Identification of a signal transduction response sequence element necessary for induction of a Dictyostelium discoideum gene by extracellular cyclic AMP.

The signal transduction pathways that lead to gene induction are being intensively investigated in Dictyostelium discoideum. We have identified by deletion and transformation analysis a sequence element necessary for induction of a gene coding for uridine diphosphoglucose pyrophosphorylase (UDPGP1) of D. discoideum in response to extracellular cyclic AMP (cAMP). This regulatory element is located 380 base pairs upstream of the transcription start site and contains a G+C-rich partially palindromic sequence. It is not required for transcription per se but is required for induction of the gene in response to the stimulus of extracellular cAMP. The cAMP response sequence is also required for induction of the gene during normal development. A second A+T-rich cis-acting region located immediately downstream of the cAMP response sequence appears to be essential for the basal level of expression of the UDPGP1 gene. The position of the cAMP response element coincides with a DNase I-hypersensitive site that is observed when the UDPGP1 gene is actively transcribed.

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Regulation of the Discoidin I gamma gene in Dictyostelium discoideum: identification of individual promoter elements mediating induction of transcription and repression by cyclic AMP

Molecular and Cellular Biology ◽

10.1128/mcb.10.8.4080-4088.1990 ◽

1990 ◽

Vol 10 (8) ◽

pp. 4080-4088

Author(s):

F Vauti ◽

P Morandini ◽

J Blusch ◽

A Sachse ◽

W Nellen

Keyword(s):

Gene Expression ◽

Cyclic Amp ◽

Dictyostelium Discoideum ◽

Base Pair ◽

Regulation Of Gene Expression ◽

Promoter Elements ◽

Down Regulation ◽

Heterologous Promoter ◽

Sequence Element ◽

Pair Sequence

We dissected the promoter of the developmentally induced and cyclic AMP-repressed discoidin I gamma gene and identified a sequence element essential for developmental induction. Transfer of the element to an inactive heterologous promoter demonstrated that this sequence is sufficient to confer expression in axenically growing cells and to induce gene activity in development after growth on bacteria. A 16-base-pair sequence within this element was shown to be sufficient for induction in the discoidin promoter context and was used to reactivate different truncated promoter constructs. This led to the localization of an element necessary for down regulation of gene expression by extracellular cyclic AMP.

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Regulation of the Discoidin I gamma gene in Dictyostelium discoideum: identification of individual promoter elements mediating induction of transcription and repression by cyclic AMP.

Molecular and Cellular Biology ◽

10.1128/mcb.10.8.4080 ◽

1990 ◽

Vol 10 (8) ◽

pp. 4080-4088 ◽

Cited By ~ 43

Author(s):

F Vauti ◽

P Morandini ◽

J Blusch ◽

A Sachse ◽

W Nellen

Keyword(s):

Gene Expression ◽

Cyclic Amp ◽

Dictyostelium Discoideum ◽

Base Pair ◽

Regulation Of Gene Expression ◽

Promoter Elements ◽

Down Regulation ◽

Heterologous Promoter ◽

Sequence Element ◽

Pair Sequence

We dissected the promoter of the developmentally induced and cyclic AMP-repressed discoidin I gamma gene and identified a sequence element essential for developmental induction. Transfer of the element to an inactive heterologous promoter demonstrated that this sequence is sufficient to confer expression in axenically growing cells and to induce gene activity in development after growth on bacteria. A 16-base-pair sequence within this element was shown to be sufficient for induction in the discoidin promoter context and was used to reactivate different truncated promoter constructs. This led to the localization of an element necessary for down regulation of gene expression by extracellular cyclic AMP.

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Isolation and characterization of the Beadex locus of Drosophila melanogaster: a putative cis-acting negative regulatory element for the heldup-a gene.

Molecular and Cellular Biology ◽

10.1128/mcb.4.7.1343 ◽

1984 ◽

Vol 4 (7) ◽

pp. 1343-1353 ◽

Cited By ~ 6

Author(s):

W W Mattox ◽

N Davidson

Keyword(s):

Drosophila Melanogaster ◽

Base Pair ◽

Regulatory Element ◽

P Element ◽

Functional Domain ◽

Lambda Phage ◽

Base Pairs ◽

Negative Regulatory Element ◽

Cis Acting ◽

Isolation And Characterization

We isolated recombinant lambda phage clones spanning 49 kilobases of DNA which contain the Beadex and heldup-a loci of Drosophila melanogaster. These cloned DNAs were used to analyze the structure of eight dominant mutant alleles of the Beadex locus which show increased gene activity. A region, only 700 base pairs in length, is altered in each of these mutants. Six of the mutations have DNA insertions within this segment. Most of these insertions resemble retrovirus-like transposable elements. In one case (Beadex2) the inserted sequences are homologous to the gypsy transposon family. The other two Beadex alleles were induced by hybrid dysgenesis and suffered deletions which included at least part of the 700-base-pair segment. These deletions appear to have resulted from imprecise excision or deletion of a nearby P element found in the wild-type parental strain. Analysis of one heldup-a allele (heldup-aD30r) indicates that a similar P element-mediated event is responsible for this lesion. In this mutant, deletion of sequences no more than 1,600 base pairs from the Beadex locus accompanies the loss of heldup-a function. The deleted sequences in heldup-aD30r include the entire 700-base-pair segment within which at least part of the Beadex locus resides, yet these flies have no Beadex phenotype. This indicates that a functional heldup-a gene is necessary for expression of the Beadex phenotype. Together, these results suggest that the Beadex functional domain is contained within a short segment of DNA near the heldup-a gene and support the hypothesis that the Beadex locus functions as a cis-acting negative regulatory element for the heldup-a gene.

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Isolation and characterization of the Beadex locus of Drosophila melanogaster: a putative cis-acting negative regulatory element for the heldup-a gene

Molecular and Cellular Biology ◽

10.1128/mcb.4.7.1343-1353.1984 ◽

1984 ◽

Vol 4 (7) ◽

pp. 1343-1353

Author(s):

W W Mattox ◽

N Davidson

Keyword(s):

Drosophila Melanogaster ◽

Base Pair ◽

Regulatory Element ◽

P Element ◽

Functional Domain ◽

Lambda Phage ◽

Base Pairs ◽

Negative Regulatory Element ◽

Cis Acting ◽

Isolation And Characterization

We isolated recombinant lambda phage clones spanning 49 kilobases of DNA which contain the Beadex and heldup-a loci of Drosophila melanogaster. These cloned DNAs were used to analyze the structure of eight dominant mutant alleles of the Beadex locus which show increased gene activity. A region, only 700 base pairs in length, is altered in each of these mutants. Six of the mutations have DNA insertions within this segment. Most of these insertions resemble retrovirus-like transposable elements. In one case (Beadex2) the inserted sequences are homologous to the gypsy transposon family. The other two Beadex alleles were induced by hybrid dysgenesis and suffered deletions which included at least part of the 700-base-pair segment. These deletions appear to have resulted from imprecise excision or deletion of a nearby P element found in the wild-type parental strain. Analysis of one heldup-a allele (heldup-aD30r) indicates that a similar P element-mediated event is responsible for this lesion. In this mutant, deletion of sequences no more than 1,600 base pairs from the Beadex locus accompanies the loss of heldup-a function. The deleted sequences in heldup-aD30r include the entire 700-base-pair segment within which at least part of the Beadex locus resides, yet these flies have no Beadex phenotype. This indicates that a functional heldup-a gene is necessary for expression of the Beadex phenotype. Together, these results suggest that the Beadex functional domain is contained within a short segment of DNA near the heldup-a gene and support the hypothesis that the Beadex locus functions as a cis-acting negative regulatory element for the heldup-a gene.

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Functional analysis of the promoter of a sea urchin metallothionein gene

Biochemistry and Cell Biology ◽

10.1139/o92-160 ◽

1992 ◽

Vol 70 (10-11) ◽

pp. 1142-1150 ◽

Cited By ~ 8

Author(s):

Peter Cserjesi ◽

Peter Fairley ◽

Bruce P. Brandhorst

Keyword(s):

Sea Urchin ◽

Regulatory Element ◽

Gene Promoter ◽

Flanking Sequence ◽

Core Element ◽

Sequence Element ◽

Enhancer Element ◽

Cis Acting ◽

Conserved Sequence ◽

Gc Box

The 5′-flanking region of the metallothionein (MT) gene LpMT1 of the sea urchin Lytechinus pictus includes three copies of a conserved sequence that includes the metal-responsive element (MRE) consensus core sequence required for heavy metal induction of other MT genes, a GC box, a G box of a putative basal level enhancer element which includes another MRE core element, and a poly(C) tract. A fragment of LpMT1 DNA from nucleotides + 31 to −309 fused to a chloramphenicol acetyltransferase reporter gene was inducible with cadmium after injecton into L. pictus embryos. This induced activity was greatly reduced in a deletion mutant which retained only 195 base pairs of 5′-flanking sequence, including the proximal pair of MREs and the G box, but excluding the poly(C) tract, GC box, and distal MRE. A potent human hMT-IIA gene promoter is marginally functional in L. pictus embryos. In contrast, the LpMT1 promoter is active in HeLa cells and in embryos of the sea urchin Strongylocentrotus purpuratus. The hMT-IIA gene may lack a cis-acting sequence element required for expression of MT genes in L. pictus embryos. The LpMT1 promoter is a powerful, inducible, promiscuous promoter useful for driving the expression of heterologous genes in sea urchin embryos.Key words: metallothionein, sea urchin, transcriptional regulation, induction by heavy metals, metal regulatory element.

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Different sequence requirements for expression in erythroid and megakaryocytic cells within a regulatory element upstream of the GATA-1 gene

Development ◽

10.1242/dev.126.12.2799 ◽

1999 ◽

Vol 126 (12) ◽

pp. 2799-2811 ◽

Cited By ~ 2

Author(s):

P. Vyas ◽

M.A. McDevitt ◽

A.B. Cantor ◽

S.G. Katz ◽

Y. Fujiwara ◽

...

Keyword(s):

Transcription Factor ◽

Base Pair ◽

Regulatory Element ◽

Point Mutations ◽

Comparative Sequence Analysis ◽

Gata Factor ◽

Base Pairs ◽

Cis Acting ◽

E Box ◽

High Level

The lineage-restricted transcription factor GATA-1 is required for differentiation of erythroid and megakaryocytic cells. We have localized a 317 base pair cis-acting regulatory element, HS I, associated with a hematopoietic-specific DNase I hypersensitive site, which lies approx. 3.7 kilobases upstream of the murine hematopoietic-specific GATA-1 IE promoter. HS I directs high-level expression of reporter GATA-1/lacZ genes to primitive and definitive erythroid cells and megakaryocytes in transgenic mice. Comparative sequence analysis of HS I between human and mouse shows approx. 63% nucleotide identity with a more conserved core of 169 base pairs (86% identity). This core contains a GATA site separated by 10 base pairs from an E-box motif. The composite motif binds a multi-protein hematopoietic-specific transcription factor complex which includes GATA-1, SCL/tal-1, E2A, Lmo2 and Ldb-1. Point mutations of the GATA site abolishes HS I function, whereas mutation of the E-box motif still allows reporter gene expression in both lineages. Strict dependence of HS I activity on a GATA site implies that assembly of a protein complex containing a GATA-factor, presumably GATA-1 or GATA-2, is critical to activating or maintaining its function. Further dissection of the 317 base pair region demonstrates that, whereas all 317 base pairs are required for expression in megakaryocytes, only the 5′ 62 base pairs are needed for erythroid-specific reporter expression. These findings demonstrate differential lineage requirements for expression within the HS I element.

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The Stage-regulated Expression ofLeishmania mexicanaCPB Cysteine Proteases Is Mediated by an Intercistronic Sequence Element

Journal of Biological Chemistry ◽

10.1074/jbc.m108498200 ◽

2001 ◽

Vol 276 (50) ◽

pp. 47061-47069 ◽

Cited By ~ 44

Author(s):

Darren R. Brooks ◽

Hubert Denise ◽

Gareth D. Westrop ◽

Graham H. Coombs ◽

Jeremy C. Mottram

Keyword(s):

Untranslated Region ◽

Regulatory Element ◽

Cysteine Proteases ◽

Specific Gene ◽

Base Pairs ◽

Specific Expression ◽

Sequence Element ◽

Repeat Units ◽

Regulated Expression ◽

Specific Stage

The tandemly arrangedCPBgenes ofLeishmania mexicanaare polycistronically transcribed and encode cysteine proteases that are differentially stage-specific;CPB1andCPB2are expressed predominantly in metacyclics, whereasCPB3–CPB18are expressed mainly in amastigotes. The mechanisms responsible for this differential expression have been studied via gene analysis and re-integration of individualCPBgenes, and variants thereof, into aCPB-deficient parasite mutant. Comparison of the nucleotide sequences of the repeat units ofCPB1andCPB2withCPB2.8(typical ofCPB3–CPB18) revealed two major regions of divergence as follows: one of 258 base pairs (bp) corresponding to the C-terminal extension of CPB2.8; another, designated InS, of 120 bp, with insertions totaling 57 bp, localized to the intercistronic region downstream ofCPB1andCPB2. Cell lines expressingCPB2.8orCPB2with the 3′-untranslated region and intercistronic sequence ofCPB2.8showed up-regulation in amastigotes. Conversely, metacyclic-specific expression occurred withCPB2orCPB2.8with the 3′-untranslated region and intercistronic sequence ofCPB2. Moreover, the InS down-regulated expression in amastigotes of a reporter gene integrated into theCPBlocus. It is proposed that the InS mediates metacyclic-specific stage-regulated expression ofCPBby affecting the maturation of polycistronic pre-mRNA. This is the first well definedcis-regulatory element implicated in post-transcriptional stage-specific gene expression inLeishmania.

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Delimitation and characterization of cis-acting DNA sequences required for the regulated expression and transcriptional control of the chicken skeletal alpha-actin gene

Molecular and Cellular Biology ◽

10.1128/mcb.6.7.2462-2475.1986 ◽

1986 ◽

Vol 6 (7) ◽

pp. 2462-2475

Author(s):

D J Bergsma ◽

J M Grichnik ◽

L M Gossett ◽

R J Schwartz

Keyword(s):

Dna Sequences ◽

Transcriptional Control ◽

Regulatory Element ◽

Upstream Region ◽

Xenopus Laevis Oocytes ◽

Actin Gene ◽

Base Pairs ◽

Cis Acting ◽

Flanking Region ◽

Alpha Actin

We have previously observed that DNA sequences within the 5'-flanking region of the chicken skeletal alpha-actin gene harbor a cis-acting regulatory element that influences cell type and developmental stage-specific expression (J. M. Grichnik, D. J. Bergsma, and R. J. Schwartz, Nucleic Acids Res 14:1683-1701, 1986). In this report we have constructed unidirectional 5'-deletion and region-specific deletion-insertion mutations of the chicken skeletal alpha-actin upstream region and inserted these into the chloramphenicol acetyltransferase expression vector pSV0CAT. These constructions were used to locate DNA sequences that are required for developmental modulation of expression when transfected into differentiating myoblasts. With this assay we have delimited the 5' boundary of a cis-acting regulatory element to ca. 200 base pairs upstream of the mRNA cap site. In addition, we have preliminarily identified DNA sequences that may be important subcomponents within this element. A second major focus of this study was to identify those DNA signals within the regulatory element that control transcription. Toward this end, the expression phenotypes of progressive 5'-deletion and deletion-insertion mutants of the 5'-flanking region of the chicken skeletal alpha-actin gene were assayed in microinjected Xenopus laevis oocytes. These experiments defined a cis-acting transcriptional control region having a 5' border 107 base pairs preceding the alpha-actin RNA cap site. Proximal and distal functionally important regions of DNA were identified within this element. These DNA signals included within their DNA sequences the "CCAAT" and "TATA" box homologies.

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