Characterization of the promoter-regulatory region and structural organization of E1 alpha gene (BCKDHA) of human branched-chain alpha-keto acid dehydrogenase complex.
We have isolated genomic clones containing the complete exon 1 and the promoter-regulatory region of the E1 alpha gene (BCKDHA) of human branched-chain alpha-keto acid dehydrogenase complex. The cloning was achieved by amplification of a genomic library in the SRB (P2) host strain that allowed the replication of nonstandard DNA structures. The results of this and previous (Dariush, N., Fisher, C. W., Cox, R. P., and Chuang, D. T. (1991) FEBS Lett. 284, 34-38) studies showed that the human E1 alpha gene contains 9 exons, and spans at least 55 kilobases (kb). Exon 1 is 135 bp in length, and contains multiple transcription initiation sites at bases +1, +18, and +22. The complete human E1 alpha cDNA is, therefore, 1,758 bp in length excluding the poly(A)+ tail, and has 27 nucleotides in the 5'-untranslated region. Sequencing of the 5'-flanking region disclosed the absence of a canonical TATA-box in the vicinity of base -30. Several sets of "CAAT" box-like sequences and Sp1 binding-sites are present. Also present are copies of potential AP-2 binding, fat-specific element 1, fat-specific element 2, glucocorticoid-responsive element, and cAMP-responsive element sequences, as well as multiple sets of direct and inverted repeats. The promoter-regulatory region was characterized using deletion constructs and the luciferase reporter assay. The human hepatoma cells (Hep-G2) and Chinese hamster ovary (CHO) cell lines were used as hosts. The results obtained with Hep-G2 cells indicate that the region for high level transcription is located between bases -320 and -115. Extension of the 5'-end of the insert to beyond base -320 markedly reduces promoter activity. The results strongly suggest the presence of inhibitory elements in the region upstream of base -320. Assays in CHO cells show that the region for high level transcription lies between bases -909 and -115. The variation in the region for high level transcription in Hep-G2 and CHO cells may represent cell-type specific differences in the E1 alpha gene promoter function.
Chuang
J L
JL
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.
Cox
R P
RP
Chuang
D T
DT
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D015342:DNA Probes; D005091:Exons; D015698:Genomic Library; D006801:Humans; D007658:Ketone Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D016329:Sp1 Transcription Factor / Q000378:metabolism; D016385:TATA Box
D015342:DNA Probes; D009097:Multienzyme Complexes; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D004247:DNA; D008156:Luciferases; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)