Rigby, Jacqueline Susan; (1990) Structure and characterization of cytrochrome b5 genes. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Cytochrome b5 is an amphipathic protein with a prosthetic haem group. It is found in various tissues such as liver, kidney, heart and brain as well as in erythrocytes. Liver microsomal cytochrome b5 is 133 amino acids in length. The carboxy-terminal 36 amino acids constitute a hydrophobic tail and embed the protein in the membrane. Microsomal cytochrome b5 functions in the cytochrome P450 mono-oxygenase system, acting to metabolise both exogenous and endogenous substances. Erythrocyte cytochrome b5 is truncated after amino acid 97 and the protein is cytosolic. It functions in erythrocytes to reduce methaemoglobin. An absence of functional cytochrome b5 in erythrocytes has been shown to lead to the medical disorder, methaemoglobinaemia. Within a species, amino acids 1 to 96 are identical between microsomal and erythrocyte cytochrome b5 - only amino acid 97 differs, in all species except bovine. It was not known whether there were one or two genes coding for the two forms of cytochrome b5. If there was only one gene, the primary transcript may undergo differential RNA splicing to produce two different mature cytochrome b5 mRNAs. Rat cytochrome b cDNA probes were used to screen a rat genomic library. Six different genomic clones were isolated which contained regions homologous to cytochrome b5. These clones were analysed by restriction mapping, hybridization studies and DNA sequencing. Two of the genomic clones contained sequence for the same rat cytochrome b5 processed, truncated pseudogene. From analysis of the other genomic clones, the functional microsomal cytochrome b gene is more than 23 kb in length and consists of at least five exons. Only four exons from the functional rat cytochrome b5 gene were isolated - the exon or exons containing amino acids 1-42 inclusive were not located. Due to this fact and the complicated pattern of hybridizing bands on a genomic Southern blot, it is still not clear whether there are one or two cytochrome b5 genes.

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