Nucleic Acids Research, 1994, Vol. 22, No. 15 3210-3217
© 1994
MOLECULAR BIOLOGY |
Transcriptional regulation of the Papilio polyxenes CYP6B1 gene
1Departments of Biochemistry, University of Illinois Urbana, IL 61801, USA 2Departments of Entomology, University of Illinois Urbana, IL 61801, USA 3Departments of Biochemistry and Plant Biology University of Illinois Urbana, IL 61801, USA
*To whom correspondence should be addressed at: Department of Plant Biology, University of Illinois, Urbana, XL 61801, USA
Received January 24, 1994. Revised June 3, 1994. Accepted June 16, 1994.
Detoxification of host plant defensive compounds by larval Lepidoptera is mediated by cytochrome P450 monooxygenases (P450s) such as CYP6B1, which is expressed in Papilio polyxenes (black swallowtail) larvae in response to xanthotoxin, a linear furanocoumarin. Baculovirus-mediated expression of two cloned CYP6B1 cDNAs in lepidopteran cell lines has demonstrated that CYP6B1 isozymes primarily metabolize the linear furanocoumarins, xanthotoxin and bergapten, and not angular furanocoumarins. To characterize the regulatory features of the CYP6B1 transcription unit, we have isolated the first full-length CYP6B1v3 genomic DNA clone from P.polyxenes. The open reading frame of this gene is interrupted by a single intron and is virtually identical to the previously characterized CYP6B1 cDNAs. Primer extension and ribonuclease protection analyses have localized the transcription initiation site to a point 28 nucleotides upstream from the AUG initiation codon. RNase protection analyses on RNA from larvae induced by linear and angular furanocoumarins indicate that transcription of the CYP6B1 gene is induced in insects significantly in response to xanthotoxin and only slightly in response to bergapten. Angular furanocoumarins, such as angelicin, which are not appreciably metabolized by the CYP6B1 gene product, do not significantly induce transcription of this gene. We conclude that this P450 gene is transcriptionally regulated in vivo by at least one of the substrates which the encoded protein metabolizes. Transient expression of CAT fusion constructs in transfected Sf9 lepidopteran cells demonstrates that nucleotides -1 to -838 upstream from the CYP6B1v3 transcription initiation site retain basal and xanthotoxin-inducible transcriptional activities in this heterologous cell line. These data clearly indicate that P.polyxenes has adapted to the presence of furanocoumarins in its host plants by evolving P450 isozymes and regulatory cascades which respond to specific toxins.
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