Isolation and Sequencing of cDNA Clones Coding for Juvenile ...

THEJOURNAL OF BrOLOGICAL

Vol. 264, No. 21, Igaue of July 25,pp. 12419-12425,1989 Printed in U.S.A.

CHEMISTRY

0 1989 by The American Society for Biochemistry end Molecular Biology, h e .

Isolation and Sequencingof cDNA Clones Codingfor Juvenile Hormone Esterase from ~ e Z i o ~uirescens ~is EVIDENCE FOR A CATALYTIC MECHANISM FOR THESERINE CARBOXYLESTERASES DIFFERENT FROM THAT OF THE SERINE PROTEASES* (Received for publication, November 18, 1988)

Terry N. Hanzlik, Yehia A. I. Abdel-Aal, Lawrence G.Harshman, and Bruce D. Hammock$ From the Departmentsof Entomology and Environmental Toxicology, University of California, Davis, California95616

Three cDNA clones containing the entirecoding sequence for JH esterase from the noctuid moth Heliothis uirescens have been isolated from an expression library using specific antibodies and oligonucleotide probes. The complete sequence of one of the clones shows a 2989-base pair insert that is approximately the length of the single 3.0-kilobase pair mRNA transcript detected by Northern blotting. The clone has an open reading frameof 1714 base pairs that predictsa mature protein (minus signal peptide 19 residues long) of 61,012 Da. The 3’-nontranslated region has three signals for polyadenylation although only one apparently is used. Edman degradation of purified juvenile hormone lesterase indicates two slightly different proteins (one being 75%of the total)while the threecDNA clones differ at three bases in their 5’ region causing their predicted sequence to differ at one or two residues of the 35 amino acids sequenced from the major form. Comparison of the predicted protein sequence to other carboxylesterase sequences indicates extensive similarity in the NHz-terminal half of the protein and the active site serine to be at position 201. The lack of conserved histidine and aspartate residues and the residues presence of conserved arginine and glutamate in appropriate positions in the NHz-terminal half of the homologous serine carboxylesterases suggests a catalytic mechanism for the serine carboxylesterases that is different from thatof the serineproteases.

iors. For m e ~ m o ~ h o stoi soccur, the JHlevel must decrease during the last larval growth stage (3). J H esterase, amember of the carboxylesterases (EC 3.1.1.1), is a protein that plays a crucial role in the decrease of J H activity in lepidopteran insects by hydrolyzing the methyl ester of J H a tthe appropriate times (4). Because of its action on JH, a studyof the regulation of J H esterase will increase ouru n d e r s t a n ~ n gof how metamorphosis is initiated, The sustained secretion of J H esterase into the hemolymph is one of the first known physiological signals that metamorphosis has begun. Later in the development of some larvae, J H itself appears to induce the enzyme’s action which eliminates the hormone from circulation. This negative feedback loop can thus serve as a valuable tool in the inquiry of how J H manifests its actions. A practical extensionof this work is that of insect control ( 5 ) . As it is crucial to these endeavors to have the gene for J H esterase, we undertook to clone the cDNA encoding the enzyme. We used the moth Heliothis virescens (Lepid0ptera:Noctuidae) as the organism from which to clone J H esterase. Recent studies of the enzyme in different lepidopteran insects indicated that JHesterase in this animal hadhighly desirable catalytic properties and the fewest apparent number of isoforms (5-8). In addition, the economic importance of the insect as an agricultural pest makes it a suitablemodel for the development of novel insecticides. In this paper we report the first successful cloning of J H esterase and find multiple but highly similar forms of the enzyme to be present. We have isolated and characterized three cDNA clones that contain slight differences in their 5‘ Juvenile hormone (methyl [ZE,6E]-[lOR]-lO,ll-epoxy-sequence that are homologous to the NH2-terminals of two 3,7,ll-trimethyl-Z,6-dodecadienoateand homologs) is a hor- highly similar proteins found in purified J H esterase. The mone intimately involved in theregulation of development in complete sequence of one of the clones predictsaserine insects such as the Lepidoptera (moths, butterflies) (1).Al- hydrolase of similar molecular weight to J H esterase. though the detailed mechanism(s) of its action remains unknown (a), it is recognized that precise modulation of JH’ EXPERIMENTAL PROCEDURES~ levels is required for the proper occurrence of oogenesis, metamorphosis, and other developmental events and behavRESULTS AND DISCUSSION ”

* This work was supported in partby National Science Foundation Grant DCB-8518697 and United States Department of Agriculture Grant 85-CRCR-1-11715. The costs of publication of this articlewere defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely t o indicate this fact. T h e nucleotide sequence(s)reported in this paper has been submitted to theGenBankTM/EMBL Data Bank with accession number(s) 504955. $Supported by the Burroughs Wellcome Toxicology Scholar award. The abbreviationsused are: JH, juvenile hormone; bp, base pairs; kb, kilobase pairs; HPLC, high performance liquid chromatography; PTH, phenyithioh~dant,oin.

’

Amino Acid Sequence of the NH2-terminal End of JH Esterase-When we purified J H esterase by affinity chromatography from the hemolymph of H. virescens and subjected it to Edman degradation, two proteins were indicated to be in our preparationeven though it was characterized as one band when subjected to electrophoresis and isoelectric focusing (8). The initial cycles had two signals existing at roughly a 3:l -__-

Portions of this paper(including “Experimental Procedures,” Figs. 1-5, and Table 1) are presented in miniprintat the endof this paper. Miniprint is easily read with the aidof a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journalt,hat is available from Waverly Press.

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Cloning of JH Esterase from H. virescens

ratio when quantified (Table1).From themajor form we were quence of the ultimate 35 amino acids derived from Edman able to obtaina sequence of 35 residues. Edman degradation degradation of the major form of J H esterase and that preindicated the minor form to have a 2-residue extension of dicted by the cDNA sequence match except at two sites. The Ser-Ala followed by a sequence of five residues identical to residues Val-10 and Phe-33 predicted by the sequence of clone the five NH2-terminal residues of the major form. The pres- 3hv21 are indicated to be Leu and Pro, respectively, on the ence of isoforms of J H esterase in H. uirescens is consistent sequenced protein (Table 1).In addition, the serine present with observations of the enzyme in other insects(5-8). at the NHn-terminal end of the minor form of J H esterase Isolation of cDNA Clones-Our strategy to isolate a cDNA protein that was sequenced is indicated to be Leu -2 on the clone of J H esterase mRNAwas to screen a cDNA expression cDNA. To approach the question of whether the differences library initially with antisera then rescreen the positive clones were due to thecloning processor were genuine, we sequenced by hybridization to a mixture of the 15-mer oligonucleotides the 5’ region of clones 3hvl and 3hv16 which were isolated complementary to theregion of the mRNA transcriptcoding from the same unamplified library as 3hv21. We found slight for the NH2 terminusof the secreted protein. Thisincreased differences among all three clones (Fig. 4). The sequence of the likelihood of obtaining a full length clone. Initially we clone 3hvl translates identically in the NHz-terminalregion screened a Xgtll library that was constructed with oligo(dT) as clone 3hv21 but differs at base 94, the last position of a priming and no size selection. Screening 900,000 clones of codon for Ser-6. Clone 3hv16 differsat two bases (50 and 104) this libraryyielded 4 consistently immunoreactive clones that from clone 3hv21in the areacoding forthe NHz-terminal end failed to hybridize to theoligonucleotides. Using one of these one of which causes a substitution of a phenylalanine for Leuclones as a probe on a Northern blot, we observed a single 9 and aleucinefor Val-10. These data indicate that both band migrating at 3.0 kilobase pairs. We reasonedthis library cloning artifacts aswell as multiplegenes may be responsible had a strong 3’ bias anddid not contain full length copies of for the differences between the protein sequence and the transcripts. T o avoid this problem in a second cDNA expres- sequence of the three cDNA clones. However, the fact that sion library, we used both random and oligo(dT) priming of two out of three clones (3hvl and3hv21) have a valineinstead the first strand. In addition we used cDNA selected fora size of a leucine a t position 10 strongly suggests this particular greater than 1,350bpforligation to the vector. Screening difference is due to dissimilar J H esterase genes in H. uiresof clone 3hv16 has 400,000 clones of this library produced 25 immunoreactive cens populations. Notably, the translation clones, 5 of which hybridized to the oligonucleotide probes. a leucine at position 10which makes it match 34 of 35 amino Three of these clones, designated 3hv1, 3hv16 and 3hv21, acid residues determined from the purified protein. Perhaps were subcloned into plasmids. All three clones contained a contributing to the heterogeneitybetween the cDNA and were protein sequences is the fact that the protein and RNA 3,000-bp insert that had identical restriction patterns when incubated with EcoRI, XhoI, and Hind111 (data not shown). extracted from two different colonies of H. uirescens. Another When the clone 3hv21 was used as a probe on a Northern indication of variability in forms of J H esterase is the someblot, it hybridized at low stringency to a single band of 3.0- what surprising amount of interspecies variability in protein kilobase pairs (Fig. 2). structure, post-translational modifications and catalytic paThe amountof screening required to isolate positive clones rameters in a protein believed to conduct a critically imporindicates that the frequency of t h e J H esterase message during tant function in thephysiology of lepidopteran insects (5-7). the period of its secretion into thehemolymph during the last This is true even between the two closely related heliothine instar is relatively low. Assuming all positiveclones were species such as H. uirescens and Helicouerpa zea (formerly Heliothis zea) (8). An additional observation concerning this detectedinthescreening of thefirst unamplifiedcDNA library thatwas not size-selected (65%of the clones contained heterogeneity is that it has been found in two other carboxinserts), 0.004% of the isolated poly(A) RNAwas J H esterase ylesterases cloned from an insect. Discrepancies between sequenced amino acids and those predictedfrom a cDNA clone mRNA. Sequence Analysis-At the beginning of our effort to deter- occurred with Drosophila esterase-6 (25) and differences in mine the sequence of t h e J H esterase cDNA we considered base sequences among cDNA clones were detected for Drothe three clones to be identical due to their identical length sophila acetylcholine esterase (the protein sequence was not and restriction patterns. We selected clone 3hv21 to sequence, determined) (26). There are three consensus poly(A) signal sequences (AAthe strategy for which is shown in Fig. 3 together with a TAAA) that start at bases 2299,2315, and 2951. The presence restrictionmap.The clonewassequenced 100% inboth of three signals for polyadenylation may signify alternative directions. Fig. 4 shows the DNAsequence andpredicted 3‘ region (27).Strong primary structure of J H esterase. The dataderived from Fig. processing of thetranscriptinthe 2 indicate that the 2,989-bp cDNA cloneis nearlya fulllength evidence from studies of another noctuid moth shows that copy of the mRNA transcript. There is a short 19-base se- there is a constitutively expressed intracellular form of J H quence prior ( 5 ’ ) to the first ATG. The position and compo- esterase located in the cytosol throughout its larval stage and sition of the bases immediatelyprior to the first ATG matches thus presumably the larval stage of H. uirescens (28). Hence, the consensus for an insect ribosome binding site (23) except a means of producing an intracellular as well as a secreted at position -3 where a G (the second most frequent base a t form of J H esterase is indicated to exist. However, as shown this site) replaces an A. After the first ATG, there isa 1,714- in Fig. 2, we found evidence for a single poly(A) RNA species bp open reading frame followed by an untranslated 1256-bp migrating at 3.0 kilobase pairs and nonefor species migrating are region including a 12-base poly(A) tail. Translation of the at sizes where theother two polyadenylationsignals of polyadenylation). A 2open reading frame predicts a 563-residue protein. The se- located (assuming the same amount quence and composition of the 19 residues prior to the NH,- day longer exposure of the blot than that shown in Fig. 2 terminal Trpof the secretedmajor form of J H esterase match failed to indicate other bands (data not shown). This obserwell with the consensusfor signal peptides for secretion (24). vation suggests that processing at alternate polyadenylation case at thedevelopmental stage of H. uirescens The molecular weight of the mature protein (minus signal sites is not the peptide) is predicted to be 61,012 which is in agreement with from which the RNA was isolated. The protein translated from the cDNA clone contains 4 the M, of 62,000 derived from electrophoresis ( 5 ) . The se-


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asparagine residues, Asn-62, Asn-161,Asn-383, and Asn-496, carboxylesterases employed an identical catalytic mechanism, that are candidates for glycosylation (29). However, prelimi- the residues involved would be similarly conserved. Indeed nary evidence indicatesthat should this modification be for one of the components of the catalytic triad, the active present on the secreted J H esterase from H. uirescens, man- site serines of the proteases and mostof the esterases correnose and glucose are not present (5). spond in the samegeneral region. Ser-201 of J H esterase and Computer Analysis of JH Esterase Sequence-Comparison correspondent serines of most of the esterases (Fig. 6) are at of the translation of clone 3hv21 to protein sequences in the positions similar to theactive site serine of chymotrypsin as protein data bank of the NationalBiomedical Research Foun- well as to Ser-183 and Ser-188 of bovine trypsinogen and dation and to translationsof proteins characterized as ester- porcine elastase,respectively (38).An exception to this is the ases, lipases andserine hydrolases inGenBank revealed putative active site Ser-276 of Drosophila acetylcholine esterstrong similarity to five proteins. Identical residue matches ase which has a higher position number because of an insert (gaps were counted as one substitution regardless of length) of26 amino acids in the NHz-terminal half not found in of 24.2,23.8, 23.2, and 23.2%, respectively, were noted to electric ray acetylcholine esterase. It is evident in Fig. 6 that human pseudocholine esterase (30), Drosophila melanogaster although there is strong conservationof primary structure in acetylcholine esterase (26),electric ray acetylcholine esterase theNHB-terminal half of all five esterases,thereareno (31), and D. melanogaster esterase-6 (25). In addition, simi- conserved histidines or aspartates in positions corresponding larity to a region situated toward the carboxyl-terminal end to the positions of the components of the serine protease bovine catalytic triad. There are,however, conserved aspartates and (residues 2229-2470) of the thyroid hormone precursor thyroglobulin (32) was noted as with othercarboxylesterases histidines at positions Asp-173, Asp-351, and His-446 of J H (25, 26, 30, 31). Analysis of the comparison data reveals the esterase. Two of these residues are situated on the COOHextensive similarity among the esterases is situated in the terminal side of Ser-201 ina region where there is little other NHz-terminal halves (Fig 6). Little or significant no homology homology among thefive proteins. Itis possible that esterases was noted to human esterase D (33), serine proteases, or to have adopted a radically different conformation for the conserved histidine and aspartate residues to function as comlipases. The homology of the region Asn-161 to Thr-248 of J H ponents of a catalytic triad ina manner similar to the serine esterase to similarregions containing Ser-200 and Ser-198of proteases. However the otherpossibility exists that esterases electric eel acetylcholine esterase and human pseudocholine have adopted a distinctly different mechanism of hydrolysis esterase, respectively, indicates Ser-201 of JH esterase to be of ester substrates. Thispossibility is supported by the presits active site serine (Fig. 6). Previous work has shown Ser- ence of highly conserved pentapeptide sequences from man, conserved the residuesthat 200 and Ser-198of these enzymes to be in theactive sites (30, teleosts, and insects that surround 31). The pentapeptide containing Ser-201 of JH esterase at correspond to Arg-47, Glu-88, and Ser-201 of J H esterase. its centershows the motif Gly-X-Ser-X-Gly,which is common The conserved residues are at positions similar to those of the to the active sites of all serine hydrolases (34). There are no conserved catalytic triad of the serine proteases and in the other amino acid sequences translated from 3hv21 that have region most conserved among esterases that bind dissimilar this motif, indicating there is only one active site serine for in vivo substrates (alarge, neutral, lipophilic substrate for J H esterase while acetylcholine esterase has a smaller charged J H esterase. substrate). It is possible to imagine the free carboxylic acidof We analyzed t h e J H esterase primary structure with the algorithm of Garnier et al. (35) and for hydrophobicity with glutamicacid actingin a similarcatalyticmechanismto the algorithm of Kyte and Doolittle (36) (Fig. 5). The data aspartate; however, one would not anticipate that the very high pK, of arginine would allow it to function inanalogous an suggest that no particular structural feature dominates the manner to histidine’sproposed role in catalysis by serine J H esterase conformation. Possibility of a Different Catalytic Mechanism-As noted proteases (39). Possibly this arginine could function to order or to activate a water molecule in the catalytic site or to previously, there is limited homology between the primary very least, it seems structures between proteins characterized as serine proteases activate another amino acid(s). At the and those characterized as serine carboxylesterases (25, 26, reasonable to consider the possibility that these differences 30, 31). However, numerous workers have hypothesized that in conserved motifs between serine carboxylesterases and classes of enzymes arose froma common ancestral protein as proteases suggest that thetwo classesof enzymes have evolved discussed recently by Brenner (37) in the case of enzymes different mechanisms of catalyzing similar reactions or have employing an active site serine. Since both groups of enzymes a very different pattern of folding. These observations may employ a catalytically active serine and are able tohydrolyze lead to an explanation of why most serine proteases have esters, it iswidely assumed that the mechanismsof catalysis esterase activity but most esterases have negligible protease employed by the two groups are also identical. However, our activity. Our comparison of the sequence of J H esterase tosequences analysis of the similarities among thecarboxylesterases sugreveals an additional gests that this may not be the case. Both groups of proteins of proteins contained in the data banks have conserved motifs in the same general regions of their reason for the further study of this protein. J H esterase is only one of two well characterized enzymes in the data banks primary structures; however, they are not the same. The serine proteases that have been sequenced and ana- with defined in vivo carboxylester substrates, and thus it is lyzed with x-ray crystallography all have a“catalytic triad”of only one of two enzymes that can be defined as an esterase serine, aspartate, and histidineresidues that have been pro- on a physiological basis (40). Acetylcholine esterase from posed to be integral in the catalysis by these enzymes (38). electric ray and Drosophila is the only other esterase in the For example, the reactivity of Ser-195 of chymotrypsin is databanks whose physiological role is knownwithsome believed to be enhanced because of its juxtaposition with His- precision. Study of the three-dimensional structure of J H is requiredfor esterolytic 57 and Asp-102 in theactive site.These residues are conserved esterase will shed light on what in similar positions in trypsin Streptomyces from griseus(His- catalysis from a physiological perspective. As active enzymes 37 and Asp-80) and pig elastase (His-45 and Asp-93) and exposed to the hemocoel of an organism, esterases must not other serine proteases. One would expect that if the serine be amidases able to hydrolyze peptide bonds. An interesting


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14. Aviv, H., and Leder, P. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, 1408-1412 15. Gubler, U., and Hoffman, B. J. (1983) Gene (Amst.) 25,263-269 16. Huynh, T. V., Young, R. A., and Davis, R. W. (1984) in D N A (Glover, D. M., ed) pp. 49-78, Cloning: A Practical Approach .. IRL Press, Oxford 17. Saneer. F.. Nicklen. F., and Coulson, A. R. (1977) Proc.Natl. Acad: Sci. U. S. A,' 7 4 , 5463-5467 18. Toneguzzo, F., Glynn, S., Levi, E., Mjolsness, S., and Hayday, A. (1988) BioTechniques 6 , 460-469 19. Yanisch-Perron, C., Vieira, J., and Messing, J . (1985) Gene (Amst.) 33, 103-119 Acknowledgments-We thank Dr.Susumu Maeda of the Entomol- 20. Henikoff, S. (1984) Gene (Amst.) 28, 351-359 ogy Department and Alan Smith, Jack Presley, and Dena Dillon of 21. Pustell, J., and Kafatos, F. C. (1984) Nucleic Acids Res. 1 2 , 643655 the Biochemistry and Biophysics Department at the University of California, Davis, for advice, the oligonucleotide synthesis, and per- 22. Queen, C., and Korn, L. J. (1984) Nucleic Acids Res. 12,581-599 forming the automated Edman degradations. We also wish to thank 23. Cavener, D. R. (1987) Nucleic Acids Res. 1 5 , 1353-1361 24. von Heijne, G. (1985) J. Mol. Biol. 184,99-I05 the Shell Oil Company and Dow Chemical Company for permitting 25. Oakeshott, J. G., Collet, C., Phillis, R. W., Nielsen, K. M., Russel, OUI' access to their insect colonies. R. J., Chambers, G. K., Ross, V., and Richmond, R. C. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 3359-3363 REFERENCES 26. Hall, L. M. C., and Spierer, P. (1986) EMBO J. 5 , 2949-2954 1. Sehnal, F. (1985) in Comprehensiue Insect Physiology, Biochem- 27. Capetanaki, Y. G., Ngai, J., Flytzanis, C. N., and Lazarides, E. (1983) Cell 35,411-420 istry and Pharmacology (Kerkut, G. A., and Gilbert, L. I., eds) 28. Hanzlik, T. N., and Hammock, B. D. (1988) Arch. Insect Biochem. Vol. 2, pp. 17-102, Pergamon Press, Oxford Physiol. 9, 135-156 2. Willis, J. H. (1986) Arch. Insect Biochem. Physiol. 3 , 47-57 3. Riddiford, L. M. (1980) in Progress in Ecdysone Research (Hoff- 29. Bause, E., and Legler, G. (1981) Biochem. J . 1 9 5 , 639-644 mann, J . A,, ed) pp. 409-430, Elsevier Science Publishers B. 30. Lockridge, O., Bartels, C.F., Vaughan, T. A., Wong, C. K., Norton, S. E., and Johnson, L. L. (1987) J. Biol. Chem. 2 6 2 , V., Amsterdam 549-557 4. Hammock, B. D., Abdel-Aal, Y. A. I., Hanzlik, T. N., Jones, D., Jones, G., Roe, R. M., Rudnicka, M., and Sparks, T. C. (1984) 31. Schumacher, M., Camp, S., Maulet, Y., Newton, M., MacPheeQuigley, K., Taylor, S., Friedman, T., and Taylor, P. (1986) in Biosynthesis, Metabolism and Modeof Action of Inuertebrate Nature 319,407-409 Hormones (Hoffmann, J., and Porchet, M., eds) pp. 416-425, 32. Mercken, L., Simons, M-J., Swillens, S., Massaer, M., and VasSpringer-Verlag, Heidelburg sart, G. (1985) Nature 3 1 6 , 647-651 5. Hammock, B. D., Harshman, L. G., Philpott, M. L., Szekacs, A., 33. Lee, Y. H. P., and Lee, W.-H. (1986) Proc. Natl. Acad.Sci. U. 5'. A. Ottea, J. A., Newitt, R. A., Wroblewski, V. J., Halarnkar, P.P., 83,6337-6341 and Hanzlik, T. N. (1987) in Biomechanisms Regulating Growth 34. Dayhoff, M. O., Barker, W. C., and Hardman, J. K. (1972) in and Development (Steffens, G. L., and Rumsey, T. S., eds) pp. Atlas of Protein Sequence and Structure (Dayhoff, M. O., ed) 137-173, Kluwer, Dordrecht Vol. 5, pp. 53-66, National Biomedical Research Foundation, Silver Springs, MD. 6. Abdel-Aal, Y. A. I., and Hammock, B. D. (1985) Arch. Biochem. 35. Garnier,J., Osguthorpe, D. J., and Robson, B. (1978) J. Mol. Biophys. 243,206-219 Biol. 120.97-120 7. Hanzlik, T. N., and Hammock, B. D. (1987) J . Biol. Chem. 262, 36. Kyte, J., and Doolittle, R. F. (1982) J . Mol. Biol. 1 5 7 , 105-132 13584-13591 37. Brenner, S. (1988) Nature 334, 528-530 8. Ahdel-Aal, Y. A. I., Hanzlik, T. N., Hammock, B. D., Harshman, L. G., and Prestwich, G. (1988) Comp. Biochem. Physiol. 9 0 B , 38. Young, C. L., Barker, W. C., Tomaselli, C. M., and Dayhoff, M. 0. (1978) in Atlas of Protein Sequence and Structure(Dayhoff, 117-124 M. O., ed) Vol. 5, Suppl. 3, pp. 73-93, National Biomedical 9. Abdel-Aal, Y. A. I., and Hammock, B. D. (1986) Science 233, Research Foundation, Silver Springs, MD 1073-1076 39. James, M. N. G., Sielecki, A. R., Brayer, G. D., and Delhaere, L. Laemmli, U. K. (1970) Nature 227,680-685 10. T. J. (1980) J. Mol. Biol. 1 4 4 , 43-88 11. Vaitukaitis, J. L. (1981) Methods Enzymol. 73, 46-52 413 _l.Heymann, E. (1980) in Enzymatic Basis of Detoxication (Jakoby, 12. Zeff, R. A., and Geliehter, J. (1987) B R L Focus 9 , l - 2 W. B., ed) Vol. 11, pp. 291-323, Academic Press, New York 13. Turpen, T. H., and Griffith, 0. M. (1986) BioTechniques 4 , 1141. Yen, T. J., Machlin, P. S., and Cleveland, D. W. (1988) Nature 15 334,580-585

question to ask is: What are the structural features of the (physiologically defined) carboxylesterases thatcausethe physiologically required selectivity for esters? Cloning of JH esterase cDNA will allow the efforts mentioned in the introduction proceed to as well as a forthcoming analysis of the JH esterase gene(s). The precise temporal control of this important regulatory enzyme as well as its RNA transcript's structural features may make it a valuable model for the study of message stability as well (41).

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Cloning of JH Esterase fromH. virescens

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EXPERIMENTAL PROCEWRES

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