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

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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Molecular characterization of the Drosophila melanogaster urate oxidase gene, an ecdysone-repressible gene expressed only in the malpighian tubules

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5114-5127.1990 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5114-5127

Author(s):

L L Wallrath ◽

J B Burnett ◽

T B Friedman

Keyword(s):

Drosophila Melanogaster ◽

Amino Acid ◽

Regulatory Element ◽

Malpighian Tubule ◽

Amino Acid Sequences ◽

Malpighian Tubules ◽

P Element ◽

Urate Oxidase ◽

Base Pairs ◽

Oxidase Gene

The urate oxidase (UO) gene of Drosophila melanogaster is expressed during the third-instar larval and adult stages, exclusively within a subset of cells of the Malpighian tubules. The UO gene contains a 69-base-pair intron and encodes mature mRNAs of 1,224, 1,227, and 1,244 nucleotides, depending on the site of 3' endonucleolytic cleavage prior to polyadenylation. A direct repeat, 5'-AAGTGAGAGTGAT-3', is the proposed cis-regulatory element involved in 20-hydroxyecdysone repression of the UO gene. The deduced amino acid sequences of UO of D. melanogaster, rat, mouse, and pig and uricase II of soybean show 32 to 38% identity, with 22% of amino acid residues identical in all species. With use of P-element-mediated germ line transformation, 826 base pairs 5' and approximately 1,200 base pairs 3' of the D. melanogaster UO transcribed region contain all of the cis elements allowing for appropriate temporal regulation and Malpighian tubule-specific expression of the UO gene.

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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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Molecular characterization of the Drosophila melanogaster urate oxidase gene, an ecdysone-repressible gene expressed only in the malpighian tubules.

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5114 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5114-5127 ◽

Cited By ~ 31

Author(s):

L L Wallrath ◽

J B Burnett ◽

T B Friedman

Keyword(s):

Drosophila Melanogaster ◽

Amino Acid ◽

Regulatory Element ◽

Malpighian Tubule ◽

Amino Acid Sequences ◽

Malpighian Tubules ◽

P Element ◽

Urate Oxidase ◽

Base Pairs ◽

Oxidase Gene

The urate oxidase (UO) gene of Drosophila melanogaster is expressed during the third-instar larval and adult stages, exclusively within a subset of cells of the Malpighian tubules. The UO gene contains a 69-base-pair intron and encodes mature mRNAs of 1,224, 1,227, and 1,244 nucleotides, depending on the site of 3' endonucleolytic cleavage prior to polyadenylation. A direct repeat, 5'-AAGTGAGAGTGAT-3', is the proposed cis-regulatory element involved in 20-hydroxyecdysone repression of the UO gene. The deduced amino acid sequences of UO of D. melanogaster, rat, mouse, and pig and uricase II of soybean show 32 to 38% identity, with 22% of amino acid residues identical in all species. With use of P-element-mediated germ line transformation, 826 base pairs 5' and approximately 1,200 base pairs 3' of the D. melanogaster UO transcribed region contain all of the cis elements allowing for appropriate temporal regulation and Malpighian tubule-specific expression of the UO gene.

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

Molecular and Cellular Biology ◽

10.1128/mcb.9.11.4660-4669.1989 ◽

1989 ◽

Vol 9 (11) ◽

pp. 4660-4669

Author(s):

J Pavlovic ◽

B Haribabu ◽

R P Dottin

Keyword(s):

Signal Transduction ◽

Cyclic Amp ◽

Dictyostelium Discoideum ◽

Regulatory Element ◽

Response Sequence ◽

Base Pairs ◽

Camp Response Element ◽

Sequence Element ◽

Cis Acting ◽

Gene Coding

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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Hypomorphic mutations in the larval photokinesis A (lphA) gene have stage-specific effects on visual system function in Drosophila melanogaster.

Genetics ◽

10.1093/genetics/139.4.1623 ◽

1995 ◽

Vol 139 (4) ◽

pp. 1623-1629

Author(s):

B Gordesky-Gold ◽

J M Warrick ◽

A Bixler ◽

J E Beasley ◽

L Tompkins

Keyword(s):

Drosophila Melanogaster ◽

Visual System ◽

Specific Aspect ◽

System Structure ◽

P Element ◽

System Function ◽

Specific Effects ◽

Isolation And Characterization ◽

In Trans ◽

The Many

Abstract Of the many genes that are expressed in the visual system of Drosophila melanogaster adults, some affect larval vision. However, with the exception of one X-linked mutation, no genes that have larval-specific effects on visual system structure or function have previously been reported. We describe the isolation and characterization of two mutant alleles that define the larval photokinesis A (lphA) gene, one allele of which is associated with a P-element insertion at cytogenetic locus 8E1-10. Larvae that express lphA mutations are, like normal animals, negatively photokinetic, but they are less responsive to white light than lphA + controls. Larvae that are heterozygous in trans for a mutant lphA allele and a deficiency that uncovers the lphA locus are blind, which indicates that the mutant allele is hypomorphic. lphA larvae respond normally to odorants and taste stimuli. Moreover, the lphA mutations do not affect adult flies' fast phototaxis or visually driven aspects of male sexual behavior, and electroretinograms recorded from the compound eyes of lphA/deficiency heterozygotes and lphA1/lphA2 females are normal. These observations suggest that the lphA gene affects a larval-specific aspect of visual system function.

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Identification of an evolutionarily conserved 110 base-pair cis-acting regulatory sequence that governs Wnt-1 expression in the murine neural plate

Development ◽

10.1242/dev.125.14.2735 ◽

1998 ◽

Vol 125 (14) ◽

pp. 2735-2746 ◽

Cited By ~ 3

Author(s):

D.H. Rowitch ◽

Y. Echelard ◽

P.S. Danielian ◽

K. Gellner ◽

S. Brenner ◽

...

Keyword(s):

Base Pair ◽

Regulatory Element ◽

Regulatory Region ◽

Neural Plate ◽

Homologous Region ◽

Regulatory Sequence ◽

Embryonic Mouse ◽

Cis Acting ◽

Evolutionarily Conserved

The generation of anterior-posterior polarity in the vertebrate brain requires the establishment of regional domains of gene expression at early somite stages. Wnt-1 encodes a signal that is expressed in the developing midbrain and is essential for midbrain and anterior hindbrain development. Previous work identified a 5.5 kilobase region located downstream of the Wnt-1 coding sequence which is necessary and sufficient for Wnt-1 expression in vivo. Using a transgenic mouse reporter assay, we have now identified a 110 base pair regulatory sequence within the 5.5 kilobase enhancer, which is sufficient for expression of a lacZ reporter in the approximate Wnt-1 pattern at neural plate stages. Multimers of this element driving Wnt-1 expression can partially rescue the midbrain-hindbrain phenotype of Wnt-1(−/−) embryos. The possibility that this region represents an evolutionarily conserved regulatory module is suggested by the identification of a highly homologous region located downstream of the wnt-1 gene in the pufferfish (Fugu rubripes). These sequences are capable of appropriate temporal and spatial activation of a reporter gene in the embryonic mouse midbrain; although, later aspects of the Wnt-1 expression pattern are absent. Genetic evidence has implicated Pax transcription factors in the regulation of Wnt-1. Although Pax-2 binds to the 110 base pair murine regulatory element in vitro, the location of the binding sites could not be precisely established and mutation of two putative low affinity sites did not abolish activation of a Wnt-1 reporter transgene in vivo. Thus, it is unlikely that Pax proteins regulate Wnt-1 by direct interactions with this cis-acting regulatory region. Our analysis of the 110 base pair minimal regulatory element suggests that Wnt-1 regulation is complex, involving different regulatory interactions for activation and the later maintenance of transgene expression in the dorsal midbrain and ventral diencephalon, and at the midbrain-hindbrain junction.

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A negative regulatory element with properties similar to those of enhancers is contained within an Alu sequence

Molecular and Cellular Biology ◽

10.1128/mcb.9.2.355-364.1989 ◽

1989 ◽

Vol 9 (2) ◽

pp. 355-364

Author(s):

J D Saffer ◽

S J Thurston

Keyword(s):

Nuclear Protein ◽

Regulatory Element ◽

Simian Virus 40 ◽

Bidirectional Promoter ◽

Simian Virus ◽

Beta Globin ◽

Base Pairs ◽

Negative Regulatory Element ◽

Alu Sequence ◽

Green Monkey

A negative regulatory element has been found within a member of the African green monkey Alu family of interspersed repeated sequences. This "reducer" element decreased transcription in both directions from a cellular simian virus 40-like bidirectional promoter independently of both orientation and position. The reducer was not promoter specific since it also decreased expression from the simian virus 40 early and human beta-globin promoters. In addition, the reducer decreased transcription from a polymerase III promoter. The reducer was contained in 38 base pairs of an Alu family member and interacted specifically with a monkey cell nuclear protein.

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Sequences involved in temperature and ecdysterone-induced transcription are located in separate regions of a Drosophila melanogaster heat shock gene.

Molecular and Cellular Biology ◽

10.1128/mcb.6.2.663 ◽

1986 ◽

Vol 6 (2) ◽

pp. 663-673 ◽

Cited By ~ 32

Author(s):

E Hoffman ◽

V Corces

Keyword(s):

Drosophila Melanogaster ◽

Heat Shock ◽

Dna Sequences ◽

Germ Line ◽

Initiation Site ◽

P Element ◽

Heat Shock Gene ◽

Base Pairs ◽

Consensus Sequences ◽

Shock Gene

The transcriptional regulation of the Drosophila melanogaster hsp27 (also called hsp28) gene was studied by introducing altered genes into the germ line by P element-mediated transformation. DNA sequences upstream of the gene were defined with respect to their effect on steroid hormone-induced and heat-induced transcription. These two types of control were found to be separable; the sequences responsible for 80% of heat-induced expression were located more than 1.1 kilobases upstream of the RNA initiation site, while the sequences responsible for the majority of ecdysterone induction were positioned downstream of the site at -227 base pairs. We have determined the DNA sequence of the intergenic region separating hsp23 and hsp27 and have located putative heat shock and ecdysterone consensus sequences. Our results indicate that the heat shock promoter of the hsp27 gene is organized quite differently from that of hsp70.

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A negative regulatory element in the bcl-2 5'-untranslated region inhibits expression from an upstream promoter.

Molecular and Cellular Biology ◽

10.1128/mcb.13.6.3686 ◽

1993 ◽

Vol 13 (6) ◽

pp. 3686-3697 ◽

Cited By ~ 132

Author(s):

R L Young ◽

S J Korsmeyer

Keyword(s):

B Cell ◽

Cell Lines ◽

Untranslated Region ◽

Regulatory Element ◽

Cell Development ◽

Negative Control ◽

B Cell Development ◽

Negative Regulatory Element ◽

Cis Acting ◽

B Lineage

bcl-2 mRNA is present at high levels in pre-B-cell lines but is down-regulated in most mature B-cell lines. To investigate the mechanisms responsible for its developmental control, we studied the regulation of bcl-2 expression in human B-lineage cell lines. Using nuclear run-on assays, we found that bcl-2 transcription decreases in parallel with levels of steady-state mRNA during B-cell development. To define cis-acting elements that regulate bcl-2 transcription, we analyzed the expression of transiently transfected promoter-reporter constructs. We identified a novel negative regulatory element (NRE) in the bcl-2 5'-untranslated region that decreased expression from the bcl-2 P1 promoter or heterologous promoters in a position-dependent fashion. The NRE functions in either orientation but contains distinct orientation-dependent subfragments. Additional analyses demonstrated that multiple, functionally redundant sequence elements mediate NRE activity. Though the bcl-2 NRE is active in pre-B- and mature B-cell lines, chromatin structure of the endogenous NRE differs in these cells, suggesting that its activity or effect may vary during B-cell development. Our results indicate that negative control of transcription initiated at the P1 promoter is an important determinant of the differential expression of bcl-2.

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