cytochrome p450 cyp6l1 is speciﬁcally …scott.entomology.cornell.edu/107.pdfcytochrome p450...

Insect Biochemistry and Molecular Biology 31 (2001) 179–187www.elsevier.com/locate/ibmb

Cytochrome P450CYP6L1is specifically expressed in thereproductive tissues of adult male German cockroaches,Blattella

germanica(L.)

Zhimou Wena, Jeffrey G. Scotta,*

a Department of Entomology, Comstock Hall, Cornell University, Ithaca, NY14853, USA

Received 7 February 2000; received in revised form 8 June 2000; accepted 12 June 2000

Abstract

A full-length cDNA encoding a new cytochrome P450, CYP6L1, was cloned from German cockroaches,Blattella germanica.CYP6L1has an open reading frame of 1509 nucleotides with a deduced protein of 503 amino acids and molecular mass of 57 Kd.CYP6L1 is most similar to CYP6H1, a putative ecdysone 20-hydroxylase fromLocusta migratoria. CYP6L1 mRNA was notdetected in embryos nor nymphs, nor in adult females. CYP6L1 mRNA was detected only in the testes and accessory glands ofmale adult German cockroaches. Given that the testes and accessory glands are the most important components of the reproductivesystem in male insects, the expression of CYP6L1 mRNA exclusively in these tissues strongly suggests thatCYP6L1has a rolein reproduction. Possible substrates for CYP6L1 are discussed. 2001 Elsevier Science Ltd. All rights reserved.

Keywords:Cytochrome P450 monooxygenase; Sex-specific expression; Male reproductive system;Blattella germanica; Insecta

1. Introduction

Cytochrome P450s (P450s) are an important enzy-matic system found in all organisms examined, includingbacteria, fungi, plants and animals. Each animal hasapproximately 80 (Caenorhabditis elegans,(Consortium, 1998)) to 90 (Drosophila melanogaster,(Adams et al., 2000)) P450s, and some can metabolizenumerous substrates (Bernhardt, 1995; Guengerich,1995; Nebert and Gonzalez, 1987). P450s are namedCYP followed by a number, letter and number indicatingthe family, subfamily and individual isoform (Nelson etal., 1996). In this system, individual P450s with.40%amino acid sequence identity are usually grouped intothe same family, and those with.55% identity groupedinto the same subfamily. However, there are severalexceptions. For example, CYP6A1 and CYP6B2 are,40% identical, but are both grouped into family 6because “sequences surrounding the conserved cysteine

* Corresponding author. Tel.:+1-607-255-7340; fax:+1-607-255-0939.

E-mail address:[email protected] (J.G. Scott).

0965-1748/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved.PII: S0965-1748 (00)00116-8

residue make it clear that these two genes are evol-utionarily related” (Nelson et al., 1993). Given that thisnomenclature system is based on the overall sequencesimilarity, and that a minor change in a single aminoacid can alter substrate specificity (Lindberg and Negi-shi, 1989), substrate preference cannot be assumed fora certain P450 based on its name.

Insect P450s catalyze the metabolism of plant allelo-chemicals and insecticides, as well as physiologicallyimportant endogenous compounds including juvenilehormones (JHs), ecdysteroids and pheromones(Berenbaum, 1999; Feyereisen, 1999; Hodgson, 1985;Scott et al., 1998). Among the insect P450s, CYP6B1(Ma et al., 1994) and CYP6B4 (Hung et al., 1997) wereshown to metabolize plant allelochemicals(furanocoumarins). CYP6A1 (Andersen et al., 1994),CYP6A2 (Dunkov et al., 1997), CYP6D1 (Wheelockand Scott, 1992; Zhang and Scott, 1996) and CYP12A1(Guzov et al., 1998) have been shown to metabolizeinsecticides. CYP4C7 was demonstrated to metabolizeJHs and JH precursors (Sutherland et al., 1998).CYP6A1 also metabolizes JHs and their analogs(Andersen et al., 1997). The cloning of these P450soffers a great opportunity to better understand the roles

180 Z. Wen, J.G. Scott / Insect Biochemistry and Molecular Biology 31 (2001) 179–187

of P450s in herbivore–plant interactions, insecticideresistance, insect development and physiology.

P450s have been implicated in insect reproduction(Hodgson, 1985; Yu and Terriere, 1974). However, noindividual insect P450 has yet been associated withreproduction. Herein, we report cloning of the full-lengthcDNA of the first sex-specific insect P450, CYP6L1,which is exclusively expressed in the reproductive sys-tem of male adult German cockroaches. This finding hasimportant implications for understanding the roles ofP450s in insect reproduction and may offer a potentialtarget for the development of novel insect control agents.

2. Materials and methods

2.1. Insects

Two strains of German cockroaches were used: Bay-gon-R (Siegfried and Scott, 1991) and CSMA (Scott andMatsumura, 1981). Cockroaches were reared asdescribed previously (Siegfried and Scott, 1991).

2.2. Dissection

To examine the expression ofCYP6L1 in differentbody regions of male and female adult cockroaches theabdomens were separated from the remainder of the bod-ies on dry ice using forceps. The expression ofCYP6L1in the abdomens of adult male cockroaches was alsoexamined. For this, cockroaches were anesthetized onice and their wings removed. Then cockroaches werepinned in a Sylgard dish, opened dorsal-ventrally andthe testes and/or accessory glands (Cornwell, 1968;Snodgrass, 1937) were quickly removed under a binocu-lar dissection scope at 24°C. The heads and thoraceswere then cut off and the dissected abdomens put on dryice for subsequent RNA isolation.

2.3. RNA isolation

Total RNA was isolated using 5 M guanidine thio-cyanate solution based on the method of Chirgwin et al.(Chirgwin et al., 1979). Messenger RNA was isolatedfrom total RNA using Oligotex suspension (QIAGEN)or directly from tissues using a QuickPrep mRNA puri-fication kit (Pharmacia) as described by the manufac-turers.

2.4. cDNA synthesis

Superscript II (GIBCO/BRL) was used to synthesizethe first strand cDNA following the manufacturer’sinstructions. For the first strand cDNA synthesis, C3PT

(Danielson and Fogleman, 1997) (Table 1) was used asprimer and|500 ng mRNA from abdomens of maleadult Baygon-R strain of German cockroaches as tem-plate. After RNAase H (2 units) treatment at 37°C for30 min, the first strand cDNA was isolated using a QIA-quick PCR purification kit (QIAGEN) to remove the pri-mers and short cDNA (eluted with 100µl H2O). Thepurified cDNA was used as a template for 39 and 59RACEs (Frohman et al., 1988) as described below.

2.5. 39 RACE

The first strand cDNA (5µl) was used as a template.A degenerate primer based on the P450 heme signaturemotif GPRNCIG (Danielson et al., 1999) and a C3primer (Danielson and Fogleman, 1997) (Table 1) wereused to amplify putative 39 cDNA sequences of P450genes by PCR. The PCR reaction (100µl) was heatedto 95°C for 3 min, followed by 35 cycles of amplifi-cation (95°C, 50°C and 72°C sequentially each for 30s) and a final extension at 72°C for 10 min. After 1.5%agarose gel fractionation of the PCR products, a regioncorresponding to about 300–700 bp based on DNA sizemarkers (100 bp ladder, GIBCO/BRL) was cut and theDNA purified using a QIAquick gel extraction kit(QIAGEN). The purified PCR products were cloned intopCR2.1 vector (Invitrogen) following the manufac-turer’s instructions. Plasmids were isolated from positiveclones (white colonies) and the inserts sequenced by theDNA Sequencing Facility at Cornell University. PutativeP450s were identified based on BLAST analysis(Altschul et al., 1997) using the deduced amino acidsequences. The inserts of six clones (MCH1, MCH6,MCH11, MCH45, MCH46 and MCH75) had identicalsequences with a length of 371 bp excluding poly (A).The gene these sequences represented was namedMCHA.

2.6. 59 RACE

To obtain the 59 sequence of MCHA, the purified firststrand cDNA was tailed with dCTP by terminal deoxyn-ucleotidyl transferase (GIBCO/BRL) following themanufacturer’s instructions with the following modifi-cation. Instead of using the manufacturer’s 5× reactionbuffer, we used a 5× tailing buffer (500 mM sodiumcacodylate, 1 mMβ-mercaptoethanol, 20 mM magnes-ium chloride, pH 7.2) as suggested by P. Danielson(University of Denver, Colorado). The tailed cDNA waspurified using a PCR purification kit as described above(eluted with 100µl H2O) and 5µl was used as templatefor 59 RACE.

An MCHA specific antisense primer MCHAA1(Table 1 and Fig. 1) was designed based on the 39sequence information of MCHA. Two rounds of PCRwere performed for the 59 RACE. The first round PCR

181Z. Wen, J.G. Scott / Insect Biochemistry and Molecular Biology 31 (2001) 179–187

Table 1Primers used for cloning of CYP6L1 cDNA

Primer Sequence

C3PT 59-GACTCGAGTCGGATCCATCGTTTTTTTTTTTTTTTT-39C3 59-GACTCGAGTCGGATCCATCG-39Heme 59-GGICCI(A/C)GIAA(C/T)TG(C/T)ATIGG-39AAP 59-GGCCACGCGTCGACTAGTACGGGGGGGGGGGGGGGG-39AUAP 59-GGCCACGCGTCGACTAGTAC-39MCHAA1 59-AACAAACATCAGAGGAAGTCGTCC-39MCHAA9 59-CTCTGAACCCTTCAGTTTCACC-39MCHAS3 59-CAACGTCAAGAAGTAGCCAACC-39MCHAS4 59-TTCAGTCCTGTGGTTCCTTTC-39

(100 µl reaction) used MCHAA1/AAP (GIBCO, Table1) as the primer set and dCTP tailed first strand cDNA(see above) as template. The conditions for the firstround PCR were 95°C for 3 min followed by 32 cyclesin the order of 95°C for 1.5 min, 50°C for 1.5 min and72°C for 4 min. The PCR reaction had a final extensionfor 15 min at 72°C. After electrophoresis (1.5% agarosegel) of the first round PCR products, a region corre-sponding to 1–2 kb was cut and the PCR products pur-ified (eluted with 100µl H2O). The purified first roundPCR products (5µl) then served as templates for thesecond round PCR using MCHAA1/AUAP (GIBCO,Table 1) as the primer set. Products of the second roundPCR (conditions were the same with that of the firstround PCR, except the annealing temperature was raisedto 55°C and cycles increased to 35) corresponding to aregion of 1–2 kb on agarose gel was again purified andcloned into pCR 2.1 vector. Positive clones werescreened by colony PCR (Gussow and Clackson, 1989)using single putatively positive colonies (white colonies)as templates and heme primer/MCHAA1 as the primerset. The insert lengths of those positive clones werefurther checked by colony PCR using MCHAA1/AUAPas the primer set. Plasmids were isolated from twoclones (A11 and A180) with the longest inserts and theinserts were sequenced. The newly obtained sequenceinformation from A11 and A180 was used to design pri-mers for another round of 59 RACE. After two roundsof 59 RACE, the 59 sequence beyond the translationalstart codon was obtained. All the adjacent sequencesobtained by 39 and 59 RACEs had at least 150 bp overlapwith each other. The putative full-length MCHAsequence MCHAMER was obtained by integrating over-lapping sequences from the 39 RACEs and 59 RACEs.

2.7. Cloning of full-length cDNA

Two sense primers upstream of the start codon(MCHAS3 and MCHAS4, Table 1 and Fig. 1) and oneantisense primer downstream of the stop codon(MCHAA9, Table 1 and Fig. 1) were designed basedon the sequence of MCHAMER. The first round PCR

products using first strand cDNA as template andMCHAS4/MCHAA9 as primer set were used as tem-plates to perform the second round PCR withMCHAS3/MCHAA9 as primer set. The conditions forthe first round PCR were 95°C to denature for 3 minfollowed by 32 cycles in the order of 95°C for 1 min,50°C for 1 min and 72°C for 2 min. The PCR reactionended with a final extension for 15 min at 72°C. Theconditions for the second round PCR were the same asthe first round PCR, except that the cycles were 35instead of 32. A 1.6 kb band on agarose gel was purifiedand cloned into pCR2.1 vector. A positive clone(FA13) was identified by colony PCR and its insertentirely sequenced.

2.8. Northern blots

Northern blot analysis of CYP6L1 mRNA expressionwas performed as described by Sambrook et al.(Sambrook et al., 1989). Briefly, 10µg of total RNA wasfractionated on 1% denaturing formaldehyde agarose gelcontaining ethidium bromide. After washing in distilledwater for about 3–4 h with several changes, the RNAwas transferred to a GeneScreen Plus hybridizationtransfer membrane (NEN Life Science Products, Inc.).Equal loading was monitored by comparing the densityof the 18S ribosomal RNA (rRNA) band (Savonet et al.,1997; Spiess and Ivell, 1998) on the agarose gel beforetransfer and/or on the membrane after transfer under UV.A 1.6 kb CYP6L1 fragment was amplified by PCR(using plasmids isolated from FA13 as templates andMCHAS3/MCHAA9 as the primer set), labeled with [α-32P] dCTP using the RadPrame labeling system(GIBCO/BRL), and used as a hybridization probe. Themembrane was hybridized to the probe in QuickHyb sol-ution (Stratagene) at 68°C. Washing was done at highstringency (i.e. three 15 min washes with 2×SSC+0.1%SDS at room temperature, followed by a 30 min washwith 0.2×SSC+0.1% SDS at 60°C). The membrane wasair dried and exposed to a BioMax MR film (EastmanKodak). All Northern analyses were repeated at leastthree times using independent preparations of RNA.


Fig. 1. CYP6L1 full-length cDNA and deduced amino acid sequence. Amino acids (AA) are numbered on the left and nucleotides (NT), on theright. Primers (Table 1) are thickly underlined and conserved regions thinly underlined. Putative polyadenylation signals are indicated by bold font.

2.9. Southern blots

Genomic DNA was isolated from the abdomens ofmale adult German cockroaches of Baygon-R strainusing standard methods (Sambrook et al., 1989). South-ern blots were performed by standard methods(Sambrook et al., 1989) with some modifications. Four

restriction enzymes (BamHI, NotI, SmaI, andXhoI), hav-ing no cutting site within the CYP6L1 cDNA sequencewere used to digest 2.5µg of genomic DNA overnightas described by the manufacturer (GIBCO/BRL). Fiftyunits of each enzyme was used for each reaction in avolume of 100µl. The digested DNA was then ethanolprecipitated and fractionated by electrophoresis on 1%


agarose gel containing ethidium bromide. After electro-phoresis, the gel was placed in 0.25 M HCl with gentleshaking until 10 min after the dyes had changed color.After being rinsed in H2O, the gel was denatured in asolution containing 1.5 M NaCl and 0.5 M NaOH withgentle shaking for 30 min. The gel was then rinsed withH2O and neutralized in a solution containing 1.5 MNaCl, 0.5 M Tris–HCl (pH 7.2) and 0.001 M EDTA.After rinsing in H2O, the DNA was transferred to aGeneScreen Plus membrane (NEN Life Science Pro-ducts, Inc.). Probe preparation, membrane hybridizationand film exposure were performed exactly as describedin Section 2.8. Southern analyses were repeated threetimes.

3. Results

3.1. cDNA cloning and characterization

The full-length cDNA sequence (accession #:AF227531) of MCHA is shown in Fig. 1. MCHA wasnamed CYP6L1 by the P450 Nomenclature Committee.CYP6L1has an open reading frame of 1509 nucleotideswith a deduced protein of 503 amino acids and a molecu-lar mass of 57 kDa. A BLAST search (Altschul et al.,1997) with the deduced amino acid sequence shows thatCYP6L1 is most similar to the members of P450 family6 from insects. A comparison of CYP6L1 with somemembers of family 6 is shown in Table 2. CYP6L1 hasthe highest percent amino acid identity (37%) withCYP6H1, a putative ecdysone 20-hydroxylase from thegrasshopper,Locusta migratoria(Winter et al., 1999).

CYP6L1 is a typical microsomal P450 and its deduced

Table 2Comparison of CYP6L1 fromB. germanicawith other related P450sa

Isoforms Species % identityb Life stage specific expression Sex specific expression Source of tissuesd

Embryos Immaturec Adults Males Females

CYP6L1 B. germanica 100 – – + +e – Many tissues examined(see text)

CYP6H1 L. migratoria 37 – + + NI NI Malpighian tubulesCYP6A1 M. domestica 28 + + + + + Gut, fat bodyCYP6A2 D. melanogaster 31 NI NI + NI NI Fat body and other tissuesCYP6A9 D. melanogaster 29 NI NI + NI NI NICYP6B1 P. polyxenes 28 NI + NI NI NI MidgutCYP6B2 H. armigera 28 – + – NI NI Midgut, fat body,

head/foregutCYP6D1 M. domestica 26 – – + + + Many tissues

a References: CYP6A1 (Carino et al., 1992, 1994); CYP6A2 (Brun et al., 1996); CYP6A9 (Maitra et al., 1996); CYP6B1 (Cohen et al., 1992);CYP6B2 (Ranasinghe et al., 1997; Xiao-Ping and Hobbs, 1995); CYP6D1 (Scott, 1999) and CYP6H1 (Winter et al., 1999) NI=not investigated.

b The per cent amino acid identity was calculated based on the Clustal method of MEGALIGN program (DNASTAR Inc., Madison, Wisconsin).c Nymphs forB. germanicaandL. migratoria, larvae for all others.d Tissues where the expression was investigated or from which the cDNA was cloned.e Specifically in testes and accessory glands (see text).

amino acid sequence shares a number of commoncharacteristics with other members of the P450 super-family. The N-terminal region is strongly hydrophobic,with hydrophobic residues accounting for|70% (16 outof the first 23 amino acid residues), which is importantfor membrane-anchoring (Wachenfeldt and Johnson,1995). The sequence FGDGPRNCIG at positions 431–440 matches the signature motif FxxGxxxCxG, which isthe heme-binding region and is highly conserved amongP450s. AGFET at positions 298–302 is highly conserved(consensus (A/G)GxxT) within the I helix where T302

may function as a proton donor and/or an acid-base cata-lyst in oxygen scission (Wachenfeldt and Johnson,1995). The charge pair at positions 356–359 is highlyconserved (EVLR; consensus ExxR) within the K helixwhich is assumed to be hydrogen-bonded to the‘meander’, a region N-terminus to the heme-bindingregion (Peterson and Graham-Lorence, 1995). Otherconserved regions or residues include: WRQIR(consensus WxxxR) at positions 129–133 in the C helix,P318 exactly 16 residues from T302 and YPDP (consensus(aromatic)xx(P/D)) at positions 404–407 followed fiveresidues later by PERF at positions 412–415 (Nelson,1998).

3.2. Expression in adults

CYP6L1 mRNA expression was examined in six lifestages (mixed sexes): eggs (egg cases), first and secondinstars, third and fourth instars, fifth instars, sixth instarsand adults. CYP6L1 mRNA (~1.8 kb long signal) wasdetected only in adults, but not in eggs nor in any nym-phal instars (Fig. 2).


Fig. 2. Life stage-specific expression of CYP6L1. Total RNA was prepared from approximately 1 g of cockroaches (mixed sexes) for each lifestage and 10µg of total RNA was loaded for each lane. Northern hybridization with the CYP6L1 cDNA probe is shown on the top panel. RNAloading was standardized by ethidium bromide staining of 18S rRNA (bottom panel).

3.3. Expression in adult male abdomens

CYP6L1 mRNA expression was examined in theabdomens (A) versus the remainder of the bodies (H+T)in adult males or females. CYP6L1 mRNA was detectedin the abdomens of males, but not in the abdomens offemales nor in the remainder of the bodies of males orfemales (Fig. 3).

3.4. Expression in reproductive tissues

Given that CYP6L1 mRNA was expressed exclus-ively in the abdomens of adult males, expression in the

Fig. 3. Sex-specific expression of CYP6L1. Total RNA was prepared from abdomens (A) or the remainder (H+T) of the bodies (excludingabdomens) of 15 male or female adult German cockroaches. Ten micrograms of total RNA was loaded for each lane. Northern hybridization withthe CYP6L1 cDNA probe is shown on the top panel. RNA loading was standardized by ethidium bromide staining of 18S rRNA (bottom panel).

male reproductive system was examined. Fig. 4 showsthat CYP6L1 mRNA was detected in the abdomens aftereither testes or accessory glands were removed, butcould not be detected when both the testes and accessoryglands were removed. These results indicate thatCYP6L1 was specifically expressed in the testes (germcells) and accessory glands (somatic cells) of male adultGerman cockroaches. The greater reduction in CYP6L1signal following the removal of accessory glands com-pared to removal of testes may simply be a reflection ofthe greater mass of the accessory glands. CYP6L1mRNA was also detected by Northern blot using totalRNA from testes, but could not be detected using total


Fig. 4. Tissue-specific expression of CYP6L1. Total RNA was pre-pared from abdomens of 15 male adult German cockroaches excludingcertain tissues. Ten micrograms of total RNA was loaded for eachlane. Lane 1: Abdomens with testes removed; Lane 2: Abdomens withaccessory glands removed; Lane 3: Abdomens with both testes andaccessory glands removed; Lane 4: Whole abdomens. Northernhybridization with the CYP6L1 cDNA probe is shown on the toppanel. RNA loading was standardized by ethidium bromide stainingof 18S rRNA (bottom panel).

RNA from guts or abdomen remains (excluding testes,accessory glands and guts) (results not shown). Forunknown reasons, we were unable to isolate enoughRNA from dissected accessory glands for analysis.

3.5. Southern blots

Genomic DNA digested withBamHI, NotI, SmaI, andXhoI was used in a Southern blot to evaluate the speci-ficity of our CYP6L1 probe. A single band was observedin all cases (data not shown), suggesting that the probewas specific for CYP6L1. The relatively large fragmentsthat were detected (ranging from approximately 10–20kb) make it impossible to conclude whetherCYP6L1exists as a single copy gene or not.

4. Discussion

Although expression patterns for most insect P450sare not yet clear, available information shows that insectP450s have great diversity in their expression patternswith regard to life stages and tissues (Scott et al., 1998)(Table 2).CYP6L1is unique because it is the first sex-specific insect P450 reported. Furthermore,CYP6L1stands out because, although a number of mammalianP450s are expressed at different levels in males andfemales (Waxman and Chang, 1995),CYP6L1is the firstP450 found to be specifically expressed in the testes andaccessory glands of the male reproductive system(Table 2).

Substrates for CYP6L1 cannot be predicted based onits sequence similarity with other P450s of family 6.However, gene expression patterns can provide a “strongclue as to its biological role” (DeRisi et al., 1997), as

has been demonstrated forCYP4C7(Sutherland et al.,1998). Given that testes and accessory glands are themost important components of the reproduction systemin male insects, the expression ofCYP6L1exclusivelyin these tissues suggests that it plays some role in repro-duction. What could this role be?

JHs and ecdysteroids are very important in that theycontrol insect development during larval and pupalstages (nymphal stages in cockroaches) and have gona-dotropic function in the adult stages (Hardie, 1995). Yuand Terriere suggested that insect P450s were involvedin reproduction via control of hormone titers (Hodgson,1985; Yu and Terriere, 1974). Thus, one possible role forCYP6L1 might be the regulation of JHs in the accessoryglands and ecdysteroids in the testes. Three lines ofreasoning suggest that this is possible. First, insectaccessory glands can synthesize juvenile hormone(Borovsky et al., 1994) and testes are an alternative sitefor ecdysteroid synthesis in several insects (Delbecqueet al., 1990). Although the production of hormones bythese tissues might be small, the local concentrationincrease could be significant. Second, insect P450s areinvolved in metabolism of JH and ecdysteroids(Feyereisen, 1999; Hodgson, 1985). Third, substrates fora given P450 can be structurally unrelated compounds(Andersen et al., 1994, 1997; Guengerich, 1995). There-fore, it appears possible that CYP6L1 could be involvedin ecdysteroid synthesis in testes and juvenile hormonesynthesis in the accessory glands of male adult Ger-man cockroaches.

Other possible roles for CYP6L1 exist as well. Insecttestes and accessory glands produce a complex mixtureof compounds which can modify female fecundity,receptivity and/or have other functions (Gillott, 1995).CYP6L1 might be involved in some of these processes.Alternatively, CYP6L1 could be involved in a functionyet unknown to us (e.g., regulating titers of an unknownhormone). Further study of CYP6L1 will help clarify thefunction(s) of this sex-specific P450 and improve ourunderstanding of the male reproductive system ininsects. If CYP6L1 is critical for reproduction, this willoffer a potential target for the development of novelinsect control agents.

Acknowledgements

We thank Drs P. Danielson and J. Fogleman forsuggestions with the 39 and 59 RACE techniques, DrsC. Gilbert, J. Helmann, J. Ewer and M. Wolfner for valu-able discussions. We also thank Dr D. Nelson for namingthe P450. This project was supported by a grant fromSigma Xi (to ZW) and Hatch Project 139414.


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cytochrome p450 cyp6l1 is speciﬁcally …scott.entomology.cornell.edu/107.pdfcytochrome p450...

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