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Molecular cloning, sequencing and functional study of the promoter region of the human alpha2C4-adrenergic receptor gene.

ABSTRACT: Screening of a human foetal brain genomic DNA library allowed us to isolate an EcoRI-EcoRI fragment containing 6 kb of the 5'-flanking region, the open reading frame and 4 kb of the 3'-flanking region of the alpha2C4 gene. Analysis of the sequenced region (4850 bp) revealed that the first 900 bp 5' to the start codon are very rich in GC (84%), contain several Sp1-binding sites and lack a consensus TATA box. The 5'- and 3'-ends of the alpha2C4 transcript were determined by RNase-protection assays carried out with a series of antisense probes. The data obtained with cellular RNA from HepG2 cells demonstrated that transcription is initiated 891 bases upstream of the translation-start site and that the polyadenylation site is located 550 bases downstream of the stop codon. These results are consistent with the existence of a non-conventional TATA box (TTAGAAA) and the presence of a unique polyadenylation signal (AATAAA). They also fit with the size of alpha2C4-RNA found by Northern-blot analysis (2.9 kb). The transcriptional activity of the alpha2C4 promoter region was investigated by transfecting several cell types with chimaeric constructs containing various fragments of the 5'-non-coding region and luciferase as a reporter gene. The activity of the construct containing the entire 5'-non-coding region appeared to depend on the host cell. Removal of the 5'-untranslated region resulted in loss of cell specificity and a concomitant increase in luciferase activity. Transfection of HepG2 and SK-N-MC cells with constructs deleted of additional 5'-flanking fragments permitted the definition of a minimal 200 bp promoter fragment containing the pseudo-TATA box and two putative SP1-binding sites.

SUBMITTER: Schaak S

PROVIDER: S-EPMC1218938 | biostudies-other | 1997 Dec

REPOSITORIES: biostudies-other

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Project description:Acetoacetate decarboxylase (ADC) (EC4.1.1.4) of Clostridium acetobutylicum DSM 792 was purified to homogeneity, and its first 25 N-terminal amino acids were determined. Oligonucleotide probes deduced from this sequence were used to detect positive clones in partial gene banks derived from Sau3A and HaeIII digests with following ligation into the vector pUC9. In Escherichia coli, the 2.1-kbp HaeIII clones expressed high levels of ADC activity. The expression was independent of the orientation of the insert with respect to the lac promoter of the vector and also of the addition of isopropyl-beta-D-thiogalactopyranoside, thus indicating that sequences located on the clostridial DNA controlled transcription and translation. From the E. coli clone with the recombinant plasmid pUG93 containing the 2.1-kbp HaeIII fragment, the ADC protein was purified and compared with the native enzyme. Both were indistinguishable with respect to the molecular mass of subunits and native protein as well as to activity stain. The 2.9-kbp Sau3A fragment could be shown to contain the amino terminus of the acetoacetate decarboxylase (adc) gene but did not express enzyme activity. It partially overlapped with the HaeIII fragment, spanning together 4,053 bp of the clostridial genome that were completely sequenced. Four open reading frames (ORFs) could be detected, one of which was unambiguously assigned to the acetoacetate decarboxylase (adc) gene. Amino acid sequences of the N terminus and the catalytic center as deduced from the nucleotide sequence were identical to sequences obtained from direct analysis of the protein. Typical procaryotic transcriptional and translational start and stop signals could be found in the DNA sequence. Together with these regulatory sequences, the adc gene formed a single operon. The carboxyl terminus of the enzyme proved to be rather hydrophobic. In vitro transcription-translation assays resulted in formation of ADC and ORF3 gene product; the other two ORFs were not expressed. Whereas no homology of the adc gene and ORF2 could be detected with sequences available in the EMBL or GenBank data bases, the obviously truncated ORF1 showed significant similarity to alpha-amylase of Bacillus subtilis. The restriction pattern and N-terminal amino acid sequence (as deduced from the nucleotide sequence) of ORF3 proved to be identical to those of the large subunit of acetoacetyl coenzyme A:acetate/butyrate:coenzyme A transferase.

Project description:Cyclin D1 is a cell cycle regulatory protein, which acts as a growth factor sensor to integrate extracellular signals with the cell cycle machinery, particularly during G1 phase of the cell cycle. Previous study using promotion-sensitive JB6 mouse epidermal cells, an in vitro model of the promotion stage of multistage carcinogenesis, showed that the expression of cyclin D1 is stimulated in the presence (but not in the absence) of 12-O-tetradecanoylphorbol-13-acetate (TPA) in these cells maintained under anchorage-independent culture conditions. In the present study, to explore the molecular basis of this observation, the promoter region of mouse cyclin D1 gene was cloned and sequenced (GenBank accession number AF212040). Dot matrix comparison of mouse, human and rat promoter sequences indicated that the mouse promoter is homologous to the human and more so to the rat promoters. The mouse promoter, like human and rat promoters, lacks canonical TATA-box or TATA-like sequence, but it has one or possibly two initiator (Inr) or Inr-like sequences. Energy dot plot analysis predicted that the mouse promoter consists of three domains: (1) the 3' domain contains NF-kappaB response element, cAMP-response element (CRE), Inr or Inr-like elements, Sp1 binding site and Oct 1 (2) the middle domain contains another Sp1 binding site, E-box and E2F binding site and (3) the 5' domain contains TPA-response element (TRE) and a tandem silencer element. The cyclin D1 promoter sequence of either promotion-sensitive or resistant JB6 mouse epidermal cells was, except for a few minor differences, essentially identical to the sequence determined for a mouse genomic clone. Since TPA is capable of stimulating the expression of cyclin D1 not only through TRE but also through CRE and NF-kappaB response element in the promoter, we tentatively propose a sequence of events that possibly leads to TPA-induced, anchorage-independent synthesis of cyclins D1 and A in the promotion-sensitive JB6 mouse epidermal cells.

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Molecular cloning, sequencing and functional study of the promoter region of the human alpha2C4-adrenergic receptor gene.

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