Molecular Insect Biology (2000) 9(5), 441449
Blackwell Science, Ltd
Expression and activity of a house-y cytochrome P450, CYP6D1, in Drosophila melanogaster
P. J. Korytko, R. J. Maclntyre and J. G. Scott
1
1
2
1,3
Field of Environmental Toxicology, Rice Hall, Cornell University, 2Department of Molecular Biology and Genetics, Cornell University, and 3Department of Entomology, Cornell University, Ithaca, NY, USA
Abstract
The cytochrome P450 system of animals comprises many individual cytochromes P450 in addition to a single cytochrome P450 reductase and cytochrome b5. Although individual genes of the cytochrome P450 superfamily are highly diverged, the P450 reductase and cytochrome b5 remain more conserved across taxa. Here, we describe the transformation of Drosophila melanogaster with a house-y-specic cytochrome P450, CYP6D1. Functional activity of ectopically expressed cytochromes P450 requires successful interaction between the transgenic P450 and the requisite coenzymes of the host organism. Transformed Drosophila, but not controls, contained CYP6D1 protein as identied by protein immunoblotting, elevated total P450 and elevated CYP6D1 enzymatic activity. These data demonstrate that house-y CYP6D1 can interact with low to moderate efciency with Drosophila P450 reductase and cytochrome b5. Keywords: cytochrome P450 monooxygenases, house y, Drosophila, expression, activity, P450 reductase, cytochrome b5. Introduction Cytochromes P450 (P450s) comprise a gene superfamily (Nelson et al., 1996) with each cytochrome P450 designated CYP followed by a family, subfamily and isoform number (Nelson et al., 1996). P450s metabolize xenobiotics and endogenous compounds (Sipes & Gandol, 1991; Mansuy, 1998). Most species have multiple P450 isoforms (eighty-six
Received 28 February, 2000; accepted following revisions 15 May 2000. Correspondence: Dr Jeffrey G. Scott, Comstock Hall, Department of Entomology, Cornell University, Ithaca, NY 14853, USA. Tel.: [phone]; fax: [phone]; e-mail: [e-mail]
in Drosophila melanogaster (Adams et al., 2000)) and each P450 may have several substrates (Rendic & Di Carlo, 1997). Since the P450s in any one species may have overlapping substrate specicity, it remains difcult to identify the functions of individual P450s. Transgenic technology allows for examination of metabolic contributions of individual P450s in vitro and possibly in vivo in a system where the enzyme may be absent. P450 isoforms are the catalytic agents of the P450 monooxygenase system. For functional activity, all P450 isoforms require the cofactor NADPH-dependent P450 oxidoreductase (P450 reductase) and sometimes cytochrome b5. P450 reductase transfers one or more electrons to the P450 (Backes, 1993; Lewis & Pratt, 1998). In a substrate-specic manner, some P450s also require cytochrome b5 to either donate the second electron or to allosterically interact with the P450 (Bonls et al., 1989; Schenkman, 1989; Zhang & Scott, 1996). For example, CYP6D1, a house-yspecic P450 (Wheelock et al., 1991), requires cytochrome b5 for aryl hydrocarbon hydroxylase activity (AHH activity, i.e. metabolism of benzo[a]pyrene), but not for methoxyresorunO-demethylase (MROD activity) (Wheelock & Scott, 1992). Although P450 reductase and cytochrome b5 are well conserved (Hovemann et al., 1997), the conservation does not guarantee that P450s of one species will interact with the P450 reductase or cytochrome b5 of any other species. Ectopic expression (i.e. expression where it is not normally present) and activity of P450s in transgenic organisms provides evidence that P450s from one species can interact with the P450 reductase of closely as well as distantly related taxa. For example, human CYP3A7 is active in transgenic mice (Li et al., 1996), while mammalian CYP1A1 expressed in plants is metabolically active with or without fusion to P450 reductase (Shiota et al., 1994; Inui et al., 1999). In yeast, house-y CYP6D1 and Drosophila CYP6A2 are active when coexpressed with P450 reductase of yeast (Smith & Scott, 1997) and humans (Saner et al., 1996), respectively. Furthermore, the microsomal activity of baculovirus-expressed Drosophila CYP6A2 is enhanced when incubated with puried P450 reductase of house ies (Dunkov et al., 1997). These studies indicate that P450s can interact with the P450 reductase of other species. Less is known of the interactions of P450s and the cytochrome b5 of other species. In one study, house-y CYP6D1 441
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did not interact efciently or at all in the presence of endogenous yeast cytochrome b5 (Smith & Scott, 1997); while in another study baculovirus-expressed Drosophila CYP6A2 activity was enhanced by the addition of puried house-y cytochrome b5 (Dunkov et al., 1997). Drosophila remains the most practical transgenic insect system (Ashburner et al., 1998), and P-element-mediated transformation of Drosophila is an extensively used method (Rubin & Spradling, 1982). This transformation system provides potential for identifying the specic functions of P450 isoforms (particularly for the growing number of cloned insect P450s), because ectopic P450s can be studied in vivo in a eukaryote that may lack that isoform. Evidence from transgenic studies indicates that the P450 reductase of Drosophila can interact with two P450s of distantly related species; rat CYP2B1 was directly shown to be active in Drosophila microsomes (Jowett et al., 1991), while indirect evidence demonstrates that canine CYP1A1 was active in Drosophila (Komori et al., 1993). It remains unclear if the cytochrome b5 of Drosophila could also interact with ectopically expressed P450s. Here, we describe the genetic transformation of Drosophila with the cDNA of a well-studied house-y-specic P450, CYP6D1 (Scott et al., 1998; Scott, 1999a,b). We used two enzyme activity assays to determine if CYP6D1 was active in Drosophila (i.e. interacts with the P450 reductase and cytochrome b5 of Drosophila). The utility of this type of expression system is discussed in light of the many insect P450s that are presently known. Results Embryo microinjections of Drosophila with the pUASTCYP6D1 plasmid resulted in at least three individual strains transformed with UASCYP6D1 in which CYP6D1 could be expressed after activation of the upstream activator sequence (UAS) elements by GAL4, a transcriptional activator (Brand & Perrimon, 1993). DNA blots (Fig. 1) revealed that the CYP6D1 insert was in the transformed
Figure 1. DNA blots of strains GAL4/CyO, z1w11e4 and CYP6D1transformed strains A, B and C. Genomic DNA (3 g) was restriction digested with EcoRI prior to Southern blotting (see text) using a 800 bp probe specic to CYP6D1. Intensity of bands was not reproducibly different between strains. Approximate sizes of DNA fragments (kb) shown to right of the gure.
strains, but not control strains. The insertion position of the CYP6D1 gene was different between transgenic strains A, B and C because the band size was different for each strain. Also, the single band identied in the DNA of the CYP6D1-transformed strains indicated that there was probably only one CYP6D1 insert each for strains AC. Linkage experiments veried that strains A, B, and C were genetically distinct because the insertion positions were on chromosomes 2, 3 and X (Table 1). CYP6D1 can be expressed in the transformed Drosophila via the GAL4UAS expression system only if GAL4 is expressed in ies transformed with the UASCYP6D1 element. Crossing strains A, B and C with GAL4/CyO resulted in F1 animals with either a complete or incomplete expression system that were either capable of expressing CYP6D1 or not, respectively (Table 1). Protein immunoblotting (Western blotting) with a CYP6D1-specic antibody (Wheelock & Scott, 1990) conrmed that no CYP6D1 was present in Drosophila with an incomplete expression system regardless of heat-shock treatment (Fig. 2). The blots also showed that low levels of CYP6D1 were present in
b Genotype of F 1
Strain A B C z1w11e4
a b
Chromosomal linkagea 2 3 X
Complete expression systemc
Incomplete expression systemd
Table 1. Chromosomal linkage of CYP6D1 insertion in three Drosophila strains and genotypes of the F1
GAL4/UAS-CYP6D1 GAL4/+; UAS-CYP6D1/+ UAS-CYP6D1/+; GAL4/+
CyO/UAS-CYP6D1 CyO/+; UAS-CYP6D1/+ UAS-CYP6D1/+; CyO/+ GAL4/+OR; CyO/+
Linkage of UASCYP6D1 insert in transformed strains. F1 are progeny of females of the transgenic lines (strains AC) or z1w11e4 and males of GAL4/CyO. c All Drosophila with a complete expression system should have enhanced CYP6D1 expression after a heat shock. d All Drosophila with an incomplete expression system (i.e. lack either GAL4 or UASCYP6D1 element) should not be capable of CYP6D1 expression regardless of heat-shock treatment.
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Figure 2. Protein immunoblotting with a CYP6D1-specic antibody. The antibody bound to only protein bands from microsomes of house ies or transformed Drosophila with a complete expression system. Microsomes from LPR and CS house ies are shown in each panel as a positive control. Microsomes of Drosophila are designated complete (C) or incomplete (I) regarding the expression system. Drosophila with an incomplete expression system were incapable of expressing CYP6D1 because they lacked either the GAL4 component of the expression system or UASCYP6D1, the target gene. Protein content in each lane is expressed as micrograms of microsomal protein/ lane. Heat shock (HS) is indicated by + or (control). Panels A, B, C and D show immunoblots of microsomes of progeny of strains A, B, C, or z1w11e4, respectively, crossed with GAL4/CyO (see Table 1 for genotype of each group of Drosophila). All proteins that cross-reacted with the antibody were approximately 59.5 kDa, the estimated size of CYP6D1 (Korytko & Scott, 1998). Each blot was replicated at least twice with similar results.
Drosophila with a complete GAL4/UASCYP6D1 expression system, and that CYP6D1 expression was dramatically enhanced in the Drosophila with a complete expression system that were heat shocked. The low level of CYP6D1 in Drosophila with a complete expression system that were not heat shocked is probably due to the leaky nature of the hsp70 promoter, while the enhanced expression following heat shock is clearly a result of the induced GAL4/UASCYP6D1 expression system. More immuno 2000 Blackwell Science Ltd, Insect Molecular Biology, 9, 441 449
detectable CYP6D1 was found in heat-shocked Drosophila with a complete expression system than in CS house ies, and this was about one- to two-thirds the amount of CYP6D1 found in LPR house ies. AHH activity is a known metabolic marker activity of CYP6D1 (Wheelock & Scott, 1992). Microsomes from Drosophila with an incomplete CYP6D1 expression system had no or barely detectable AHH activity (< 20 pmol/ min/ mg protein; data only shown for GAL4/CyO genotype),
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Figure 3. AHH activity of microsomes from Drosophila with or without heat shock. AHH activity of microsomes from Drosophila that could express CYP6D1 was signicantly greater than AHH activity of microsomes that lacked CYP6D1 (GAL4/+), at P < 0.05 (*) and P < 0.01 (**) (t-test).
Figure 4. Total P450 levels in microsomes of Drosophila with and without heat-shock treatment. Total P450 levels declined after heat shock in microsomes of Drosophila that could not express CYP6D1 (GAL4/+) relative to sibling controls that were not heat shocked (t-test, P < 0.01 (**)).
but AHH activity was readily detected in all strains with a complete CYP6D1 expression system (Fig. 3). Also, there were 2.67.6-fold higher levels of AHH activity in Drosophila with a complete expression system that were heat shocked relative to non-heat-shocked siblings. Since AHH is a known activity of CYP6D1 (Wheelock & Scott, 1992) and substantial AHH activity was detected only in microsomes that had high levels of CYP6D1 (as determined by the protein immunoblots above), the AHH activity appears due to CYP6D1 expression. The AHH activity in Drosophila with a complete CYP6D1 expression system demonstrates that CYP6D1 is active in Drosophila microsomes; it interacts at some level of efciency with P450 reductase and cytochrome b5. However, the maximum AHH activity in microsomes of Drosophila was only about 50% of the AHH activity of microsomes from CS house ies (CS strain: 1034 230; mean SD pmol/min /mg protein). Total P450 is the sum of all P450 isozymes in the microsomes of an organism, including ectopically expressed P450s. After heat shock, a control strain that could express
GAL4, but not CYP6D1 (GAL4/+) had lower total microsomal P450 levels relative to siblings that were not heat shocked (Fig. 4). However, the strains with a complete expression system did not suffer the reduction of total microsomal P450 relative to sibling controls that were not heat shocked (Fig. 4). These data indicate that the heat shock in ies that express GAL4 reduces total P450 levels, but some of the total P450 appeared to be replaced by CYP6D1 in Drosophila that have a complete CYP6D1 expression system (GAL4 and CYP6D1 expression). In Drosophila that were not heat shocked, there was no difference in total P450 between ies with a complete or incomplete expression system (data not shown). MROD is a second CYP6D1-mediated activity in LPR house-y microsomes (Wheelock & Scott, 1992). Unfortunately, the background MROD activity of the normal Drosophila P450s and the general decrease of total P450 after heat shock gave a declining trend of MROD activity in all Drosophila after heat shock. The moving baseline of this activity made the CYP6D1-specic component of MROD activity impossible to measure. The decline in total P450 was not a problem for measuring AHH activity because
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there was no P450-mediated background AHH activity in control ies. Discussion We demonstrated that the house-y-specic P450, CYP6D1, was active when expressed in Drosophila, yet the activity was lower than found in wild-type house ies (CS strain). This remains true even when protein immunoblotting (Western) demonstrated that the microsomes of Drosophila with induced CYP6D1 expression contained similar levels or more CYP6D1 protein than the microsomes of CS house ies. We did not quantify the amount of CYP6D1 in the microsomes of Drosophila using rocket immunoelectrophoresis (Hagedorn et al., 1978; Korytko & Scott, 1998) because the amount of CYP6D1 in these samples was near the limit of detection using this analysis method. The limited activity of CYP6D1 in the transgenic system may be due in part to suboptimal interactions of CYP6D1 with the Drosophila P450 cofactors or to a limited supply of cofactors in the microsomes of Drosophila relative to the LPR strain of house y (i.e. house-y strains having elevated CYP6D1 also have elevated P450 reductase and b5). Alternatively, a difference in endoplasmic reticulum membrane structure in Drosophila relative to house y may not fully support P450s of house ies (Stewart & Strother, 1999). Furthermore, the direct comparison of microsomes of house ies and Drosophila as equal is not valid because house-y microsomes are prepared from only abdomens where P450s (Scott et al., 1998) including CYP6D1 (Lee & Scott, 1992; Korytko & Scott, 1998) are concentrated. In contrast, the microsomes of Drosophila were prepared from whole animals. The preparation of microsomes from the abdomens avoids dilution of the microsomes with proteins from body regions that are high in protein but relatively low in P450 such as the muscular thorax. For these reasons, the microsomes that we isolated from Drosophila that express CYP6D1 may have a lower level of total P450, CYP6D1 protein and CYP6D1-mediated activity than if we prepared microsomes exclusively from Drosophila abdomens. Unfortunately, production of microsomes from only Drosophila abdomens proved intractable. The moderate ectopic activity of CYP6D1 demonstrated in these experiments encourages our pursuit of further enhancing the CYP6D1 activity in Drosophila. At present, the best method to increase the activity of ectopic CYP6D1 is to express more CYP6D1 in Drosophila. This may be accomplished by altering the heat-shock regime to further increase CYP6D1 production, using different strains that express GAL4 to higher levels, and by increasing the copy number of CYP6D1 and GAL4 genes in the Drosophila from one copy each to two or more each. The ability of P450 reductase and cytochrome b5 of Drosophila to support the activity of P450s from other
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species has telling implications for the evolution of the P450 system. Although P450 gene sequences are widely divergent (Nelson et al., 1996), the P450 reductase (Hovemann et al., 1997) and cytochrome b5 are much more conserved in sequence and can still function as cofactors with P450s of different species. The evolution of the structures of P450 reductase and b5 may be constrained because domains of these cofactors must interact with several P450s. Small changes in P450 reductase or cytochrome b5 may have dramatic effects on the activity of many or all P450s in an organism potentially resulting in diminished tness. In contrast, P450s may be less constrained to evolve because there is a pool of P450 monooxygenases that have overlapping substrate specicity. Furthermore, many P450 monooxygenases may function as general detoxifying enzymes with no specic substrate. Mutations of one P450 merely alter that P450, not a subset or all of the P450s, and there may be no deleterious effect. For example, humans that lack a functional CYP2D6 gene (Dalen et al., 1998) or mice that lack either a CYP1A1, 1A2, 1B1 or 2E1 gene (Buters et al., 1999), but have an otherwise normal complement of P450 genes, are developmentally, physiologically and pathologically normal. Drosophila remains a practical transgenic insect system (Ashburner et al., 1998) for the study of P450s. Since the P450 reductase and b5 of this species support the general activities of ectopically expressed P450s to at least a moderate degree, this system could be useful to discover the functions of a variety of P450s, particularly those of insects (Dombrowski et al., 1998). Using a Drosophila expression system to examine the in vivo functions of insect P450s could be useful in identifying P450s that are involved in insect hormone metabolism, homeostasis or xenobiotic metabolism.
Experimental procedures
Strains and reagents
J. Lis and M. Wolfner (Cornell University) supplied the z1w11e4 and GAL4/CyO strains. The pUAST plasmid (Brand & Perrimon, 1993) was a gift from M. Wolfner. Unless noted, all reagents were from Sigma-Aldrich Co. (St Louis, MO) or Fisher Scientic (Pittsburgh, PA). All Drosophila were reared at 25 C on media provided by the Cornell Drosophila media services, while houseies, Musca domestica, were reared as described (Scott et al. 2000). CS is a wild-type strain of house-y (Scott et al., 1996). LPR is a mutant strain of house-y that overexpresses CYP6D1 (Liu & Scott, 1996).
GAL4UAS expression system
Brand & Perrimon (1993) developed a two-component GAL4 UAS expression system that allows for selective activation of any cloned gene or cDNA in transformed Drosophila (Brand & Perrimon, 1993). Excellent and detailed descriptions of this system are
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strain that lacks transposase. Of these lines, twenty-four appeared to have stable P-elements after four generations of crossing back to the z1w11e4 strain. The z1w11e4 strain was used as a control strain for the remaining experiments. Since the transformed strains were crossed back to z1w11e4 for several generations, this strain is genetically most similar to the experimental strains, but lacks the UASCYP6D1 genetic element.
available (Brand & Perrimon, 1993; Kaiser, 1993; Brand & Dormand, 1995; OKane, 1998). The rst component is a Drosophila strain transformed with a vector that contains a transcription element coupled to a GAL4 gene. Many strains have been developed, each with different transcription elements (Brand & Dormand, 1995; Kaiser, 1993) that allow for tissue-specic, cell-specic and /or temporal expression of GAL4. In the GAL4/CyO strain, hsp70 heat-shock elements enhance GAL4 expression such that there is a high level of GAL4 protein after a heat shock. The second component of the expression system the contains UAS DNA element. The Drosophila transformation vector pUAST (Brand & Perrimon, 1993) is commonly used for GAL4-mediated expression of exogenous genes. Within the anking transposable P-elements, this plasmid contains a w+ gene, ve optimized UAS elements, an hsp70 transcriptional start site, and a multiple cloning site. Even potentially lethal genes (Brand & Perrimon, 1993; Kaiser, 1993) could be expressed in Drosophila using this GAL4UAS expression system. When GAL4 is expressed in cells that have a UAS element, the GAL4 binds the element and strongly enhances the expression of the target gene controlled by the UAS element (Brand & Perrimon, 1993; Kaiser, 1993; OKane, 1998).
DNA hybridization
Genomic DNA was isolated (Jowett, 1998) from mixed sex Drosophila. DNA concentration was determined spectrophotometrically (Sambrook et al., 1989a). Genomic DNA (3 g) was completely digested (Sambrook et al., 1989b) with EcoRI restriction endonuclease enzymes (Life Technologies, Grand Island, NY), separated by agarose gel electrophoresis (Smith, 1996) using a 1% agarose gel, and transferred (Smith & Murphy, 1996) to a GeneScrene Plus membrane (NEN Life science Products, Inc., Boston, MA). The membrane was probed with an 800-bp DNA probe that was prepared by PCR amplication of a portion of the CYP6D1 cDNA with the pUAST-6D1 plasmid as template (sense primer 5-TAT GGC ATG ACG T TG AGT CG, antisense primer 5CCT CAT TAA AGG CAT TCC ACC). The probe was puried by gel electrophoresis (QIAGEN gel extraction kit, QIAGEN, Valencia, CA) and labelled with a random primer labelling kit (Life Technologies) with -32P-dCTP (Amersham Life Science, Arlington, IL). The DNA hybridization procedure was completed using a Stratagene QuikHyb solution according to manufacturers instructions (La Jolla, CA) followed by exposure of the membrane to Kodak BioMax MR lm (Eastman Kodak Co., Rochester, NY). The size of the detected DNA bands was estimated with DNA size standards (Life Technologies).
Subcloning of CYP6D1 and Drosophila transformation
A cDNA of CYP6D1 was created from messenger RNA (mRNA) of the LPR house-y strain (Scott & Georghiou, 1985, 1986a) by reverse transcription followed by polymerase chain reaction (PCR) amplication. Both primers contained a 5 GG clamp, a restriction site (Not I or XhoI underlined) and base pairs specic to the 5 and 3 ends of the CYP6D1 cDNA (sense primer: 5-GGGCGGCCGC ATG TTG TTA TTA CTG CTG ATT; antisense primer: 5-GGCTCGAG TCA CCA CCT CTT CGA CAG GCC CAC). The PCR product was digested with NotI and XhoI and inserted into the multiple cloning site of the pUAST vector. Competent Top10 Escherichia coli cells (Invitrogen, Carlsbad, CA) were transformed with the new plasmid construct and incubated on LB-amp agar plates. Plasmid DNA from the resultant colonies were amplied by culturing the bacterial colonies, puried, then screened by PCR in addition to restriction mapping. The PCR screen used primers specic to pUAST on either side of the multiple cloning site (sense primer: 5-AGA ATC TGA ATA GGG AAT TGG G; antisense primer: 5-CCT CAT TAA AGG CAT TCC ACC). The second screen used NotI and XhoI restriction enzymes to digest the plasmid. Both screens were evaluated by assessing the size of the products using 1% agarose gel electrophoresis. Of the 12 plasmids that contained a cDNA insert, one plasmid contained the correct CYP6D1 sequence (100% identity, Cornell DNA Sequencing Facility). This plasmid, pUAST CYP6D1, was amplied by culturing the bacterial colony and extensively puried; purication steps included three extractions each with phenol : chloroform, chloroform and ethyl ether followed by ethanol precipitation. Plasmid DNA was resuspended (0.1 mM sodium phosphate pH 7.8, 5 mM potassium chloride) to a concentration of 0.7 g/l. J. Werner of Cornell University microinjected 343 embryos of the w;P(23) Drosophila strain with puried pUASTCYP6D1 plasmid using an established transformation protocol (Rubin & Spradling, 1982). Seventy insects pupated, sixty-ve eclosed and sixty were fertile. Two G0 ies were transformants; together, they produced fty transformed G 1 ies that were identied + by the w eye marker. Stable lines of the transformants were developed by crossing each of these fty ies with the z1w11e4
Linkage of CYP6D1 insert
Chromosomal linkage of the CYP6D1 inserts were determined by crossing each line with ywf; CyO/Gla and w;TM6,Tb/XBB70 stocks (Karess, 1985). CyO and Gla represent balancers for chromosome 2; TM6,Tb and XBB70 represent balancers for chromosome 3. These balancer stocks were also used to make those strains with inserts on the second and third chromosome homozygous for CYP6D1 (Karess, 1985). Homozygous lines of the X-linked transgenes were developed by single pair sibling crosses for three generations.
Genetic crosses to establish a complete CYP6D1 expression system
Expression of CYP6D1 in the transformed strains requires combining a GAL4 component with the UASCYP6D1 genetic component. This was accomplished by crossing the GAL4 /CyO strain with virgin female homozygous UASCYP6D1 transformants. The GAL4/CyO strain has a lethal insertion of the GAL4 gene on chromosome 2, which is balanced with a CyO chromosome. Expression of GAL4 protein is enhanced in this strain with hsp70 promoter elements upstream of the GAL4 gene. The progeny of this cross (Table 1) will have either a complete expression system (GAL4 and UASCYP6D1) that should express CYP6D1 or an incomplete expression system (lacks either GAL4 or UAS CYP6D1) that should not express CYP6D1. Those ies with an incomplete expression system were easily separated from the ies with a complete expression system 2000 Blackwell Science Ltd, Insect Molecular Biology, 9, 441 449
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because the former were marked with Curly wings or were progeny of z1w11e4. Here, ies with an incomplete expression system are the best controls because they are similar to the experimental animals except that one chromosome is different (i.e. GAL4 insert or CyO balancer). Although the heat-shock enhancer elements are leaky and should enhance GAL4 expression in all strains with the GAL4 element, we increased CYP6D1 expression by heat shocking adult ies for 90 min at 37 C two times daily for 3 consecutive days. Preliminary evidence showed that this heat-stress regime provided strong expression of CYP6D1. On day 4, microsomes were prepared from the adult ies.
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adding 0 0.8 l (in 0.2-l increments) of 2 mM benzo[a]pyrene to a reaction mixture that did not contain NADPH. MROD is also a marker of CYP6D1 activity in LPR microsomes (Wheelock & Scott, 1992), but this activity requires only P450 reductase and not cytochrome b5 (Zhang & Scott, 1994). The MROD activity was measured as described for the AHH assays except for the following: (i) excitation and emission wavelengths were set at 530 (slit 4) and 580 (slit 0.5), respectively; (ii) 4.0 l of 1 mM methoxyresorun was used as the substrate; and (iii) the linear standard curve was developed by adding 0 5 l of 0.005 mM resorun to reaction buffer.
Protein preparation
To prepare microsomal protein fractions from Drosophila, 200 300 mixed-sex adult ies were homogenized in 5 ml of homogenization buffer (10% glycerol, 1 mM EDTA, 0.1 mM dithiothreitol, 1 mM phenylmethyl sulphonyl uoride, 1 mM phenylthiocarbamide, 0.1 M sodium phosphate, pH 7.5) (Lee & Scott, 1989a) in a glass / Teon homogenizer by ten strokes of a motorized homogenizer (Caframo, Wiarton, Ontario). The homogenate was centrifuged for 20 min at 10 000 g (13 000 rpm) in a JA-20 rotor with a Beckman (Fullerton, CA) J-21C centrifuge. The supernatant was centrifuged for 60 min at 100 000 g (37 000 rpm) in a Beckman 70-ti rotor with a Beckman L8-M ultracentrifuge. The resulting pellet of microsomes was resuspended in 0.5 ml resuspension buffer (20% glycerol, 1 mM EDTA, 0.1 mM dithiothreitol, 1 mM phenylmethyl sulphonyl uoride, 0.1 M sodium phosphate, pH 7.5) (Lee & Scott, 1989a). Microsomes of LPR (Scott & Georghiou, 1986b) and CS (Scott et al., 1996) house-y strains were prepared as above, but from abdomens of females that were previously frozen. Protein was assayed (Bradford, 1976) with bovine serum albumin (BSA) as the standard. Total P450 and cytochrome b5 was quantied spectrophotometrically (Omura & Sato, 1964).
Acknowledgements We thank Dr J. T. Lis for his helpful comments and assistance. We thank Dr G. Zhao for his assistance in subcloning the CYP6D1 gene and J. Werner (Cornell University) for performing the microinjection procedure. We thank J. Rice and V. Vargas for technical assistance. This work was supported by a grant from the National Institute of Health (GM47835), the Andrew W. Mellon Research Grant Program, and Sigma-Xi (National and Cornell University of Chapter). An NIEHS toxicology training grant (ES07052) partially supported PJK.
References
Adams, M.D., Celniker, S.E., Holt, R.A. et al. (2000) The genome sequence of Drosophila melanogaster. Science 287: 2185 2195. Ashburner, M., Hoy, M.A. and Peloquin, J.J. (1998) Prospects for the genetic transformation of arthropods. Insect Mol Biol 7: 201 213. Backes, W.L. (1993) NADPH-Cytochrome P450 Reductase: Function. Cytochrome P450 (Schenkman, J.B. and Greim, H., eds), pp. 15 34. Springer-Verlag, New York. Bonls, C., Sadana, J.L., Balny, C. and Maurel, P (1989) Electron . Transfer from Cytochrome b5 to Cytochrome P450. Molecular Aspects of Monooxygenases and Bioaccumulation of Toxic Compounds (Arinc, E., Schenkman, J.B. and Hodgson, E., eds), pp. 171183. Plenum Press, New York. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Chem 72: 248 254. Brand, A.H. and Dormand, E.-L. (1995) The GAL4 system as a tool for unraveling the mysteries of the Drosophila nervous system. Curr Opin Neurobiol 5: 572 578. Brand, A.H. and Perrimon, N. (1993) Targeted gene expression as a means of altering cell fates and dominant phenotypes. Development 118: 401 415. Buters, J.T.M., Doehmer, J. and Gonzalez, F.J. (1999) Cytochrome P450-null mice. Drug Metab Rev 31: 437 447. Dalen, P., Dahl, M.-L., Ruiz, M.L.B., Nordin, J. and Bertilsson, L. (1998) 10-Hydroxylation of nortiptyline in white persons with 0, 1, 2, 3, and 13 functional CYP2D6 genes. Clin Pharmacol Therap 64: 444 452. Dombrowski, S.M., Krishnan, R., White, M., Maitra, S., Diesing, C., Waters, L.C. and Ganguly, R. (1998) Constitutive and barbitalinduced expression of the Cyp6a2 allele of a high producer strain of CYP6a2 in the genetic background of a low producer strain. Gene 221: 69 77.
Protein immunoblotting
The microsomal protein was separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using a Mini-PROTEAN II apparatus (Bio-Rad, Hercules, CA) with 4% and 10% acrylamide for the stacking and separation gels, respectively (Laemmli, 1970) and transferred to nitrocellulose membrane (Kao et al., 1986). Immunoblotting with the CYP6D1-specic antibody 7735 was completed as previously described (Wheelock & Scott, 1990).
AHH and MROD activities of CYP6D1 in Drosophila
To determine if the CYP6D1 expressed in Drosophila was active and could interact with Drosophila P450 reductase and cytochrome b5, the CYP6D1-specic (Wheelock & Scott, 1992) AHH activity was examined in the Drosophila microsomes. AHH activity was assayed with an Aminco SPF 500 spectrouorometer (excitation 387, slit 2 nm; emission 407, slit 0.2 nm). Each assay consisted of 0.2 mg microsomal protein in a total of 200 l resuspension buffer (above), 1.8 ml of reaction buffer (0.1 M potassium phosphate pH 7.8, 0.1 mM EDTA and 0.5 mM MgCl2) (Lee & Scott, 1989b), 0.8 l of 2 mM benzo[a]pyrene in DMSO (substrate), and 10 l of 10 mM NADPH. For each assay, NADPH was added after verifying that the background activity was zero. Then, each reaction was assayed for 6 8 min at 28 C. To quantify the activity, a linear standard curve of benzo[a]pyrene was developed by 2000 Blackwell Science Ltd, Insect Molecular Biology, 9, 441 449
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