Characterization of the transcription-initiation site and of the promoter region within the 5' flanking region of the human aldolase C gene

The trout metallothionein (MT) genes consist of two members. We describe the structure of the first fish MT (tMT-B) gene which shows an overall resemblance but some remarkable differences with mammalian MT genes. The similarities included (i) tripartite structure of the gene, (ii) conservation of cysteine residues, and (iii) a TATAAA signal and two copies of metal-responsive elements (MREs). The differences consisted of (i) an AT-rich tMT-B promoter compared with highly GC-rich mammalian MT promoters and (ii) a lack of SP1-binding sites in the tMT-B promoter. Functional analysis of the tMT-B 5'-flanking region following fusion with the bacterial chloramphenicol acetyltransferase gene and its transfection into the rainbow trout hepatoma cell line revealed that sequences from positions -600 to +8 are sufficient for regulation by metals. Further deletion analyses of this fragment suggested that a minimum of 100 nucleotides upstream of the transcription initiation site are required for induction by cadmium and zinc. The tMT-B promoter was also functional in the human hepatoblastoma cell line, suggesting that an MT regulatory factor(s) is conserved in phylogenetically distant species like humans and fish.

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Structure of the rainbow trout metallothionein B gene and characterization of its metal-responsive region.

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4469 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4469-4476 ◽

Cited By ~ 71

Author(s):

M Zafarullah ◽

K Bonham ◽

L Gedamu

Keyword(s):

Rainbow Trout ◽

Cell Line ◽

Binding Sites ◽

Transcription Initiation ◽

Hepatoma Cell ◽

Initiation Site ◽

Hepatoma Cell Line ◽

Flanking Region ◽

Chloramphenicol Acetyltransferase Gene

The trout metallothionein (MT) genes consist of two members. We describe the structure of the first fish MT (tMT-B) gene which shows an overall resemblance but some remarkable differences with mammalian MT genes. The similarities included (i) tripartite structure of the gene, (ii) conservation of cysteine residues, and (iii) a TATAAA signal and two copies of metal-responsive elements (MREs). The differences consisted of (i) an AT-rich tMT-B promoter compared with highly GC-rich mammalian MT promoters and (ii) a lack of SP1-binding sites in the tMT-B promoter. Functional analysis of the tMT-B 5'-flanking region following fusion with the bacterial chloramphenicol acetyltransferase gene and its transfection into the rainbow trout hepatoma cell line revealed that sequences from positions -600 to +8 are sufficient for regulation by metals. Further deletion analyses of this fragment suggested that a minimum of 100 nucleotides upstream of the transcription initiation site are required for induction by cadmium and zinc. The tMT-B promoter was also functional in the human hepatoblastoma cell line, suggesting that an MT regulatory factor(s) is conserved in phylogenetically distant species like humans and fish.

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Genomic footprinting of a yeast tRNA gene reveals stable complexes over the 5'-flanking region.

Molecular and Cellular Biology ◽

10.1128/mcb.9.8.3244 ◽

1989 ◽

Vol 9 (8) ◽

pp. 3244-3252 ◽

Cited By ~ 37

Author(s):

J M Huibregtse ◽

D R Engelke

Keyword(s):

Stationary Phase ◽

Transcription Initiation ◽

Initiation Site ◽

Dnase I ◽

Transcription Initiation Site ◽

Upstream Region ◽

Stable Complex ◽

Coding Region ◽

Flanking Region ◽

Genomic Footprinting

We have shown by genomic footprinting that the 5'-flanking region of the Saccharomyces cerevisiae tRNASUP53 gene is protected from DNase I digestion. The protected region has a 5' boundary at -40 (relative to the transcription initiation site) and extends into the coding region of the gene, with a 3' boundary at approximately +15. Although the DNase I protection over this region was much greater than at the A- and B-box internal promoters, point mutations within the A or B box that reduced transcription in vitro eliminated the upstream DNase I protection. This implies that formation of a stable complex over the 5'-flanking region is dependent on interaction of the gene with transcription factor IIIC but that stability of the complex may not require continued interaction with this factor. The DNase I protection under varied growth conditions further suggested that the upstream complex is composed of two or more components. The region over the transcription initiation site (approximately +15 to -10) was less protected in stationary-phase cultures, whereas the more upstream region (approximately -10 to -40) was protected in both exponential- and stationary-phase cultures.

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Characterization of the rat NHE3 promoter

AJP Renal Physiology ◽

10.1152/ajprenal.1996.271.3.f629 ◽

1996 ◽

Vol 271 (3) ◽

pp. F629-F636 ◽

Cited By ~ 20

Author(s):

A. Cano

Keyword(s):

Transcriptional Regulation ◽

Transcription Initiation ◽

Transmembrane Protein ◽

Initiation Site ◽

Proximal Tubule Cell ◽

Specific Expression ◽

Genomic Libraries ◽

Sequence Elements ◽

Flanking Region

NHE3, a transmembrane protein involved in transcellular ion transport, is expressed in the apical membrane of renal and gastrointestinal epithelia. Chronic regulation by multiple stimuli, including glucocorticoid-induced transcriptional regulation, has been demonstrated. To study the tissue-specific expression and transcriptional regulation of NHE3, the 5' flanking region of the rat NHE3 gene was cloned. Two genomic libraries were screened with the 5' end of the NHE3 cDNA. The 5' flanking region and first exon were isolated. Primer extension mapped a single transcription start site in stomach, colon and kidney. The NHE3 promoter near the transcription initiation site is characterized by the absence of TATA and CAAT sequences. Two Sp1 sites, one Egr-1 site, and an initiator with the sequence GGGATTAAA mark the area of transcription initiation. Upstream sequences include multiple DNA sequence elements recognized by the glucocorticoid and thyroid receptors, Sp1, atriopeptin-2, and several other transcription factors. Transcriptional regulation by glucocorticoids and chronic acidosis was demonstrated. Promoter activity was present in OKP cells, a renal proximal tubule cell line, but not in fibroblasts. This suggests that the NHE3 promoter contains elements conferring epithelial cell-specific expression.

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Gene structure and functional analysis of the human Na+/phosphate co-transporter

Biochemical Journal ◽

10.1042/bj3240927 ◽

1997 ◽

Vol 324 (3) ◽

pp. 927-934 ◽

Cited By ~ 35

Author(s):

Yutaka TAKETANI ◽

Ken-ichi MIYAMOTO ◽

Keiko TANAKA ◽

Kanako KATAI ◽

Mika CHIKAMORI ◽

...

Keyword(s):

Vitamin D ◽

Vitamin D Receptor ◽

Transcription Initiation ◽

Thyroid Hormone Receptor ◽

Direct Repeat ◽

Initiation Site ◽

Transcription Initiation Site ◽

Receptor Expression ◽

Specific Gene ◽

Flanking Region

Three λ phage clones encompassing the Na+/phosphate co-transporter (NaPi-3) gene and its 5′ flanking region were isolated from a human genomic DNA library. The gene comprises 13 exons and 12 introns and spans approx. 14 kb. All exon–intron junctions conform to the GT/AG rule. The major transcription-initiation site was determined by primer-extension analysis and is an adenosine residue 57 bp upstream of the 3′ end of the first exon. There is a typical TATA box 28 bp upstream of the major transcription-initiation site and various cis-acting elements, including a cAMP-responsive element, AP-1, AP-2 and SP-1 sites in the 5′ flanking region. This region also contains three direct-repeat-like sequences that resemble the consensus binding sequence for members of the steroid–thyroid hormone receptor superfamily, including vitamin D. Deletion analysis suggests that the region from nt-2409 to nt-1259 in the 5′ flanking region may be involved in kidney-specific gene expression. Vitamin D responsiveness of the NaPi-3 promoter was also detected in COS-7 cells co-transfected with a human vitamin D receptor expression vector. The presence of the three vitamin D receptor-responsive elements in the NaPi-3 promoter may be important in mediating the enhanced expression of the gene by 1,25-dihydroxyvitamin D3.

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Genomic footprinting of a yeast tRNA gene reveals stable complexes over the 5'-flanking region

Molecular and Cellular Biology ◽

10.1128/mcb.9.8.3244-3252.1989 ◽

1989 ◽

Vol 9 (8) ◽

pp. 3244-3252

Author(s):

J M Huibregtse ◽

D R Engelke

Keyword(s):

Stationary Phase ◽

Transcription Initiation ◽

Initiation Site ◽

Dnase I ◽

Transcription Initiation Site ◽

Upstream Region ◽

Stable Complex ◽

Coding Region ◽

Flanking Region ◽

Genomic Footprinting

We have shown by genomic footprinting that the 5'-flanking region of the Saccharomyces cerevisiae tRNASUP53 gene is protected from DNase I digestion. The protected region has a 5' boundary at -40 (relative to the transcription initiation site) and extends into the coding region of the gene, with a 3' boundary at approximately +15. Although the DNase I protection over this region was much greater than at the A- and B-box internal promoters, point mutations within the A or B box that reduced transcription in vitro eliminated the upstream DNase I protection. This implies that formation of a stable complex over the 5'-flanking region is dependent on interaction of the gene with transcription factor IIIC but that stability of the complex may not require continued interaction with this factor. The DNase I protection under varied growth conditions further suggested that the upstream complex is composed of two or more components. The region over the transcription initiation site (approximately +15 to -10) was less protected in stationary-phase cultures, whereas the more upstream region (approximately -10 to -40) was protected in both exponential- and stationary-phase cultures.

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Sp1 and Sp3 control constitutive expression of the human NHE2 promoter by interactions with the proximal promoter and the transcription initiation site

Biochemical Journal ◽

10.1042/bj20070364 ◽

2007 ◽

Vol 407 (1) ◽

pp. 101-111 ◽

Cited By ~ 9

Author(s):

Ian Pearse ◽

Ying X. Zhu ◽

Eleanor J. Murray ◽

Pradeep K. Dudeja ◽

Krishnamurthy Ramaswamy ◽

...

Keyword(s):

Transcription Initiation ◽

Promoter Region ◽

Promoter Activity ◽

Critical Role ◽

Constitutive Expression ◽

Regulatory Elements ◽

Initiation Site ◽

Transcription Initiation Site ◽

Proximal Promoter ◽

Gc Box

We have previously cloned the human Na+/H+ exchanger NHE2 gene and its promoter region. In the present study, the regulatory elements responsible for the constitutive expression of NHE2 were studied. Transient transfection assays revealed that the −40/+150 promoter region contains the core promoter responsible for the optimal promoter activity. A smaller fragment, −10/+40, containing the TIS (transcription initiation site) showed minimal activity. We identified a palindrome that overlaps the TIS and binds to the transcription factors Sp1 and Sp3. Mutations in the 5′ flank of the palindrome abolished the Sp1/Sp3 interaction and reduced promoter activity by approx. 45%. In addition, a conserved GC-box centered at −25 was found to play a critical role in basal promoter activity and also interacted with Sp1 and Sp3. An internal deletion in the GC-box severely reduced the promoter activity. Sp1/Sp3 binding to these elements was established using gel-mobility shift assays, confirmed by chromatin immunoprecipitation and co-transfections in Drosophila SL2 cells. Furthermore, we identified two positive regulatory elements in the DNA region corresponding to the 5′-UTR (5′-untranslated region). The results in the present study indicate that Sp1 and Sp3 are required for constitutive NHE2 expression and that the positive regulatory elements of the 5′-UTR may co-operate with the 5′-flanking region to achieve the optimal promoter activity.

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Characterization of the Promoter of PRS1 inSaccharomyces cerevisiae Identifies Three Regions Potentially Involved in Control of Expression

Journal of Bacteriology ◽

10.1128/jb.183.2.795-799.2001 ◽

2001 ◽

Vol 183 (2) ◽

pp. 795-799 ◽

Cited By ~ 1

Author(s):

Yolanda Hernando ◽

Andrew T. Carter ◽

Stefan Sickinger ◽

Michael Schweizer

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcription Initiation ◽

Initiation Site ◽

Transcription Initiation Site ◽

Galactosidase Activity ◽

Shift Analysis ◽

Gel Shift ◽

Control Of Expression

ABSTRACT The transcription initiation site of the Saccharomyces cerevisiae PRS1 gene was mapped at −179 bp. Measurement of β-galactosidase activity of the successively deleted PRS1promoter linked to lacZ and integrated at theura3 locus defined three DNA regions involved in the control of PRS1 expression. Gel shift analysis confirmed the data.

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