation of maize mitochondrial gene expression

1991 ◽  
Vol 11 (1) ◽  
pp. 533-543
Author(s):  
R M Mulligan ◽  
P Leon ◽  
V Walbot
Keyword(s):  
Dna Sequences ◽  
Transcription Initiation ◽  
Posttranscriptional Regulation ◽  
Rank Order ◽  
Mitochondrial Gene ◽  
Initiation Site ◽  
Gene Copy ◽  
Promoter Strength ◽  
Protein Coding

Lysed maize mitochondria synthesize RNA in the presence of radioactive nucleoside triphosphates, and this assay was utilized to compare the rates of transcription of seven genes. The rates of incorporation varied over a 14-fold range, with the following rank order: 18S rRNA greater than 26S rRNA greater than atp1 greater than atp6 greater than atp9 greater than cob greater than cox3. The products of run-on transcription hybridized specifically to known transcribed regions and selectively to the antisense DNA strand; thus, the isolated run-on transcription system appears to be an accurate representation of endogenous transcription. Although there were small differences in gene copy abundance, these differences cannot account for the differences in apparent transcription rates; we conclude that promoter strength is the main determinant. Among the protein coding genes, incorporation was greatest for atp1. The most active transcription initiation site of this gene was characterized by hybridization with in vitro-capped RNA and by primer extension analyses. The DNA sequences at this and other transcription initiation sites that we have previously mapped were analyzed with respect to the apparent promoter strengths. We propose that two short sequence elements just upstream of initiation sites form at least a portion of the sequence requirements for a maize mitochondrial promoter. In addition to modulation at the level of transcription, steady-state abundance of protein-coding mRNAs varied over a 20-fold range and did not correlate with transcriptional activity. These observations suggest that posttranscriptional processes are important in the modulation of mRNA abundance.

Ligand-dependent, Pit-1/growth hormone factor-1 (GHF-1)-independent transcriptional stimulation of rat growth hormone gene expression by thyroid hormone receptors in vitro

1993 ◽  
Vol 13 (3) ◽  
pp. 1719-1727
Author(s):  
C S Suen ◽  
W W Chin
Keyword(s):  
Gene Expression ◽  
Growth Hormone ◽  
Thyroid Hormone ◽  
Promoter Activity ◽  
Hormone Receptors ◽  
Nuclear Extract ◽  
In Vitro Transcription ◽  
Stimulation Of ◽  
Transcriptional Stimulation

The expression of the rat growth hormone (rGH) gene in the anterior pituitary gland is modulated by Pit-1/GHF-1, a pituitary-specific transcription factor, and by other more widely distributed factors, such as the thyroid hormone receptors (TRs), Sp1, and the glucocorticoid receptor. Thyroid hormone (T3)-mediated transcriptional stimulation of rGH gene expression has been extensively studied in vivo and in vitro including the measurements of (i) rGH mRNA by blot hybridization, (ii) transcriptional rate of rGH gene by nuclear run-on, and (iii) reporter gene expression in which a chimeric plasmid containing 5'-flanking sequences of the rGH gene linked to a reporter gene has been transfected either stably or transiently into pituitary and/or nonpituitary cells. From these studies, it has been suggested that the Pit-1/GHF-1 binding site is necessary for full T3 action. We developed a cell-free in vitro transcription system to examine further the roles of the TRs and Pit-1/GHF-1 in rGH gene activation. Using GH3 nuclear extract as a source of TRs and Pit-1/GHF-1, this in vitro transcription assay showed that T3 stimulation of rGH promoter activity is dependent on the addition of T3 to the GH3 nuclear extract. This transcriptional stimulation was augmented with increasing concentrations of ligand and was T3, but not T4 or reverse T3, specific. T3-mediated stimulation of rGH promoter activity was completely abolished by preincubation of the nuclear extract with rGH-thyroid hormone response element (-200 to -160) but not with Pit-1/GHF-1 (-137 to -65) oligonucleotides. Further, neither deletion of both Pit-1/GHF-1 binding sites nor mutation of the proximal Pit-1/GHF-1 binding site from the rGH promoter abrogated the T3 effect. These results provide evidence that T3-stimulated rGH promoter activity is independent of Pit-1/GHF-1 and raise the possibility that the stimulation of rGH gene expression by T3 might involve direct interaction of TRs with the general transcriptional apparatus.

scholarly journals In vitro analysis of a transcription termination site for RNA polymerase II.

1990 ◽  
Vol 10 (11) ◽  
pp. 5782-5795 ◽  
Author(s):  
D K Wiest ◽  
D K Hawley
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Nuclear Extract ◽  
Transcription Unit ◽  
Dependent Manner ◽  
Wide Range ◽  
Vitro Analysis

Transcription from the adenovirus major late (ML) promoter has previously been shown to pause or terminate prematurely in vivo and in vitro at a site within the first intron of the major late transcription unit. We are studying the mechanism of elongation arrest at this site in vitro to define the DNA sequences and proteins that determine the elongation behavior of RNA polymerase II. Our assay system consists of a nuclear extract prepared from cultured human cells. With standard reaction conditions, termination is not observed downstream of the ML promoter. However, in the presence of Sarkosyl, up to 80% of the transcripts terminate 186 nucleotides downstream of the start site. Using this assay, we showed that the DNA sequences required to promote maximal levels of termination downstream of the ML promoter reside within a 65-base-pair region and function in an orientation-dependent manner. To test whether elongation complexes from the ML promoter were functionally homogeneous, we determined the termination efficiency at each of two termination sites placed in tandem. We found that the behavior of the elongation complexes was different at these sites, with termination being greater at the downstream site over a wide range of Sarkosyl concentrations. This result ruled out a model in which the polymerases that read through the first site were stably modified to antiterminate. We also demonstrated that the ability of the elongation complexes to respond to the ML termination site was promoter specific, as the site did not function efficiently downstream of a heterologous promoter. Taken together, the results presented here are not consistent with the simplest class of models that have been proposed previously for the mechanism of Sarkosyl-induced termination.

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