Genetic analysis of cytochrome b5 from arachidonic acid-producing fungus, Mortierella alpina 1S-4:: Cloning, RNA editing and expression of the gene in Escherichia coli, and purification and characterization of the gene product

Information on the amino acid sequences of the internal peptide fragments of cytochrome b(5) from Mortierella hygrophila was used to prepare synthetic oligonucleotides as primers for the polymerase chain reaction. A 100-base DNA fragment was thus amplified, by using a genomic gene from Mortierella alpina 1S-4 as a template, which produced polyunsaturated fatty acids such as arachidonic acid. The amplified DNA fragment was used as the probe to clone both a 523-base cDNA fragment and a 2.1-kilobase SalI-NruI genomic fragment coding for the whole M. alpina 1S-4 cytochrome b(5) On the basis of nucleotide sequences of both cytochrome b(5) genomic gene and cDNA, the genomic cytochrome b(5) gene was found to consist of four exons and three introns, A novel type of RNA editing, in which the cDNA included either guanine insertion or adenine-->guanine substitution at one base upstream of poly(A), was interestingly observed, The deduced amino acid sequence of M. alphia 1S-4 cytochrome b(5) showed significant similarities with those of cytochrome b(5)s from other organisms such as rat, chicken, and yeast. The soluble form of the cytochrome b(5) gene was expressed to 16% of the total soluble protein in Escherichia coli, The holo-cytochrome b(5) accounted for 8% of the total cytochrome b(5) in the transformants. The purified cytochrome b(5) showed the oxidized and reduced absorbance spectra characteristic of fungal microsomal cytochrome b(5).