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

1999 ◽  
Vol 339 (3) ◽  
pp. 775-775
Author(s):  
Y.-S. HUANG ◽  
D. T. CHUANG
Keyword(s):  
Acid Dehydrogenase ◽  
Basal Expression ◽  
Branched Chain

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 Hepatic Branched-Chain .ALPHA.-Keto Acid Dehydrogenase Complex in Female Rats: Activation by Exercise and Starvation.

1999 ◽  
Vol 45 (3) ◽  
pp. 303-309 ◽  
Author(s):  
Rumi KOBAYASHI ◽  
Yoshiharu SHIMOMURA ◽  
Taro MURAKAMI ◽  
Naoya NAKAI ◽  
Megumi OTSUKA ◽  
...  
Keyword(s):  
Female Rats ◽  
Keto Acid ◽  
Acid Dehydrogenase ◽  
Branched Chain ◽  
Acid Dehydrogenase Complex ◽  
Dehydrogenase Complex
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