scholarly journals Structural organization of the rat branched-chain 2-oxo-acid dehydrogenase kinase gene and partial characterization of the promoter-regulatory region

1996 ◽  
Vol 313 (2) ◽  
pp. 603-609 ◽  
Author(s):  
Yi-shuian HUANG ◽  
David T. CHUANG
Keyword(s):  
Rat Kidney ◽  
Regulatory Region ◽  
Luciferase Reporter ◽  
Gene Encoding ◽  
Kinase Gene ◽  
Cis Acting ◽  
Acid Dehydrogenase ◽  
Frame Sequence ◽  
Flanking Region ◽  
Branched Chain

The gene encoding the rat branched-chain 2-oxo-acid dehydrogenase kinase (EC 2.7.1.115) has been isolated and partially characterized. The entire gene, including the promoter-regulatory region, spans 6 kb and contains 11 exons. The 5ʹ-untranslated region comprising 264 bp is interrupted by intron 1 which is 581 bp in size. The complete in-frame sequence of intron 7 encodes the 49 amino acid insert previously reported to be present in the larger isoform of the rat kinase (Harris, Popov, Shimomura, Zhao, Jaskiewicz, Nanaumi and Suzuki (1992) Adv. Enzyme Regul. 32, 267-284). Sequencing of the 679 bp of the 5ʹ-flanking region showed the absence of a canonical TATA box, similar to other branched-chain 2-oxo-acid dehydrogenase-complex genes. Several candidate cis-acting elements are present. These include CAAT boxes, Sp-1-binding sites, GCN-4 sites, CCAAT enhancer binding-protein sites (C/EBP) and glucocorticoid-responsive element (GRE) sites. Also present are a pair of direct repeats of unknown function. The luciferase-reporter assay showed that promoter activity is markedly higher in normal rat kidney (NRK-52E) cells than in rat hepatoma (FTO-2B) cells, and that the 5ʹ-flanking region between bases -449 and +264 is both necessary and sufficient for basal transcription of the kinase gene.

scholarly journals Mechanism for basal expression of rat mitochondrial branched-chain-2-oxo-acid dehydrogenase

1998 ◽  
Vol 334 (3) ◽  
pp. 713-722 ◽  
Author(s):  
Yi-Shuian HUANG ◽  
David T. CHUANG
Keyword(s):  
Dna Sequences ◽  
Binding Sites ◽  
Promoter Activity ◽  
Nuclear Protein ◽  
Luciferase Reporter ◽  
Kinase Gene ◽  
Acid Dehydrogenase ◽  
Basal Promoter Activity ◽  
Basal Expression ◽  
Branched Chain

The rat branched-chain-2-oxo-acid dehydrogenase (BCOD) kinase mRNA is transcribed from a TATA-less promoter that has GC-rich sequences and two putative Sp1 binding sites near the transcription start site. We demonstrated previously that the 5´ region of the kinase gene, base pairs -128 to +264, contained promoter activity when assayed using luciferase as a reporter (Huang and Chuang (1996) Biochem. J. 313, 603–609). To define DNA elements required for efficient expression of the kinase gene, nested deletion constructs of the above promoter region fused with a luciferase reporter gene were transfected into cultured H4IIE (hepatoma) and NRK-52E (kidney) cells. The results showed that the region between nucleotides -58 and +21 was indispensable for the kinase basal promoter activity. Methylation-interference and mutagenesis-promoter assays identified nucleotides -50 to -40 (ACAACTCCCA) as cis-acting DNA sequences that are required for nuclear protein binding and efficient promoter activity. Gel-supershift analysis with anti-Sp1 antibody suggested that the nuclear protein capable of binding to the -58 oligonucleotide (bp -58 to -34) was immunologically related to the Sp1 protein. The -58 oligonucleotide formed a DNA–protein complex with recombinant Sp1 protein with an affinity approximately ten-fold lower than that of the consensus Sp1 oligonucleotide. Co-transfection of the Sp1 expression plasmid and the -58 promoter construct into Drosophila Schneider cells revealed that Sp1 contributed to the kinase basal promoter activity by binding to the non-consensus site in the -58 region. Deletion of two consensus Sp1 binding sites (bases -150 to -140 and bases +29 to +38) in the kinase gene did not affect the basal promoter activity. Therefore binding of Sp1 or Sp1-like proteins to the above single non-consensus Sp1 sequence in the -58 region plays a major role of transactivating basal expression of the BCOD kinase.

scholarly journals Multiple regions within the promoter of the murine Ifnar-2 gene confer basal and inducible expression

2002 ◽  
Vol 365 (2) ◽  
pp. 355-367 ◽  
Author(s):  
Matthew P. HARDY ◽  
Paul J. HERTZOG ◽  
Catherine M. OWCZAREK
Keyword(s):  
Splice Variants ◽  
Regulatory Region ◽  
Distal Region ◽  
Inducible Expression ◽  
Luciferase Reporter ◽  
Type I ◽  
Type I Ifn ◽  
Mrna Isoforms ◽  
Gene Encoding ◽  
Multiple Regions

The (murine) type I interferon (IFN) receptor, muIfnar-2, is expressed ubiquitously, and exists as both transmembrane and soluble forms. In the present study we show that the gene encoding muIfnar-2 spans approx. 33kb on mouse chromosome 16, and consists of nine exons and eight introns. The three mRNA splice variants resulting in one transmembrane (muIfnar-2c) and two soluble (muIfnar-2a/2a′) mRNA isoforms are generated by alternative RNA processing of the muIfnar-2 gene. Treatment of a range of murine cell lines with a combination of type I and II IFN showed that the muIfnar-2a and −2c mRNA isoforms were up-regulated independently of each other in L929 fibroblasts and hepa-1c1c7 hepatoma cells, but not in M1 myeloid leukaemia cells. Analysis of the 5′ flanking region of muIfnar-2 using promoter—luciferase reporter constructs defined three regulatory regions: a region proximal to exon 1, conferring high basal expression, a distal region conferring inducible expression, and a negative regulatory region between the two. These data represent the first promoter analysis of a type I IFN receptor and, taken together with our previous data demonstrating high expression levels and dual biological functions for muIfnar-2a protein, suggests that the regulation of muIfnar-2 isoform expression may be an important way of modulating type I IFN responses.

scholarly journals Purification of rat kidney branched-chain oxo acid dehydrogenase complex with endogenous kinase activity

1982 ◽  
Vol 204 (1) ◽  
pp. 353-356 ◽  
Author(s):  
R Odessey
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Time Course ◽  
Kinase Activity ◽  
Rat Kidney ◽  
Pyruvate Dehydrogenase Complex ◽  
Acid Dehydrogenase ◽  
Endogenous Protein ◽  
Branched Chain ◽  
Acid Dehydrogenase Complex ◽  
Dehydrogenase Complex

A method was devised to purify branched-chain oxo acid dehydrogenase (BCOAD) from rat kidney which retains endogenous kinase activity. Incorporation of 32P into purified enzyme parallels the time course of enzyme inhibition by ATP. Phosphorylation occurs on a serine residue(s) of the 46000-mol.wt. subunit of the enzyme complex. Endogenous phosphatase activity is not present after purification, and added pyruvate dehydrogenase phosphate phosphatase does not re-activate BCOAD or liberate 32P from previously labelled enzyme. These results demonstrate that BCOAD can be regulated by an endogenous protein kinase and that the phosphorylation-cycle enzymes regulating BCOAD appear to be distinct from those associated with pyruvate dehydrogenase complex.

scholarly journals Suppression of erythro-megakaryocytopoiesis and the induction of reversible thrombocytopenia in mice transgenic for the thymidine kinase gene targeted by the platelet glycoprotein alpha IIb promoter.

1995 ◽  
Vol 181 (6) ◽  
pp. 2141-2151 ◽  
Author(s):  
D Tronik-Le Roux ◽  
V Roullot ◽  
A Schweitzer ◽  
R Berthier ◽  
G Marguerie
Keyword(s):  
Transgenic Mice ◽  
Thymidine Kinase ◽  
Progenitor Cells ◽  
Regulatory Region ◽  
Platelet Glycoprotein ◽  
Gene Encoding ◽  
Kinase Gene ◽  
Myeloid Progenitor Cells ◽  
Simplex Virus

The mechanisms that regulate the commitment of a totipotent stem cell to the megakaryocytic lineage are largely unknown. Using a molecular approach to the study of megakaryocytopoiesis and platelet production, mice in which thrombocytopoiesis could be controlled were produced by targeting the expression of the herpes simplex virus thymidine kinase toxigene to megakaryocytes using the regulatory region of the gene encoding the alpha subunit of the platelet integrin alpha IIb beta 3. The programmed eradication of the megakaryocytic lineage was induced by treating transgenic mice bearing the hybrid construct (alpha IIbtk) with the antiherpetic drug ganciclovir (GCV). After 10 d of treatment, the platelet number was reduced by > 94.6%. After discontinuing GCV, the bone marrow was repopulated with megakaryocytes and the platelet count was restored within 7 d. Prolonged GCV treatment induced erythropenia in the transgenic mice. Assays of myeloid progenitor cells in vitro demonstrated that the transgene was expressed in early erythro-megakaryocytic progenitor cells. The reversibility and facility of this system provides a powerful model to determine both the critical events in megakaryocytic and erythroid lineage development and for evaluating the precise role that platelets play in the pathogenesis of a number of vascular occlusive disorders.

scholarly journals Metabolism of valine and 3-methyl-2-oxobutanoate by the isolated perfused rat kidney

1984 ◽  
Vol 224 (1) ◽  
pp. 109-116 ◽  
Author(s):  
R H Miller ◽  
A E Harper
Keyword(s):  
Amino Acids ◽  
High Activity ◽  
Rat Kidney ◽  
Linear Increase ◽  
Isolated Perfused Kidney ◽  
Isolated Perfused Rat Kidney ◽  
Acid Dehydrogenase ◽  
Perfused Rat Kidney ◽  
Branched Chain ◽  
Perfused Kidney

Metabolism of branched-chain amino and 2-oxo acids was studied in the isolated perfused kidney. Significant amounts of 2-oxo acids were released by perfused kidney with all concentrations of amino acids tested (0.1-1.0 mM each), despite the high activity of branched-chain 2-oxo acid dehydrogenase in kidney. As perfusate valine concentration was increased from 0.2 to 1.0 mM, [1-14C]valine transamination (2-oxo acid oxidized + released) increased roughly linearly; [1-14C]valine oxidation, however, increased exponentially. Increasing perfusate concentration of 3-methyl-2-oxo[1-14C]butanoate from 0 to 1.0 mM resulted in a linear increase in the rate of its oxidation and a rise in perfusate valine concentration; at the same time significant decreases occurred in perfusate isoleucine and leucine concentrations, with corresponding increases in rates of release of their respective 2-oxo acids. Comparison of rates of oxidation of [1-14C]valine and 3-methyl-2-oxo[1-14C]butanoate suggests that 2-oxo acid arising from [1-14C]valine transamination has freer access to the 2-oxo acid dehydrogenase than has the 2-oxo acid from the perfusate. The observations indicate that, when branched-chain amino and 2-oxo acids are present in perfusate at near-physiological concentrations, rates of transamination of the amino and 2-oxo acids by isolated perfused kidney are greater than rates of oxidation.

Transcriptional regulation of string (cdc25): a link between developmental programming and the cell cycle

Development ◽  
1994 ◽  
Vol 120 (11) ◽  
pp. 3131-3143 ◽  
Author(s):  
B.A. Edgar ◽  
D.A. Lehman ◽  
P.H. O'Farrell
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Regulatory Region ◽  
Spatiotemporal Pattern ◽  
Regulatory Sequences ◽  
Gene Encoding ◽  
Drosophila Cell ◽  
Cell Cycles ◽  
Cis Acting ◽  
Spatial Domains

During postblastoderm embryogenesis in Drosophila, cell cycles progress in an invariant spatiotemporal pattern. Most of these cycles are differentially timed by bursts of transcription of string (cdc25), a gene encoding a phosphatase that triggers mitosis by activating the Cdc2 kinase. An analysis of string expression in 36 pattern-formation mutants shows that known patterning genes act locally to influence string transcription. Embryonic expression of string gene fragments shows that the complete pattern of string transcription requires extensive cis-acting regulatory sequences (> 15.3 kb), but that smaller segments of this regulatory region can drive proper temporal expression in defined spatial domains. We infer that string upstream sequences integrate many local signals to direct string's transcriptional program. Finally, we show that the spatiotemporal progression of string transcription is largely unaffected in mutant embryos specifically arrested in G2 of cycles 14, 15, or 16, or G1 of cycle 17. Thus, there is a regulatory hierarchy in which developmental inputs, not cell cycle inputs, control the timing of string transcription and hence cell cycle progression.

Two Novel Mutations in theBCKDK(Branched-Chain Keto-Acid Dehydrogenase Kinase) Gene Are Responsible for a Neurobehavioral Deficit in Two Pediatric Unrelated Patients

2014 ◽  
Vol 35 (4) ◽  
pp. 470-477 ◽  
Author(s):  
Angels García-Cazorla ◽  
Alfonso Oyarzabal ◽  
Joana Fort ◽  
Concepción Robles ◽  
Esperanza Castejón ◽  
...  
Keyword(s):  
Keto Acid ◽  
Novel Mutations ◽  
Kinase Gene ◽  
Acid Dehydrogenase ◽  
Branched Chain
Sign in / Sign up

Export Citation Format

Share Document