Molecular cloning of the pheromone biosynthesis-activating ... - PNAS

Proc. Nati. Acad. Sci. USA Vol. 89, pp. 142-146, January 1992 Neurobiology

Molecular cloning of the pheromone biosynthesis-activating neuropeptide in Helicoverpa zea (gene/insect/neurohormone/sex pheromone)

MINH-TAM B. DAVIS*t, VIKRAM N. VAKHARIAt, JACQUELYN HENRY§, TOMAS G. KEMPE¶, AND ASHOK K. RAINA* *Insect Neurobiology and Hormone Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705; tCenter for

Agricultural Biotechnology of Maryland Biotechnology Institute and Virginia-Maryland Regional College of Veterinary Medicine, and IDepartment of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742; and §Department of Surgery, Morehouse School of Medicine, 720 West View Drive, Atlanta, GA 30310

Communicated by E. F. Knipling, September 25, 1991

family of genes with pheromonotropic and/or myotropic activity. Knowledge of the PBAN gene sequence will be a complementary addition to the recently advanced, but still relatively small, pool of known insect neuropeptide genes (12-17). Using two oligonucleotide mixed probes spanning two overlapping amino acid regions of PBAN, we report the isolation and characterization of a PBAN gene sequence from a genomic library of H. zea.

Pheromone biosynthesis-activating neuABSTRACT ropeptide (PBAN) regulates sex pheromone biosynthesis in female Helicoverpa (Heliothis) zea. Two oligonudeotide probes representing two overlapping amino acid regions ofPBAN were used to screen 2.5 X 1i0 recombinant plaques, and a positive recombinant clone was isolated. Sequence analysis of the isolated clone showed that the PBAN gene is interrupted after the codon encoding amino acid 14 by a 0.63-kilobase (kb) intron. Preceding the PBAN amino acid sequence is a 10-amino acid sequence containing a pentapeptide Phe-Thr-Pro-ArgLeu, which is followed by a Gly-Arg-Arg processing site. Immediately after the PBAN amino acid sequence is a Gly-Arg processing site and a short stretch of 10 amino acids. This 10-amino acid sequence contains a repeat of the PBAN C-terminal pentapeptide Phe-Ser-Pro-Arg-Leu and is terminated by another Gly-Arg processing site. It is suggested that the PBAN gene in H. zea might carry, besides PBAN, a 7- and an 8-residue amidated peptide, which share with PBAN the core C-terminal pentapeptide Phe-(Ser or Thr)-Pro-Arg-Leu-NH2. The C-terminal pentapeptide sequence of PBAN represents the minimum sequence required for pheromonotropic activity in H. zea and also bears a high degree of homology to the pyrokinin family of insect peptides with myotropic activity. It is possible that the putative heptapeptide and octapeptide might be new members of the pyrokinin family, with pheromonotropic and/or myotropic activities. Thus, the PBAN gene products, besides affecting sexual behavior, might have broad influence on many biological processes in H. zea.

EXPERIMENTAL PROCEDURES Oligonucleotide Probes: Design, Purification, and Labeling. Two mixed oligonucleotide probes spanning two overlapping amino acid regions of PBAN were designed. The first mixed probe, representing amino acids 3-15, contained 38 nucleotides and five deoxyinosine residues (Fig. 1). Deoxyinosine residues were inserted at positions of four-fold degeneracy. Both possible bases were used at positions of two-fold degeneracy. The second mixed probe, representing amino acids 11-21, was designed by a random method and contained five 33-mers (Fig. 1). The probes were purified by gel electrophoresis with 2% NuSieve low-melting temperature agarose (FMC). Purified oligonucleotides in gel slices were then directly labeled with terminal deoxynucleotidyltransferase (TdT) and [a-32P]dATP. To prepare a probe, the gel slices were melted in boiling water for 3-5 min, mixed, and incubated at 37°C for 5-30 min prior to labeling. DNA (1-2.5 ,ul or about 10 ng) was added to TdT buffer [final concentrations, 0.1 M potassium cacodylate, pH 7.2/2 mM CoCl2/0.2 mM dithiothreitol/0.1 mg of bovine serum albumin per ml containing 5 ,l (100 ,tCi; 3700 kBq) of [a-32P]dATP (New England Nuclear; 5000 Ci/mmol)]. The final volume was 10 ,ul. One microliter of TdT (11 units) (Bethesda Research Laboratories) was then added to the reaction mixture and thoroughly mixed. After incubation at 37°C for 1-2 hr, 89 ,ul of STE (10 mM Tris chloride, pH 8.0/100 mM NaCI/1 mM EDTA, pH 8.0) was added to the reaction mixture, and labeled DNA was purified from unreacted [32P]dATP by centrifugation through a Sephadex G-50 spun column (18). The column was preequilibrated with 100 ,ul of STE containing 250 ,ug of salmon sperm DNA per ml and then with 100

The pheromone biosynthesis-activating neuropeptide (PBAN) of the corn earworm Helicoverpa (Heliothis) zea, Hez-PBAN, is a 33-amino acid neurohormone that controls sex pheromone biosynthesis in females of many species of moths (1-8). The amino acid sequence of Hez-PBAN is as follows: Leu-Ser-Asp-Asp-Met-Pro-Ala-Thr-Pro-Ala-Asp-

Gln-Glu-Met-Tyr-Arg-Gln-Asp-Pro-Glu-Gln-Ile-Asp-SerArg-Thr-Lys-Tyr-Phe-Ser-Pro-Arg-Leu-NH2. PBAN is among the first few neuropeptides with known amino acid sequences that influence sexual behavior in moths. A PBANlike activity is even demonstrated in evolutionarily distant insects such as the locust (9). Thus, elucidation of the PBAN gene sequence will provide insight into molecular evolution of PBAN and be useful for studying its prohormone processing and regulation of expression. In addition, since PBAN in H. zea and Bombyx mori shares with the pyrokinin family in locust and cockroach the core C-terminal pentapeptide Phe-(Ser, Thr, or Val)-Pro-Arg-Leu-NH2 (10, 11), it would be interesting to know whether the PBAN gene belongs to a

Abbreviations: PBAN, pheromone-biosynthesis-activating neuropeptide; Hez-PBAN, Helicoverpa (Heliothis) zea PBAN; TdT, terminal deoxynucleotidyltransferase. tTo whom reprint requests should be addressed at: Insect Neurobiology and Hormone Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Building 467, BARC-East, 10300 Baltimore Avenue, Beltsville, MD 20705-2350. "The sequence reported in this paper has been deposited in the GenBank data base (accession no. M80588).

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Neurobiology: Davis et al. A Tyr 5' B TA 5' C TA D Gin 5' E CTG 5'

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Proc. Natl. Acad. Sci. USA 89 (1992)

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FIG. 1. Design of two oligonucleotide mixed probes used for screening a H. zea genomic library. Rows: A, peptide sequence of PBAN amino acids 3 (Asp) to 15 (Tyr); B, cloned PBAN sequence encoding PBAN amino acids 3-15. C, oligonucleotide mixed probes designed with deoxyinosines, representing PBAN amino acids 3-15; D, peptide sequence ofPBAN amino acids 11 (Asp) to 21 (Gln); E, cloned PBAN sequence encoding PBAN amino acids 11-21; and F, oligonucleotide mixed probes designed by a random method, representing PBAN amino acids 11-21.

,ul of STE. The specific activity of the labeled DNA was 1-2 x 101 cpm/,ug of DNA. Construction and Screening of a Genomic Library. A genomic library was constructed from DNA of dewinged adult H. zea bodies. Total genomic DNA was partially digested with Mbo I, fractionated by sucrose gradient centrifugation, and the resultant 19-22 kilobase (kb) fragments were selected (19). These fragments were ligated to phage A Dash II DNA (Stratagene), packaged with Gigapack II Gold extract, propagated, and screened on Escherichia coli strain P2392. E. coli strain P2PLK-17 was used in all subsequent manipulations of the positive PBAN clone. To screen the genomic library, 2.5 x 105 plaques were blotted onto Optibind nitrocellulose filters (Schleicher & Schuell) and then baked at 80°C in vacuum for 2 hr. Prehybridization was carried out at 50°C for 2 hr in 6x standard sodium citrate (SSC; 1 x SSC = 0.15 M NaCl/15 mM sodium citrate, pH 7.0) containing 5x Denhardt's solution (lx = 0.02% polyvinyl pyrrolidone/0.02% Ficoll/0.02% bovine serum albumin), 0.6% sodium dodecyl sulfate (SDS), and 100 ,ug of denatured salmon sperm DNA and 100 ,g of poly(A) per ml. The labeled probes were brought up to a final volume of 0.5 ml in 50 mM sodium phosphate (pH 7.5j containing 1 mg of yeast tRNA per ml, incubated in boiling water for 3 min, and then cooled in ice before being added to the hybridization solution. The final probe concentration in the hybridization solution was 4 or 10 x 106 cpm/ml for 38-basepair (bp) and 33-bp probes, respectively. For 38-bp probes, hybridization was carried out at 50°C for 2 days in prehybridization solution containing 3.2 M tetramethylammonium chloride and 50 mM sodium phosphate (pH 7.0) but without 6x SSC (20, 21). For 33-bp probes, hybridization was carried out overnight at 42°C in prehybridization solution containing 20% formamide. Filter wash was in 2x SSC/0.5% SDS at room temperature for 5 min and then in 3.2 M tetramethylammonium chloride/50 mM Tris, pH 8.0/1% SDS for 30 min at 55°C and 60°C for 38-bp and 33-bp probes, respectively. The washed filters were exposed to x-ray film at -80°C for 2 days. Polymerase Chain Reaction (PCR) Methods. Details of PCR methods will be described elsewhere. Briefly, the sense primer was as follows:

for 1 min, followed by four cycles at 94°C for 1.5 min, 42°C for 1 min, linear increase for 2.5 min, and 72°C for 1 min, and finally 25 cycles at 94°C for 1.5 min, 55°C for 1 min, and 72°C for 1 min. The 100 ,ul of PCR incubation mixture contained 5 ,ul of plate lysate of the genomic PBAN clone, 1 ,uM of each primer, 3.0 mM MgCl2, 0.5 ,ul (2.5 units) of Taq polymerase (Perkin-Elmer/Cetus) and 200 ,M of each of four deoxynucleotidyl triphosphates including 7-deaza-dGTP:dGTP (3:1 ratio). Nucleotide Sequence Analysis. To obtain sequencing data, PCR products were purified by gel electrophoresis and Qiagen tip 5 column (Qiagen, Studio City, CA) using as equilibration and wash buffers 850 mM NaCl/50 mM Mops/15% ethanol, pH 7.0 and as elution buffer 1.2 M NaCI/50 mM Mops/15% ethanol, pH 8.0. The purified PCR product was then digested with EcoRI and HindIII and subcloned into phage M13 mpl9 vector (Bethesda Research Laboratories). The single-stranded DNA from the M13 clone was sequenced by using adenosine 5'-[y-(35S)thio]triphosphate and a Sequenase kit (United States Biochemical). A 7-kb BamHI fragment from the genomic PBAN clone was subcloned into pBluescript II (Stratagene), and a 1.3-kb Cla I fragment from the 7-kb BamHI fragment was subcloned into pGEM-7Z (Promega) for double-stranded sequencing with Sequenase. The sequences of some regions were determined with synthetic internal oligonucleotide primers and the degenerate 38-bp probes representing amino acids 3-15. Structural analysis of the PBAN gene was performed with a PC/gene software (IntelliGenetics). Southern Analysis of Hez-PBAN Genomic Clone. Restriction digests of 7-kb BamHI fragment (1 jig of DNA) were separated in 0.8% agarose and transferred to Optibind nitrocellulose filters. Conditions for prehybridization and hybridization with the degenerate 38-bp probes and the PCR probes were as previously described for library screening with the 38-bp probes. The PCR products were labeled with TdT and [a-32P]dATP as described for oligonucleotide probes. The final filter wash was in 3.2 M tetramethylammonium chloride/50 mM Tris, pH 8.0/1% SDS for 1 hr at 57°C.

T A A 5 '-A-AAGCTT-AC1-CC1-GC1-GAC-CAG-GAG-ATG-TA-3 '.

RESULTS Isolation and Identification of the DNA Sequence of HezPBAN. To isolate the PBAN clone, 2.5 x 105 nonamplified recombinant plaques were screened with 33-bp probes representing PBAN amino acids 11-21. One hundred and fifty positive clones were obtained. A second screen of these clones with 38-bp probes representing PBAN amino acids 3-15 resulted in one positive clone. This clone was further identified as containing a PBAN gene sequence by PCR.

The antisense primer was as follows: A T G CA 5'TGACC-G- _A-T-T-G-' Conditions for PCR were as follows:

5

one

cycle at 96WC for

min, 42°C for 1 min, linear increase for 2.5 min, and 72°C

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Sequencing the PCR product yielded a stretch of 81 nucleotides corresponding to amino acids 8-33 of PBAN. To obtain further PBAN sequence information, a 7-kb BamHI fragment from the positive PBAN A clone was identified as containing PBAN by its hybridization to the radiolabeled PCR product (Fig. 2A). With internal oligonucleotide primers designed from the 81-nucleotide PCR sequence, a region of the 7-kb BamHI fragment was sequenced. However, the sequencing data only matched PBAN amino acids 15-33. To identify the region containing PBAN amino acids 1-14, two approaches were performed simultaneously. First, restriction-digested DNAs of whole PBAN A DNA and of the 7-kb BamHI fragment were hybridized to the degenerate 38-bp probes spanning amino acids 3-15 and the PCR product spanning amino acids 8-33. The results showed that only the 7-kb BamHI fragment from the whole PBAN A DNA hybridized to the 38-bp probes (Fig. 2A), indicating that the 7-kb BamHI fragment contained both regions spanning amino acids 3-14 and 15-33. In addition, from four Cla I fragments within the 7-kb BamHI fragment, only a 1.3-kb Cla I fragment hybridized to the PCR product (Fig. 2B). Similarly, only this fragment hybridized to the 38-bp probes (Fig. 2B), indicating that only the 1.3-kb Cla I fragment contained both regions spanning amino acids 3-14 and 15-33. The 1.3-kb Cla I fragment was then subcloned into pGEM for sequencing. As a second approach, internal oligonucleotide primers representing amino acids 8-14 were designed from sequencing data of the PCR product and used for sequencing the 7-kb BamHI fragment. No sequence data could be obtained from these primers. However, the degenerate 38-bp probes gave high quality sequencing data when used as primers for sequencing 7-kb BamHI and 1.3-kb Cla I fragments. Sequencing data from the 38-bp probes were identical for both

BamHI and Cla I fragments and showed the presence of a TATA homology. In addition, some sequencing data from 38-bp probes were overlapped with those from SP6 primers. From these sequencing data, internal oligonucleotide primers were designed for sequencing downstream of the TATA homology, and PBAN amino acids 1-14 were identified. Organization of Hez-PBAN Gene. The DNA sequence of Hez-PBAN is shown in Fig. 3. The first putative initiation codon GTG was found 92 nucleotides after a TATA homology. It has been reported that GTG, when used as initiation signal, is translated as methionine instead of the usual valine (22). Therefore, Met was written as the first amino acid of the proposed open reading frame for PBAN gene. The initiation signal GTG is relatively rare as compared with the initiation signal ATG. A putative cap site, TCACAGTC, is situated 15 nucleotides downstream of TATA homology. Following the putative initiator GTG is a 9-amino acid sequence before the NH2 terminus of the known first amino acid of PBAN. This 9-amino acid sequence contains a pentapeptide sequence Phe-Thr-Pro-Arg-Leu and is terminated by Gly-Arg-Arg. The presence of a signal peptide following the initiation signal is not found. The PBAN gene is interrupted by an intron after PBAN amino acid 14. The intron is 0.63-kb in length and begins and ends with the usual GT/AG doublets (23). PBAN amino acids 15-33 are followed by Gly-Arg, a common cleavage site of prohormone (24). After Gly-Arg, which is followed by the tripeptide Thr-Met-Asn, there is a repeat of five C-terminal PBAN amino acids Phe-Ser-Pro-Arg-Leu. Again, this pentapeptide is followed by Gly-Arg. A termination codon TGA was found 15 amino acids after the last Gly-Arg site. A putative polyadenylylation signal AATAAA was found 231 nucleotides downstream from the end of the open reading frame. The PBAN gene sequence is not significantly related to any of the gene sequences listed in EMBL and GenBank data bases.

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FIG. 2. (A) Southern hybridization of DNA of PBAN A clone, which was digested with BamHI and hybridized to the 32P-labeled PCR product representing PBAN amino acids 8-33. Identical results (not shown) were observed when 32P-labeled 38-bp probes, representing PBAN amino acids 3-15, were used instead of the labeled PCR product. DNA size markers (kilobases) are indicated to the right. (B) Southern hybridization of DNA of Bluescript containing PBAN BamHI fragment. The cloned DNAs were digested with various restriction enzymes and hybridized to the 32P-labeled PCR product, representing PBAN amino acids 8-33. Identical results (not shown) were observed when 32P-labeled 38-bp probes were used instead of the labeled PCR product. Lanes: 1, Sma I; 2, HincII; 3, EcoRI; 4, Pst I; 5, Xho I; 6, Cla I. DNA size markers (kilobases) are indicated to the left.

DISCUSSION We have isolated a genomic clone of Hez-PBAN using two mixed probes, representing two overlapping amino acid regions of PBAN. A genomic library rather than a cDNA library was screened because it might not be possible to obtain a mRNA population in which PBAN mRNA are adequately represented in the total population. The results showed that, even with a highly degenerate amino acid region, the use of a 38-mer containing five centrally located deoxyinosine residues was successful for isolating the PBAN clone. In addition, we were able to use this degenerate 38-mer to obtain reliable sequence data. Although sequencing with degenerate primers has been published (25), this is the first report of using long degenerate probes containing deoxyinosines for sequencing. We used PCR to confirm the identification of the positive clone and to obtain a readily identifiable sequence from the positive clone. Surprisingly, the PCR sequence data matched PBAN amino acids 8-33 without interruption after amino acid 14, as if the intron did not exist. However, sequencing data (Fig. 3) show that only a single PBAN gene block containing a 0.63-kb intron exists within the 1.3-kb Cla I fragment. In addition, Southern hybridization data of the whole PBAN A clone and of the PBAN BamHI fragment (Fig. 2) rules out the possibility of the presence of any PBAN DNA sequences, whether with or without intron, situated outside the 1.3-kb Cla I fragment. The results suggest that probably the PCR sense primers fortuitously bound to the intron sequence just before PBAN amino acids 15-33. The organization of the Hez-PBAN gene is somewhat similar to that of the gene for Manduca sexta eclosion hormone (13). Different from the gene for M. sexta adipokinetic hormone (12), both the genes of M. sexta eclosion



145

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tataatttggaaOM~ajcaaacaaaaaaatcgat FIG. 3. Genomic DNA sequence of Hez-PBAN gene and the deduced amino acid sequence of the peptide precursor. Underlined are: PBAN peptide sequence and pentapeptide sequence Phe-(Ser or Thr)-Pro-Arg-Leu. Marked in boxes are: TATA homology, TCACAGTC as a possible cap site, GTG as a possible initiator, Arg-Arg and Gly-Arg as possible processing sites, GT/AG doublets, TGA as a possible termination codon, and AATAAA as a possible polyadenylylation signal homology. Identified restriction sites are shown above the DNA sequences.

hormone and Hez-PBAN are interrupted by an intron located very close to the 5' coding terminus. We propose that the Hez-PBAN gene might encode, besides PBAN, two insect neuropeptides possibly with pheromonotropic and/or myotropic activities. All three peptides share the core C-terminal pentapeptide sequence Phe-(Ser or Thr)-Pro-Arg-Leu-NH2, are amidated, and probably are released separately during prohormone processing. The proposed structures of the two putative peptides are Met-GluPhe-Thr-Pro-Arg-Leu-NH2 and Thr-Met-Asn-Phe-Ser-ProArg-Leu-NH2. The above speculation is based upon the following observations. (i) The first 10 amino acids encoded by the PBAN gene contain the pentapeptide Phe-Thr-ProArg-Leu, which is followed immediately by Gly-Arg-Arg. This dibasic Arg-Arg, a common prohormone processing site (24), situates just before the NH2-terminal amino acid 1 of PBAN. Glycine is known to provide the amino group for amidation (26). Therefore, the presence of Gly-Arg-Arg suggests that the putative heptapeptide Met-Glu-Phe-Thr-ProArg-Leu-NH2 is probably cleaved from PBAN and then amidated during prohormone processing. (ii) The C-terminal amino acid 33 of PBAN is followed by Gly-Arg. Peptide-Glybasic amino acid is a widely used cleavage site in the prohormone (24). It has been shown that after prohormone proteolytic cleavage of this site by an endopeptidase, followed by carboxypeptidase activities, the COOH-terminal end is frequently amidated. Glycine probably provides the amino group for amidation (26), a process necessary for PBAN bioactivity. Immediately after the PBAN cleavage site is a short stretch of 10 amino acids containing a repeat of five amino acids, Phe-Ser-Pro-Arg-Leu. This pentapeptide again is followed by Gly-Arg. The presence of possible processing sites Arg-Arg and peptide-Gly-Arg suggests that PBAN and the octapeptide are probably released separately and ami-

dated during prohormone processing. (iii) Prior to having information on the PBAN gene structure, the C-terminal, amidated pentapeptide Phe-Ser-Pro-Arg-Leu-NH2, was determined to be the minimal, critical sequence necessary for pheromonotropic activity in H. zea (27). It is not known whether the putative octapeptide containing PBAN C-terminal pentapeptide acts in concert with PBAN to enhance pheromone production or controls pheromone production independently of PBAN and at a different time than PBAN does. (iv) The sequence Phe-(Ser, Thr, or Val)-Pro-Arg-LeuNH2 is the minimal C-terminal amino acid sequence that is shared by members of the pyrokinin family and is necessary for myotropic activity (10, 11). Neuropeptides of this family stimulate contraction of hindgut or foregut and/or oviduct in locust and cockroach. However, the structures of the aminoterminal ends of the two proposed peptides are different from those of the pyrokinin family. Thus, the two proposed peptides probably constitute new members of the pyrokinin family. It is suggested that the octapeptide encoded within the PBAN gene might act in concert with or independently of PBAN for controlling such activities as pheromone production and ovipositor movement in H. zea females. In this respect, the Hez-PBAN gene is different from the M. sexta eclosion hormone gene. The product of the eclosion hormone gene is a single peptide that is responsible for coordinated behavioral responses in diverse tissues such as the nervous system, muscle, and epidermis (13). In conclusion, the PBAN gene products, besides affecting sexual behavior, might have broad influence on many biological processes in H. zea. We are grateful to Drs. T. Kelly and R. Davis for use of the Perkin-Elmer/Cetus thermal cycler, P. Thomas for insect rearing, G. Edwards for technical assistance, and S. Cohen and M. Weinrich for typing the manuscript. This research was supported in part by a

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competitive grant from the U.S. Department of Agriculture (9037153-5557), awarded to M.-T.B.D. and A.K.R. and by a contract grant from the U.S. Department of Agriculture (58-1275-9-030) awarded to V.N.V. 1. Raina, A. K. & Klun, J. A. (1984) Science 225, 531-533. 2. Raina, A. K., Jaffe, H., Kempe, T. G., Kiem, P., Blacher, R. W., Fales, H. M., Klun, J. A., Ridgway, R. L. & Hayes, D. K. (1989) Science 244, 7%-798. 3. Altstein, M., Harel, M. & Dunkleblum, E. (1989) Insect Biochem. 19, 645-649. 4. Bestman, H. J., Herrig, M., Attygalle, A. B. & Hupe, M. (1989) Experientia 45, 778-781. 5. Kitamura, A., Nagasawa, H., Kataoka, H., Inoue, T., Matsumoto, S., Ando, T. & Suzuki, A. (1989) Biochem. Biophys. Res. Commun. 163, 520-526. 6. Martinez, T. & Camps, F. (1988) Arch. Insect Biochem. Physiol. 5, 211-220. 7. Rafaeli, A. & Soroker, V. (1989) J. Chem. Ecol. 15, 447-455. 8. Tang, J. D., Charlton, R. E., Jurenka, R. A., Wolf, W. A., Phelan, P. L., Sreng, L. & Roelofs, W. L. (1989) Proc. Nati. Acad. Sci. USA 86, 1806-1810. 9. Sreng, L., Moreau, R. & Girardie, A. (1990) J. Insect Physiol. 36, 719-726. 10. Holman, G. M., Nachman, R. J., Schoofs, L., Hayes, T. K., Wright, M. S. & DeLoof, A. (1991) Insect Biochem. 21, 107112. 11. Nachman, R. J., Roberts, V. A., Dyson, H. J., Holman, G. M. & Tainer, J. A. (1991) Proc. Nati. Acad. Sci. USA 88, 45184522. 12. Bradfield, J. Y. & Keely, L. L. (1989) J. Biol. Chem. 264, 12791-12793.

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