Forrest, Lesley Anne; (1994) Expression and regulation of members of the CYP2B and CYP1A subfamilies. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The expression and xenobiotic-induction of members of the CFP2B and CYP1A sub families in the liver and extrahepatic tissues of the male Sprague Dawley rat was investigated by western and immuno-slot blot quantification. The concentration of CYP2B proteins in liver microsomal membranes isolated from untreated animals was ten-fold higher than that in lung and kidney microsomal membranes, whilst CYP1A1 was only detectable in lung. Treatment of animals with isosafrole increased the amount of CYP2Bs, 22-fold in liver microsomal membranes and 1.5-fold in microsomal membranes isolated from lung. The more potent inducer, phenobarbital increased the concentration of CYP2Bs 40-fold in liver microsomal membranes and two-fold in lung and kidney microsomal membranes. β-Naphthoflavone treatment induced CYP1A1 to values higher than that observed in liver, lung and kidney microsomal membranes isolated from PB-treated animals. The CYP1A1 protein was also induced, but to a lesser extent in all three tissues, by isosafrole. To investigate the molecular mechanisms responsible for the phenobarbital induction of CYP2B gene expression, in the absence of a suitable phenobarbital-responsive cell line, in vitro techniques of Bandshift and DNase footprinting were used. A CYP2B2 rat genomic clone was isolated from a Charon 4A library. Small overlapping DNA fragments that covered the entire 1.4kb sequence immediately upstream of the transcriptional start site were used in a detailed study in determining the binding capacities of nuclear extracts isolated from the livers of phenobarbital-treated and untreated animals. Numerous sites were identified by bandshift assays that bound proteins of equal concentration in both nuclear extracts. Two DNA sequences located in the promoter region between nts -217 to -178 and nts -85 to -31 bound proteins that were either more abundant or active in liver nuclear extracts isolated from PB-treated animals. A third sequence situated between nts -33 to -2 bound a protein that was less abundant or active in nuclear extracts from PB-treated animals. The boundaries of the DNA sequences to which these protein factors bound was determined more precisely by DNase footprinting assays. This study identified several DNA sequences to which proteins bind which may be involved in the regulation of expression of the CYP2B2 gene by phenobarbital.

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