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Isolation and characterization of cytochrome P450s from Papilio polyxenes and Papilio glaucus

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Title: Isolation and characterization of cytochrome P450s from Papilio polyxenes and Papilio glaucus
Author(s): Hung, Chien-Fu
Doctoral Committee Chair(s): Berenbaum, May R.
Department / Program: Entomology
Discipline: Entomology
Degree Granting Institution: University of Illinois at Urbana-Champaign
Degree: Ph.D.
Genre: Dissertation
Subject(s): Biology, Molecular Biology, Entomology
Abstract: Previous studies have isolated two cytochrome P450 monooxygenase cDNAs designated CYP6B1v1 and CYP6B1v2 from the oligophagous Papilio polyxenes. Baculovirus-mediated expression of these P450s demonstrated that both enzymes metabolize substantial amounts of linear furanocoumarins and low, if any, amount of angular furanocoumarins. The polyphagous Papilio glaucus rarely encounters furanocoumarin-containing plants and yet it contains low constitutive levels of cytochrome P450s capable of metabolizing furanocoumarins, which significantly induce these cytochrome P450 activities when present furanocoumarins in the diet. The purposes of this thesis were to isolate and characterize cDNAs and genes encoding an angular furanocoumarin-metabolizing P450 from P. polyxenes and furanocoumarin-metabolizing P450s from P. glaucus.Metabolism of several furanocoumarins by cytochrome P450s of P. polyxenes is significantly and selectively induced by two linear furanocoumarins, xanthotoxin and bergapten, as well as the angular furanocoumarin, angelicin but not by the angular furanocoumarin sphondin. In Northern analysis, CYP6B1 and related mRNA are highly induced by xanthotoxin, bergapten, and angelicin but not sphondin. In RNase protection analysis, CYP6B1v3 transcripts are specifically induced by xanthotoxin, bergapten and angelicin, and additional CYP6B1-related transcripts are induced in response to angelicin.The CYP6B3v1 cDNA was isolated from an angelicin-induced cDNA library of P. polyxenes and shown to be 88% identical to CYP6B1 cDNA at the amino acid level. Reverse transcription-PCR Southern analyses demonstrated that CYP6B3 transcripts are highly induced by linear (xanthotoxin and bergapten) and angular (angelicin and sphondin) furanocoumarins. Comparison of constitutive levels of these transcripts indicates that CYP6B1 transcripts are expressed at a higher level than CYP6B3 transcripts.The CYP6B3v2 genomic DNA clone was isolated from P. polyxenes and CYP6B4v2 and CYP6B5v1 genomic clones were isolated from P. glaucus. CYP6B3v2, CYP6B4v2, CYP6B5v1 and CYP6B1v3 genes all contain one intron at the same amino acid position. CYP6B3v2, CYP6B4v2 and CYP6B5v1 contain sequences similar to the xanthotoxin-responsive element identified from $-$146 to $-$97 in CYP6B1v3 promoter region shown to be required for transcriptional regulation by xanthotoxin. In addition, putative xenobiotic-responsive elements (XRE-AhR) are present in all four gene promoters; Barbie boxes (Bar) are present in the CYP6B1v3 and CYP6B4v2/CYP6B5v1 promoters; Sequences resembling antioxidant-responsive elements (ARE) are found in the CYP6B1v3 and CYP6B4v2/CYP6B5v1 promoters. Shared xanthotoxin-responsive elements, as well as conserved intron positions, suggest that these insect CYP6B genes are derived from a common ancestral gene likely associated with furanocoumarin detoxification. (Abstract shortened by UMI.)
Issue Date: 1996
Type: Text
Language: English
URI: http://hdl.handle.net/2142/22188
ISBN: 9780591199086
Rights Information: Copyright 1996 Hung, Chien-Fu
Date Available in IDEALS: 2011-05-07
Identifier in Online Catalog: AAI9712318
OCLC Identifier: (UMI)AAI9712318
 

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