Po1y(A)+ RNA was isolated from membrane-bound polysomes of the livers of 3- methylcholanthrene (MC)-treated rats, and was partially purified by sucrose density gradient centrifugation. The mRNA was translated in an in vitro rabbit reticulocyte Jysate system, and assayed for the synthesis of MC-inducible forms of cytochrome P-450 (cytochrome P-450MC) using anti-cytochrome P-450c antibody which reacted with two types of cytochrome P-450MC, P-450c, and P-450d. The mRNA activity for cytochrome P-450MC was located at around 18S, accounting for approximately 5% of total mRNA activity. The double-stranded complementary DNA which had been synthesized from the partially purified mRNA by avian myeloblastosis virus reverse transcriptase and DNA polymerase I (Klenow enzyme) was cloned in Escherichia coli %1776, using plasmid pBR322 as a cloning vector. After differential colony hybridization using [32P]cDNA's synthesized from mRNA preparation of MC-treated or untreated rat liver as a probe, a clone (3-9-1) carrying cytochrome P-450MC cDNA sequence was identified by a positive hybridization-translation assay. The specific mRNA hybridized with plasmid 3-9-1 DNA showed an enriched synthesis of a protein with apparent molecular weight of 56,000 daltons, which was immunoprecipitable with anti-P-450c antibody. In RNA blot analysis with MC-, polychlorinated biphenyls (PCB)-, and phenobarbital (PB)-induced mRNA as well as uninduced mRNA, a longer cDNA (P-34) which had been isolated by hybridiza tion with the insertion of clone 3-9-1, and the previously isolated PB-inducible cytochrome P-450b cDNA (Fujii-Kuriyama et al. (1982) Proc. Natl. A cad. Sci. U.S. 79, 2793-2797) hybridized with mRNA preparations in an inducer-specific manner. The former cDNA strongly hybridized with MC- and PCB-induced mRNA, while the latter one did so with PB- and PCB-induced mRNA. The mRNA hybridized with P-450MC cDNA showed a single band with almost the same mobility as observed with the mRNA hybridized with P-450b cDNA, indicating that the mRNA for P-450MC is approximately 2,000 bases long, as is the case with the P-450b mRNA. From these results, we conclude that the isolated cDNA clones carry a comple mentary sequence to the cytochrome P-450MC mRNA.A preliminary sequence analysis of the longer cDNA insert (P-34) revealed that the longer cDNA insert indeed contained the coding nucleotide sequence for the reported NH sequence of 30 amino acids of one of the two MC- inducible cytochrome P-450's, P-450d (Botelho et al. (1982) Biochemistry 21, 1152-1155) (data to be published elsewhere).
|Number of pages||9|
|Journal||Journal of biochemistry|
|Publication status||Published - 1983 Nov|
ASJC Scopus subject areas
- Molecular Biology