Mutagenesis of the rat insulin II 5'-flanking region defines sequences important for expression in HIT cells.

To define the cis-acting elements important for rat insulin II gene expression, we analyzed the effects of 5' deletions and linker-scanning mutations on the expression of a rat insulin II reporter gene in an insulinoma cell line (HIT). The reporter gene contained 448 base pairs of 5'-flanking sequence joined to the bacterial chloramphenicol acetyltransferase gene. Expression of the 5' deletion mutations indicated that the minimal sequence requirement for efficient expression was 218 base pairs of 5'-flanking sequence, and at least three regions downstream from - 218 were important for transcription. A more precise localization of these elements and the cis-acting sequences in the promoter was achieved by analysis of the expression of 18 linker-scanning mutations. In these studies at least four other regions important for expression of the rat insulin II gene were identified. These findings suggest that the sequences important for rat insulin II and rat insulin I expression may differ significantly despite the high degree of sequence similarity in their 5'-flanking regions.

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Two Medfly Promoters That Have Originated by Recent Gene Duplication Drive Distinct Sex, Tissue and Temporal Expression Patterns

Genetics ◽

10.1093/genetics/156.1.173 ◽

2000 ◽

Vol 156 (1) ◽

pp. 173-182

Author(s):

George K Christophides ◽

Ioannis Livadaras ◽

Charalambos Savakis ◽

Katia Komitopoulou

Keyword(s):

Gene Expression ◽

Reporter Gene ◽

Fat Body ◽

Sequence Similarity ◽

Expression Patterns ◽

Gene Promoters ◽

Adult Males ◽

Specific Expression ◽

Cis Acting ◽

Genes Encoding

Abstract Genes encoding predominantly male-specific serum polypeptides (MSSPs) in the medfly Ceratitis capitata are members of a multigene family that are structurally similar to the genes encoding odorant binding proteins of insects. To study the transcriptional regulation of the genes MSSP-α2 and MSSP-β2, overlapping fragments of their promoters, containing the 5′ UTRs and 5′ flanking regions, were fused to the lacZ reporter gene and introduced into the medfly genome via Minos-mediated germline transformation. Transgenic flies were functionally assayed for β-galactosidase activity. Despite their extensive sequence similarity, the two gene promoters show distinct expression patterns of the reporter gene, consistent with previously reported evidence for analogous transcriptional activity of the corresponding endogenous genes. The MSSP-α2 promoter drives gene expression specifically in the fat body of the adult males, whereas the MSSP-β2 promoter directs gene expression in the midgut of both sexes. In contrast, similar transformation experiments in Drosophila melanogaster showed that both promoters drive the expression of the reporter gene in the midgut of adult flies of both sexes. Thus, the very same MSSP-α2 promoter fragment directs expression in the adult male fat body in Ceratitis, but in the midgut of both sexes in Drosophila. Our data suggest that through the evolution of the MSSP gene family a limited number of mutations that occurred within certain cis-acting elements, in combination with new medfly-specific trans-acting factors, endowed these recently duplicated genes with distinct sex-, tissue-, and temporal-specific expression patterns.

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Hematopoietic regulatory domain of gata1 gene is positively regulated by GATA1 protein in zebrafish embryos

Development ◽

10.1242/dev.128.12.2341 ◽

2001 ◽

Vol 128 (12) ◽

pp. 2341-2350

Author(s):

Makoto Kobayashi ◽

Keizo Nishikawa ◽

Masayuki Yamamoto

Keyword(s):

Gene Expression ◽

Reporter Gene ◽

Regulatory Region ◽

Ectopic Expression ◽

Transgenic Fish ◽

Functional Domain ◽

Cis Acting ◽

Gfp Reporter ◽

Gata Motif

Expression of gata1 is regulated through multiple cis-acting GATA motifs. To elucidate regulatory mechanisms of the gata1 gene, we have used zebrafish. To this end, we isolated and analyzed zebrafish gata1 genomic DNA, which resulted in the discovery of a novel intron that was unknown in previous analyses. This intron corresponds to the first intron of other vertebrate Gata1 genes. GFP reporter analyses revealed that this intron and a distal double GATA motif in the regulatory region are important for the regulation of zebrafish gata1 gene expression. To examine whether GATA1 regulates its own gene expression, we microinjected into embryos a GFP reporter gene linked successively to the gata1 gene regulatory region and to GATA1 mRNA. Surprisingly, ectopic expression of the reporter gene was induced at the site of GATA1 overexpression and was dependent on the distal double GATA motif. Functional domain analyses using transgenic fish lines that harbor the gata1-GFP reporter construct revealed that both the N- and C-terminal zinc-finger domains of GATA1, hence intact GATA1 function, are required for the ectopic GFP expression. These results provide the first in vivo evidence that gata1 gene expression undergoes positive autoregulation.

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Remarkable intron and exon sequence conservation in human and mouse homeobox Hox 1.3 genes

Molecular and Cellular Biology ◽

10.1128/mcb.9.5.2273-2278.1989 ◽

1989 ◽

Vol 9 (5) ◽

pp. 2273-2278

Author(s):

E Tournier-Lasserve ◽

W F Odenwald ◽

J Garbern ◽

J Trojanowski ◽

R A Lazzarini

Keyword(s):

Sequence Similarity ◽

Gene Clusters ◽

Binding Studies ◽

Base Pairs ◽

Cis Element ◽

Multiple Transcripts ◽

Exon Sequence ◽

Dna Binding Studies ◽

High Degree ◽

Sequence Similarities

A high degree of conservation exists between the Hox 1.3 homeobox genes of mice and humans. The two genes occupy the same relative positions in their respective Hox 1 gene clusters, they show extensive sequence similarities in their coding and noncoding portions, and both are transcribed into multiple transcripts of similar sizes. The predicted human Hox 1.3 protein differs from its murine counterpart in only 7 of 270 amino acids. The sequence similarity in the 250 base pairs upstream of the initiation codon is 98%, the similarity between the two introns, both 960 base pairs long, is 72%, and the similarity in the 3' noncoding region from termination codon to polyadenylation signal is 90%. Both mouse and human Hox 1.3 introns contain a sequence with homology to a mating-type-controlled cis element of the yeast Ty1 transposon. DNA-binding studies with a recombinant mouse Hox 1.3 protein identified two binding sites in the intron, both of which were within the region of shared homology with this Ty1 cis element.

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High levels of ripening-specific reporter gene expression directed by tomato fruit polygalacturonase gene-flanking regions

Plant Molecular Biology ◽

10.1007/bf00020391 ◽

1995 ◽

Vol 28 (3) ◽

pp. 423-435 ◽

Cited By ~ 56

Author(s):

Fiona J. Nicholass ◽

Christopher J.S. Smith ◽

Wolfgang Schuch ◽

Colin R. Bird ◽

Donald Grierson

Keyword(s):

Gene Expression ◽

Reporter Gene ◽

Tomato Fruit ◽

Reporter Gene Expression ◽

Flanking Regions ◽

Polygalacturonase Gene

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A Ty1 cell-type-specific regulatory sequence is a recognition element for a constitutive binding factor.

Molecular and Cellular Biology ◽

10.1128/mcb.8.12.5299 ◽

1988 ◽

Vol 8 (12) ◽

pp. 5299-5309 ◽

Cited By ~ 17

Author(s):

M Company ◽

B Errede

Keyword(s):

Gene Expression ◽

Binding Site ◽

Reporter Gene ◽

Base Pair ◽

Adjacent Gene ◽

Cell Type ◽

Cis Acting ◽

Cell Extracts ◽

Diploid Cells ◽

Constitutive Factor

Ty transposable-element insertion mutations of Saccharomyces cerevisiae can cause cell-type-dependent activation of adjacent-gene expression. Several cis-acting regulatory regions within Ty1 are responsible for the effect of Ty1 on adjacent-gene expression. One of these is the block II sequence that was defined by its homology to mammalian enhancers and to the yeast a1-alpha 2 control site. Tandem copies of a 57-base-pair region encompassing block II caused an additive increase in expression of the CYC7 reporter gene in the absence of other Ty1 sequences. The activation of gene expression by the multiple repeats was abolished in a/alpha diploid cells. A specific complex between a constitutive factor in whole-cell extracts and the DNA regulatory element was observed. The protein-binding site for the constitutive factor coincided with the block II element. Base-pair substitutions within the binding site abolished the ability of the block II element to function as a component of the Ty1 activator and to form the factor-DNA complex. The correlation between complex formation and reporter gene expression indicates that factor binding to the cis-acting element is essential for this element to function as a component of the Ty1 activator.

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Identification, Characterization, and Functional Analysis of a Gene Encoding the Ferric Uptake Regulation Protein inBartonella Species

Journal of Bacteriology ◽

10.1128/jb.183.19.5751-5755.2001 ◽

2001 ◽

Vol 183 (19) ◽

pp. 5751-5755 ◽

Cited By ~ 8

Author(s):

Sarah Y. Park ◽

Karen L. Kelminson ◽

Anthea K. Lee ◽

Peng Zhang ◽

Rachel E. Warner ◽

...

Keyword(s):

Gene Expression ◽

Functional Analysis ◽

Vibrio Cholerae ◽

Reporter Gene ◽

Isoelectric Point ◽

Promoter Activity ◽

Iron Acquisition ◽

Bartonella Henselae ◽

Gene Encoding ◽

Flanking Regions

ABSTRACT Environmental iron concentrations coordinately regulate transcription of genes involved in iron acquisition and virulence via the ferric uptake regulation (fur) system. We identified and sequenced the fur gene and flanking regions of threeBartonella species. The most notable difference betweenBartonella Fur and other Fur proteins was a substantially higher predicted isoelectric point. No promoter activity or Fur autoregulation was detected using a gfp reporter gene fused to the 204 nucleotides immediately upstream of the Bartonella fur gene. Bartonella henselae fur gene expression complemented a Vibrio cholerae fur mutant.

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A Ty1 cell-type-specific regulatory sequence is a recognition element for a constitutive binding factor

Molecular and Cellular Biology ◽

10.1128/mcb.8.12.5299-5309.1988 ◽

1988 ◽

Vol 8 (12) ◽

pp. 5299-5309

Author(s):

M Company ◽

B Errede

Keyword(s):

Gene Expression ◽

Binding Site ◽

Reporter Gene ◽

Base Pair ◽

Adjacent Gene ◽

Cell Type ◽

Cis Acting ◽

Cell Extracts ◽

Diploid Cells ◽

Constitutive Factor

Ty transposable-element insertion mutations of Saccharomyces cerevisiae can cause cell-type-dependent activation of adjacent-gene expression. Several cis-acting regulatory regions within Ty1 are responsible for the effect of Ty1 on adjacent-gene expression. One of these is the block II sequence that was defined by its homology to mammalian enhancers and to the yeast a1-alpha 2 control site. Tandem copies of a 57-base-pair region encompassing block II caused an additive increase in expression of the CYC7 reporter gene in the absence of other Ty1 sequences. The activation of gene expression by the multiple repeats was abolished in a/alpha diploid cells. A specific complex between a constitutive factor in whole-cell extracts and the DNA regulatory element was observed. The protein-binding site for the constitutive factor coincided with the block II element. Base-pair substitutions within the binding site abolished the ability of the block II element to function as a component of the Ty1 activator and to form the factor-DNA complex. The correlation between complex formation and reporter gene expression indicates that factor binding to the cis-acting element is essential for this element to function as a component of the Ty1 activator.

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Amylase cis-acting sequences mediate the alleviation of glucose repression by cAMP in Drosophila

Biochemistry and Cell Biology ◽

10.1139/o92-114 ◽

1992 ◽

Vol 70 (9) ◽

pp. 751-757 ◽

Cited By ~ 4

Author(s):

Charalambos Magoulas ◽

Ada Loverre-Chyurlia ◽

Donal A. Hickey

Keyword(s):

Gene Expression ◽

Glucose Repression ◽

Transcriptional Start Site ◽

Minimal Length ◽

Upstream Sequence ◽

Wild Type ◽

Amylase Gene ◽

Glucose Effect ◽

Base Pairs ◽

Cis Acting

α-Amylase gene expression is highly repressed by dietary glucose in Drosophila melanogaster. This glucose effect can be alleviated by exogenous adenosine 3′:5′-cyclic monophosphate (cAMP). Here, we show that the relief of glucose repression by cAMP occurs at the level of amylase mRNA abundance. Furthermore, exogenous cAMP was shown to alleviate glucose repression of the transient expression of an amylase gene construct in transformed Amynull larvae. This construct contains only 109 base pairs of the promoter region; this is the minimal length of upstream sequence which is necessary for wild-type levels of amylase gene expression. These results indicate that cis-acting promoter elements located close to the transcriptional start site of the Drosophila amylase gene mediate both glucose repression and the cAMP-derepression effects.Key words: Drosophila, glucose repression, cAMP effect, amylase gene.

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Cis-acting elements regulating the placenta-specific promoter of the bovine Cyp19 gene

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0250265 ◽

2000 ◽

Vol 25 (3) ◽

pp. 265-273 ◽

Cited By ~ 7

Author(s):

C Kalbe ◽

R Furbass ◽

M Schwerin ◽

J Vanselow

Keyword(s):

Reporter Gene ◽

Point Mutations ◽

Regulatory Elements ◽

Flanking Sequence ◽

Mobility Shift ◽

Enhancer Element ◽

Cis Acting ◽

Bovine Placenta ◽

Cyp19 Gene ◽

Key Enzyme

Cyp19 encodes aromatase cytochrome P450, the key enzyme of oestrogen biosynthesis. In the bovine placenta, the majority of Cyp19 transcripts include a 5' untranslated region which is encoded by exon 1.1; this suggests that its 5'-flanking region is the predominant placental promoter. The aim of the present investigation was to examine the promoter activity of this region and to map cis-acting regulatory elements in order to improve our understanding of the complex regulation of this gene within the placenta. As an initial approach, human JEG-3 choriocarcinoma cells were transiently transfected with reporter-gene constructs consisting of different 5'-flanking sequences of exon 1.1 fused to the luciferase gene as a reporter. To localise and further characterise functional cis-acting elements, targeted point mutations and electrophoretic mobility-shift experiments were used. The data demonstrate, for the first time, (1) that the bovine exon 1.1 5'-flanking sequence is an active promoter, (2) that 404 bp of this region are sufficient for constitutive reporter-gene expression in JEG-3 cells and (3) that the region includes at least two different enhancer elements; the data also suggest (4) that one of these elements consists of the E-box motif CATGTG and that the second enhancer element includes the half-site hexameric sequence AGGTCA and additional nucleotides flanking this element upstream.

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