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• W2018496182 abstract "The protein ES20, derived from earthworm shock secretion, is a vomeronasally mediated chemoattractant for garter snakes (Jiang, X. C., Inouchi, J., Wang, D., and Halpern, M. (1990) J. Biol. Chem. 265, 8736–8744). Based on its 15-residue N-terminal amino acid sequence, degenerative oligodeoxynucleotide probes were synthesized and used to screen a cDNA library that was constructed in sense orientation using a Uni-ZAPTM XR vector and XL1-Blue MRF′ host. A gene was cloned from a polymerase chain reaction as well as from the cDNA library. A combination of the forward degenerative primer and T7 primer was used to obtain gene-specific DNA fragments, from which probes were synthesized and successfully used in screening the cDNA library. The ES20 gene is about 700 base pairs long and encodes 208 amino residues. The ES20 gene was excised from a recombinant plasmid pSK-ES20, ligated to pQE30 expression vector, and transformed into Escherichia coli strain JM109. The selected recombinant plasmids were transformed into expression host cell, E. coli M15[pREP4]. Three transformants were selected, induced with isopropyl-1-thio-ॆ-d-galactopyranoside for fusion gene expression and an expressed 20-kDa fusion protein purified under denaturing conditions. This protein was refolded and gave a positive reaction against ES20-specific polyclonal antibodies. The fusion protein that had not been denatured remained as an aggregate and was an active chemoattractant for garter snakes.AF019234 The protein ES20, derived from earthworm shock secretion, is a vomeronasally mediated chemoattractant for garter snakes (Jiang, X. C., Inouchi, J., Wang, D., and Halpern, M. (1990) J. Biol. Chem. 265, 8736–8744). Based on its 15-residue N-terminal amino acid sequence, degenerative oligodeoxynucleotide probes were synthesized and used to screen a cDNA library that was constructed in sense orientation using a Uni-ZAPTM XR vector and XL1-Blue MRF′ host. A gene was cloned from a polymerase chain reaction as well as from the cDNA library. A combination of the forward degenerative primer and T7 primer was used to obtain gene-specific DNA fragments, from which probes were synthesized and successfully used in screening the cDNA library. The ES20 gene is about 700 base pairs long and encodes 208 amino residues. The ES20 gene was excised from a recombinant plasmid pSK-ES20, ligated to pQE30 expression vector, and transformed into Escherichia coli strain JM109. The selected recombinant plasmids were transformed into expression host cell, E. coli M15[pREP4]. Three transformants were selected, induced with isopropyl-1-thio-ॆ-d-galactopyranoside for fusion gene expression and an expressed 20-kDa fusion protein purified under denaturing conditions. This protein was refolded and gave a positive reaction against ES20-specific polyclonal antibodies. The fusion protein that had not been denatured remained as an aggregate and was an active chemoattractant for garter snakes.AF019234 We have been studying chemosignal transduction in the vomeronasal system using garter snakes (Thamnophis sp.) as model subjects. As is the case for most terrestrial vertebrates, garter snakes possess dual olfactory systems, a main olfactory apparatus and a vomeronasal system. However, unlike most other vertebrates, the vomeronasal systems of most snakes are better developed than their main olfactory systems (2Gabe M. Saint Girons H. Mem. Mus. Nat. Hist. Nat. Paris Ser. A. 1976; 98: 1-87Google Scholar). Snakes appear to be dependent on vomeronasal input for a wide variety of behaviors requiring responses to chemical signals (3Parson T.S. Gans C. Parsons T.S. Biology of the Reptilia. 2. Academic Press, New York1970: 99-191Google Scholar). In addition, the two olfactory sensory systems play distinct functional roles in detecting environmental stimuli. The olfactory system detects mainly volatile compounds while the vomeronasal system detects primarily nonvolatile substances. Garter snakes detect prey, such as earthworms (Lumbricus terrestris), with their vomeronasal systems (4Burghardt G.M. Pruitt C.H. Physiol. Behav. 1975; 14: 185-194Crossref PubMed Scopus (64) Google Scholar, 5Halpern M. Frumin N. Physiol. Behav. 1979; 22: 1183-1189Crossref PubMed Scopus (92) Google Scholar, 6Kubie J.L. Halpern M. J. Comp. Physiol. Psychol. 1979; 93: 648-667Crossref Scopus (82) Google Scholar). Garter snakes respond to earthworm preparations by rapid tongue flicking and attack (5Halpern M. Frumin N. Physiol. Behav. 1979; 22: 1183-1189Crossref PubMed Scopus (92) Google Scholar, 6Kubie J.L. Halpern M. J. Comp. Physiol. Psychol. 1979; 93: 648-667Crossref Scopus (82) Google Scholar, 7Wilde W.S. J. Exp. Zool. 1938; 77: 445-465Crossref Scopus (51) Google Scholar, 8Burghardt G.M. Psychol. Sci. 1966; 4: 37-38Google Scholar, 9Kubie J.L. Halpern M. J. Comp. Physiol. Psychol. 1978; 92: 362-373Crossref PubMed Scopus (32) Google Scholar). Several chemoattractive proteins to garter snakes have been isolated from earthworms (1Jiang X.C. Inouchi J. Wang D. Halpern M. J. Biol. Chem. 1990; 265: 8736-8744Abstract Full Text PDF PubMed Google Scholar, 10Wang D. Chen P. Jiang X.C. Halpern M. Arch. Biochem. Biophys. 1988; 267: 459-466Crossref PubMed Scopus (27) Google Scholar, 11Wang D. Chen P. Halpern M. Doty R.L. Muller-Schwarze D. Chemical Signals in Vertebrates. VI. Plenum Publishing Corp., New York1992: 107-114Google Scholar). A chemoattractive protein, ES20, derived from electric shock-induced earthworm secretion, was isolated, purified, and characterized to a considerable extent (1Jiang X.C. Inouchi J. Wang D. Halpern M. J. Biol. Chem. 1990; 265: 8736-8744Abstract Full Text PDF PubMed Google Scholar). When the vomeronasal organ is irrigated with an aqueous solution of ES20, it induces an increase in the firing rate of mitral cells in the accessory olfactory bulb, the postsynaptic target of receptor cells of the vomeronasal epithelium (1Jiang X.C. Inouchi J. Wang D. Halpern M. J. Biol. Chem. 1990; 265: 8736-8744Abstract Full Text PDF PubMed Google Scholar, 12Inouchi J. Wang D. Jiang X.C. Kubie J. Halpern M. Brain Behav. Evol. 1993; 41: 171-182Crossref PubMed Scopus (32) Google Scholar, 13Li C.S. Wang D. Chen P. Halpern M. Soc. Neurosci. 1993; 23 (Abstr. 54.8): 122Google Scholar). The chemoattractive protein, ES20, binds to vomeronasal epithelial membrane in a saturable and reversable manner with a K d value of 0.3 ॖm (1Jiang X.C. Inouchi J. Wang D. Halpern M. J. Biol. Chem. 1990; 265: 8736-8744Abstract Full Text PDF PubMed Google Scholar). The binding of ES20 is tissue-specific, and both the binding and the bioactivity of this chemoattractive protein require the presence of Ca2+ (14Luo Y. Lu S. Chen P. Wang D. Halpern M. J. Biol. Chem. 1994; 269: 16867-16877Abstract Full Text PDF PubMed Google Scholar). When ES20 binds to its G protein-coupled receptors on the vomeronasal receptor cell membranes, the intracellular level of inositol 1,4,5-trisphosphate is increased while that of cAMP is decreased (14Luo Y. Lu S. Chen P. Wang D. Halpern M. J. Biol. Chem. 1994; 269: 16867-16877Abstract Full Text PDF PubMed Google Scholar, 15Wang D. Chen P. Liu W. Li C.S. Halpern M. Arch. Biochem. Biophys. 1997; (in press)Google Scholar). The level of cAMP is regulated mainly by calcium ions rather than by inhibitory G protein or cAMP-phosphodiesterase (15Wang D. Chen P. Liu W. Li C.S. Halpern M. Arch. Biochem. Biophys. 1997; (in press)Google Scholar). To facilitate the elucidation of the nature and mechanism of interaction between chemoattractant and its receptors that generate a chemoreceptive signal, it is necessary to have molecular and structural information concerning the chemoattractive protein as well as its receptors. Since a partial amino acid sequence of ES20 protein was previously reported (1Jiang X.C. Inouchi J. Wang D. Halpern M. J. Biol. Chem. 1990; 265: 8736-8744Abstract Full Text PDF PubMed Google Scholar), degenerative oligonucleotide primers were synthesized and utilized to clone the gene that encodes this chemoattractive protein. In this communication, we report the cloning of the gene encoding ES20 and its deduced amino acid sequence. The fusion ES20 protein was recognized by polyclonal antibodies specific for the native ES20 protein and was active in a bioassay for chemoattractivity to garter snakes.MATERIALS AND METHODSThe reagents were obtained from various suppliers: TRIzol and reverse transcriptase from Life Technologies, Inc.; UniZAPTM XR vector and XL1-Blue MRF′ host from Stratagene; poly(A)+ RNA isolation kit and pCRTM II from Invitrogen; [32P]dCTP, DNA sequence kit, and ECL kit (horseradish peroxidase-labeled anti-rabbit antibody) from Amersham Corp.; Taq polymerase from Boehringer Mannheim; pQE30 expression vector and M15[pREP4] cells from QIAGEN; and earthworms from Connecticut Valley Biological Supply; garter snakes from various suppliers.Poly(A)+ mRNA IsolationTotal RNA was prepared from 5 g of earthworm tissues as follows. Freshly dissected earthworm tissues were pulverized by grinding under liquid nitrogen. To the ground tissues, 10 ml of TRIzol were added and allowed to thaw at room temperature. The thawed mixture was incubated at room temperature for 30 min. It was transferred into a Falcon 2059 plastic tube, mixed with 2 ml of chloroform, and then centrifuged. The supernatant was carefully transferred into a new tube, and the RNA was precipitated by mixing with 5 ml of isopropyl alcohol. Poly(A)+ mRNA was isolated from total RNA according to the procedures described in the manual for poly(A)+mRNA isolation kit (Invitrogen).cDNA Library ConstructionThe construction of a cDNA library was performed according to standard procedures of Sambrook et al. (16Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York1989: 8.3-8.81Google Scholar) using UniZAPTM XR vector (Stratagene) and XL1-Blue MRF′ host. A cDNA library containing 5 × 106 recombinants in sense orientation was achieved. One fifth of the recombinants (1 × 106) were amplified to 1 × 1011 plaque-forming units/ml.PCR 1The abbreviations used are: PCR, polymerase chain reaction; bp, base pair(s); IPTG, isopropyl-1-thio-ॆ-d-galactopyranoside. 1The abbreviations used are: PCR, polymerase chain reaction; bp, base pair(s); IPTG, isopropyl-1-thio-ॆ-d-galactopyranoside. Amplification of ES20 Gene from cDNA LibraryThe ES20 cDNA from the recombinants (1 × 106) was cloned by performing degenerative oligonucleotides-primed amplification. Two oligonucleotides, GCI CTI GTI TGT CCI CCI GGI TTT ACI TAT and GTA ATA CGA CTC ACT ATA GGG C, were used, respectively, as a sense degenerative primer and antisense T7 primer. The sense primer corresponds to the amino acid sequence ALVCPPGFTY of chemoattractive protein ES20. PCR was performed in 50 ॖl of PCR buffer (10 mm Tris-HCl, pH 8.3, 50 mm KCl, 1.5 mm MgCl2, 0.0017 gelatin) containing 200 ॖm each of dATP, dCTP, dGTP, and dTTP, 2.5 units of Taq polymerase (Boehringer Mannheim), and 2 ॖm of each primer. Amplifications were carried out according to the following schedule: 94 °C for 1 min, 38 °C for 3 min, and 72 °C for 3 min (four cycles); 94 °C for 1 min, 50 °C for 2 min and 72 °C for 2 min (30 cycles). PCR products were analyzed with 100-bp ladder DNA markers by agarose (1.47) gel electrophoresis. A 700-bp PCR product was gel-isolated and cloned into pCRTM II (Invitrogen). The clones were identified by restriction enzyme digestion and sequencing. The recombinant is referred to as pCR-ES20.Determination of the cDNA Sequence of ES20 ClonesThe cDNA of ES20 clones was sequenced by dideoxy-mediated chain termination according to the method of Sanger et al.(17Sanger F. Nicklen S. Coulsen A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52310) Google Scholar).Northern Blot AnalysisA 200-bp EcoRI fragment of pCR-ES20 was purified, labeled with [32P]dCTP, and used as a probe for Northern blot analysis. Earthworm total RNA (10 ॖg) and mRNA (50 ng) were size-fractionated on a 1.37 agarose formaldehyde gel, blotted onto a nylon membrane, and cross-linked to the membrane under UV irradiation. Prehybridization was performed in 507 formamide, 5 × SSC, 5 × Denhardt's solution, 0.47 SDS, and 100 ॖg/ml denatured salmon sperm DNA at 45 °C for 4 h and hybridization at 45 °C overnight in a similar solution but containing the ES20probe. Hybridized membrane was washed under high stringency conditions.cDNA Library ScreeningThe 200-bp EcoRI fragment of pCR-ES20 labeled with 32P was used as a gene-specific probe for screening the cDNA library. A total of 1 × 106 plaques were screened under high stringency conditions. Four positive clones were isolated following secondary and tertiary screenings. They were excised as pSK(−) from UniZAPTM XR, and referred to as pSK-ES20. A 702-bp sequence was determined, and the complete amino acid sequence of 208 residues was deduced from the coding region.ES20 Gene Expression in E. coli CellsThe ES20gene was excised from pSK-ES20 and amplified by PCR using a gene-specific sense primer containing a BamHI site and an antisense primer containing a HindIII site. Both the pQE30 expression vector (QIAGEN) and PCR-amplified ES20 DNA were digested by BamHI and HindIII, purified, ligated, and subsequently transformed into JM 109 cells for the purpose of selecting the correct insertion of the coding fragment. The selected recombinant plasmids were transformed into expression host cell, M15[pREP4], in a medium containing appropriate antibiotics for selection. Three transformants were selected and induced with IPTG for recombinant gene expression. An expressed 20-kDa fusion protein (linked with 6 residues of His) was purified under denaturing conditions by affinity chromatography with nickel-nitrilotriacetic acid resin. The purified protein was refolded by dialyzing against stepwise dilutions of the denaturant. Such refolded recombinant protein was analyzed by Western immunoblot against ES20-specific polyclonal antibodies using an horseradish peroxidase-labeled anti-rabbit antibody (Amersham Corp.) for detection with an ECL kit (Amersham Corp.).Protein DeterminationThe concentration of protein was estimated using the Micro BCA protein assay reagent and bovine serum albumin as a standard.Snake BioassayThe snake bioassay was conducted by following the procedures of Jiang et al. (1Jiang X.C. Inouchi J. Wang D. Halpern M. J. Biol. Chem. 1990; 265: 8736-8744Abstract Full Text PDF PubMed Google Scholar). Briefly, test samples (200 ॖl of 53.8 ॖm fusion ES20 protein or native ES20 protein) or control solution (200 ॖl of a similar fraction prepared from uninduced Escherichia coli cells) was evenly applied to the surface of a piece of artificial plastic earthworm, which had been glued to a circular plastic disk and presented to a snake in its home cage. The response of the snake to the test material was monitored visually. A positive response was recorded when the snake approached and attacked the worm bit. If the snake approached the dish twice without attacking the worm bit, the response was considered negative.DISCUSSIONEarthworms, a primary prey of garter snakes, contain a number of proteins attractive to garter snakes. The recognition of and response to these chemoattractive proteins is mediated by the garter snake's vomeronasal system (5Halpern M. Frumin N. Physiol. Behav. 1979; 22: 1183-1189Crossref PubMed Scopus (92) Google Scholar, 6Kubie J.L. Halpern M. J. Comp. Physiol. Psychol. 1979; 93: 648-667Crossref Scopus (82) Google Scholar, 9Kubie J.L. Halpern M. J. Comp. Physiol. Psychol. 1978; 92: 362-373Crossref PubMed Scopus (32) Google Scholar). Several of these chemoattractants have been isolated, purified, and characterized (1Jiang X.C. Inouchi J. Wang D. Halpern M. J. Biol. Chem. 1990; 265: 8736-8744Abstract Full Text PDF PubMed Google Scholar, 10Wang D. Chen P. Jiang X.C. Halpern M. Arch. Biochem. Biophys. 1988; 267: 459-466Crossref PubMed Scopus (27) Google Scholar, 11Wang D. Chen P. Halpern M. Doty R.L. Muller-Schwarze D. Chemical Signals in Vertebrates. VI. Plenum Publishing Corp., New York1992: 107-114Google Scholar, 18Wang D. Chen P. Halpern M. Comp. Biochem. Physiol. 1992; 102B: 601-610Google Scholar, 19Wang D. Chen P. Jiang X.C. Inouchi J. Halpern M. Brain Behav. Evol. 1993; 41: 246-254Crossref PubMed Scopus (16) Google Scholar). The recognition of the prey is presumably initiated by the binding of one or several chemoattractants to their receptors on vomeronasal sensory neurons. Two of the purified chemoattractive proteins, ES20 and EW3, when delivered as aqueous washes to the vomeronasal sensory epithelium, have been shown to generate action potentials in the mitral cell layer of the accessory olfactory bulb of garter snakes (1Jiang X.C. Inouchi J. Wang D. Halpern M. J. Biol. Chem. 1990; 265: 8736-8744Abstract Full Text PDF PubMed Google Scholar, 12Inouchi J. Wang D. Jiang X.C. Kubie J. Halpern M. Brain Behav. Evol. 1993; 41: 171-182Crossref PubMed Scopus (32) Google Scholar, 13Li C.S. Wang D. Chen P. Halpern M. Soc. Neurosci. 1993; 23 (Abstr. 54.8): 122Google Scholar). Furthermore, the binding of ES20 and its G protein-coupled receptor(s) results in generation of second messengers (14Luo Y. Lu S. Chen P. Wang D. Halpern M. J. Biol. Chem. 1994; 269: 16867-16877Abstract Full Text PDF PubMed Google Scholar) and appears to involve cross-talk between two second messenger pathways (15Wang D. Chen P. Liu W. Li C.S. Halpern M. Arch. Biochem. Biophys. 1997; (in press)Google Scholar).Since earthworms contain many proteins attractive to garter snakes, whether these proteins bind to a single type of receptor or to different subtypes of receptors is not yet known. Do these chemoattractive proteins possess a similar type of receptor-binding domain or different types of domains? To answer these questions, a knowledge of structure-function relationships between ligands and receptors is needed. It is therefore necessary to elucidate the molecular structures of both the ligands and their specific receptors. Toward this end, we have successfully cloned the gene for one chemoattractive protein, the ES20 gene. This clone is the first and only chemoattractive gene that has become available and undoubtedly more chemoattractive genes are needed to map their binding domains. We have been studying chemosignal transduction in the vomeronasal system using garter snakes (Thamnophis sp.) as model subjects. As is the case for most terrestrial vertebrates, garter snakes possess dual olfactory systems, a main olfactory apparatus and a vomeronasal system. However, unlike most other vertebrates, the vomeronasal systems of most snakes are better developed than their main olfactory systems (2Gabe M. Saint Girons H. Mem. Mus. Nat. Hist. Nat. Paris Ser. A. 1976; 98: 1-87Google Scholar). Snakes appear to be dependent on vomeronasal input for a wide variety of behaviors requiring responses to chemical signals (3Parson T.S. Gans C. Parsons T.S. Biology of the Reptilia. 2. Academic Press, New York1970: 99-191Google Scholar). In addition, the two olfactory sensory systems play distinct functional roles in detecting environmental stimuli. The olfactory system detects mainly volatile compounds while the vomeronasal system detects primarily nonvolatile substances. Garter snakes detect prey, such as earthworms (Lumbricus terrestris), with their vomeronasal systems (4Burghardt G.M. Pruitt C.H. Physiol. Behav. 1975; 14: 185-194Crossref PubMed Scopus (64) Google Scholar, 5Halpern M. Frumin N. Physiol. Behav. 1979; 22: 1183-1189Crossref PubMed Scopus (92) Google Scholar, 6Kubie J.L. Halpern M. J. Comp. Physiol. Psychol. 1979; 93: 648-667Crossref Scopus (82) Google Scholar). Garter snakes respond to earthworm preparations by rapid tongue flicking and attack (5Halpern M. Frumin N. Physiol. Behav. 1979; 22: 1183-1189Crossref PubMed Scopus (92) Google Scholar, 6Kubie J.L. Halpern M. J. Comp. Physiol. Psychol. 1979; 93: 648-667Crossref Scopus (82) Google Scholar, 7Wilde W.S. J. Exp. Zool. 1938; 77: 445-465Crossref Scopus (51) Google Scholar, 8Burghardt G.M. Psychol. Sci. 1966; 4: 37-38Google Scholar, 9Kubie J.L. Halpern M. J. Comp. Physiol. Psychol. 1978; 92: 362-373Crossref PubMed Scopus (32) Google Scholar). Several chemoattractive proteins to garter snakes have been isolated from earthworms (1Jiang X.C. Inouchi J. Wang D. Halpern M. J. Biol. Chem. 1990; 265: 8736-8744Abstract Full Text PDF PubMed Google Scholar, 10Wang D. Chen P. Jiang X.C. Halpern M. Arch. Biochem. Biophys. 1988; 267: 459-466Crossref PubMed Scopus (27) Google Scholar, 11Wang D. Chen P. Halpern M. Doty R.L. Muller-Schwarze D. Chemical Signals in Vertebrates. VI. Plenum Publishing Corp., New York1992: 107-114Google Scholar). A chemoattractive protein, ES20, derived from electric shock-induced earthworm secretion, was isolated, purified, and characterized to a considerable extent (1Jiang X.C. Inouchi J. Wang D. Halpern M. J. Biol. Chem. 1990; 265: 8736-8744Abstract Full Text PDF PubMed Google Scholar). When the vomeronasal organ is irrigated with an aqueous solution of ES20, it induces an increase in the firing rate of mitral cells in the accessory olfactory bulb, the postsynaptic target of receptor cells of the vomeronasal epithelium (1Jiang X.C. Inouchi J. Wang D. Halpern M. J. Biol. Chem. 1990; 265: 8736-8744Abstract Full Text PDF PubMed Google Scholar, 12Inouchi J. Wang D. Jiang X.C. Kubie J. Halpern M. Brain Behav. Evol. 1993; 41: 171-182Crossref PubMed Scopus (32) Google Scholar, 13Li C.S. Wang D. Chen P. Halpern M. Soc. Neurosci. 1993; 23 (Abstr. 54.8): 122Google Scholar). The chemoattractive protein, ES20, binds to vomeronasal epithelial membrane in a saturable and reversable manner with a K d value of 0.3 ॖm (1Jiang X.C. Inouchi J. Wang D. Halpern M. J. Biol. Chem. 1990; 265: 8736-8744Abstract Full Text PDF PubMed Google Scholar). The binding of ES20 is tissue-specific, and both the binding and the bioactivity of this chemoattractive protein require the presence of Ca2+ (14Luo Y. Lu S. Chen P. Wang D. Halpern M. J. Biol. Chem. 1994; 269: 16867-16877Abstract Full Text PDF PubMed Google Scholar). When ES20 binds to its G protein-coupled receptors on the vomeronasal receptor cell membranes, the intracellular level of inositol 1,4,5-trisphosphate is increased while that of cAMP is decreased (14Luo Y. Lu S. Chen P. Wang D. Halpern M. J. Biol. Chem. 1994; 269: 16867-16877Abstract Full Text PDF PubMed Google Scholar, 15Wang D. Chen P. Liu W. Li C.S. Halpern M. Arch. Biochem. Biophys. 1997; (in press)Google Scholar). The level of cAMP is regulated mainly by calcium ions rather than by inhibitory G protein or cAMP-phosphodiesterase (15Wang D. Chen P. Liu W. Li C.S. Halpern M. Arch. Biochem. Biophys. 1997; (in press)Google Scholar). To facilitate the elucidation of the nature and mechanism of interaction between chemoattractant and its receptors that generate a chemoreceptive signal, it is necessary to have molecular and structural information concerning the chemoattractive protein as well as its receptors. Since a partial amino acid sequence of ES20 protein was previously reported (1Jiang X.C. Inouchi J. Wang D. Halpern M. J. Biol. Chem. 1990; 265: 8736-8744Abstract Full Text PDF PubMed Google Scholar), degenerative oligonucleotide primers were synthesized and utilized to clone the gene that encodes this chemoattractive protein. In this communication, we report the cloning of the gene encoding ES20 and its deduced amino acid sequence. The fusion ES20 protein was recognized by polyclonal antibodies specific for the native ES20 protein and was active in a bioassay for chemoattractivity to garter snakes. MATERIALS AND METHODSThe reagents were obtained from various suppliers: TRIzol and reverse transcriptase from Life Technologies, Inc.; UniZAPTM XR vector and XL1-Blue MRF′ host from Stratagene; poly(A)+ RNA isolation kit and pCRTM II from Invitrogen; [32P]dCTP, DNA sequence kit, and ECL kit (horseradish peroxidase-labeled anti-rabbit antibody) from Amersham Corp.; Taq polymerase from Boehringer Mannheim; pQE30 expression vector and M15[pREP4] cells from QIAGEN; and earthworms from Connecticut Valley Biological Supply; garter snakes from various suppliers.Poly(A)+ mRNA IsolationTotal RNA was prepared from 5 g of earthworm tissues as follows. Freshly dissected earthworm tissues were pulverized by grinding under liquid nitrogen. To the ground tissues, 10 ml of TRIzol were added and allowed to thaw at room temperature. The thawed mixture was incubated at room temperature for 30 min. It was transferred into a Falcon 2059 plastic tube, mixed with 2 ml of chloroform, and then centrifuged. The supernatant was carefully transferred into a new tube, and the RNA was precipitated by mixing with 5 ml of isopropyl alcohol. Poly(A)+ mRNA was isolated from total RNA according to the procedures described in the manual for poly(A)+mRNA isolation kit (Invitrogen).cDNA Library ConstructionThe construction of a cDNA library was performed according to standard procedures of Sambrook et al. (16Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York1989: 8.3-8.81Google Scholar) using UniZAPTM XR vector (Stratagene) and XL1-Blue MRF′ host. A cDNA library containing 5 × 106 recombinants in sense orientation was achieved. One fifth of the recombinants (1 × 106) were amplified to 1 × 1011 plaque-forming units/ml.PCR 1The abbreviations used are: PCR, polymerase chain reaction; bp, base pair(s); IPTG, isopropyl-1-thio-ॆ-d-galactopyranoside. 1The abbreviations used are: PCR, polymerase chain reaction; bp, base pair(s); IPTG, isopropyl-1-thio-ॆ-d-galactopyranoside. Amplification of ES20 Gene from cDNA LibraryThe ES20 cDNA from the recombinants (1 × 106) was cloned by performing degenerative oligonucleotides-primed amplification. Two oligonucleotides, GCI CTI GTI TGT CCI CCI GGI TTT ACI TAT and GTA ATA CGA CTC ACT ATA GGG C, were used, respectively, as a sense degenerative primer and antisense T7 primer. The sense primer corresponds to the amino acid sequence ALVCPPGFTY of chemoattractive protein ES20. PCR was performed in 50 ॖl of PCR buffer (10 mm Tris-HCl, pH 8.3, 50 mm KCl, 1.5 mm MgCl2, 0.0017 gelatin) containing 200 ॖm each of dATP, dCTP, dGTP, and dTTP, 2.5 units of Taq polymerase (Boehringer Mannheim), and 2 ॖm of each primer. Amplifications were carried out according to the following schedule: 94 °C for 1 min, 38 °C for 3 min, and 72 °C for 3 min (four cycles); 94 °C for 1 min, 50 °C for 2 min and 72 °C for 2 min (30 cycles). PCR products were analyzed with 100-bp ladder DNA markers by agarose (1.47) gel electrophoresis. A 700-bp PCR product was gel-isolated and cloned into pCRTM II (Invitrogen). The clones were identified by restriction enzyme digestion and sequencing. The recombinant is referred to as pCR-ES20.Determination of the cDNA Sequence of ES20 ClonesThe cDNA of ES20 clones was sequenced by dideoxy-mediated chain termination according to the method of Sanger et al.(17Sanger F. Nicklen S. Coulsen A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52310) Google Scholar).Northern Blot AnalysisA 200-bp EcoRI fragment of pCR-ES20 was purified, labeled with [32P]dCTP, and used as a probe for Northern blot analysis. Earthworm total RNA (10 ॖg) and mRNA (50 ng) were size-fractionated on a 1.37 agarose formaldehyde gel, blotted onto a nylon membrane, and cross-linked to the membrane under UV irradiation. Prehybridization was performed in 507 formamide, 5 × SSC, 5 × Denhardt's solution, 0.47 SDS, and 100 ॖg/ml denatured salmon sperm DNA at 45 °C for 4 h and hybridization at 45 °C overnight in a similar solution but containing the ES20probe. Hybridized membrane was washed under high stringency conditions.cDNA Library ScreeningThe 200-bp EcoRI fragment of pCR-ES20 labeled with 32P was used as a gene-specific probe for screening the cDNA library. A total of 1 × 106 plaques were screened under high stringency conditions. Four positive clones were isolated following secondary and tertiary screenings. They were excised as pSK(−) from UniZAPTM XR, and referred to as pSK-ES20. A 702-bp sequence was determined, and the complete amino acid sequence of 208 residues was deduced from the coding region.ES20 Gene Expression in E. coli CellsThe ES20gene was excised from pSK-ES20 and amplified by PCR using a gene-specific sense primer containing a BamHI site and an antisense primer containing a HindIII site. Both the pQE30 expression vector (QIAGEN) and PCR-amplified ES20 DNA were digested by BamHI and HindIII, purified, ligated, and subsequently transformed into JM 109 cells for the purpose of selecting the correct insertion of the coding fragment. The selected recombinant plasmids were transformed into expression host cell, M15[pREP4], in a medium containing appropriate antibiotics for selection. Three transformants were selected and induced with IPTG for recombinant gene expression. An expressed 20-kDa fusion protein (linked with 6 residues of His) was purified under denaturing conditions by affinity chromatography with nickel-nitrilotriacetic acid resin. The purified protein was refolded by dialyzing against stepwise dilutions of the denaturant. Such refolded recombinant protein was analyzed by Western immunoblot against ES20-specific polyclonal antibodies using an horseradish peroxidase-labeled anti-rabbit antibody (Amersham Corp.) for detection with an ECL kit (Amersham Corp.).Protein DeterminationThe concentration of protein was estimated using the Micro BCA protein assay reagent and bovine serum albumin as a standard.Snake BioassayThe snake bioassay was conducted by following the procedures of Jiang et al. (1Jiang X.C. Inouchi J. Wang D. Halpern M. J. Biol. Chem. 1990; 26" @default.
• W2018496182 created "2016-06-24" @default.
• W2018496182 creator A5027091822 @default.
• W2018496182 creator A5030978586 @default.
• W2018496182 creator A5059331575 @default.
• W2018496182 creator A5068974304 @default.
• W2018496182 date "1997-10-01" @default.
• W2018496182 modified "2023-09-26" @default.
• W2018496182 title "Cloning and Expression of a Gene Encoding a Protein Obtained from Earthworm Secretion That Is a Chemoattractant for Garter Snakes" @default.
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