|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1990 : ¥2,000,000 (Direct Cost : ¥2,000,000)
Maple syrup urine disease (MSUD) is an autosomal recessive inherited disorder due to a deficiency of any subunits, E_1 alpha, E_1 beta or E_2 of branched chain alpha-ketoacid dehydrogenase complex (BCKDH). We performed following study to seek a molecular basis of the MSUD.
1. We isolated and sequenced a cDNA insert of E_2 subunit of BCKDH. It consists of 2,649 base pairs with and open reading frame of 1,431 base pairs, which can be translated into 477 amino acids, and 3'-untranslated region of 1,205 base pairs. The deduced amino acid residues were a lipoyl-bearing domain, a E_3-binding domain and innor core domain.
2. We analyzed cDNA and genomic cDNA structure of E_1beta subunit of human BCKDH. Isolated cDNA clone contained a 5'-untranslated sequence of 4 nucleotides, the translated sequence of 1.176 uncleotides and a 3'-untranslated sequence of 169 nucleotides. The cDNA encodes for a 342 amino acid subunit with a Mr=37,585, which includes a leader sequence of 50 amino acids. The gene of E_1beta subunit is over 150 kb long and splits into 10 exons. All of the splice donor and acceptor sites confirm to the GT/AG rule. The transcription initiation site was located 47 base upstream of the initiation codon. A "CAAT" bax and its reverse complement sequence were present at 37 bases and 47 bases upstream from the cap sites, but there was no "TATA" box like sequence.
3. We found 3 different mutant genes ; A T-to-A substitution in the E_1alpha subunit gene in Mennonite MSUD, a deletion of an 11-base pair repeat sequence, which encodes a mitochondrial ratgetting leader peptide, in a Japanese MSUD family and a 78 base pair deletion in E_2 subunit genedue to splicing abnormalities in another Japanese MSUD family.