Molecular genetic aspects of human pyruvate dehydrogenase and its defect

Genomic clones encompassing the entire genes for the human pyruvate dehydrogenase alpha and beta subunits (PDH alpha or beta) have been isolated by screening the leukocyte genomic libraries with a nick-translated human foreskin fibroblast PDH alpha or beta cDNA probe. These genomic clones were characterized by restriction enzyme analysis. extensive DNA sequencing and primer extension analysis. The PDH alpha gene spans 17.08 kilobases and is composed of 11 exons and 10 introns within its coding region. The 18-kilobase clone of PDH beta gene is composed of 10 exons and 9 introns. All intron-exon splice junctions of two genes follow the GT/AG rule. A total of seven Alu repeats in the PDH alpha gene were found in five introns and two Alu family in the PDH beta gene were found in intron 2 and 8. The 5'-flanking region of the PDH alpha gene contains typical CCAAT and TATA-like consensus promoter sequence and two Sp1 binding sequences. That of the PDH beta gene contains a TCAAT sequence but no TATA sequence. Primer extension analyses indicated that the PDH alpha and beta genes transcription start sites are thymine and adenine residues located 124 and 132 bases upstream from initiation codon in exon 1, respectively. Genomic DNA of patient, died 93 hours after birth with acidemia and defect of PDH activity, was isolated and all of the exons of PDH alpha and beta genes were amplified by PCR.(ABSTRACT TRUNCATED AT 250 WORDS)