RESUMEN
The cDNA encompassing the complete coding sequence of human liver short-chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD) was isolated and characterized. Screening of a cDNA library combined with rapid amplification of 5' cDNA ends resulted in a SCHAD cDNA sequence of 1877 bp. It encodes a protein of 314 amino acids with a calculated molecular weight of 34.3 kDA containing a mitochondrial import signal peptide of 12 amino acids and 302 amino acids of mature SCHAD protein. The deduced amino acid sequence of the mature protein shows a 92 percent identity with SCHAD from pig heart. Northern blot analysis reveals SCHAD mRNA to be expressed in liver, kidney, pancreas, heart and skeletal muscle. The human SCHAD gene was mapped by fluorescence in situ hybridization to chromosome 4q22-26.
Asunto(s)
3-Hidroxiacil-CoA Deshidrogenasas/biosíntesis , 3-Hidroxiacil-CoA Deshidrogenasas/genética , Cromosomas Humanos Par 4 , Mitocondrias Hepáticas/enzimología , 3-Hidroxiacil-CoA Deshidrogenasas/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Cartilla de ADN , Biblioteca de Genes , Humanos , Hibridación Fluorescente in Situ , Mitocondrias Cardíacas/enzimología , Datos de Secuencia Molecular , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Porcinos , Transcripción GenéticaRESUMEN
The third step in the mitochondrial beta-oxidation spiral of short-chain fatty acids is catalyzed by short-chain L-3-hydroxyacyl-CoA dehydrogenase (HADHSC; EC 1.1.1.35). We have determined the structural organization of the human HADHSC gene by sequencing of cloned genomic amplification products, obtained using HADHSC-specific cDNA-based primers, as well as by direct sequencing of an isolated PAC clone containing the HADHSC gene. Upon comparison with the HADHSC cDNA sequence, HADHSC was shown to encompass at least eight exons, ranging in size from 73 to 158 bp, and 7 introns. The total HADHSC gene spans approximately 49 kb. The HADHSC 5'-flanking region was characterized with an AluI plasmid library constructed from a partially AluI-digested PAC clone containing the human HADHSC gene. Several typical promoter elements such as a CAAT-box, Sp1, AP1, and AP2 sites were found, while a TATA-box was apparently absent. Among other putative regulatory elements, a NRRE-1 site was identified. By radiation hybrid panel, assisted fine-mapping HADHSC was linked to marker AFM070TH5, corresponding to Chromosome (Chr) 4q22-26, and a putative HADHSC pseudogene was linked to marker D15S1324, located at Chr 15q17-21. Knowledge of the genomic organization and 5'-flanking region of HADHSC will enable genomic mutation analysis of patients suspected of HADHSC deficiency, as well as facilitate the investigation into the transcriptional regulation of short-chain fatty acid oxidizing gene products in general and HADHSC expression in particular.