Ayub, M. and Levell, M. (1987) J. Steroid Hiochem. 14. Mason ... Ernest Hodgson,* Randy L. Rose,* Doreen K. S. Goh,t George C. Rockt and R. Michael Roet.
Biochemical Society Transactions
or in stably transfected cell models facilitate those developments.
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[22]are likely to
This work was supported. in part, by grants AGO8175 and HLI- 1 1 149 from the National Institutes of Health, 1)HHS.
S.,Kan, P. H., Hirst, M. A., Marcus, R. A. and Feldman, L). A. (1983) J. Clin. Invest. 71, 1495- I499 Santen, K. J., van den Bossche, H., Symoens, J., Hrugmans, J. and de Coster, R. (1 983) J. Clin. Endocrinol. Metab. 57,732-736 Schurmeyer, Th. and Nieschlag, E. (1984) Acta Endocrinol. 105,275-280 Sheets. J. J. and Mason, J. 1. (1984) Drug Metab. 1)ispos. 12,603-606 Mason, J. I., Murry, H. A., Olcott, M. and Sheets, J. J. (1985) Hiochem. I’harmacol. 34, 1087-1092 Thompson, E. A. and Siiteri, P. K. (1974) J. Biol. Chem. 239,5364-5372 Lloody, K. M., Murry, B. A. and Mason, J. I. (1990) J. Enzyme Inhib. 4, 153-158 Hrodie, A. M. H., Schwargel. W. C., Shaikh, A. A. and Hrodie, H. J. (1988) Endocrinology (Baltimore) 100, 1684- 1695 Covey. L). F., Hood, W. F.and I’arikh, V. 1).(1981)J. Hiol. Chem. 256, 1076- 1079 Nakajin, S..Takahashi, K. and Shinoda, M. (1991) J. Steroid Hiochem. Mol. Hiol. 38, 95-99 Santen, K. J., van den Hossche, H., Symoens, J., Hrugmans, J. and de Coster. K. (1983) J. Clin. Endocrinol. Metab. 57. 732-736
1. Loose, 1).
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12. Kan, 1’. H., Hirst, M. A. and Feldman, L). (1985) J. Steroid Hiochem. 23, 1023-1029 13. Ayub, M. and Levell, M. (1987) J. Steroid Hiochem. 28,521-531 14. Mason, J. I., Carr, B. K. and Murry. H. A. (1987) Steroids 50, 179- 189 15. Swart, P., Swart, A. C., Waterman, M. K., Estabrook. K. W. and Mason, J. I. (1993) J. Clin. Endocrinol. Metab.. in the press 16. Gazdar, A. F., Oie, H. K., Shackleton. C. H., Chen, T. R.. Triche, T. J., Myers, C. E., Chrousos, G. I’., Brennan, M. F., Stein, C. A. and La Kocca, X. (1990) Cancer Res. 50,5488-5496 17. Kainey, W. E., Bird, I. M., Sawetawan, C., Hanley, N. A., McCarthy, J. I,., McGee, E. A., Wester, K. and Mason, J. 1. (1993) J. Clin. Endocrinol. Metab., in the press 18. Bird, I. M., Hanley, N. A,, McCarthy, J. I,., Mathis, J. M., Word. K. A,, Mason, J. 1. and Kainey, W. E. (1 993) Endocrinology (Baltimore).in the press 19. Staels, H.. Hum, D. W. and Miller, W. I,. (1993) Mol. Endocrinol. 7,423-433 20. Capdevila, J.? Gil, I,., Orellana, M., Marnett, I,. J., Mason, J. I., Yadagiri, P. and Falck, J. K. (1988) Arch. Riochem. Hiophys. 261,257-263 21. van den Hossche, H. (1992) J. Steroid Hiochem. Mol. Hiol. 43. 1003-1021 22. Zhou, I).. Pompon, L). and Chen, S. (1090) Cancer Kes. 50.6949-6954
Received 13 August 1993
Insect cytochrome P-450: metabolism and resistance to insecticides Ernest Hodgson,* Randy L. Rose,* Doreen K. S. Goh,t George C. Rockt and R. Michael Roet Departments of *Toxicology and tEntomoIogy, Box 7633, North Carolina State University, Raleigh, NC 27695, U.S.A.
Introduction Cytochrome P-450-dependent mono-oxygenase systems (P-450) occur in most if not all organisms, from bacteria to mammals and higher plants. T h e diversity of reactions catalysed by P-450 make it important, not only for the metabolism of toxic substances and secondary plant chemicals found in food, but also for the metabolism of endogenous substrates, including hormones and fatty acids. Insect mono-oxygenase systems are known to be involved in many different processes, including the synthesis and degradation of insect hormones and fatty acids, insecticide resistance and in the adaptation of insects to multiple host plants [ 1-31. Due to the importance of P-450 in host plant adaptations, as well as in insecticide metabolism and resistance,
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efforts have been made recently in several laboratories to characterize these mono-oxygenase enzymes and to understand their regulation and expression in susceptible and in resistant populations of insects.
Cytochrome P-450 in adaptation to secondary plant chemicals For millenia, insects have undergone constant adaptation to a wide array of secondary plant chemicals directed against herbivores. T h e evolutionary significance of such a relationship between secondary plant chemicals and phytophagous insects was first suggested by Fraenkel [4]. Gordon [5] suggests that highly polyphagous insects were more resistant
P-450 Biotechnology
to synthetic pesticides than less polyphagous species, as a result of their adaptations to a wide array of plant toxins. As evidence of this, Krieger et al. [6] demonstrated a correlation between the number of plant families fed on by the species and the monooxygenase activity in the larval gut. Although these generalizations have since been criticized [7, 81, the suggestion led to numerous studies examining not only the influence of a wide variety of host plants upon enzyme metabolism and insecticide toxicity, but also the role of individual host plant alleochemicals in these interactions (see [ 1, 31 for reviews). Because the induction of insect metabolic enzymes by host plants has been shown to increase pesticide detoxication, several studies have been conducted in this area. One of the best examples involved the variegated cutworm, which was shown to have much higher levels of mono-oxygenase activity when fed on peppermint than when fed on beans [9]. Monoterpenes such as pulegone, menthone and menthol were shown to be active components [lo]. In the tobacco budworm, Heliothzli virescens (F), Riskallah et al. [ l l , 121 demonstrated that P-450 content was increased 2-3-fold in midgut microsomes from insects fed on wild tomato leaves compared with insects feed on an artificial diet. This induction was accompanied by a more than 4-fold increase in tolerance to diazinon. Diazinon was degraded faster in induced animals by means of desulphuration, side-chain hydroxylation and oxidative dearylation. The active component in tomato leaves, identified previously as 2-tridecanone [ 131, was demonstrated to be primarily responsible for these effects [ 11, 121. Recent studies in our laboratory involved insect populations that had been selected for resistance to secondary host plant chemicals. H. virescens larvae possessing low levels of resistance either to 2-tridecanone or to nicotine were shown to have substantially greater levels of P-450 and associated mono-oxygenase activity than the susceptible populations from whch they were derived [ 141. In the 2-tridecanone-resistant population, the increases found in metabolism appeared to be tied closely to general increases in P-450 content. In contrast, larvae resistant to nicotine exhibited isozymespecific increases in metabolism, since, for two of the six substrates measured, metabolism was significantly greater than could be accounted for strictly on the basis of increased P-450 content. In a third population that were quercetin-resistant, no increase in P-450 content was seen, although for one substrate significant increases in oxidative ability were observed, suggesting the presence of
specific increases in one or more P-450 isozymes. An interesting observation from the above study is that, although in all cases resistance was associated with increases in oxidative potential, cross-resistance between these particular populations was not observed, suggesting the possibility that particular isozymes may have been necessary for each case of resistance. Two other important metabolic systems examined (esterases and glutathione transferases) did not appear to be involved in resistance to these compounds, although for quercetin, superoxide dismutase may have an important role.
Cytochrome P-450 in insecticide metabolism Insect mono-oxygenases, like those of mammals and of other animal groups, catalyse a wide variety of oxidations that resuk in the detoxication and/or activation of pesticides. These reactions have been classified as aliphatic and aromatic hydroxylation, epoxidation, 0-,N- and S-dealkylation, P-, S- and N-oxidation, deamination, desulphuration and ester cleavage, and methylenedioxy ring cleavage [ 151. Since some insecticides are activated by these enzymes, toxicity can depend not only on the rate and extent of activation, but also on the degree of detoxication by the same enzymes.
Cytochrome P-450 in insecticide resistance Resistance of insects to insecticides is a world-wide problem and an increasing threat to the production of food and of fibre. Resistance is held in general to be due to selection by the toxic substance for genes present at low levels in the susceptible population. Many different mechanisms of resistance have been demonstrated including those based on the metabolism of the toxic substance, modified receptors at the site of action, decreased cuticular penetration and behavioural avoidance of the toxic substance. However, increased metabolism of the toxic substance is the most common, with P-4.50, glutathione transferase and esterases being the most important enzymes. Frequently, resistant insects have more than one mechanism of resistance. The initial indication that mono-oxygenases are involved in the resistance to pesticides came from observations that the P-450 inhibitor sesamex could reverse resistance to carbaryl [16]. It had been demonstrated previously that, in vivo, methylenedioxyphenyl compounds, such as sesamex, were inhibitory to P-450 [ 171 and confirmed subsequently in vitro [ 181. Indeed, these compounds, particularly piperonyl butoxide, have proved useful for
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three decades, not only for the demonstration of oxidative metabolism in resistant populations, but also in the control of resistant populations in field situations. Other early studies demonstrated that microsomes from carbamate-selected strains of the housefly showed an increased capacity for hydroxylation, N-dealkylation, 0-dealkylation, epoxidation and oxidative desulphuration, compared with susceptible strains [19, 201. Several early studies examining P-450 involvement in resistance, however, used only a single substrate, such as aldrin, to assay mono-oxygenase activity. Because of the many types of oxidations carried out by P-450s, it is important when studying resistant populations to examine several different substrates, as different isozymes may possess large differences in substrate specificity. In a recent example, 0-dealkylation of methoxyresorufin and ethoxyresorufin were highly elevated in a pyrethroid-resistant strain of houseflies, while much smaller elevations in activities of four other mono-oxygenase substrates were observed [21]. This data suggests that resistance is associated with the selection of specific isozymes of P-450, rather than the selection for several contributing isozymes. The known involvement of insect P-450 in insecticide resistance mechanisms suggests that research should focus on understanding mechanisms of P-450 gene regulation and inheritance. In mammalian systems, one of the most extensively studied areas involves gene regulation and P-450 inducibility by various xenobiotics, including their own substrates. The principal mode of P-450 regulation for many isozymes involves transcription [22]. P-450 Genes 1Al and 1A2 are activated by inducers such as 3-methylcholanthrene and 2,3,7,8tetrachlorodibenzo-p-dioxin. Transcription of the 1Al gene is governed by interaction of trans-acting factors with DNA sequence elements upstream from the RNA polymerase I1 start site. These elements have been identified as inducible enhancers that increase transcription of the 1Al gene only in the presence of the inducer. Multiple copies of these elements result in an additive augmentation of 1Al promoter activity in the presence of their inducers ~31. For other known mammalian inducers of P450, less is known about molecular mechanisms of gene regulation. Transcriptional regulation of P-450 families inducible by phenobarbital, by dexamethasone and by clofibrate has been demonstrated for families 2, 3 and 4, respectively [24-261. In contrast, acetone induction of 2E1 does not increase mRNA,
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yet P-450 protein is increased 6-fold as a result of protein stabilization [27]. Because so many studies of insecticide resistance and of P-4.50 have been conducted on the housefly, it is, perhaps, the best understood model for future studies of insect P-450s. Many resistant and susceptible strains are available, many with visible mutant markers that allow for studies of gene regulation. Studies on the genetics of resistance in the housefly led to the conclusion that much of the resistance in houseflies was due to semi-dominant genes, primarily located on chromosome 2. Studies on the inheritance of P-450 in resistance, using mutant strains [28, 291 indicated that multiple isozymes with genes located on chromosomes 2 and 5 were involved in resistance and that these were functionally different, as indicated by the importance of the type I binding site. Progress has been made recently in the characterization, at the molecular level, of the P450s involved in resistance. The first insect P-450 gene sequence was identified in an insecticideresistant strain of housefly that had been induced with phenobarbital [30]. Two other P-450 families have been isolated recently from Drosophila, one belongmg to family 6 [31] and the other to family 4 [321. In the one case where resistant and susceptible P-450 sequences could be compared directly within the species, it was found that the susceptible sequence differed by the presence of a long terminal repeat of a transposable element, along with other minor differences in sequence [31]. Features of this long terminal repeating element suggest that a posttranscriptional mechanism involving mRNA stability is involved in the regulation of this P-450 gene [31]. Other insect P-450 genes sequenced recently include one from the cockroach (family 4) [32], one from PupiZio polpenes (family 6) [33] and six from a susceptible population of houseflies [34]. Studies like those above relating to the mechanisms of insecticide resistance at the molecular level are necessary to provide information that can be used in future work to monitor and curtail the development of resistance more effectively. It is particularly important to conduct these studies in insects of economic importance, since the information obtained from such studies may be applied directly to these agricultural pest complexes.
Cytochrome P-450 in insecticide resistance in H. virescens The genus Heliothk is, arguably, the most important insect pest genus in agriculture, many of its
P-450 Biotechnology
members can feed on a wide variety of food plants, and resistance both to plant allelochemicals and to synthetic organic chemicals is common within the group [ 14,351. Several resistance mechanisms have been documented in tobacco budworm strains resistant to organochlorines, organophosphates and carbamate insecticides, including differences in penetration, altered target sites and increases in metabolism [36]. With the arrival of pyrethroids, previous difficulties with resistant populations were alleviated; however, in recent years, the advent of pyrethroid-resistant populations has become an area of increasing concern in Australia and in the south-eastern United States [ 37-39]. Studies of pyrethroid resistance indicate that resistance is due to several factors, including reduced penetration, insensitivity and increases in metabolism [40, 411. In one resistant population, the application of a mono-oxygenase inhibitor, piperonyl butoxide, increased toxicity of cypermethrin by 520-fold, suggesting a role for mono-oxygenases in the detoxication of this compound. Furthermore, the major metabolite of cypermethrin (2,4-OH-tertcypermethrin) in resistant H. virescens is the product of an oxidative reaction [40]. In related studies of a flucythrinate-resistant population, hydrolysis was found to be the primary resistance mechanism [41]. Our most recent studies have involved the biochemistry and molecular biology of resistance in a field-collected resistant population of H virescens. The Hebert population, collected from an area where multiple pesticides have been in use, was subjected to additional selection pressure in the laboratory with the carbamate insecticide, thiodicarb. Bioassays conducted nine generations after the initiation of laboratory selection pressure demonstrated 90-fold resistance to cypermethrin and greater than 150-fold resistance to thiodicarb in comparison with a susceptible laboratory population (Wake). Comparative biochemical assays conducted on microsomal and cytosolic fractions from various tissues demonstrated increased levels of P450 and esterase and glutathione transferase activities compared with the susceptible laboratory population. Significant increases in P-450 content were accompanied by particularly high levels of activity toward certain mono-oxygenase substrates (Figure l), particularly for methoxyresorufin and benzo[ alpyrene. The former substrate had been shown previously to be associated with high monooxygenase activity in pyrethroid-resistant strains of housefly [21]. The extremely high mono-oxygenase activity observed for particular substrates as well as high
Figure I Cytochrorne P-450-dependent mono-oxygenase activity in insecticide-resistant and insecticide susceptible strains of Heliothis virescens Abbreviations used: BENZ, benzphetamine; PNA, p-nitroanisole; B(A)P, benzo[o]pyrene; MRR, rnethoxyresorufin; R, resistant: S, susceptible.
s
2 .-0 2 Y
60
50 40
P-450
h
H BENZ
Y
Y
30
PNA
tzI B(A)P
m
.-"
0 MRR
20
n 0
9
2
10
0 Gut
Fat body
Carcass
Tissue
esterase activity suggested that biochemical assays might be used to monitor the development of resistance in field populations. Although several biochemical assays have been developed to monitor esterase and acetylcholinesterase levels in fieldcollected individuals, no assays have been described in the monitoring of mono-oxygenase activity associated with resistance. In microtitre plate assays using p-nitroanisole as the substrate, individual larval homogenates from the Hebert population were compared with those of the Wake population (Figure 2). Using this method, only 11% of the Wake individuals had a discernible activity; in contrast to 96% of the resistant individuals. The relative difference between the populations in p-nitroanisol o-dealkylase activity approximated 100-fold. A similar assay conducted using p-nitrophenyl acetate to monitor esterases was also capable of discriminating between resistant and susceptible individuals. In this case, the mean difference between populations was 5-fold. Results obtained from microtitre plate analysis suggest that these assays might allow useful estimations of the numbers of resistant individuals to be made in given field populations. Not only will these assays allow for estimation of the frequency of resistance, but will also provide a quantitative assessment of the levels of resistance in each individual. Such estimations of resistance frequency and level could provide growers with information about the types of resistance present in fields before pesticide application so that alternative insecticides
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Figure 2
Figure 3
Distribution of p-nitroanisole o-dealkylase (a) and f-nitrophenyl acetate hydrolase (b) activities of individuals in insecticide-resistant and insecticide-susceptible strains of Heliothis virescens
N-terminal sequence comparison of cytochrome P-450s from different insect species at the haembinding region
Abbreviations used: R, resistant: S,susceptible.
-
.EL
-C-
p-Nitroanisole activity
H Wake(S) Hebert(R) U ul ._
; 5 C
1-5
v
M B
0-1
.-* Y
.-U>
0
Y
10
20
30
40
Number of individuals
h
C ._
p-Nitrophenyl acetate activity
E
. E
H W a k e (S) H Hebert(R)
-La
5-10
v
g
0-1
E
.-Lx >
._ Y
0
Y
10
20
30
40
Number of individuals
could be used that might circumvent further selection for the resistant genes. SDSIPAGE of resistant and susceptible tissues was conducted to characterize further differences in the P-450 profile between these populations. P-450 Proteins typically fall within a molecular mass range of 45-60 kDa. In the fat body and midgut, considerable induction of approx. four proteins 50-60 kDa was observed. The additional proteins found in the Hebert population including one at 55 kDa in fat body and one at 60 kDa in the midgut were notable. Immunoblotting with a monoclonal antibody derived from Drosophila P-450 resulted in significant cross-reactivity with a 59 kDa protein found in the fat body of the resistant population. This protein was not present in the Wake population, however, other proteins were immunoreactive in midgut microsomes both from resistant and from susceptible populations. T o take advantage of this cross-reactivity, fat body mRNA was prepared from last-instar larvae of the Hebert population on day 3 of the instar. A Volume 21
H. virescens Drosophila rnelonogaster Musca dornestica Blaberus discoidalis P. polyxenes
Q P F T Y F P E C L G P R N C I G S R E A E
s v
E
w
L P F G D G P R N
c
I G
n n E
G
D S V D W L G E G D G P R N C I G H R E G H P Y A Y V P E S A G P R N C I G Q K P A H P C A Y L P E S A G P R N C L G H R E A
cDNA expression library was conducted using the Uni-Zap XR vector system (Stratagene) that allows the insertion of the cDNA into the Lambda Zap vector in a unidirectional orientation (EcoRI-XhoI). Screening of the library was conducted using the Lkosophila antibody, and three positive clones were plaque-purified through two rounds of screening. The pBluescript vector was excised from Lambda Zap and recircularized in the presence of the helper phage R408. Restriction analysis of these phagemids using EcoRI and XhoI indicated that the sizes of the inserts were 1400, 1300 and 800 bases. Restriction mapping of these clones revealed a PstI site about 500 bases from XhoI. To facilitate sequencing, the EcoRI-PstI region (around 900 bases in size) and the PstI-XhoI fragment (500 bases) were subcloned in to pUC 19. Positive clones were sequenced using the dideoxy-mediated chaintermination method [&]. The reactions were primed with both forward and reverse universal M13 primers of 17 base synthetic nucleotides. The sequence obtained was analysed using the University of Wisconsin Genetics Computer Group (GCG) sequence analysis software package. Sequence data from the 3’ end of all three clones revealed a conserved amino acid sequence containing the haem-binding region (GPRNCIG) common to most P-450s. The two longest clones had identical sequences, while the shortest of the three clones had several regions where significant differences in sequence were observed. Comparisons of this partial sequence with sequences from Drosophila, house fly, cockroach and the black swallowtail butterfly revealed several similarities, particularly in the haem-binding region (Figure 3). Although it is tempting to speculate about the classification of this particular P-450, it is difficult to do so in the absence of complete sequence data. Current efforts are directed to completing the sequence analysis and characterizing the mRNA regulation in different populations, tissues and life-history stages.
Summary and future directions In contrast with studies of mammalian P-450s.
P-450 Biotechnology
studies of insect P-450s are still in their infancy. Although insect P-450s are induced readily by host plant allelochemicals as well as by the traditional inducers of mammalian P-450s, such as phenobarbital and 3-methylcholanthrene, isozyme differences have yet to be characterized. Mechanisms of regulation likewise await future studies. Sufficient sequence data will be available soon to permit generalizations about the role of P-450 in insecticide resistance, in host plant adaptations and in the metabolism of endogenous substrates. In particular, relationships between resistance, induction and the inheritance of constitutively expressed genes needs to be clarified. Earlier studies carried out at NSCIJ supported in part by 1WS grant numbers ES-00044 and ES-07046, and by Ciba-Geigy Agricultural Chemicals. Current studies are supported in part by the North Carolina IPM Center (NC Agricultural Research Service) and by Khone-Poulenc Agricultural Chemicals.
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18. Hodgson, E. and Casida, J. E. (1961) Biochem. Pharmacol. 8,179-191 19. Schonbrod, R. D., Philleo, W. W. and Terriere, L.C. (1965) J. Econ. Entomol. 58,74-77 20. Hammock, B. D., Mumby, S. M. and Lee, 1’. W. (1977) Pestic. Biochem. Physiol. 7,261-272 21. Lee, S. S. T. and Scott, J. G. (1989) Pestic. Biochem. Physiol. 35, 1-10 22. Nebert, D. W. and Gonzalez, F. J. (1987) Annu. Rev. Biochem. 56,945-993 23. Gonzalez, F. J. (1990) Pharmacol. Rev. 40,243-288 24. Hardwick, J. P., Gonzalez, F. J. and Kasper, C. B. (1983) J. Biol. Chem. 258,8081-8085 25. Hardwick, J. P., Song, B.-J., Huberman, E. and Gonzalez, F.J. (1987) J. Biol. Chem. 262,80 1-8 10 26. Simmons, D. I,.? McQuiddy, P. and Kasper, C. €3. (1987) J. Biol. Chem. 262, 326-332 27. Song, B.-G., Veech, R. L., Park, S. S., Gelboin, H. V. and Gonzalez, F. J. (1989) J. Biol. Chem. 264, 3568-3 572 28. Tate, I,. G., Plapp, I;. W. and Hodgson, E. (1974) Chem. Biol. Interac. 6,237-247 29. Tate, I,. G., Plapp, F. W. and Hodgson, E. (1974) Riochem. Gene. 11,49-63 30. Feyereisen, R., Koener, J. F., Farnsworth, I). E. and Nebert, D. W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 1465-1 469 31. Waters, L. C., Zelhof, A. C., Shaw, B. J. and Ch’ang, I,. Y. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4855-4859 32. Bradfield, J. Y., Lee, Y. H. and Keeley, I,. I,. (1991) Proc. Natl. Acad. Sci. U.S.A. 88,4558-4562 33. Cohen, M. B., Schuler, M. A. and Berenbaum, M. R. (1992) Proc. Natl. Acad. Sci. USA. 89,10920-10924 34. Cohen, M. €3.and Feyereisen, R. (1993) Second lnternational Symposium on Insect Science, Flagstaff, Arizona, p. 43 35. Sparks, T. C. (1981) Hull. Entomol. SOC.Am. 27, 186- 192 36. Hull, L). 1,. (1981) ESA Bull. 27, 193-197 37. Plapp, F.W., Jr. and Campanhola, C. (1986) in Proc. Beltwide Cotton Production Research Conf., Natl. Cotton Council, Memphis, Tennessee, pp. 167- 169 38. Leonard, B. K., Graves, J. H., Sparks, T. C. and Pavloff, A. M. (1988) J. Econ. Entomol. 81, 1521-1528 39. Gunning, R. V., Easton, C. S., Greenup, I,. K. and Edge, V. H. ( 1984)J. Econ. Entomol. 77, 1283- 1291 40. Little, E. J., McCaffery, A. R. and Walker, C. H. (1989) in Proc. Beltwide Cotton Production Research Conf. Nat. Cotton Council Amer. Nashville, Tennesee, pp. 335-337 41. Dowd, P. F., Gagne, C. C. and Sparks, T. C. (1987) I’estic. Hiochem. Physiol. 28, 9- 10 42. Sanger, F., Nicklen, S. and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5463-5467 Received 13 August 1993
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