RFLP for a human cytochrome P-450 gene at 19q13.1-qter (HGM8 provisional designation CYPI).
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Chromosomes, Human, 19-20↗
Biomedical subjects
I R Phillips
Publications and source records attributed to I R Phillips.
Isolation and sequence of a human cytochrome P-450 cDNA clone.
A previously reported cDNA clone [pP450(1)] coding for a phenobarbital-inducible cytochrome P-450 variant of rat liver microsomal membranes, designated P-450e(U.C.), was used as a specific hybridization probe to screen a human liver cDNA library. Restriction mapping showed that two of the colonies isolated contained plasmids coding for overlapping regions of the same cDNA sequence. The clone [pHP450(1)] having the longer cDNA insert (1.25 kilobase pairs) was sequenced. The homology between the rat and human cDNAs is 62% in their coding regions but is only random (24%) in the 3'-noncoding nucleotides. The amino acid sequence deduced from the human cDNA is 50% identical to that of P-450e(U.C.). The homology increases to 72% if conservative changes in amino acid residues are permitted. The hydropathy profile of the polypeptide encoded by pHP450(1) is almost identical to that of P-450e(U.C.). Regions known to be highly conserved in cytochrome P-450 isozymes isolated from rat, rabbit, and mouse were found to be conserved in the amino acid sequence derived from pHP450(1). Analysis by Southern blotting indicated that the human cytochrome P-450 encoded by pHP450(1) is part of a multigene family.
Amino Acid Sequence↗
A cytochrome P-450 gene family mapped to human chromosome 19.
We have recently isolated a cloned cDNA coding for a cytochrome P-450 of human liver microsomal membranes, which corresponds to a major phenobarbital-inducible cytochrome P-450 of rat liver. This human cytochrome P-450 is encoded by a member of a multigene family. DNA extracted from a panel of 12 independent human-rodent somatic cell hybrids was analysed by Southern blot hybridization with the cloned cDNA. The results indicate that all components of this cytochrome P-450 gene family are located on chromosome 19. Evidence from hybrids derived from an individual carrying a balanced translocation suggests a regional localization of 19p13.2----qter. Analysis of human metaphase chromosomes by in situ hybridization localizes this cytochrome P-450 gene family further to the long arm of chromosome 19 in the region q13.1----qter. We propose the designation P450PB for this locus.
Animals↗
Cloning of cDNA coding for a rat liver protein whose expression is subject to gender-specific regulation by xenobiotics.
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Animals↗
Hormonal control of carbonic anhydrase III.
Using radioimmunoassay, the concentration of carbonic anhydrase III (CA III) in the livers of adult male rats was found to be approximately 30 times greater than that observed in mature females. Castration of male rats led to a marked reduction in liver CA III concentrations that could be partially restored to control levels by testosterone replacement. Administration of testosterone to ovariectomized female rats induced about a 5-fold increase in liver CA III concentration. Immunoprecipitational analysis of the products of liver mRNA translation in vitro with antiserum specific for CA III showed that hormonal control of the levels of CA III in rat liver is mediated by changes in the amount of translatable CA III mRNA. Marked changes in liver CA III concentrations were also observed in developing and aging male rats. Different control mechanisms appear to operate in mouse and man.
Aging↗
Sexual differentiation of rat liver carbonic anhydrase III.
Using radioimmunoassay, the concentration of carbonic anhydrase III in the livers of adult male rats was found to be approx. 30-times greater than that observed in mature females. Castration of male rats led to a marked reduction in liver carbonic anhydrase III concentrations which could be partially restored to control levels by testosterone replacement. Administration of testosterone to ovariectomised female rats induced about a 5-fold increase in liver carbonic anhydrase III concentration. Immunoprecipitation analysis of the products of liver mRNA translation in vitro with antiserum specific for carbonic anhydrase III showed that hormonal control of the levels of carbonic anhydrase III in liver is mediated by changes in the amount of translatable carbonic anhydrase III mRNA. Marked changes in liver carbonic anhydrase III concentrations were also observed in developing and ageing male rats.
Age Factors↗
Quantification of NADPH: cytochrome P-450 reductase in liver microsomes by a specific radioimmunoassay technique.
We have developed a specific radioimmunoassay to quantify NADPH: cytochrome P-450 reductase. The assay is based on the use of 125I-labelled NADPH: cytochrome P-450 reductase as the radiolabelled antigen and can detect quantities of this protein in amounts as low as 30 pg. The results of the radioimmunoassay demonstrates that the 2.7-fold increase in enzyme activity in rat liver microsomal membranes after phenobarbital treatment is due to increased amounts of the protein. beta-Naphthoflavone treatment, however, did not alter the activity or the quantity of this enzyme in microsomes. The quantification of NADPH: cytochrome P-450 reductase in the microsomes isolated from control and phenobarbital- and beta-naphthoflavone-treated animals permits the calculation of the ratio of this protein to that of total cytochromes P-450. A molar ratio of 15:1 (cytochromes P-450/NADPH: cytochrome P-450 reductase) was calculated for control and phenobarbital-treated animals. This ratio increased to 21:1 after beta-naphthoflavone treatment. Thus the molar ratio of these proteins in liver microsomes can vary with exposure of the animals to particular xenobiotics.
Animals↗
Induction and repression of the major phenobarbital-induced cytochrome P-450 measured by radioimmunoassay.
Two independent radioimmunoassay techniques for the major phenobarbital-inducible cytochrome P-450 (PB P-450) of rat liver microsomal membranes are described. The first technique employs as the source of radiolabelled antigen the products of translation in vitro labelled with [35S]methionine. The second technique employs purified antigen labelled with 125I and is quicker, less expensive and more precise. Both assays are highly specific for PB P-450 and can detect quantities of this variant as small as 1 ng. This is several orders of magnitude more sensitive than any method described previously for the quantification of cytochromes P-450, and consequently the technique is particularly well suited for the quantification of so-called constitutive cytochrome P-450 variants that are present in very low amounts. The results of the radioimmunoassays demonstrate that the apparent 2.6-fold induction of total cytochromes P-450 after phenobarbital treatment is due to a 43-fold increase in Pb P-450. Although beta-naphthoflavone increases the total content of cytochrome P-450 of microsomal membranes 1.4-fold, it actually causes a 55% decrease in the amount of PB P-450. Thus different xenobiotics can have differential effects on the expression of the genes for specific cytochrome P-450 variants.
Animals↗
Androgen-linked control of rat liver carbonic anhydrase III.
The concentration of carbonic anhydrase III (CAIII) in male rat liver was found to be 30 times greater than that in the female. Castration of male rats led to marked reduction in liver CAIII concentrations which could be partially restored to control levels by testosterone replacement. Marked developmental and senescence changes in liver CAIII were also observed in male rats.
Aging↗
A rapid one-step purification of NADPH-cytochrome c (P-450) reductase from rat liver microsomes.
NADPH-cytochrome c (P-450) reductase from liver microsomes of phenobarbital-treated rats has been purified in a single step by affinity chromatography on agarose-hexane-adenosine 2',5'-diphosphate. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme assay, and radioimmunoassay the protein obtained by this single step procedure is as pure as that isolated by multicolumn procedures.
Animals↗
Cloning and sequence analysis of a rat liver cDNA coding for a phenobarbital-inducible microheterogenous cytochrome P-450 variant: regulation of its messenger level by xenobiotics.
A rat liver cDNA library was prepared from total polyribosomal poly(A)+ RNA extracted from phenobarbital-treated animals. A cDNA clone coding for a phenobarbital-inducible cytochrome P-450 (PB P-450) was identified by differential colony hybridization to cDNAs synthesized from liver poly(A)+RNAs isolated from phenobarbital-treated rats for positive selection and cDNAs from either untreated rats or beta-naphthoflavone-treated rats as negative controls, followed by hybrid-selected translation and analysis of the translation products by immunoprecipitation. As the cloning and screening strategies involve no prior enrichment for specific mRNAs, they also permit the identification of sequences coding for phenobarbital-induced proteins other than cytochromes P-450. This relatively straightforward approach is generally applicable to the molecular cloning of sequences coding for other inducible cytochromes P-450. Nucleic acid sequencing data indicated that the cloned PB P-450 cDNA codes for a cytochrome P-450 variant [designated P-450e(U.C.)] that is very similar, but not identical, to P-450e. Sequence analysis of the section of cDNA specifying the 3'-non-coding region of the mRNA revealed that it lacked the usual poly(A) addition site signal sequence but contained three inverted repeat structures. Solution hybridization analysis demonstrated that PB P-450 mRNA is increased 20-fold by phenobarbital treatment and decreased 3-fold by beta-naphthoflavone treatment.
Animals↗
Factors controlling the expression of genes coding for drug-metabolizing enzymes.
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Animals↗
Differential effect of phenobarbital and beta-naphthoflavone on the mRNAs coding for cytochrome P450 and NADPH cytochrome P450 reductase.
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Animals↗
Complexes of cytochrome P450 with metyrapone. A convenient method for the quantitative analysis of phenobarbital-inducible cytochrome P450 in rat liver microsomes.
Phenobarbital-inducible cytochrome P450 variants have a higher affinity (app. Ks = 0.6 microM). The difference in affinity has enabled us to quantitate, by metyrapone-induced difference spectral analysis, phenobarbital-inducible cytochromes P450 in liver microsomal membranes. The result obtained by this simple, rapid technique compare well with those found by more sophisticated techniques such as radioimmunoassay and immunoprecipitation.
Animals↗
Induction by phenobarbital of the mRNA for a specific variant of rat liver microsomal cytochrome P-450.
The treatment of rats for 4 days with phenobarbital causes an apparent 3-fold increase in the amount of total liver cytochrome P-450. By sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, metyrapone binding and immunoprecipitation, this increase was found to be due to a much larger increase in a restricted number of specific cytochrome P-450 variants. A radioimmunoassay technique demonstrated that the major phenobarbital-inducible variant, of molecular weight 52 000, is induced 24-fold by phenobarbital. Immunoprecipitation analysis of products of translation in vitro with an antibody specific to the 52 000-mol.wt. cytochrome P-450 showed that phenobarbital induces the mRNA in polyribosomes for this variant 20-fold. Evidence is presented for the action of phenobarbital at the transcriptional and translational levels.
Animals↗
Phosphorylation and dephosphorylation of chromosomal proteins during in vitro transcription.
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Cell Nucleus↗
Representation of mRNA sequences in normal and SV40 transformed human diploid fibroblasts.
The representation of mRNA sequences in normal and SV40-transformed WI-38 human diploid fibroblasts was examined. Variations in the hybridization of polysomal RNAs to 3H-labeled DNAs complementary to poly A+ mRNA of normal and SV40-transformed cells suggest differences in the mRNA populations. a comparison of the representation of histone RNA sequences of normal and SV40-transformed human diploid cells did not reveal significant differences.
Base Sequence↗
Multiple forms of H4 histone mRNA in human cells.
Two species of H4 histone mRNA were isolated from the polysomes of S phase HeLa S3 cells. Electrophoresis under denaturing and nondenaturing conditions indicates that the two H4 mRNAs differ in size. Both mRNAs translate H4 histones in vitro, lack poly(A) at their 3' termini, and are capped at their 5' termini. Polyacrylamide gel electrophoresis and tryptic peptide analysis suggest that the polypeptides synthesized by the two mRNAs are indistinguishable.
Cell Cycle↗