scholarly journals Short repeated elements in the upstream regulatory region of the SUC2 gene of Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (7) ◽  
pp. 2324-2333 ◽  
Author(s):  
L Sarokin ◽  
M Carlson
Keyword(s):  
Carbon Catabolite Repression ◽  
Consensus Sequence ◽  
Regulatory Region ◽  
Lacz Fusion ◽  
Upstream Region ◽  
Upstream Regulatory Region ◽  
Heterologous Promoter ◽  
Suc2 Gene ◽  
High Level ◽  
Repeated Motif

Expression of secreted invertase from the SUC2 gene is regulated by carbon catabolite repression. Previously, an upstream regulatory region that is required for derepression of secreted invertase was identified and shown to confer glucose-repressible expression to the heterologous promoter of a LEU2-lacZ fusion. In this paper we show that tandem copies of a 32-base pair (bp) sequence from the upstream regulatory region activate expression of the same LEU2-lacZ fusion. The level of expression increased with the number of copies of the element, but was independent of their orientation; the expression from constructions containing four copies of the sequence was only twofold lower than that when the entire SUC2 upstream regulatory region was present. This activation was not significantly glucose repressible. The 32-bp sequence includes a 7-bp motif with the consensus sequence (A/C)(A/G)GAAAT that is repeated at five sites within the upstream regulatory region. Genetic evidence supporting the functional significance of this repeated motif was obtained by pseudoreversion of a SUC2 deletion mutant lacking part of the upstream region, including two copies of the 7-bp element. In three of five pseudorevertants, the mutations that restored high-level SUC2 expression altered one of the remaining copies of the 7-bp element.

Short repeated elements in the upstream regulatory region of the SUC2 gene of Saccharomyces cerevisiae

1986 ◽  
Vol 6 (7) ◽  
pp. 2324-2333
Author(s):  
L Sarokin ◽  
M Carlson
Keyword(s):  
Carbon Catabolite Repression ◽  
Consensus Sequence ◽  
Regulatory Region ◽  
Lacz Fusion ◽  
Upstream Region ◽  
Upstream Regulatory Region ◽  
Heterologous Promoter ◽  
Suc2 Gene ◽  
High Level ◽  
Repeated Motif

Expression of secreted invertase from the SUC2 gene is regulated by carbon catabolite repression. Previously, an upstream regulatory region that is required for derepression of secreted invertase was identified and shown to confer glucose-repressible expression to the heterologous promoter of a LEU2-lacZ fusion. In this paper we show that tandem copies of a 32-base pair (bp) sequence from the upstream regulatory region activate expression of the same LEU2-lacZ fusion. The level of expression increased with the number of copies of the element, but was independent of their orientation; the expression from constructions containing four copies of the sequence was only twofold lower than that when the entire SUC2 upstream regulatory region was present. This activation was not significantly glucose repressible. The 32-bp sequence includes a 7-bp motif with the consensus sequence (A/C)(A/G)GAAAT that is repeated at five sites within the upstream regulatory region. Genetic evidence supporting the functional significance of this repeated motif was obtained by pseudoreversion of a SUC2 deletion mutant lacking part of the upstream region, including two copies of the 7-bp element. In three of five pseudorevertants, the mutations that restored high-level SUC2 expression altered one of the remaining copies of the 7-bp element.

scholarly journals Expression System for High Levels of GAG Lyase Gene Expression and Study of the hepA Upstream Region in Flavobacterium heparinum

2002 ◽  
Vol 184 (12) ◽  
pp. 3242-3252 ◽  
Author(s):  
Françoise Blain ◽  
A. Lydia Tkalec ◽  
Zhongqi Shao ◽  
Catherine Poulin ◽  
Marc Pedneault ◽  
...  
Keyword(s):  
Transcriptional Start Site ◽  
Expression System ◽  
Regulatory Region ◽  
Start Codon ◽  
Upstream Region ◽  
Wild Type ◽  
Heparinase I ◽  
High Level ◽  
Flavobacterium Heparinum ◽  
Transcriptional Start Sites

ABSTRACT A system for high-level expression of heparinase I, heparinase II, heparinase III, chondroitinase AC, and chondroitinase B in Flavobacterium heparinum is described. hepA, along with its regulatory region, as well as hepB, hepC, cslA, and cslB, cloned downstream of the hepA regulatory region, was integrated in the chromosome to yield stable transconjugant strains. The level of heparinase I and II expression from the transconjugant strains was approximately fivefold higher, while heparinase III expression was 10-fold higher than in wild-type F. heparinum grown in heparin-only medium. The chondroitinase AC and B transconjugant strains, grown in heparin-only medium, yielded 20- and 13-fold increases, respectively, in chondroitinase AC and B expression, compared to wild-type F. heparinum grown in chondroitin sulfate A-only medium. The hepA upstream region was also studied using cslA as a reporter gene, and the transcriptional start site was determined to be 26 bp upstream of the start codon in the chondroitinase AC transconjugant strain. The transcriptional start sites were determined for hepA in both the wild-type F. heparinum and heparinase I transconjugant strains and were shown to be the same as in the chondroitinase AC transconjugant strain. The five GAG lyases were purified from these transconjugant strains and shown to be identical to their wild-type counterparts.

RUNX1 Directly Regulates Band 3 Transcription.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1735-1735
Author(s):  
Jingping Xie ◽  
Scott W. Hiebert ◽  
Mark J. Koury ◽  
Stephen J. Brandt
Keyword(s):  
Histone Acetylation ◽  
Gene Knockout ◽  
Regulatory Region ◽  
Erythroid Differentiation ◽  
Band 3 ◽  
Upstream Region ◽  
Pcr Analysis ◽  
Upstream Regulatory Region ◽  
Terminal Erythroid Differentiation ◽  
Chip Analysis

Abstract RUNX1 (AML1 or CBFA2) regulates the expression of a number of genes important to hematopoiesis. Gene knockout studies demonstrated that a heterodimeric complex of RUNX1 and its DNA binding partner, core binding factor-beta (CBFbeta), is essential for definitive hematopoiesis. Here, we report that RUNX1 directly represses expression of the Band 3 gene prior to terminal erythroid differentiation. Band 3 is one of four major components of the erythrocyte membrane skeleton and is important for maintenance of cytoskeletal architecture and electroneutral Cl-/HCO3− exchange across the red cell membrane. Band 3 expression, like that of beta-globin, increases dramatically with terminal erythroid differentiation. In a previous study, we identified an upstream region in the mouse Band 3 gene designated as B3URE (for Band 3 upstream regulatory region) bound by multiple transcription factors, including TAL1 (also known as SCL), RUNX1, Ldb1, and GATA1, that acts as an orientation- and position-independent and tissue-specific repressive element. Chromatin immunoprecipitation (ChIP) analysis showed that RUNX1 was associated with the B3URE in intact MEL cells and electrophoretic mobility shift analysis confirmed specific RUNX1 interaction with RUNX1 binding sites in the B3URE. Together with CBFbeta, RUNX1 inhibited reporter activity from a construct linking the B3URE with 1 kb of Band 3 promoter in transiently transfected MEL but not COS cells. DNA affinity precipitation analysis with wild-type and mutant oligos established that RUNX1 and CBFbeta in MEL cell nuclear extracts could interact with the B3URE in vitro and suggested that RUNX1 recruits TAL1 and Ldb1 to DNA. Northern blot and quantitative real-time PCR analysis demonstrated that enforced expression of RUNX1 dramatically inhibited dimethylsulfoxide (DMSO)-induced Band 3 gene expression. Quantitative ChIP analysis showed that histone acetylation in the B3URE increased more than 4-fold, while histone methylation decreased ~50% after 3 days of DMSO-induced differentiation. Over the same time frame, the promoter region underwent significantly less acetylation but more extensive demethylation. Finally, changes in B3URE acetylation and methylation were attenuated and inhibited, respectively, in RUNX1-transfected MEL cells relative to vector controls. In sum, these results demonstrate that the Band 3 gene is a direct target of RUNX1 in erythroid cells and indicate that the B3URE contributes to the tightly regulated expression of this gene in differentiating erythroid progenitors. One mechanism by which RUNX1 regulates Band 3 transcription may be by influencing histone acetylation/methylation in this upstream regulatory region.

Upstream region required for regulated expression of the glucose-repressible SUC2 gene of Saccharomyces cerevisiae

1984 ◽  
Vol 4 (12) ◽  
pp. 2750-2757
Author(s):  
L Sarokin ◽  
M Carlson
Keyword(s):  
Gene Expression ◽  
Glucose Repression ◽  
Transcriptional Start Site ◽  
Upstream Region ◽  
Yeast Genome ◽  
Base Pairs ◽  
Regulated Expression ◽  
Suc2 Gene ◽  
High Level

The SUC2 gene produces two mRNAs with different 5' ends that encode two forms of invertase. The 1.9-kilobase mRNA encoding secreted invertase is regulated by glucose repression (carbon catabolite repression), and the 1.8-kilobase mRNA encoding intracellular invertase is produced constitutively at low levels. To identify 5' noncoding sequences essential for regulated expression of SUC2, we constructed in vitro a series of deletions and inserted them into the yeast genome at the chromosomal SUC2 locus. Analysis of the effects of each deletion on SUC2 gene expression identified an upstream region required for derepression of secreted invertase synthesis. The 3' boundary of this region is near -418. The 5' boundary does not appear to be sharply defined, but lies ca. 100 base pairs upstream. A deletion extending from -418 to -140 allowed high-level derepression, indicating that no essential sequences lie between the upstream region and the TATA box at -133 and that the upstream region can be moved 279 base pairs closer to the transcriptional start site. Interactions between the deletions and several unlinked mutations affecting the regulation of SUC2 gene expression were examined. Sequences between -1,900 and -86 are dispensable for expression of the 1.8-kilobase mRNA.

scholarly journals Upstream region required for regulated expression of the glucose-repressible SUC2 gene of Saccharomyces cerevisiae.

1984 ◽  
Vol 4 (12) ◽  
pp. 2750-2757 ◽  
Author(s):  
L Sarokin ◽  
M Carlson
Keyword(s):  
Gene Expression ◽  
Glucose Repression ◽  
Transcriptional Start Site ◽  
Upstream Region ◽  
Yeast Genome ◽  
Base Pairs ◽  
Regulated Expression ◽  
Suc2 Gene ◽  
High Level

The SUC2 gene produces two mRNAs with different 5' ends that encode two forms of invertase. The 1.9-kilobase mRNA encoding secreted invertase is regulated by glucose repression (carbon catabolite repression), and the 1.8-kilobase mRNA encoding intracellular invertase is produced constitutively at low levels. To identify 5' noncoding sequences essential for regulated expression of SUC2, we constructed in vitro a series of deletions and inserted them into the yeast genome at the chromosomal SUC2 locus. Analysis of the effects of each deletion on SUC2 gene expression identified an upstream region required for derepression of secreted invertase synthesis. The 3' boundary of this region is near -418. The 5' boundary does not appear to be sharply defined, but lies ca. 100 base pairs upstream. A deletion extending from -418 to -140 allowed high-level derepression, indicating that no essential sequences lie between the upstream region and the TATA box at -133 and that the upstream region can be moved 279 base pairs closer to the transcriptional start site. Interactions between the deletions and several unlinked mutations affecting the regulation of SUC2 gene expression were examined. Sequences between -1,900 and -86 are dispensable for expression of the 1.8-kilobase mRNA.

scholarly journals Molecular architecture of the hsp70 promoter after deletion of the TATA box or the upstream regulation region.

1997 ◽  
Vol 17 (7) ◽  
pp. 3799-3808 ◽  
Author(s):  
J A Weber ◽  
D J Taxman ◽  
Q Lu ◽  
D S Gilmour
Keyword(s):  
Transcriptional Activation ◽  
Regulatory Region ◽  
Dnase I ◽  
Tata Box ◽  
Upstream Region ◽  
Upstream Regulatory Region ◽  
Gaga Factor ◽  
Transcription Start ◽  
Hsp70 Promoter ◽  
Promoter Construct

GAGA factor, TFIID, and paused polymerase are present on the hsp70 promoter in Drosophila melanogaster prior to transcriptional activation. In order to investigate the interplay between these components, mutant constructs were analyzed after they had been transformed into flies on P elements. One construct lacked the TATA box and the other lacked the upstream regulatory region where GAGA factor binds. Transcription of each mutant during heat shock was at least 50-fold less than that of a normal promoter construct. Before and after heat shock, both mutant promoters were found to adopt a DNase I hypersensitive state that included the region downstream from the transcription start site. High-resolution analysis of the DNase I cutting pattern identified proteins that could be contributing to the hypersensitivity. GAGA factor footprints were clearly evident in the upstream region of the TATA deletion construct, and a partial footprint possibly caused by TFIID was evident on the TATA box of the upstream deletion construct. Permanganate treatment of intact salivary glands was used to further characterize each promoter construct. Paused polymerase and TFIID were readily detected on the normal promoter construct, whereas both deletions exhibited reduced levels of each of these factors. Hence both the TATA box and the upstream region are required to efficiently recruit TFIID and a paused polymerase to the promoter prior to transcriptional activation. In contrast, GAGA factor appears to be capable of binding and establishing a DNase I hypersensitive region in the absence of TFIID and polymerase. Interestingly, purified GAGA factor was found to bind near the transcription start site, and the strength of this interaction was increased by the presence of the upstream region. GAGA factor alone might be capable of establishing an open chromatin structure that encompasses the upstream regulatory region as well as the core promoter region, thus facilitating the binding of TFIID.

scholarly journals Control of Transposon-mediated Activation of theglpFKOperon ofEscherichia coliby two DNA binding Proteins

2016 ◽  
Author(s):  
Zhongge Zhang ◽  
Milton H. Saier
Keyword(s):  
Insertion Sequence ◽  
Gene Activation ◽  
Regulatory Region ◽  
Receptor Protein ◽  
Upstream Region ◽  
Upstream Regulatory Region ◽  
Wild Type ◽  
Transposon Insertion ◽  
Glycerol Utilization ◽  
Camp Receptor

AbstractEscherichia colicells deleted for the cyclic AMP (cAMP) receptor protein (Crp) gene (Δcrp) cannot utilize glycerol because cAMP-Crp is a required positive activator of glycerol utilization operonglpFK. We have previously shown that a transposon, Insertion Sequence 5 (IS5), can reversibly insert into the upstream regulatory region of the operon so as to activateglpFKand enable glycerol utilization. GlpR, which repressesglpFKtranscription, binds to theglpFKupstream region near the site of IS5insertion, and prevents insertion. We here show that the cAMP-Crp complex, which also binds to theglpFKupstream regulatory region, also inhibits IS5hopping into the activating site. This finding allowed us to identify conditions under which wild type cells can acquireglpFK-activating IS5insertions. Maximal rates of IS5insertion into the activating site require the presence of glycerol as well as a non-metabolizable sugar analogue that lowers cytoplasmic cAMP concentrations. Under these conditions, IS5insertional mutants accumulate and outcompete the wild type cells. Because of the widespread distribution of glucose analogues in nature, this mechanism of gene activation could have evolved by natural selection.

scholarly journals Transvection at the End of the Truncated Chromosome in Drosophila melanogaster

Genetics ◽  
2003 ◽  
Vol 163 (4) ◽  
pp. 1375-1387
Author(s):  
Mikhail Savitsky ◽  
Tatyana Kahn ◽  
Ekaterina Pomerantseva ◽  
Pavel Georgiev
Keyword(s):  
Drosophila Melanogaster ◽  
Homologous Chromosome ◽  
Regulatory Region ◽  
Proximal Region ◽  
The Other ◽  
Upstream Region ◽  
Upstream Sequence ◽  
Transcription Start ◽  
Promoter Proximal Region

Abstract The phenomenon of transvection is well known for the Drosophila yellow locus. Thus enhancers of a promoterless yellow locus in one homologous chromosome can activate the yellow promoter in the other chromosome where the enhancers are inactive or deleted. In this report, we examined the requirements for trans-activation of the yellow promoter at the end of the deficient chromosome. A number of truncated chromosomes ending in different areas of the yellow regulatory region were examined in combination with the promoterless y alleles. We found that trans-activation of the yellow promoter at the end of a deficient chromosome required ∼6 kb of an additional upstream sequence. The nature of upstream sequences affected the strength of transvection: addition of gypsy sequences induced stronger trans-activation than addition of HeT-A or yellow sequences. Only the promoter proximal region (within -158 bp of the yellow transcription start) was essential for trans-activation; i.e., transvection did not require extensive homology in the yellow upstream region. Finally, the yellow enhancers located on the two pairing chromosomes could cooperatively activate one yellow promoter.

Mutations Causing Constitutive Invertase Synthesis in Yeast: Genetic Interactions with snf Mutations

Genetics ◽  
1987 ◽  
Vol 115 (2) ◽  
pp. 247-253
Author(s):  
Lenore Neigeborn ◽  
Marian Carlson
Keyword(s):  
Regulatory Region ◽  
Constitutive Expression ◽  
Genetic Interactions ◽  
The Third ◽  
Recessive Mutations ◽  
Constitutive Mutation ◽  
Complementation Groups ◽  
High Level ◽  
Yeast Genetic ◽  
Very High

ABSTRACT We have selected 210 mutants able to grow on sucrose in the presence of 2-deoxyglucose. We identified recessive mutations in three major complementation groups that cause constitutive (glucose-insensitive) secreted invertase synthesis. Two groups comprise alleles of the previously identified HXK2 and REG1 genes, and the third group was designated cid1 (constitutive invertase derepression). The effect of cid1 on SUC2 expression is mediated by the SUC2 upstream regulatory region, as judged by the constitutive expression of a SUC2-LEU2-lacZ fusion in which the LEU2 promoter is under control of SUC2 upstream sequences. A cid1 mutation also causes glucose-insensitive expression of maltase. The previously isolated constitutive mutation ssn6 is epistatic to cid1, reg1 and hxk2 for very high level constitutive invertase expression. Mutations in SNF genes that prevent derepression of invertase are epistatic to cid1, reg1 and hxk2; we have previously shown that ssn6 has different epistasis relationships with snf mutations. The constitutive mutation tup1 was found to resemble ssn6 in its genetic interactions with snf mutations. These findings suggest that CID1, REG1 and HXK2 are functionally distinct from SSN6 and TUP1.

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