Transcriptional regulation of CYP6B1v3, a furanocoumarin-inducible P450 gene from black swallowtail (Papilio polyxenes)
Prapaipong, Hataichanoke
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Permalink
https://hdl.handle.net/2142/21913
Description
Title
Transcriptional regulation of CYP6B1v3, a furanocoumarin-inducible P450 gene from black swallowtail (Papilio polyxenes)
Author(s)
Prapaipong, Hataichanoke
Issue Date
1995
Doctoral Committee Chair(s)
Schuler, Mary A.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Biology, Entomology
Chemistry, Biochemistry
Language
eng
Abstract
Black swallowtail (Papilio polyxenes) is an oligophagous insect feeding exclusively on plant species which contain toxic furanocoumarins. P. polyxenes owes its tolerance to furanocoumarins to at least one cytochrome P450 isozyme, CYP6B1, which is capable of metabolizing linear furanocoumarins at high efficiency. CYP6B1 is autoregulated as it is transcriptionally induced by xanthotoxin, which the protein is capable of metabolizing.
In order to understand transcriptional regulation of this furanocoumarin-inducible system, a CYP6B1v3 gene encoding this P450 was isolated from a P. polyxenes genomic library. The open reading frame of this gene, interrupted by a single intron, is virtually identical to the previously characterized CYP6B1v1 and CYP6B1v2 cDNAs. Northern and RNase protection analyses have demonstrated that CYP6B1v1-3 transcripts are induced in P. polyxenes midguts in response to xanthotoxin and bergapten, two linear furanocoumarins, and to a lesser extent by angelicin, an angular furanocoumarin. To identify regulatory elements in the CYP6B1v3 promoter responsible for xanthotoxin inducibility, chimeric constructs containing the CYP6B1v3 5$\sp\prime$ flanking region linked to CAT reporter sequences were transiently transfected into lepidopteran Sf9 cell cultures. CAT activity analysis indicates that the CYP6B1v3 promoter and the xanthotoxin response cascade are functional in this heterologous system. Transfection of deletion and mutant derivatives of the CYP6B1v3 promoter has identified a positive xanthotoxin-responsive element (XRE) located from $-$136 to $-$119, relative to the transcription start site. This 18-bp region containing a canonical CAAT sequence and T-rich sequences is required for both basal expression and xanthotoxin inducibility of the CYP6B1v3 promoter. Gel shift assays conducted with the XRE-containing probe and Sf9 nuclear extracts have identified a heat-labile protein(s) which binds specifically to this region of the promoter. I have demonstrated that this factor is constitutively present in both control and xanthotoxin-treated Sf9 nuclear and P. polyxenes cell extracts and have termed it constitutive XRE-binding factor (CXF). Phosphorylation of CXF has shown to be partially responsible for its DNA-binding activity. In toto, these data suggest that binding of a constitutively expressed protein(s) is required for both basal and inducible transcription from this promoter and that additional transcription factors are required for transcriptional activation (or derepression) by xanthotoxin.
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