Characterization of Dna Complementary to Rabbit Cytochrome P-450 Messenger-Rnas and Studies on Their Differential Regulation by Phenobarbital (Gene, Expression)

In order to understand the regulated biosynthesis of cytochrome P-450 I have prepared a clone bank of mRNA extracts from the liver of a rabbit treated with phenobarbital. Plasmid cDNAs were selected as potential P-450 clones by plus-minus hybridization, in vitro translation of hybrid-selected mRNA, and dot blot RNA hybridization. One plasmid of 500 base pairs was selected by these techniques. A rescreen of the clone bank with this clone revealed an additional 20 plasmids hybridizing to this probe. Three classes of plasmids were found in these 20 plasmids by restriction analysis. Sequencing of these plasmids revealed that these clones were 50-60% homologous to rat phenobarbital-type cytochrome P-450. Based on comparison to the rat sequence, two of the cDNAs are missing sequence coding for the first 10 amino acids and the third approximately 80 amino acids. Two of the cDNAs have a poly A tail but one uses an altered recognition signal, AATAGA. A detailed study of the effects of phenobarbital on the synthesis of these mRNAs was undertaken. Poly A RNA was isolated from the livers of control rabbits and rabbits sacrificed 6, 12, 18, and 24 hours after phenobarbital injection. Poly A RNA was also isolated from kidney and lung tissue. Analysis of the three classes revealed that the levels of P-450PBc3 was not altered by phenobarbital. This RNA was not found in kidney tissue. P-450PBc2 mRNA was increased 3-fold by 24 hours over control levels of RNA in both liver and kidney although levels of RNA in the kidney were 15% of these in the liver. P-450PBc1 was not detected in control animals or in the kidney but were increased after phenobarbital administration. None of these three were detected in the lung. Northern analysis revealed the presence of one species of RNA for P-450PBc3 and multiple species for the other two. These data demonstrate that related P-450s are differentially regulated in the liver and that differences in expression between the liver and kidney exist for these mRNAs.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Physiology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2014

Author dc:creator

• Leighton, John Kevin

• Biology, General

Identifier

(UMI)AAI8502218

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/71433