cDNA Clones for Liver Cytochrome P-450s from Individual Aroclor-Treated Rats: Constitutive Expression of a New P-450 Gene Related to Phenobarbital-Inducible Forms

Abstract

Differential hybridization and screening with cloned inserts was used to identify two families of cytochrome P-450 cDNA clones in libraries prepared from total liver poly(A)+RNA of individual Aroclor-treated rats. One family has cDNA inserts for the major phenobarbital-inducible P-450s, P-450b and P-450e. Two types of P-450e inserts were identified. In addition, irregular inserts were characterized from two clones (PB23 and PB24) of this group. The other family has cDNA inserts for the major 3-methylcholanthrene-inducible species, P-450c and P-450d. No coding sequence restriction site variants were detected among 26 P-450d and P-450c inserts analyzed. The restriction map of the irregular 2.2-kb PB23 insert has a P-450b-like portion, followed by a 3' extension that hybridizes to RNAs of 2.7 and 4.8 kb, which are also detectable with a classical P-450b probe. The PB23 insert and the 2.7- and 4.8-kb RNAs presumably represent 3' extensions of P-450b/P-450e mRNAs, polyadenylated at downstream sites. The 858-bp sequence of the PB24 insert encodes the carboxy-terminal portion of a P-450b/P-450e-like protein. There is approximately 20% divergence at the polypeptide level between the PB24 and P-450b/P-450e sequences; nevertheless, they share many essential features. A PB24-specific probe hybridizes to a 1.9-kb RNA species which is present in the liver of untreated rats and which is not appreciably induced by phenobarbital or Aroclor. The PB24 cDNA most likely represents a constitutive cytochrome P-450, related to phenobarbital-inducible forms.

Heme may not be a positive regulator of cytochrome‐P450 gene expression

“…la). An additional faint band at 4.8 kb was also induced by phenobarbitone and 2-allyl-2-isopropylacetamide and most likely results from the use of a downstream polyadenylation site in the P450IIB1 gene [20]; there was no detectable effect of succinylacetone treatment on the level of this mRNA. /?-Naphthoflavone induces P450IA1 mRNA of size 2.7 kb and this was not affected by succinylacetone (Fig.…”

Phenobarbital, dexamethasone and benzanthracene induce several cytochrome P450 mRNAs in rat hepatoma cells

“…1). This synergism would result if, as has been found for rat liver [8,9], PB induces in Fao cells an increase in the intracellular concentration of receptor molecules for PAH compounds.…”

“…For each RNA gel, 10 or 15/~g of total RNA were loaded per lane. cDNA probes, generously provided by A. Anderson and M. Adesnik, include: C6 [8], which corresponds to P450 c mRNA; R17 [9] to P450 b/e; and pTF2 [10] to P450 PBI. The probes do not crosshybridize to one another and reveal mRNAs of 3 kb (C6), 2.1 kb (Rl7) and 1.9 kb (pTF2) long (not shown).…”

Aryl Hydrocarbon Receptor Activation and Cytochrome P450 1A Induction by the Mitogen-Activated Protein Kinase Inhibitor U0126 in Hepatocytes

“…Membranes were washed with 3ϫ SSC/0.1% SDS (v/v) for 30 min and then twice with 1ϫ SSC/0.1% SDS (v/v) for 10 min at 65°C. The two probes used were the human CYP1A1-specific cDNA probe, a gift from I. de Waziers (INSERM U490, Paris, France), and the P450 C6 cDNA probe complementary to rat CYP1A1 mRNA (Affolter et al, 1986). After hybridization, filters were stripped of the previous probes and rehybridized with nick-translated genomic 18S probe used as control.…”