scholarly journals Competitive titration in living sea urchin embryos of regulatory factors required for expression of the CyIIIa actin gene

Development ◽  
1990 ◽  
Vol 110 (1) ◽  
pp. 31-40 ◽  
Author(s):  
R.R. Franks ◽  
R. Anderson ◽  
J.G. Moore ◽  
B.R. Hough-Evans ◽  
R.J. Britten ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Sea Urchin ◽  
Binding Sites ◽  
Nuclear Dna ◽  
Fusion Gene ◽  
Actin Gene ◽  
Regulatory Domain ◽  
Regulatory Factors

Previous studies have located some twenty distinct sites within the 2.3 kb 5′ regulatory domain of the sea urchin CyIIIa cytoskeletal actin gene, where there occur in vitro high-specificity interactions with nuclear DNA-binding proteins of the embryo. This gene is activated in late cleavage, exclusively in cells of the aboral ectoderm cell lineages. In this study, we investigate the functional importance in vivo of these sites of DNA-protein interaction. Sea urchin eggs were coinjected with a fusion gene construct in which the bacterial chloramphenicol acetyltransferase (CAT) reporter gene is under the control of the entire CyIIIa regulatory domain, together with molar excesses of one of ten nonoverlapping competitor subfragments of this domain, each of which contains one or a few specific site(s) of interaction. The exogenous excess binding sites competitively titrate the available regulatory factors away from the respective sites associated with the CyIIIa.CAT reporter gene. This provides a method for detecting in vivo sites within the regulatory domain that are required for normal levels of expression, without disturbing the structure of the regulatory domain. We thus identify five nonoverlapping regions of the regulatory DNA that apparently function as binding sites for positively acting transcriptional regulatory factors. Competition with a subfragment bearing an octamer site results in embryonic lethality. We find that three other sites display no quantitative competitive interference with CyIIIa.CAT expression, though as shown in the accompanying paper, two of these sites are required for control of spatial expression. We conclude that the complex CyIIIa regulatory domain must assess the state of many distinct and individually necessary interactions in order to properly regulate CyIIIa transcriptional activity in development.

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.

Concerted action of the transcriptional activators REB1, RAP1, and GCR1 in the high-level expression of the glycolytic gene TPI

1993 ◽  
Vol 13 (1) ◽  
pp. 543-550
Author(s):  
E W Scott ◽  
H V Baker
Keyword(s):  
Binding Site ◽  
Reporter Gene ◽  
Binding Sites ◽  
Triosephosphate Isomerase ◽  
Noncoding Region ◽  
Lacz Gene ◽  
Primary Role ◽  
Fold Reduction

In Saccharomyces cerevisiae, the TPI gene product, triosephosphate isomerase, makes up about 2% of the soluble cellular protein. Using in vitro and in vivo footprinting techniques, we have identified four binding sites for three factors in the 5' noncoding region of TPI: a REB1-binding site located at positions -401 to -392, two GCR1-binding sites located at positions -381 to -366 and -341 to -326, and a RAP1-binding site located at positions -358 to -346. We tested the effects of mutations at each of these binding sites on the expression of a TPI::lacZ gene fusion which carried 853 bp of the TPI 5' noncoding region integrated at the URA3 locus. The REB1-binding site is dispensable when material 5' to it is deleted; however, if the sequence 5' to the REB1-binding site is from the TPI locus, expression is reduced fivefold when the site is mutated. Because REB1 blocks nucleosome formation, the most likely function of its binding site in the TPI controlling region is to prevent the formation of nucleosomes over the TPI upstream activation sequence. Mutations in the RAP1-binding site resulted in a 10-fold reduction in expression of the reporter gene. Mutating either GCR1-binding site alone had a modest effect on expression of the fusion. However, mutating both GCR1-binding sites resulted in a 68-fold reduction in the level of expression of the reporter gene. A LexA-GCR1 fusion protein containing the DNA-binding domain of LexA fused to the amino terminus of GCR1 was able to activate expression of a lex operator::GAL1::lacZ reporter gene 116-fold over background levels. From this experiment, we conclude that GCR1 is able to activate gene expression in the absence of REB1 or RAP1 bound at adjacent binding sites. On the basis of these results, we suggest that GCR1 binding is required for activation of TPI and other GCR1-dependent genes and that the primary role of other factors which bind adjacent to GCR1-binding sites is to facilitate of modulate GCR1 binding in vivo.

scholarly journals Concerted action of the transcriptional activators REB1, RAP1, and GCR1 in the high-level expression of the glycolytic gene TPI.

1993 ◽  
Vol 13 (1) ◽  
pp. 543-550 ◽  
Author(s):  
E W Scott ◽  
H V Baker
Keyword(s):  
Binding Site ◽  
Reporter Gene ◽  
Binding Sites ◽  
Triosephosphate Isomerase ◽  
Noncoding Region ◽  
Lacz Gene ◽  
Primary Role ◽  
Fold Reduction

In Saccharomyces cerevisiae, the TPI gene product, triosephosphate isomerase, makes up about 2% of the soluble cellular protein. Using in vitro and in vivo footprinting techniques, we have identified four binding sites for three factors in the 5' noncoding region of TPI: a REB1-binding site located at positions -401 to -392, two GCR1-binding sites located at positions -381 to -366 and -341 to -326, and a RAP1-binding site located at positions -358 to -346. We tested the effects of mutations at each of these binding sites on the expression of a TPI::lacZ gene fusion which carried 853 bp of the TPI 5' noncoding region integrated at the URA3 locus. The REB1-binding site is dispensable when material 5' to it is deleted; however, if the sequence 5' to the REB1-binding site is from the TPI locus, expression is reduced fivefold when the site is mutated. Because REB1 blocks nucleosome formation, the most likely function of its binding site in the TPI controlling region is to prevent the formation of nucleosomes over the TPI upstream activation sequence. Mutations in the RAP1-binding site resulted in a 10-fold reduction in expression of the reporter gene. Mutating either GCR1-binding site alone had a modest effect on expression of the fusion. However, mutating both GCR1-binding sites resulted in a 68-fold reduction in the level of expression of the reporter gene. A LexA-GCR1 fusion protein containing the DNA-binding domain of LexA fused to the amino terminus of GCR1 was able to activate expression of a lex operator::GAL1::lacZ reporter gene 116-fold over background levels. From this experiment, we conclude that GCR1 is able to activate gene expression in the absence of REB1 or RAP1 bound at adjacent binding sites. On the basis of these results, we suggest that GCR1 binding is required for activation of TPI and other GCR1-dependent genes and that the primary role of other factors which bind adjacent to GCR1-binding sites is to facilitate of modulate GCR1 binding in vivo.

In vitro and in vivo evaluation of a green fluorescent protein-HSV1-TK dual-function pet reporter gene

2001 ◽  
Vol 44 (S1) ◽  
pp. S339-S341
Author(s):  
K. E. Luker ◽  
G. D. Luker ◽  
C. M. Pica ◽  
J. L. Dahlheimer ◽  
T. J. Fahrner ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Reporter Gene ◽  
Fluorescent Protein ◽  
Dual Function ◽  
In Vivo Evaluation ◽  
Green Fluorescent

Metabolic importance of Na+/K+-ATPase activity during sea urchin development.

1997 ◽  
Vol 200 (22) ◽  
pp. 2881-2892 ◽  
Author(s):  
P Leong ◽  
D Manahan
Keyword(s):  
Enzyme Activity ◽  
Atpase Activity ◽  
Sea Urchin ◽  
Sodium Pump ◽  
Sea Water ◽  
Strongylocentrotus Purpuratus ◽  
Lytechinus Pictus ◽  
Stimulation Of

Early stages of animal development have high mass-specific rates of metabolism. The biochemical processes that establish metabolic rate and how these processes change during development are not understood. In this study, changes in Na+/K+-ATPase activity (the sodium pump) and rate of oxygen consumption were measured during embryonic and early larval development for two species of sea urchin, Strongylocentrotus purpuratus and Lytechinus pictus. Total (in vitro) Na+/K+-ATPase activity increased during development and could potentially account for up to 77 % of larval oxygen consumption in Strongylocentrotus purpuratus (pluteus stage) and 80 % in Lytechinus pictus (prism stage). The critical issue was addressed of what percentage of total enzyme activity is physiologically active in living embryos and larvae and thus what percentage of metabolism is established by the activity of the sodium pump during development. Early developmental stages of sea urchins are ideal for understanding the in vivo metabolic importance of Na+/K+-ATPase because of their small size and high permeability to radioactive tracers (86Rb+) added to sea water. A comparison of total and in vivo Na+/K+-ATPase activities revealed that approximately half of the total activity was utilized in vivo. The remainder represented a functionally active reserve that was subject to regulation, as verified by stimulation of in vivo Na+/K+-ATPase activity in the presence of the ionophore monensin. In the presence of monensin, in vivo Na+/K+-ATPase activities in embryos of S. purpuratus increased to 94 % of the maximum enzyme activity measured in vitro. Stimulation of in vivo Na+/K+-ATPase activity was also observed in the presence of dissolved alanine, presumably due to the requirement to remove the additional intracellular Na+ that was cotransported with alanine from sea water. The metabolic cost of maintaining the ionic balance was found to be high, with this process alone accounting for 40 % of the metabolic rate of sea urchin larvae (based on the measured fraction of total Na+/K+-ATPase that is physiologically active in larvae of S. purpuratus). Ontogenetic changes in pump activity and environmentally induced regulation of reserve Na+/K+-ATPase activity are important factors that determine a major proportion of the metabolic costs of sea urchin development.

UHF-1, a factor required for maximal transcription of early and late sea urchin histone H4 genes: analysis of promoter-binding sites

1991 ◽  
Vol 11 (2) ◽  
pp. 1048-1061
Author(s):  
I J Lee ◽  
L Tung ◽  
D A Bumcrot ◽  
E S Weinberg
Keyword(s):  
Binding Site ◽  
Sea Urchin ◽  
Transcriptional Start Site ◽  
Sodium Dodecyl ◽  
Nuclear Extract ◽  
Dnase I ◽  
Histone H4 ◽  
Band Shift

A protein, denoted UHF-1, was found to bind upstream of the transcriptional start site of both the early and late H4 (EH4 and LH4) histone genes of the sea urchin Strongylocentrotus purpuratus. A nuclear extract from hatching blastulae contained proteins that bind to EH4 and LH4 promoter fragments in a band shift assay and produced sharp DNase I footprints upstream of the EH4 gene (from -133 to -106) and the LH4 gene (from -94 to -66). DNase I footprinting performed in the presence of EH4 and LH4 promoter competitor DNAs indicated that UHF-1 binds more strongly to the EH4 site. A sequence match of 11 of 13 nucleotides was found within the two footprinted regions: [sequence: see text]. Methylation interference and footprinting experiments showed that UHF-1 bound to the two sites somewhat differently. DNA-protein UV cross-linking studies indicated that UHF-1 has an electrophoretic mobility on sodium dodecyl sulfate-acrylamide gels of approximately 85 kDa and suggested that additional proteins, specific to each promoter, bind to each site. In vitro and in vivo assays were used to demonstrate that the UHF-1-binding site is essential for maximal transcription of the H4 genes. Deletion of the EH4 footprinted region resulted in a 3-fold decrease in transcription in a nuclear extract and a 2.6-fold decrease in expression in morulae from templates that had been injected into eggs. In the latter case, deletion of the binding site did not grossly disrupt the temporal program of expression from the injected EH4 genes. LH4 templates containing a 10-bp deletion in the consensus region or base substitutions in the footprinted region were transcribed at 14 to 58% of the level of the wild-type LH4 template. UHF-1 is therefore essential for maximal expression of the early and late H4 genes.

Interspersion of an unusual GCN4 activation site with a complex transcriptional repression site in Ty2 elements of Saccharomyces cerevisiae

1993 ◽  
Vol 13 (4) ◽  
pp. 2091-2103
Author(s):  
S Türkel ◽  
P J Farabaugh
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Transcriptional Repression ◽  
Physiological Control ◽  
Reading Frame ◽  
Negative Region ◽  
Activation Site

Transcription of the Ty2-917 retrotransposon of Saccharomyces cerevisiae is modulated by a complex set of positive and negative elements, including a negative region located within the first open reading frame, TYA2. The negative region includes three downstream repression sites (DRSI, DRSII, and DRSIII). In addition, the negative region includes at least two downstream activation sites (DASs). This paper concerns the characterization of DASI. A 36-bp DASI oligonucleotide acts as an autonomous transcriptional activation site and includes two sequence elements which are both required for activation. We show that these sites bind in vitro the transcriptional activation protein GCN4 and that their activity in vivo responds to the level of GCN4 in the cell. We have termed the two sites GCN4 binding sites (GBS1 and GBS2). GBS1 is a high-affinity GCN4 binding site (dissociation constant, approximately 25 nM at 30 degrees C), binding GCN4 with about the affinity of a consensus UASGCN4, this though GBS1 includes two differences from the right half of the palindromic consensus site. GBS2 is more diverged from the consensus and binds GCN4 with about 20-fold-lower affinity. Nucleotides 13 to 36 of DASI overlap DRSII. Since DRSII is a transcriptional repression site, we tested whether DASI includes repression elements. We identify two sites flanking GBS2, both of which repress transcription activated by the consensus GCN4-specific upstream activation site (UASGCN4). One of these is repeated in the 12 bp immediately adjacent to DASI. Thus, in a 48-bp region of Ty2-917 are interspersed two positive and three negative transcriptional regulators. The net effect of the region must depend on the interaction of the proteins bound at these sites, which may include their competing for binding sites, and on the physiological control of the activity of these proteins.

Progastrin1–80stimulates growth of intestinal epithelial cells in vitro via high-affinity binding sites

2003 ◽  
Vol 284 (2) ◽  
pp. G328-G339 ◽  
Author(s):  
P. Singh ◽  
X. Lu ◽  
S. Cobb ◽  
B. T. Miller ◽  
N. Tarasova ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Binding Sites ◽  
Intestinal Epithelial Cells ◽  
Full Length ◽  
Intestinal Epithelial ◽  
Growth Effects ◽  
High Affinity ◽  
Significant Difference

Proliferation and carcinogenesis of the large intestinal epithelial cells (IEC) cells is significantly increased in transgenic mice that overexpress the precursor progastrin (PG) peptide. It is not known if the in vivo growth effects of PG on IEC cells are mediated directly or indirectly. Full-length recombinant human PG (rhPG1–80) was generated to examine possible direct effects of PG on IEC cells. Surprisingly, rhPG (0.1–1.0 nM) was more effective than the completely processed gastrin 17 (G17) peptide as a growth factor. Even though IEC cells did not express CCK1and CCK2receptors (-R), fluorescently labeled G17 and Gly-extended G17 (G-Gly) were specifically bound to the cells, suggesting the presence of binding proteins other than CCK1-R and CCK2-R on IEC cells. High-affinity ( Kd= 0.5–1.0 nM) binding sites for125I-rhPG were discovered on IEC cells that demonstrated relative binding affinity for gastrin-like peptides in the order PG ≥ COOH-terminally extended G17 ≥ G-Gly > G17 > *CCK-8 (* significant difference; P< 0.05). In conclusion, our studies demonstrate for the first time direct growth effects of the full-length precursor peptide on IEC cells in vitro that are apparently mediated by the high-affinity PG binding sites that were discovered on these cells.

Evaluation of Estrogenic Activity of Wastewater: Comparison Among In Vitro ERα Reporter Gene Assay, In Vivo Vitellogenin Induction, and Chemical Analysis

2015 ◽  
Vol 49 (10) ◽  
pp. 6319-6326 ◽  
Author(s):  
Masaru Ihara ◽  
Tomokazu Kitamura ◽  
Vimal Kumar ◽  
Chang-Beom Park ◽  
Mariko O. Ihara ◽  
...  
Keyword(s):  
Chemical Analysis ◽  
Reporter Gene ◽  
Estrogenic Activity ◽  
Reporter Gene Assay ◽  
Gene Assay
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