scholarly journals Genomic organization, 5′-flanking region and chromosomal localization of the human glutathione transferase A4 gene

1998 ◽  
Vol 336 (2) ◽  
pp. 437-442 ◽  
Author(s):  
Fabienne DESMOTS ◽  
Claudine RAUCH ◽  
Catherine HENRY ◽  
André GUILLOUZO ◽  
Fabrice MOREL
Keyword(s):  
Transcription Initiation ◽  
Glutathione Transferase ◽  
Yeast Artificial Chromosome ◽  
Stop Codon ◽  
Artificial Chromosome ◽  
Initiation Site ◽  
Pcr Analysis ◽  
Small Nuclear Rna ◽  
Exon 2 ◽  
Flanking Region

We have isolated and characterized a human glutathione transferase A4 (hGSTA4) subunit gene from a yeast artificial chromosome containing several other glutathione transferase alpha genes and pseudogenes. The homodimeric protein hGSTA4-4, is involved in the detoxification of 4-hydroxynonenal and other reactive electrophiles produced by oxidative metabolism, and may have a significant role in protecting intracellular components from oxidative damage. The hGSTA4 gene spans nearly 18 kb, contains seven exons, maps onto chromosome 6p12, and lies in close proximity to the 7SK small nuclear RNA gene in a head-to-tail orientation. The intron/exon borders conform to the standard rules, an open reading frame is present beginning at position 154 in exon 2, and the stop codon is at position 822 in exon 7. The transcription initiation site has been determined by primer extension analysis and is located 135 bp upstream of intron 1. Isolation and sequencing of the hGSTA4 gene 5´-flanking region revealed it to be devoid of TATA or CCAAT boxes but it does contain an initiator element overlapping the transcription start site, a GC box and putative binding sites for transcription factors AP1, STAT, GATA1 and NF-κB. Reverse transcription-PCR analysis revealed that hGSTA4 mRNA was present in all the tissues tested, although in low amounts, suggesting that this subunit may be ubiquitously expressed.

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

1988 ◽  
Vol 8 (10) ◽  
pp. 4469-4476
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.

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

1988 ◽  
Vol 8 (10) ◽  
pp. 4469-4476 ◽  
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.

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

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.

Transgenic Analysis of a 100-kb Human β-Globin Cluster–Containing DNA Fragment Propagated as a Bacterial Artificial Chromosome

Blood ◽  
1999 ◽  
Vol 94 (9) ◽  
pp. 3178-3184 ◽  
Author(s):  
Richard M. Kaufman ◽  
Christine T.N. Pham ◽  
Timothy J. Ley
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Yeast Artificial Chromosome ◽  
Globin Gene ◽  
Artificial Chromosome ◽  
Pcr Analysis ◽  
Expression Levels ◽  
Dna Fragment ◽  
Polymerase Chain ◽  
Gene Switching ◽  
Intermediate Expression

To date, the normal transcriptional regulation of the human β-globin gene cluster has been recapitulated most accurately in transgenic mice that carry large yeast artificial chromosome (YAC) or ligated cosmid constructs. However, these large transgenes still exhibit variegated expression levels, perhaps because they tend to rearrange upon integration, or because the cloning vectors remain attached to the globin inserts. To try to circumvent these potential problems, we investigated the transgenic properties of a 100-kb DNA fragment containing the entire human β-globin cluster propagated in a bacterial artificial chromosome (BAC). We created 9 independent mouse lines, each carrying 1 to 6 copies of the human β-globin cluster without the attached BAC vector. Five of the lines carry unrearranged copies of the cluster. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of adult F1 mice showed that 2 lines express human β globin at levels approximately equivalent to the endogenous mouse β-major genes. One line expresses no human β globin, while the remaining 6 lines show intermediate expression levels. Complete γ→β-globin gene switching occurs, but is slightly delayed with respect to the endogenous mouse embryonic→adult switch. Since these data are similar to what has been obtained using globin YACs or ligated cosmids, we conclude that (1) globin transgenes propagated in BACs are no less likely to rearrange than their cosmid or YAC counterparts, and (2) the retention of YAC vector sequences in a transgene probably has no significant impact on globin expression when using constructs of this size.

scholarly journals Hepatocyte nuclear factor 6: organization and chromosomal assignment of the rat gene and characterization of its promoter

1998 ◽  
Vol 334 (3) ◽  
pp. 565-569 ◽  
Author(s):  
Mojgan RASTEGAR ◽  
Claude SZPIRER ◽  
Guy G. ROUSSEAU ◽  
Frédéric P. LEMAIGRE
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Chromosomal Assignment ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Specific Expression ◽  
Exon 2

Hepatocyte nuclear factor 6 (HNF-6) is the prototype of a family of tissue-specific transcription factors characterized by a bipartite DNA-binding domain consisting of a single cut domain and a novel type of homeodomain. We have previously cloned rat cDNA species coding for two isoforms, HNF-6α (465 residues) and β (491 residues), which differ only by the length of the spacer between the two DNA-binding domains. We have now localized the rat Hnf6 gene to chromosome 8q24–q31 by Southern blotting of DNA from somatic cell hybrids and by fluorescence in situhybridization. Cloning and sequencing of the rat gene showed that the two HNF-6 isoforms are generated by alternative splicing of three exons that are more than 10 kb apart from each other. Exon 1 codes for the N-terminal part and the cut domain, exon 2 codes for the 26 HNF-6β-specific amino acids, and exon 3 codes for the homeodomain and the C-terminal amino acids. The transcription initiation site was mapped by ribonuclease protection and 5´ rapid amplification of cDNA ends. Transfection experiments showed that promoter activity was contained within 0.75 kb upstream of the transcription initiation site. This activity was detected by the transfection of liver-derived HepG2 cells, but not of Rat-1 fibroblasts, suggesting that the promoter is sufficient to confer liver-specific expression.

Characterization of the rat NHE3 promoter

1996 ◽  
Vol 271 (3) ◽  
pp. F629-F636 ◽  
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.

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

1997 ◽  
Vol 324 (3) ◽  
pp. 927-934 ◽  
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.

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

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.

Transgenic Analysis of a 100-kb Human β-Globin Cluster–Containing DNA Fragment Propagated as a Bacterial Artificial Chromosome

Blood ◽  
1999 ◽  
Vol 94 (9) ◽  
pp. 3178-3184 ◽  
Author(s):  
Richard M. Kaufman ◽  
Christine T.N. Pham ◽  
Timothy J. Ley
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Yeast Artificial Chromosome ◽  
Globin Gene ◽  
Artificial Chromosome ◽  
Pcr Analysis ◽  
Expression Levels ◽  
Dna Fragment ◽  
Polymerase Chain ◽  
Gene Switching ◽  
Intermediate Expression

Abstract To date, the normal transcriptional regulation of the human β-globin gene cluster has been recapitulated most accurately in transgenic mice that carry large yeast artificial chromosome (YAC) or ligated cosmid constructs. However, these large transgenes still exhibit variegated expression levels, perhaps because they tend to rearrange upon integration, or because the cloning vectors remain attached to the globin inserts. To try to circumvent these potential problems, we investigated the transgenic properties of a 100-kb DNA fragment containing the entire human β-globin cluster propagated in a bacterial artificial chromosome (BAC). We created 9 independent mouse lines, each carrying 1 to 6 copies of the human β-globin cluster without the attached BAC vector. Five of the lines carry unrearranged copies of the cluster. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of adult F1 mice showed that 2 lines express human β globin at levels approximately equivalent to the endogenous mouse β-major genes. One line expresses no human β globin, while the remaining 6 lines show intermediate expression levels. Complete γ→β-globin gene switching occurs, but is slightly delayed with respect to the endogenous mouse embryonic→adult switch. Since these data are similar to what has been obtained using globin YACs or ligated cosmids, we conclude that (1) globin transgenes propagated in BACs are no less likely to rearrange than their cosmid or YAC counterparts, and (2) the retention of YAC vector sequences in a transgene probably has no significant impact on globin expression when using constructs of this size.

Glucocorticoids stimulate human sgk1 gene expression by activation of a GRE in its 5′-flanking region

2002 ◽  
Vol 283 (5) ◽  
pp. E971-E979 ◽  
Author(s):  
Omar A. Itani ◽  
Kang Z. Liu ◽  
Kristyn L. Cornish ◽  
Jason R. Campbell ◽  
Christie P. Thomas
Keyword(s):  
Transcription Initiation ◽  
Collecting Duct ◽  
A549 Cells ◽  
Initiation Site ◽  
Specific Protein ◽  
Upstream Region ◽  
Mobility Shift ◽  
Cell Extracts ◽  
Flanking Region ◽  
Gel Mobility Shift

In lung and collecting duct epithelia, glucocorticoid (GC)-stimulated Na+ transport is preceded by an increase in the protein kinase sgk1, which in turn regulates the activity of the epithelial Na+ channel (ENaC). We investigated the mechanism for GC-regulated human sgk1 expression in lung and renal epithelia. sgk1 mRNA was increased in these epithelia by GCs, and this was inhibited by actinomycin D and superinduced by cycloheximide, consistent with a transcriptional effect that did not require protein synthesis. To understand the basis for transcriptional regulation, the transcription initiation site was mapped and the 5′-flanking region cloned by PCR. A 3-kb fragment of the upstream region was coupled to luciferase and transfected into A549 cells. By deletion analysis, an imperfect GC response element (GRE) was identified that was necessary and sufficient for GC responsiveness. When tested with cell extracts, a specific protein recognized by an anti-GC receptor (GR) antibody bound the GRE in gel mobility shift assays. We conclude that GCs stimulate sgk1 expression in human epithelial cells via activation of a GRE in the 5′-flanking region of sgk1.

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