Characterization and Transcriptional Control of the Rabbit Cytochrome P450pbii Genes (Evolution, Isozymes)
Govind, Shubha
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Permalink
https://hdl.handle.net/2142/77644
Description
Title
Characterization and Transcriptional Control of the Rabbit Cytochrome P450pbii Genes (Evolution, Isozymes)
Author(s)
Govind, Shubha
Issue Date
1986
Department of Study
Biology
Discipline
Biology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, General
Language
eng
Abstract
The phenobarbital (PB)-inducible cytochrome P-450(P-450) family is tentatively divided into the PBI (including rat P-450s b and e, and rabbit P-450 (form 2)), and PBII (including rabbit P-450s PBc1, PBc2, PBc3 (form 3b), PBc4 and (form 1)) subfamilies that are 55-60% homologous in their protein coding regions. The structures of the P-450PBII genes have been determined by restriction mapping and DNA sequencing lambda phage genomic clones corresponding to the genes for P-450PBc2, P-450PBc3 and P-450 (form 1). Comparison of the cloned genomic sequences with cDNA sequences established that genomic clone 6 contained the 5'-flanking region and the first six exons of the P-450PBc2 gene. Genomic clones 25 and 31 contained exons 7 through 9, and 3'-flanking regions of P-450PBc3 and P-450 (form 1) genes respectively. The locations of intron-exon junctions relative to the coding sequence in these genes are identical to the genes of the PBI subfamily, but completely different from the 3-methyl-cholanthrene inducible genes, suggesting that genes within the PB-inducible family arose by divergent evolution. The derived sequences of the first 20-22 N-terminal amino acids of the P-450PBc2 is 65% similar to the rabbit isozymes, form1, and form 3b, and rat isozymes, f, g, h, i and PB1, indicating that the rat isozymes probably belong to the PBII subfamily. The principal transcription initiation site of P-450PBc2 is 18 bp downstream from the TATA sequence, and 23 pb upstream from the translational initiator codon.
The relative rates of cytochrome P-450PBII mRNA synthesis were measured by in vitro transcription of rabbit liver nuclei after phenobarbital administration. Isolated RNA was hybridized to immobilized plasmid DNA containing the respective P-450PBII cDNA fragments. RNA that hybridized to P450s PBc1 and PBc2 cDNAs (88% homologous in their protein coding regions) was induced by phenobarbital 8 to 9 fold within 6 hours of PB administration. These levels declined during the next 18 hours but remained higher than untreated control values. RNA levels that hybridized to P-450PBc3 (72-74% homologous to PBc1 and PBc2), remained relatively unchanged. RNA that hybridized to the P-450PBc4 probe (about 70% homologous to P-450s 1-3), is induced 5 fold at 6 hours, but its levels decline within 12 hours to uninduced values. These levels are consistent with the changes in cytoplasmic mRNA levels of each species after PB treatment. Thus, the induction of P-450 genes by PB is due to increased transcription rates.
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