scholarly journals Identification of direct-repeat-binding protein 1 (DRP-1), a DNA-binding protein that binds specifically to the ‘malic’ enzyme gene promoter direct repeat element

1995 ◽  
Vol 311 (3) ◽  
pp. 901-904
Author(s):  
K G Ford ◽  
D P Hornby ◽  
W S al Harrasy
Keyword(s):  
Malic Enzyme ◽  
Protein Complexes ◽  
Specific Binding ◽  
Binding Protein ◽  
Gene Promoter ◽  
Direct Repeat ◽  
Repeat Element ◽  
Binding Assays ◽  
Sds Page ◽  
Malic Enzyme Gene

The ‘malic’ enzyme (ME) gene promoter contains three main regulatory regions. One of these, the direct repeat element (DRE), contains tandem degenerate Sp1-binding sites separated by a 3 bp intervening sequence. We now show that a previously unreported 95 kDa protein, which we have designated DRP-1, binds strongly to the DRE region in a highly specific manner. Western-blot analysis confirms that this protein is not Sp1, which has been shown to bind to similar degenerate sites. Competitive binding assays using purified DRP-1 further reveal that neither non-specific nor Sp1-consensus-site-containing oligonucleotides can displace those complexes formed between DRP-1 and the DRE sequence, thus confirming sequence-specific binding by this protein. SDS/PAGE analysis of DRE-protein complexes isolated by direct excision and transplantation from retardation gels confirms the presence of the 95 kDa protein and, in addition, suggests that more than one binding site exists for this protein within the DRE. This is in accord with the repeated nature of the DRE DNA sequence which contains two CACC box motifs.

scholarly journals Carbohydrate Response Element Binding Protein Gene Expression Is Positively Regulated by Thyroid Hormone

Endocrinology ◽  
2009 ◽  
Vol 150 (7) ◽  
pp. 3417-3424 ◽  
Author(s):  
Koshi Hashimoto ◽  
Emi Ishida ◽  
Shunichi Matsumoto ◽  
Shuichi Okada ◽  
Masanobu Yamada ◽  
...  
Keyword(s):  
Gene Expression ◽  
Thyroid Hormone ◽  
Target Genes ◽  
Binding Protein ◽  
Gene Promoter ◽  
Direct Repeat ◽  
Transcriptional Level ◽  
Hepatic Lipogenesis ◽  
Response Element ◽  
Element Binding Protein

The molecular mechanism of thyroid hormone (TH) effects to fatty acid metabolism in liver is yet to be clear. The carbohydrate response element-binding protein (ChREBP) as well as sterol response element-binding protein (SREBP)-1c plays a pivotal role in hepatic lipogenesis. Both SREBP-1c and ChREBP are target genes of liver X receptors (LXRs). Because LXRs and TH receptors (TRs) cross talk mutually in many aspects of transcription, we examined whether TRs regulate the mouse ChREBP gene expression. In the current study, we demonstrated that TH up-regulated mouse ChREBP mRNA and protein expression in liver. Run-on and luciferase assays showed that TH and TR-β1 positively regulated the ChREBP gene transcription. The mouse ChREBP gene promoter contains two direct repeat-4 sites (LXRE1 and LXRE2) and EMSAs demonstrated that LXR-α and TR-β1 prefer to bind LXRE1 and LXRE2, respectively. The direct repeat-4 deletion and LXRE2 mutants of the promoter deteriorate the positive regulation by TR-β1, indicating that LXRE2 is functionally important for the regulation. We also showed that human ChREBP gene expression and promoter activities were up-regulated by TH. These data suggest that ChREBP mRNA expression is positively regulated by TR-β1 and TH at the transcriptional level in mammals. This novel observation indicates that TH fine-tunes hepatic lipogenesis via regulating SREBP-1c and ChREBP gene expression reciprocally.

Structural and functional analysis of the rat malic enzyme gene promoter

1988 ◽  
Vol 8 (8) ◽  
pp. 3542-3545
Author(s):  
H Morioka ◽  
G E Tennyson ◽  
V M Nikodem
Keyword(s):  
Functional Analysis ◽  
Malic Enzyme ◽  
Genomic Dna ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Coding Region ◽  
Regulatory Regions ◽  
Enzyme Gene ◽  
Malic Enzyme Gene

We have characterized sequences of genomic DNA 5' to the coding region of the rat malic enzyme gene. This sequence possesses neither TATA nor CCAAT sequences in their usual positions but is rich in GC residues. Sequences similar to those found in the regulatory regions of other genes are discussed. Deletion analyses have revealed that sequences +1 to -41 are sufficient to initiate expression, although inclusion of information up to -177 is necessary for maximal promoter activity.

scholarly journals Interaction of CCAAT/enhancer-binding protein α and β with the rat caeruloplasmin gene promoter

1993 ◽  
Vol 294 (2) ◽  
pp. 473-479 ◽  
Author(s):  
C D Bingle ◽  
R E Fleming ◽  
J D Gitlin
Keyword(s):  
Rat Liver ◽  
Specific Binding ◽  
Binding Protein ◽  
Specific Interaction ◽  
Gene Promoter ◽  
Nuclear Extract ◽  
Mobility Shift ◽  
Specific Expression ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein

To determine the mechanisms of expression of the rat caeruloplasmin gene, the promoter region was analysed by DNAase I footprinting. Using nuclear extract from rat liver, a prominent site of protein-DNA interaction was detected from -93 to -48 upstream of the caeruloplasmin gene transcription start and sequence analysis of this region revealed three potential CCAAT/enhancer-binding protein (C/EBP) consensus elements. Mobility-shift analysis using an oligonucleotide encoding this region identified specific binding of proteins from rat liver nuclear extract, and some of these complexes were supershifted using antisera to the C/EBP alpha and beta family members. Mobility-shift studies using a polypeptide encoding the DNA-binding domain of C/EBP alpha also revealed a specific interaction with this region of the caeruloplasmin promoter, and DNAase I footprinting using this polypeptide protected the identical region from -93 to -48. Co-transfection of expression plasmids encoding C/EBP alpha or a related leucine-zipper factor D-binding protein (DBP) revealed a C/EBP-specific increase in reporter gene activity in HepG2 cells transfected with caeruloplasmin-chloramphenicol acetyltransferase containing the -93 to -48 region. A similar result was obtained when these constructs were co-transfected into mouse L cells which were shown not to express the endogenous caeruloplasmin gene. Taken together, these data indicate a role for C/EBP alpha and beta in mediating transcription from the caeruloplasmin gene promoter and suggest that this region of the promoter is not responsible for tissue-specific expression.

scholarly journals Solubilization and purification of a membrane-associated 3,3′,5-tri-iodo-l-thyronine-binding protein from rat erythrocytes

1990 ◽  
Vol 270 (3) ◽  
pp. 577-582 ◽  
Author(s):  
R C Angel ◽  
J A Botta ◽  
R D Morero ◽  
R N Farias
Keyword(s):  
Hormone Receptors ◽  
Specific Binding ◽  
Binding Protein ◽  
Active Form ◽  
Binding Activity ◽  
Erythrocyte Membranes ◽  
Major Protein ◽  
Sds Page ◽  
Affinity Labelling ◽  
Zwitterionic Detergent

3,3′,5-Tri-iodo-L-thyronine (L-T3) binding sites from rat erythrocyte membranes were solubilized in an active form by using the zwitterionic detergent CHAPS or the anionic detergent lauroylsarcosine. The binding protein was successively purified by Sephadex G-200 and affinity chromatography. The purified material retained its binding activity and exhibited high affinity and specificity compared with those displayed in the original membrane. Yield was about 10% of the starting activity. The specific binding activity was enriched by approx. 100-fold, which represents a purity of only 0.1%. Analysis of the purified preparation on SDS/PAGE showed two major protein bands (Mr 64,000 and Mr 50,000), but these could not represent the binding protein since the purity obtained was low. However, affinity-labelling experiments with N-bromoacetyl-L-[125I]T3 in intact membranes showed that two proteins (also with Mr values of 64,000 and 50,000) bound the hormone specifically, suggesting a co-migration of hormone receptors and contaminants on gel electrophoresis.

scholarly journals Present and future of siderophore-based therapeutic and diagnostic approaches in infectious diseases

2019 ◽  
Vol 11 (2) ◽  
Author(s):  
Gilda Tonziello ◽  
Emanuela Caraffa ◽  
Biagio Pinchera ◽  
Guido Granata ◽  
Nicola Petrosillo
Keyword(s):  
Infectious Diseases ◽  
Iron Uptake ◽  
Protein Complexes ◽  
Specific Binding ◽  
Binding Protein ◽  
Research Progress ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Uptake Pathway ◽  
Diagnostic Approaches

Iron is an essential micronutrient required for the growth of almost all aerobic organisms; the iron uptake pathway in bacteria therefore represents a possible target for novel antimicrobials, including hybrids between antimicrobials and siderophores. Siderophores are low molecular weight iron chelators that bind to iron and are actively transported inside the cell through specific binding protein complexes. These binding protein complexes are present both in Gram negative bacteria, in their outer and inner membrane, and in Gram positive bacteria in their cytoplasmic membrane. Most bacteria have the ability to produce siderophores in order to survive in environments with limited concentrations of free iron, however some bacteria synthetize natural siderophore-antibiotic conjugates that exploit the siderophore-iron uptake pathway to deliver antibiotics into competing bacterial cells and gain a competitive advantage. This approach has been referred to as a Trojan Horse Strategy. To overcome the increasing global problem of antibiotic resistance in Gram negative bacteria, which often have reduced outer membrane permeability, siderophore-antibiotic hybrid conjugates have been synthetized in vitro. Cefiderocol is the first siderophore-antibiotic conjugate that progressed to late stage clinical development so far. In studies on murine models the iron-siderophore uptake pathway has been also exploited for diagnostic imaging of infectious diseases, in which labelled siderophores have been used as specific probes. The aim of this review is to describe the research progress in the field of siderophore-based therapeutic and diagnostic approaches in infectious diseases.

scholarly journals Structural and functional analysis of the rat malic enzyme gene promoter.

1988 ◽  
Vol 8 (8) ◽  
pp. 3542-3545 ◽  
Author(s):  
H Morioka ◽  
G E Tennyson ◽  
V M Nikodem
Keyword(s):  
Functional Analysis ◽  
Malic Enzyme ◽  
Genomic Dna ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Coding Region ◽  
Regulatory Regions ◽  
Enzyme Gene ◽  
Malic Enzyme Gene

We have characterized sequences of genomic DNA 5' to the coding region of the rat malic enzyme gene. This sequence possesses neither TATA nor CCAAT sequences in their usual positions but is rich in GC residues. Sequences similar to those found in the regulatory regions of other genes are discussed. Deletion analyses have revealed that sequences +1 to -41 are sufficient to initiate expression, although inclusion of information up to -177 is necessary for maximal promoter activity.

Cell-type-specific function of the C-type natriuretic peptide gene promoter in rat anterior pituitary-derived cultured cell lines

1993 ◽  
Vol 13 (7) ◽  
pp. 4077-4086
Author(s):  
S Ohta ◽  
Y Shimekake ◽  
K Nagata
Keyword(s):  
Natriuretic Peptide ◽  
Anterior Pituitary ◽  
Promoter Activity ◽  
Cultured Cells ◽  
Specific Binding ◽  
Binding Protein ◽  
Regulatory Region ◽  
Gene Promoter ◽  
Nuclear Extract ◽  
Gh3 Cells

The promoter function of the human C-type natriuretic peptide (CNP) gene in various cultured cells was examined by transient transfection assays. The CNP promoter functioned very effectively in GH3 cells, which originated from the growth hormone-producing tumor of the rat anterior pituitary and somatomammotroph phenotype, but functioned much less effectively in GH1 cells, another type of rat pituitary-derived cell with a somatotroph phenotype, and rat primary cardiocytes. The CNP promoter did not function at all in other cells, including AtT20 cells of murine pituitary corticotroph origin. Functional analyses of the deleted promoters with various 5' deletion breakpoints revealed the existence of at least two negative and one positive regulatory regions. Within the positive regulatory region (positions -54 to -19), which conferred 90% of the promoter activity in GH3 cells, two equipotent GC-rich cis elements (positions -49 to -45 and -40 to -35) were identified. Both sites shared half of the promoter activity and binding properties to the nuclear protein in GH3 cells. Rat anterior pituitary tissue contained the binding protein of the identified cis element, which was identical or similar to that of GH3 cells. With Southwestern (DNA-protein) analysis, a 70-kDa specific binding protein distinct from known factors such as SP-1, AP-2, and Pit-1 was identified in the nuclear extract of GH3 cells.

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