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Isolation and characterisation of the human NADH-cytochrome b5 reductase gene

Bull, Peter Charles; (1990) Isolation and characterisation of the human NADH-cytochrome b5 reductase gene. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Cytochrome b5 reductase exists in soluble and membrane bound forms. The membrane bound form is predominant in most tissues. It is anchored to the membranes by an N-terminal membrane binding domain. The membrane bound enzyme is involved in the desaturation and elongation of fatty acids and in some cytochrome P450 mediated reactions. The soluble form of b5 reductase is present mainly in the erythrocytes and is involved in the reduction of methaemoglobin. This form of the enzyme has an identical primary amino acid structure to the membrane bound form apart from the fact that it lacks the N-terminal binding domain. The long term aim of this investigation is to establish the relationship between the membrane bound and soluble forms of b5 reductase. An oligonucleotide of 43bp in length, made on the basis of a previously published partial cDNA sequence of human liver b5 reductase was used as a starting point in a cloning procedure that led to the isolation of a cDNA of 1905 bp In length, whose sequence encodes the entire length of the membrane bound form of the enzyme. Southern blot hybridization analysis of DNA isolated from a panel of 11 somatic cell hybrids provided evidence supporting the hypothesis that one gene, present on chromosome 22, encodes both forms of human b5 reductase. Overlapping genomic clones of the human gene were isolated and characterised by cos end mapping, restriction endonuclease mapping and Southern blot hybridization analysis. The clones, together spanning 26kb, contained 8 exons that together encode all but the N-terminal 6 amino acids of the membrane bound form o1 the enzyme. The 5' most exon isolated (called exon A) was found to encode the putative N-terminus of the soluble form of b5 reductase. Analysis of 300bp of the sequence immediately upstream of this exon revealed two motifs that are similar to protein binding sites within the erythroid specific alternative promotor of the porphobilinogen deaminase gene. RNAse protection analysis of RNAs isolated from erythroid and non erythroid tissue, however, did not provide any evidence to support the proposal that this sequence acts as an erythroid specific promotor for the soluble form of b5 reductase. However, the possibility that an mRNA species encoding the soluble form could be generated by the splicing of the 5' end of exon A to an alternative splice donor site could not be excluded by the results of these experiments.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Isolation and characterisation of the human NADH-cytochrome b5 reductase gene
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Membrane binding
URI: https://discovery.ucl.ac.uk/id/eprint/10111093
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