Molecular cloning of a cDNA encoding rat NADH-cytochrome b₅ reductase and the corresponding gene

Rat cDNA encoding NADH-cytochrome b₅ reductase (b5R) was isolated from a rat liver cDNA library using a human b5R cDNA as a probe. The cDNA was 1, 905 nucleotides long, consisting of a 5'-terminal untranslated region of 38 nucleotides long, an open reading frame region of 903 nucleotides long encoding 301 amino acid residues, a 3 -terminal untranslated region of 952 nucleotide long, and a poly(A) tail. The amino acid sequence deduced from the cDNA sequence indicated that the rat b5R precursor contained only one extra amino acid (Met) residue at the N terminus, in comparison with the mature form of the enzyme, suggesting that no extra leader peptide is required for translocation of the enzyme to the microsome membrane. Genomic DNA encoding the b5R gene was isolated from rat genomic DNA libraries. The gene was about 17 kb long, and consisted of nine exons and eight introns. The junction between the membrane-binding and catalytic domains of the enzyme was found in the middle of exon 2, suggesting the possibility that the two forms of the enzyme, namely the membrane-bound and soluble forms, are generated through post-translational processing. The possible promoter region of the gene contained no TATA box but four GC box sequences (GGGCGG and CCGCCC), representing potential binding sites for the transcription factor, SP1. The b5R gene seems to have structural characteristics of a house-keeping gene.