scholarly journals The Drosophila even-skipped promoter is transcribed in a stage-specific manner in vitro and contains multiple, overlapping factor-binding sites.

1990 ◽  
Vol 10 (8) ◽  
pp. 4334-4344 ◽  
Author(s):  
D Read ◽  
T Nishigaki ◽  
J L Manley
Keyword(s):  
Binding Sites ◽  
In Vitro Transcription ◽  
Binding Activity ◽  
Gaga Factor ◽  
Regulation Of Expression ◽  
Specific Expression ◽  
Multiple Binding Sites ◽  
Nuclear Extracts

To investigate the factors contributing to regulation of expression of the Drosophila segmentation gene even-skipped (eve), we have analyzed both the in vitro transcription and eve-promoter-binding proteins in embryo extracts. We show that the eve promoter is accurately and efficiently expressed in nuclear extracts derived from Drosophila embryos and that transcription is more efficient in extracts prepared from embryos at early stages of development than in those from older embryos, broadly reproducing the temporal pattern of expression observed in vivo. This stage-specific expression is dependent on sequences upstream of the eve transcription start site which contain multiple binding sites for at least two distinct proteins present in embryo nuclei. One of these proteins, the binding sites for which correspond to the sequences required for stage-specific expression, appears to be the previously described GAGA factor. Although the binding activity of the GAGA factor remains constant, the level of the binding activity of the other protein, which we have called the TCCT factor, changes during the course of embryogenesis. Activity is first detected 3 to 5 h after fertilization and decreases during later stages of embryogenesis. We discuss the possibility that the TCCT factor plays a role in the maintenance or refinement of the eve expression pattern.

The Drosophila even-skipped promoter is transcribed in a stage-specific manner in vitro and contains multiple, overlapping factor-binding sites

1990 ◽  
Vol 10 (8) ◽  
pp. 4334-4344
Author(s):  
D Read ◽  
T Nishigaki ◽  
J L Manley
Keyword(s):  
Binding Sites ◽  
In Vitro Transcription ◽  
Binding Activity ◽  
Gaga Factor ◽  
Regulation Of Expression ◽  
Specific Expression ◽  
Multiple Binding Sites ◽  
Nuclear Extracts

To investigate the factors contributing to regulation of expression of the Drosophila segmentation gene even-skipped (eve), we have analyzed both the in vitro transcription and eve-promoter-binding proteins in embryo extracts. We show that the eve promoter is accurately and efficiently expressed in nuclear extracts derived from Drosophila embryos and that transcription is more efficient in extracts prepared from embryos at early stages of development than in those from older embryos, broadly reproducing the temporal pattern of expression observed in vivo. This stage-specific expression is dependent on sequences upstream of the eve transcription start site which contain multiple binding sites for at least two distinct proteins present in embryo nuclei. One of these proteins, the binding sites for which correspond to the sequences required for stage-specific expression, appears to be the previously described GAGA factor. Although the binding activity of the GAGA factor remains constant, the level of the binding activity of the other protein, which we have called the TCCT factor, changes during the course of embryogenesis. Activity is first detected 3 to 5 h after fertilization and decreases during later stages of embryogenesis. We discuss the possibility that the TCCT factor plays a role in the maintenance or refinement of the eve expression pattern.

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.

RAP1 is required for BAS1/BAS2- and GCN4-dependent transcription of the yeast HIS4 gene

1991 ◽  
Vol 11 (7) ◽  
pp. 3642-3651 ◽  
Author(s):  
C Devlin ◽  
K Tice-Baldwin ◽  
D Shore ◽  
K T Arndt
Keyword(s):  
Steady State ◽  
Binding Site ◽  
Binding Sites ◽  
Binding Activity ◽  
Micrococcal Nuclease ◽  
Mrna Levels ◽  
High Affinity ◽  
Steady State Levels

The major in vitro binding activity to the Saccharomyces cerevisiae HIS4 promoter is due to the RAP1 protein. In the absence of GCN4, BAS1, and BAS2, the RAP1 protein binds to the HIS4 promoter in vivo but cannot efficiently stimulate HIS4 transcription. RAP1, which binds adjacently to BAS2 on the HIS4 promoter, is required for BAS1/BAS2-dependent activation of HIS4 basal-level transcription. In addition, the RAP1-binding site overlaps with the single high-affinity HIS4 GCN4-binding site. Even though RAP1 and GCN4 bind competitively in vitro, RAP1 is required in vivo for (i) the normal steady-state levels of GCN4-dependent HIS4 transcription under nonstarvation conditions and (ii) the rapid increase in GCN4-dependent steady-state HIS4 mRNA levels following amino acid starvation. The presence of the RAP1-binding site in the HIS4 promoter causes a dramatic increase in the micrococcal nuclease sensitivity of two adjacent regions within HIS4 chromatin: one region contains the high-affinity GCN4-binding site, and the other region contains the BAS1- and BAS2-binding sites. These results suggest that RAP1 functions at HIS4 by increasing the accessibility of GCN4, BAS1, and BAS2 to their respective binding sites when these sites are present within chromatin.

scholarly journals Structure of a Polycomb Response Element and In Vitro Binding of Polycomb Group Complexes Containing GAGA Factor

2000 ◽  
Vol 20 (9) ◽  
pp. 3187-3197 ◽  
Author(s):  
Béatrice Horard ◽  
Christophe Tatout ◽  
Sylvain Poux ◽  
Vincenzo Pirrotta
Keyword(s):  
Polycomb Group ◽  
Gaga Factor ◽  
Consensus Sequences ◽  
Polycomb Response Element ◽  
Nuclear Extracts ◽  
Vitro Binding ◽  
Polycomb Response Elements ◽  
Polycomb Group Complexes

ABSTRACT Polycomb response elements (PREs) are regulatory sites that mediate the silencing of homeotic and other genes. The bxd PRE region from the Drosophila Ultrabithorax gene can be subdivided into subfragments of 100 to 200 bp that retain different degrees of PRE activity in vivo. In vitro, embryonic nuclear extracts form complexes containing Polycomb group (PcG) proteins with these fragments. PcG binding to some fragments is dependent on consensus sequences for the GAGA factor. Other fragments lack GAGA binding sites but can still bind PcG complexes in vitro. We show that the GAGA factor is a component of at least some types of PcG complexes and may participate in the assembly of PcG complexes at PREs.

scholarly journals The ubiquitous octamer-binding protein(s) is sufficient for transcription of immunoglobulin genes.

1990 ◽  
Vol 10 (3) ◽  
pp. 982-990 ◽  
Author(s):  
D G Johnson ◽  
L Carayannopoulos ◽  
J D Capra ◽  
P W Tucker ◽  
J H Hanke
Keyword(s):  
B Cell ◽  
Protein S ◽  
Cell Types ◽  
In Vitro Transcription ◽  
Immunoglobulin Gene ◽  
Immunoglobulin Genes ◽  
Specific Expression ◽  
Specific Regulation

All immunoglobulin genes contain a conserved octanucleotide promoter element, ATGCAAAT, which has been shown to be required for their normal B-cell-specific transcription. Proteins that bind this octamer have been purified, and cDNAs encoding octamer-binding proteins have been cloned. Some of these proteins (referred to as OTF-2) are lymphoid specific, whereas at least one other, and possibly more (referred to as OTF-1), is found ubiquitously in all cell types. The exact role of these different proteins in directing the tissue-specific expression of immunoglobulin genes is unclear. We have identified two human pre-B-cell lines that contain extremely low levels of OTF-2 yet still express high levels of steady-state immunoglobulin heavy-chain mRNA in vivo and efficiently transcribe an immunoglobulin gene in vitro. Addition of a highly enriched preparation of OTF-1 made from one of these pre-B cells or from HeLa cells specifically stimulated in vitro transcription of an immunoglobulin gene. Furthermore, OFT-1 appeared to have approximately the same transactivation ability as OTF-2 when normalized for binding activity. These results suggest that OTF-1, without OTF-2, is sufficient for transcription of immunoglobulin genes and that OTF-2 alone is not responsible for the B-cell-specific regulation of immunoglobulin gene expression.

scholarly journals Jun is phosphorylated by several protein kinases at the same sites that are modified in serum-stimulated fibroblasts.

1992 ◽  
Vol 12 (10) ◽  
pp. 4694-4705 ◽  
Author(s):  
S J Baker ◽  
T K Kerppola ◽  
D Luk ◽  
M T Vandenberg ◽  
D R Marshak ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Dna Binding ◽  
Glycogen Synthase ◽  
In Vitro Transcription ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
Serum Stimulation ◽  
Dependent Protein

c-jun is a member of the family of immediate-early genes whose expression is induced by factors such as serum stimulation, phorbol ester, and differentiation signals. Here we show that increased Jun synthesis after serum stimulation is accompanied by a concomitant increase in phosphorylation. Several serine-threonine kinases were evaluated for their ability to phosphorylate Jun in vitro. p34cdc2, protein kinase C, casein kinase II, and pp44mapk phosphorylated Jun efficiently, whereas cyclic AMP-dependent protein kinase and glycogen synthase kinase III did not. The sites phosphorylated by p34cdc2 were similar to those phosphorylated in vivo after serum induction. The major sites of phosphorylation were mapped to serines 63, 73, and 246. Phosphorylation of full-length Jun with several kinases did not affect the DNA-binding activity of Jun homodimers or Fos-Jun heterodimers. Comparison of the DNA binding and in vitro transcription properties of wild-type and mutated proteins containing either alanine or aspartic acid residues in place of Ser-63, -73, and -246 revealed only minor differences among homodimeric complexes and no differences among Fos-Jun heterodimers. Thus, phosphorylation of Jun did not produce a significant change in dimerization, DNA-binding, or in vitro transcription activity. The regulatory role of phosphorylation in the modulation of Jun function is likely to be considerably more complex than previously suggested.

scholarly journals In vitro transcription and delimitation of promoter elements of the murine dihydrofolate reductase gene.

1986 ◽  
Vol 6 (7) ◽  
pp. 2392-2401 ◽  
Author(s):  
P J Farnham ◽  
R T Schimke
Keyword(s):  
Dihydrofolate Reductase ◽  
In Vitro Transcription ◽  
S Phase ◽  
Specific Factor ◽  
Or Efficiency ◽  
Transcription System ◽  
Nuclear Extracts ◽  
Reductase Gene

We have developed an in vitro transcription system for the murine dihydrofolate reductase gene. Although transcription in vitro from a linearized template was initiated at the same start sites as in vivo, the correct ratios were more closely approximated when a supercoiled template was used. In addition, whereas the dihydrofolate reductase promoter functions bidirectionally in vivo, the initiation signals directed unidirectional transcription in this in vitro system. The dihydrofolate reductase gene does not have a typical TATA box, but has four GGGCGG hexanucleotides within 300 base pairs 5' of the AUG codon. Deletion analysis suggested that, although sequences surrounding each of the GC boxes could specify initiation approximately 40 to 50 nucleotides downstream, three of the four GC boxes could be removed without changing the accuracy or efficiency of initiation at the major in vivo site. The dihydrofolate reductase promoter initiated transcription very rapidly in vitro, with transcripts visible by 1 min and almost maximal by 2 min at 30 degrees C with no preincubation. Nuclear extracts prepared from cells blocked in the S phase by aphidicolin or from adenovirus-infected cells at 16 h postinfection had enhanced dihydrofolate reductase transcriptional activity. This increased in vitro transcription mimicked the increase in dihydrofolate reductase mRNA seen in S-phase cells and suggested the presence of a cell-cycle-specific factor(s) which stimulated transcription from the dihydrofolate reductase gene.

scholarly journals The MCP silencer of theDrosophila Abd-Bgene requires both Pleiohomeotic and GAGA factor for the maintenance of repression

Development ◽  
2001 ◽  
Vol 128 (11) ◽  
pp. 2163-2173 ◽  
Author(s):  
Ana Busturia ◽  
Alan Lloyd ◽  
Fernando Bejarano ◽  
Michael Zavortink ◽  
Hua Xin ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Binding Sites ◽  
Mutational Analysis ◽  
Regulatory Elements ◽  
Homeotic Gene ◽  
Gaga Factor ◽  
Polycomb Response Elements ◽  
Silencer Element

Silencing of homeotic gene expression requires the function of cis-regulatory elements known as Polycomb Response Elements (PREs). The MCP silencer element of the Drosophila homeotic gene Abdominal-B has been shown to behave as a PRE and to be required for silencing throughout development. Using deletion analysis and reporter gene assays, we defined a 138 bp sequence within the MCP silencer that is sufficient for silencing of a reporter gene in the imaginal discs. Within the MCP138 fragment, there are four binding sites for the Pleiohomeotic protein (PHO) and two binding sites for the GAGA factor (GAF), encoded by the Trithorax-like gene. PHO and the GAF proteins bind to these sites in vitro. Mutational analysis of PHO and GAF binding sequences indicate that these sites are necessary for silencing in vivo. Moreover, silencing by MCP138 depends on the function of the Trithorax-like gene, and on the function of the PcG genes, including pleiohomeotic. Deletion and mutational analyses show that, individually, either PHO or GAF binding sites retain only weak silencing activity. However, when both PHO and GAF binding sites are present, they achieve strong silencing. We present a model in which robust silencing is achieved by sequential and facilitated binding of PHO and GAF.

scholarly journals The iab-7 Polycomb Response Element Maps to a Nucleosome-Free Region of Chromatin and Requires Both GAGA and Pleiohomeotic for Silencing Activity

2001 ◽  
Vol 21 (4) ◽  
pp. 1311-1318 ◽  
Author(s):  
Rakesh K. Mishra ◽  
Jozsef Mihaly ◽  
Stéphane Barges ◽  
Annick Spierer ◽  
François Karch ◽  
...  
Keyword(s):  
Binding Sites ◽  
Polycomb Group ◽  
Polycomb Group Protein ◽  
Gaga Factor ◽  
Response Element ◽  
Polycomb Response Element ◽  
Free Region ◽  
Group Protein

ABSTRACT In the work reported here we have undertaken a functional dissection of a Polycomb response element (PRE) from the iab-7 cis-regulatory domain of the Drosophila melanogasterbithorax complex (BX-C). Previous studies mapped the iab-7PRE to an 860-bp fragment located just distal to the Fab-7boundary. Located within this fragment is an ∼230-bp chromatin-specific nuclease-hypersensitive region called HS3. We have shown that HS3 is capable of functioning as a Polycomb-dependent silencer in vivo, inducing pairing-dependent silencing of amini-white reporter. The HS3 sequence contains consensus binding sites for the GAGA factor, a protein implicated in the formation of nucleosome-free regions of chromatin, and Pleiohomeotic (Pho), a Polycomb group protein that is related to the mammalian transcription factor YY1. We show that GAGA and Pho interact with these sequences in vitro and that the consensus binding sites for the two proteins are critical for the silencing activity of theiab-7 PRE in vivo.

scholarly journals Genome-wide effects onEscherichia colitranscription from ppGpp binding to its two sites on RNA polymerase

2019 ◽  
Vol 116 (17) ◽  
pp. 8310-8319 ◽  
Author(s):  
Patricia Sanchez-Vazquez ◽  
Colin N. Dewey ◽  
Nicole Kitten ◽  
Wilma Ross ◽  
Richard L. Gourse
Keyword(s):  
Rna Polymerase ◽  
Dna Sequences ◽  
Binding Sites ◽  
Promoter Sequence ◽  
In Vitro Transcription ◽  
Transcriptional Responses ◽  
Transcription Analysis ◽  
Genome Wide

The second messenger nucleotide ppGpp dramatically alters gene expression in bacteria to adjust cellular metabolism to nutrient availability. ppGpp binds to two sites on RNA polymerase (RNAP) inEscherichia coli, but it has also been reported to bind to many other proteins. To determine the role of the RNAP binding sites in the genome-wide effects of ppGpp on transcription, we used RNA-seq to analyze transcripts produced in response to elevated ppGpp levels in strains with/without the ppGpp binding sites on RNAP. We examined RNAs rapidly after ppGpp production without an accompanying nutrient starvation. This procedure enriched for direct effects of ppGpp on RNAP rather than for indirect effects on transcription resulting from starvation-induced changes in metabolism or on secondary events from the initial effects on RNAP. The transcriptional responses of all 757 genes identified after 5 minutes of ppGpp induction depended on ppGpp binding to RNAP. Most (>75%) were not reported in earlier studies. The regulated transcripts encode products involved not only in translation but also in many other cellular processes. In vitro transcription analysis of more than 100 promoters from the in vivo dataset identified a large collection of directly regulated promoters, unambiguously demonstrated that most effects of ppGpp on transcription in vivo were direct, and allowed comparison of DNA sequences from inhibited, activated, and unaffected promoter classes. Our analysis greatly expands our understanding of the breadth of the stringent response and suggests promoter sequence features that contribute to the specific effects of ppGpp.

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