AU4206301A - Novel carboxylesterase nucleic acid molecules, proteins and uses thereof - Google Patents

Novel carboxylesterase nucleic acid molecules, proteins and uses thereof Download PDF

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AU4206301A
AU4206301A AU42063/01A AU4206301A AU4206301A AU 4206301 A AU4206301 A AU 4206301A AU 42063/01 A AU42063/01 A AU 42063/01A AU 4206301 A AU4206301 A AU 4206301A AU 4206301 A AU4206301 A AU 4206301A
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nucleic acid
protein
acid molecule
proteins
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Kevin S. Brandt
Gary M. Silver
Nancy Wisnewski
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Heska Corp
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Heska Corp
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AUSTRALIA
PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT
ORIGINAL
Name of Applicant/s: Actual Inventor/s: Address for Service: Invention Title: Heska Corporation Nancy Wisnewski and Gary M. Silver and Kevin S. Brandt BALDWIN SHELSTON WATERS 60 MARGARET STREET SYDNEY NSW 2000 'NOVEL CARBOXYLESTERASE NUCLEIC ACID MOLECULES, PROTEINS AND USES THEREOF' Details of Original Application No. 51774/98 dated 10 November, 1997 The following statement is a full description of this invention, including the best method of performing it known to me/us:- File: 31620AUP00 -l a- PARASITIC NEMATODE TRANSGLUTAMINASE PROTEINS, NUCLEIC ACID MOLECULES, AND USES THEREOF FIELD OF THE INVENTION The present invention relates to parasitic nematode transglutaminase nucleic acid molecules, proteins encoded by such nucleic acid molecules, antibodies raised against such proteins, and inhibitors of such proteins. The present invention also includes therapeutic compositions comprising such nucleic acid molecules, proteins, antibodies, inhibitors, and combinations thereof, as well as the use of these compositions to protect animals from diseases caused by parasitic nematodes. The present application is a divisional application of Australian Application No. 62368/98 which is incorporated in its entirety herein by reference.
BACKGROUND OF THE INVENTION Parasitic nematode infections in animals, including humans, are typically treated by chemical drugs. One disadvantage with chemical drugs is that they must be 15 administered often. For example, dogs susceptible to heartworm are typically treated monthly. Repeated administration of drugs, however, often leads to the development of resistant nematode strains that no longer respond to treatment. Furthermore, many of S•the chemical drugs caused harmful side effects in the animals being treated, and as larger doses become required due to the build-up of resistance, the side effects become even ":20 greater. Moreover, a number of drugs only treat symptoms of a parasitic disease but are unable to prevent infection by the parasitic nematode.
An alternative method to prevent parasitic nematode infection includes administering a vaccine against a parasitic nematode. Although many investigators have tried to develop vaccines based on specific antigens, it is well understood that the ability of an antigen to stimulate antibody production does not necessarily correlate with the ability of the antigen to stimulate an immune response capable of protecting an animal from infection, particularly in the case of parasitic nematodes. Although a number of prominent antigens have been identified in several parasitic nematodes, including in Dirofilaria, there is yet to be a commercially available vaccine developed for any parasitic nematode.
limited duration. Furthermore, many of the methods are often not successful in reducing arthropod populations. In particular, insecticides have been used to prevent hematophagous arthropod infestation of animals by adding such insecticides to shampoos, powders, collars, sprays, foggers and liquid bath treatments dips).
Reduction of hematophagous arthropod infestation on the pet has been unsuccessful for one or more of the following reasons: failure of owner compliance (frequent administration is required); behavioral or physiological intolerance of the pet to the pesticide product or means of administration; and the emergence of hematophagous arthropod populations resistant to the prescribed dose of pesticide. However, hematophagous arthropod populations have been found to become resistant to insecticides.
Prior investigators have described insect carboxylesterase (CE) protein biochemistry, for example, Chen et al., Insect Biochem. Molec. Biol., 24:347-355, 1994; Whyard et al., Biochemical Genetics, 32:924, 1994 and Argentine et al., Insect Biochem.
Molec Biol, 25:621-630, 1995. Other investigators have disclosed certain insect CE amino acid sequences, for example, Mouches et al., Proc Natl Acad Sci USA, 87:2574- 2578, 1990 and Cooke et al., Proc NatlAcad Sci USA, 86:1426-1430, 1989, and nucleic acid sequence (Vaughn et al., J. Biol. Chem., 270:17044-17049, 1995).
Prior investigators have described certain insect juvenile hormone esterase (JHE) nucleic acid and amino acid sequences: for example, sequence for Heliothis virescens is disclosed by Hanzlik et al., J. Biol. Chem., 264:12419-12425, 1989; Eldridge et al., App Environ Microbiol, 58:1583-1591, 1992; Bonning et al., nsect Biochem. Molec. Biol., 22:453-458, 1992; Bonning et al., Natural and Engineered Pest Management Agents, pp.
368-383, 1994 and.Harshman et al., Insect Biochem. Molec. Biol, 24:671-676, 1994; sequence fo- anduca sexta -s disclosed by Vankatesh et al., JBiol Chem, 265:21727- 21732, 1990; sequence for Trichoplusia ni is disclosed by Venkataraman et al., Dev.
Genet., 15:391-400, 1994 and Jones et al., Biochem. 302:827-835, 1994; and sequence for Lymantria dispar is disclosed by Valaitis, Insect Biochem. Molec. Biol., 22:639-648, 1992.
Identification of an esterase of the present invention is unexpected, however, because even the most similar nucleic acid sequence identified by previous investigators could not be used to identify an esterase of the present invention. In addition, identification of an esterase protein of the present invention is unexpected because a protein fraction from flea prepupal larvae that was obtained by monitoring for serine protease activity surprisingly also contained esterase proteins of the present invention.
In summary, there remains a need to develop a reagent and a method to protect animals or plants from hematophagous arthropod infestation.
SUMMARY OF THE INVENTION The present invention relates to a novel product and process for protection of animals or plants from arthropod infestation. According to the present invention there are provided arthropod esterase proteins and mimetopes thereof; arthropod nucleic acid molecules, including those that encode such proteins; antibodies raised against such esterase proteins anti-arthropod esterase antibodies); and compounds that inhibit 15 arthropod esterase activity inhibitory compounds or inhibitors).
The present invention also includes methods to obtain such proteins, mimetopes, nucleic acid molecules, antibodies and inhibitory compounds. Also included in the present invention are therapeutic compositions comprising such proteins, mimetopes, nucleic acid molecules, antibodies, and/or inhibitory compounds, as well as use of such 20 therapeutic compositions to protect animals from arthropod infestation.
Identification of an esterase of the present invention is unexpected, however, because the most similar nucleic acid sequence identified by previous investigators could not be used to identify an esterase of the present invention. In addition, identification of an esterase protein of the present invention is unexpected because a protein fraction from flea prepupal larvae that was obtained by monitoring for serine protease activity surprisingly also contained esterase proteins of the present invention.
One embodiment of the present invention is an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene comprising a nucleic acid sequence including SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76 and/or a nucleic acid molecule encoding a protein comprising amino acid sequence SEQ ID NO:74.
The present invention also includes a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule encoding a protein comprising at least one of the following amino acid sequences:SEQ ID NO:2, SEQ ID .NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID .SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:68, SEQ ID NO:73 and/or SEQ ID NO:74; and particularly a nucleic acid molecule that hybridizes with a nucleic acid sequence that is a complement of a nucleic acid sequence encoding any of the amino acid sequences. A preferred nucleic acid molecule of the present in\vention includes a nucleic acid molecule *comprising a nucleic acid sequence including SEQ ID NO:, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:1, SEQ ID NO:17, SEQ ID NO:18, SEQ QID SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59, SEQ IM SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76 and/or a nucleic acid molecule encoding a protein comprising amino acid sequence SEQ ID NO:74, and allelic variants thereof.
The present invention also includes an isolated carboxylesterase nucleic acid molecule comprising a nucleic acid sequence encoding a protein comprising an amino acid sequence including SEQ ID NO:5, SEQ ID NO:19, SEQ ID NO:39, SEQ ID SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 and/or SEQ ID NO:53. SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:44 represent N-terminal amino acid sequences of carboxylesterases isolated from prepupal flea larvae, the production of which are described in the Examples of the present application.
The present invention also relates to recombinant molecules, recombinant viruses and recombinant cells that include a nucleic acid molecule of the present invention.
Also included are methods to produce such nucleic acid molecules, recombinant molecules, recombinant viruses and recombinant cells.
Another embodiment of the present invention includes an isolated esterase protein that is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions to a nucleic acid molecule that includes at least one of the following nucleic acid sequences:SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:22, SEQ ID SNO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:52, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:69, and SEQ ID NO:71; and/or (b) a nucleic acid molecule encoding a protein including at least one of the following amino acid sequences: SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ 20 ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 and SEQ ID NO:74. One embodiment is a carboxylesterase protein encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions to a nucleic acid molecule that encodes a protein comprising at least one of the following amino acid sequences: SEQ ID NO:5, SEQ ID NO:19, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID 'O 43, SEQ ID 1NO:44 and/or SEQ ID NO:53.
Preferred proteins of the present invention are isolated flea proteins including at least one of the following amino acid sequences:SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:68, SEQ ID NO:73 and SEQ ID NO:74; also included are proteins encoded by allelic variants of nucleic acid molecules encoding proteins comprising any of the above-listed amino acid sequences.
The present invention also relates to mimetopes of arthropod esterase proteins as well as to isolated antibodies that selectively bind to arthropod esterase proteins or mimetopes thereof. Also included are methods, including recombinant methods, to produce proteins, mimetopes and antibodies of the present invention.
The present invention also includes a formulation of flea carboxylesterase proteins, in which the proteins, when submitted to 14% Tris-glycine
SDS-PAGE,
comprise a fractionation profile as depicted in Fig. 3, in which the proteins have carboxylesterase activity.
Also included in the present invention is a formulation of flea carboxylesterase proteins, in which the proteins, when submitted to IEF-PAGE, comprise a fractionation profile as depicted in Fig. 4, lane 3, lane 4, lane 5, lane 6 and/or lane 7, wherein the 15 proteins have carboxylesterase activity.
Another embodiment of the present invention is an isolated flea protein or a formulation of flea proteins that hydrolyzes a-napthyl acetate to produce a-napthol, when the protein is incubated in the presence of a-napthyl acetate contained in 20 mM Tris at pH 8.0 for about 15 minutes at about 37 0
C.
Yet another embodiment of the present invention is an isolated flea protein or a formulation of flea proteins that hydrolyzes the methyl ester group of juvenile hormone to produce a juvenile hormone acid.
Another embodiment of the present invention is a method to identify a compound capable of inhibiting flea carboxylesterase activity, the method comprising: (a) con*ac' ng an isolated flea carboxylesterase with a putative inhibitory compound under conditions in which, in the absence of the compound, the protein has carboxylesterase activity; and determining if the putative inhibitory compound inhibits the activity.
Also included in the present invention is a test kit to identify a compound capable of inhibiting flea carboxylesterase activity, the test kit comprising an isolated flea carboxylesterase protein having esterase activity and a means for determining the extent of inhibition of the activity in the presence of a putative inhibitory compound.
Yet another embodiment of the present invention is a therapeutic composition that is capable of reducing hematophagous ectoparasite infestation. Such a therapeutic composition includes at least one of the following protective compounds: an isolated hematophagous ectoparasite carboxylesterase protein or a mimetope thereof, an isolated carboxylesterase nucleic acid molecule that hybridizes under stringent hybridization conditions with a Ctenocephalidesfelis carboxylesterase gene, an isolated antibody that selectively binds to a hematophagous ectoparasite carboxylesterase protein, and an inhibitor of carboxylesterase activity identified by its ability to inhibit the activity of a flea carboxylesterase. A therapeutic composition of the present invention can also include an excipient, an adjuvant and/or a carrier. Preferred esterase nucleic acid molecule compounds of the present invention include naked nucleic acid vaccines, .recombinant virus vaccines and recombinant cell vaccines. Also included in the present 15 invention is a method to protect an animal from hematophagous ectoparasite infestation, comprising the step of administering to the animal a therapeutic composition of the present invention.
BRIEF DESCRIPTION OF THE FIGURES Fig. 1 depicts SDS-PAGE analysis ofDFP-labeled esterase proteins.
20 Fig. 2 depicts carboxylesterase activity of certain esterase proteins of the present invention.
Fig. 3 depicts SDS-PAGE analysis of carboxylesterase activity of certain esterase proteins of the present invention.
Fig. 4 depicts IEF analysis of certain esterase proteins of the present invention.
Fig. 5 depicts juvenile hormone esterase activity of certain esterase proteins of the present invention.
DETAILED DESCRIPTION OF THE INVENTION The present invention provides for isolated arthropod esterase proteins, isolated arthropod esterase nucleic acid molecules, antibodies directed against arthropod esterase proteins and other inhibitors of arthropod esterase activity. As used herein, the terms isolated arthropod esterase proteins and isolated arthropod esterase nucleic acid molecules refers to esterase proteins and esterase nucleic acid molecules derived from arthropods and, as such, can be obtained from their natural source or can be produced using, for example, recombinant nucleic acid technology or chemical synthesis. Also included in the present invention is the use of these proteins, nucleic acid molecules, antibodies and inhibitors as therapeutic compositions to protect animals from hematophagous ectoparasite infestation as well as in other applications, such as those disclosed below.
Arthropod esterase proteins and nucleic acid molecules of the present invention have utility because they represent novel targets for anti-arthropod vaccines and drugs.
The products and processes of the present invention are advantageous because they :.enable the inhibition of arthropod development, metamorphosis, feeding, digestion and reproduction processes that involve esterases. While not being bound by theory, it is believed that expression of arthropod esterase proteins are developmentally regulated, 15 thereby suggesting that esterase proteins are involved in arthropod development and/or Sreproduction. The present invention is particularly advantageous because the proteins of the present invention were identified in larval fleas, thereby suggesting the importance of the proteins as developmental proteins.
One embodiment of the present invention is an esterase formulation that includes one or more esterase proteins capable of binding to diisopropylfluorophosphate
(DFP).
A preferred embodiment of an esterase formulation of the present invention comprises one or more arthropod esterase proteins that range in molecular weight from about kilodaltons (kD) to about 200 kD, more preferably from about 40 kD to about 100 kD, and even more preferably from about 60 kD to about 75 kD, as determined by SDS- PAGE (sodium dodecyl sulfate polyacrylamide gel elec'ro )horesis). An even more preferred formulation includes one or more flea esterase proteins having elution (or migration) patterns as shown in Fig. 1.
Another embodiment of the present invention is a formulation comprising one or more hematophagous ectoparasite carboxylesterase (CE) proteins. The present invention includes the discovery that such a formulation has general CE activity. General CE activity can be identified using methods known to those of skill in the art and described in the Examples section herein. A suitable formulation of the present invention comprises one or more flea proteins capable ofhydrolyzing a-napthyl acetate to produce a-napthol when the proteins are incubated in the presence of a-napthyl acetate contained in 20 mm Tris at pH 8.0 for about 15 minutes at about 37 0 C. General CE activity can be identified following such incubation by detecting the production of from about 0.3 to about 2.5 absorbance units in the presence of Fast Blue when measured at 590 nm.
A preferred CE formulation of the present invention includes one or more flea CE proteins having acidic to neutral isoelectric points, or pi values. An isoelectric pH, or pi, value refers to the pH value at which a molecule has no net electric charge and fails to move in an electric field. A preferred formulation of the present invention includes one or more proteins having a pI value ranging from about pi 2 to about more preferably from about pi 3 to about 8, and even more preferably from about pl 4.7 to about 5.2, as determined by IEF-PAGE.
15 An esterase formulation, including a CE formulation, of the present invention can be prepared by a method that includes the steps of: preparing an extract by isolating flea tissue, homogenizing the tissue by sonication and clarifying the extract by centrifugation at a low speed spin, about 18,000 rpm for about 30 minutes; (b) recovering soluble proteins from said centrifuged extract and applying the proteins to a S 20 p-aminobenzamidine agarose bead column; recovering unbound protein from the column and clarifying by filtration; applying the clarified protein to a gel filtration column and eluting and collecting fractions with esterase activity; dilayzing the eluate against 20 mM MES buffer, pH 6.0, containing 10 mM NaC1; applying the dialysate to a cation exchange chromatography column, eluting protein bound to the column with a linear gradier. of from about 10 mM NaCI to about 1 M NaCl in 20 mM MES buffer, pH 6, and collecting fractions having esterase activity; adjusting the pH of the resulting fractions to pH 7 and applying the fractions to an anion exchange chromatography column; eluting protein bound to the column with a linear gradient of from about 0 to about 1 M NaCI in 25 mM Tris buffer, pH 6.8 and collecting fractions having esterase activity, such activity elutes from the column at about 170 mM NaC1.
Tissue can be obtained from unfed fleas or from fleas that recently consumed a blood meal blood-fed fleas). Such flea tissues are referred to herein as, respectively, unfed flea and fed flea tissue. Preferred flea tissue from which to obtain an esterase formulation of the present invention includes pre-pupal larval tissue, wandering flea larvae, 3rd instar tissue, fed adult tissue and unfed adult tissue.
In a preferred embodiment, a CE formulation of the present invention comprises a flea protein comprising amino acid sequence SEQ ID NO:5, SEQ ID NO:19, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 and/or SEQ ID NO:53.
Another embodiment of the present invention is a juvenile hormone esterase (JHE) formulation comprising one or more arthropod JHE proteins, the arthropod being of the order Hemiptera, Anoplura, Mallophaga, Diptera, Siphonaptera, Parasitiformes, Acariformes and Acarina. The present invention includes the discovery that such a formulation has JHE activity. JHE activity can be identified using methods known to 15 those of skill in the art and described in the Examples section herein. A suitable formulation of the present invention comprises one or more arthropod proteins capable ofhydrolyzing a methyl ester group of juvenile hormone to produce a juvenile hormone acid. Preferably, such a protein is capable of releasing of at least about 120 counts per *minute when such a protein is incubated in the presence of 3 H-juvenile hormone to 20 create a reaction mixture, the reaction mixture is combined with isooctane, the aqueous phase is recovered and the amount of H-juvenile hormone present in that phase is determined. Such a protein is also preferably capable of causing release of methane thiol when such protein is incubated in the presence of methyl 1-heptylthioacetothioate (HEPTAT) using the method generally disclosed in McCutchen et al., Insect Biochem.
Molec. Bio'., Vol. 25, No. 1, pg 119-126, 1995, which is incorporated in its entirety by this reference.
In one embodiment, a juvenile hormone esterase formulation of the present invention comprises a protein comprising amino acid sequence SEQ ID NO:74.
According to the present invention, an arthropod that is not of the order lepidoptera includes an arthropod of the order Hemiptera, Anoplura, Mallophaga, Diptera, Siphonaptera, Parasitiformes, Acariformes and Acarina. Preferred arthropods include Hemiuptera cirnicidae, Hemniptera. reduviidae, Anoplura pediculidae, Anoplura.
pthiridae, Diptera. culicidae, Diptera simuliidae, Diptera psychodidae, Diptera ceratopogonidae, Diptera. chaoboridae, Diptera tabanidae, Diptera rhagionidae, athericidae, Diptera. chloropidae, Diptera muscidae, Diptera hippoboscidae, Diptera callipboridae, Diptera sarcophagidae, Diptera. oestridae, Diptera gastrophilidae, Diptera cuterebridae, S iphonaptera ceratophyllidae, Siphonaptera leptopsyllidae, S iphonaptera pulicidae, Siphonaptera tungidae, Parasitiformes dermanyssidae, Acariformes tetranychidae, Acariformes cheyletide, Acari formes demodicidae, Acari forrnes erythraeidae, Acariformes trombiculidae, Acariformes psoroptidae, Acariformes sarcoptidae, Acarina argasidae and Acarina ixodidae. Preferred Diptera rnuscidae include Musca, Hydrotaea, Staioxys Haenzatobia. Preferred Siphonaptera include Ceratophyllidac nosopsyllus, Ceralopliyllidae diamanus, C'eatophyllidae cer-atophylflus, Leptopsyllidae leptopsylla, Pulicidae pulex, Pulicidae clenocephalides, Pulicidae xenopsylla, Pulicidae echidnophaga and Tuingidae tunga. Preferred Parasitiformes dermanyssidae include Ornithonysszts and Liponyssoidcs. Preferred Acarina include- ***Argasidae argas, Ar-gasidae ornithodoros, Ar-gasidae otobins, Ixodidae ixodes, Ixodidae hyalonnna, Ixodidae nosonia, Ixodidae rhipicephalus, Ixodidae boophilus, Ixodidae dei-nacentor, Ixodidae haernaphysalus. Lrodidae amnblyoinniza and Ixodidae anocentor.
One embodiment of a JHE formulation of the present invention is one or more arthropod JHE proteins that range in molecular weight from about 20 kD to about 200 sees kD, more preferably from about 40 kD to about 100 kD, and even more preferably from about 60 kD to about 75 k.D, as determined by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).
A JHE formulation of the present invention can be prepared by a method that includes the steps of: preparing soluble proteins from arthropod extracts as described above for CE purification and purifying such soluble proteins by gel filtration; (b) collecting fractions having JHE activity from the gel filtration step, loading the fractions onto a cation exchange column, eluting the cation exchange column with a linear gradient of from about 10 mM NaCI to about 1 M NaCl in 20mM MES buffer, pH 6 and -12collecting fractions having JHE activity; adjusting the pH of the collected fractions to about pH 7 are dialyzed against about 10 mM phosphate buffer (pH 7.2) containing about 10 mM NaC1; applying the dialysate to a hydroxyapatite column, eluting protein bound to the column with a linear gradient of from about 10 mM phosphate buffer (pH 7.2) containing 10 mM NaCl to about 0.5 M phosphate buffer (pH containing 10 mM NaCl and collecting fractions having JHE activity; dialyzing the fractions against 20 mM Tris buffer (pH 8.0) containing 10 mM NaCI; applying the dialysate an anion exchange chromatography column and eluting protein bound to the column with a linear gradient of from about 10 mM to about 1 M NaCI in 20 mM Tris buffer, pH 8 and collecting fractions containing JHE activity.
A THE formulation of the present invention can be prepared by a method that includes the steps of: preparing flea extracts as described herein in the Examples section and applying the extract to p-aminobenzamidine linked agarose beads and collecting protein not bound to the beads; applying the unbound protein to a 1* 15 Superdex 200 HR gel filtration column and collecting fractions having JHE activity; (c) applying the fractions to an anion exchange chromatography column, eluting the anion exchange column with a linear gradient of 0 to 1 M NaCl in 25 mM Tris buffer, pH 6.8 and collecting fractions having JHE activity; dialyzing the fractions overnight against about 1 L of 20 mM Tris buffer, pH 8.0, containing 10 mM NaCI; applying the 20 dialysate to a Poros 10 HQ anion exchange column, eluting the column with buffer containing about 120 mM NaCl and collecting fractions having JHE activity.
Suitable arthropods from which to isolate a JHE formulation of the present invention include, but are not limited to agricultural pests, stored product pests, forest pests, structural pests or animal health pests. Suitable agricultural pests of the present invention include, but are not limited to Coloradr potato beetle-., corn earworms, fleahoppers, weevils, pink boll worms, cotton aphids, beet armyworms, lygus bugs, hessian flies, sod webworms, whites grubs, diamond back moths, white flies, planthoppers, leafhoppers, mealy bugs, mormon crickets and mole crickets. Suitable stored product pests of the present invention include, but are not limited to dermestids, anobeids, saw toothed grain beetles, indian mealmoths, flour beetles, long-horn wood boring beetles and metallic wood boring beetles. Suitable forest pests of the present invention include but are not limited to southern pine bark bettles, gypsy moths, elm beetles, ambrosia bettles, bag worms, tent worms and tussock moths. Suitable structural pests of the present invention include, but are not limited to, bess beetles, termites, fire ants, carpenter ants, wasps, hornets, cockroaches, silverfish, Musca domestica and Musca autumnalis. Suitable animal health pests of the present invention include, but are not limited to fleas, ticks, mosquitoes, black flies, lice, true bugs, sand flies, Psychodidae, tsetse flies, sheep blow flies, cattle grub, mites, horn flies, heel flies, deer flies, Culicoides and warble flies. Preferred arthropods from which to isolate a JHE formulation of the present invention include fleas, midges, mosquitos, sand flies, black flies, horse flies, snipe flies, louse flies, hor flies, deer flies, tsetse flies, buffalo flies, 0 blow flies, stable flies, myiasis-causing flies, biting gnats, lice, mites, bee, wasps, ants, S9' true bugs and ticks, preferably fleas, ticks and blow flies, and more preferably fleas.
Preferred fleas from which to isolate JHE proteins include Ctenocephalides, 15 Ceratophyllus, Diamanus, Echidnophaga, Nosopsyllus, Pulex, Tunga, Oropsylla, Orchopeus and Xenopsylla. More preferred fleas include Ctenocephalides felis, Ctenocephalides canis, Ceratophyllus pulicidae, Pulex irritans, Oropsvlla (Thrassis) bacchi, Oropsylla (Diamanus) montana, Orchopeus howardi, Xenopsylla cheopis and Pulex simulans, with C. felis being even more preferred.
20 Suitable tissue from which to-isolate a JHE formulation of the present invention includes unfed fleas or fleas that recently consumed a blood meal blood-fed fleas).
Such flea tissues are referred to herein as, respectively, unfed flea and fed flea tissue.
Preferred flea tissue from which to obtain a JHE formulation of the present invention includes pre-pupal larval tissue, 3rd instar tissue, fed or unfed adult tissue, with unfed adult gut tissue bei .g more preferred than fed or unfed whole adult tissue. It is of note that a JHE formulation of the present invention obtained from pre-pupal larval tissue does not hydrolyze a-napthyl acetate.
Another embodiment of the present invention is an esterase formulation comprising a combination of one or more arthropod CE and JHE proteins of the present invention. Suitable arthropods from which to isolate a combined CE and JHE formulation include those arthropods described herein for the isolation ofa JHE formulation of the present invention. Preferred arthropods from which to isolate a combined CE and JHE formulation include fleas, midges, mosquitos, sand flies, black flies, horse flies, horn flies, deer flies, tsetse flies, buffalo flies, blow flies, stable flies, myiasis-causing flies, biting gnats, lice, bee, wasps, ants, true bugs and ticks, preferably fleas, ticks and blow flies, and more preferably fleas. Suitable flea tissue from which to isolate a combined CE and JHE formulation of the present invention includes 3rd instar tissue, fed or unfed adult tissue and unfed adult tissue, with unfed adult gut tissue being more preferred than fed or unfed whole adult tissue.
In one embodiment, a formulation of the present invention comprises an esterase having both CE and JHE activity. Preferably, a formulation of the present invention that comprises an esterase having both CE and JHE activity comprises a flea protein comprising amino acid sequence SEQ ID NO:8 and/or SEQ ID NO:37.
Another embodiment of the present invention is an isolated protein comprising 15 an arthropod esterase protein. It is to be noted that the term or "an" entity refers to one or more of that entity; for example, a protein refers to one or more proteins or at .o least one protein. As such, the terms (or "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably. Furthermore, a compound S. 20 "selected from the group consisting of' refers to one or more of the compounds in the list that follows, including mixtures combinations) of two or more of the compounds. According to the present invention, an isolated, or biologically pure, protein, is a protein that has been removed from its natural milieu. As such, "isolated" and "biologically pure" do not necessarily reflect the extent to which the protein has been purified. An isolated protein of the present invention can be obtained from its natural source, can be produced using recombinant DNA technology or can be produced by chemical synthesis.
As used herein, an isolated arthropod esterase protein can be a full-length protein or any homolog of such a protein. An isolated protein of the present invention, including a homolog, can be identified in a straight-forward manner by the protein's ability to elicit an immune response against arthropod esterase proteins, to hydrolyze anapthyl acetate, to hydrolyze the methyl ester group of juvenile hormone or bind to DFP.
Esterase proteins of the present invention include CE and JHE proteins. As such, an esterase protein of the present invention can comprise a protein capable of hydrolyzing a-napthyl acetate, hydrolyzing the methyl ester group ofjuvenile hormone and/or binding to DFP. Examples of esterase homologs include esterase proteins in which amino acids have been deleted a truncated version of the protein, such as a peptide), inserted, inverted, substituted and/or derivatized by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation and/or addition of glycerophosphatidyl inositol) such that the homolog includes at least one epitope capable of eliciting an immune response against an arthropod esterase protein. That is, when the homolog is administered to an animal as an immunogen, using techniques known to those skilled in the art, the animal will produce an immune response against at least one epitope of a natural arthropod esterase protein. The ability of a protein to effect an immune response, can be measured using techniques known to i those skilled in the art. Esterase protein homologs of the present invention also include esterase proteins that hydrolyze a-napthyl acetate and/or that hydrolyze the methyl ester group of juvenile hormone.
Arthropod esterase protein hornologs can be the result of natural allelic variation or natural mutation. Esterase protein homologs of the present invention can also be produced using techniques known in the art including, but not limited to, direct modifications to the protein or modifications to the gene encoding the protein using, for example, classic or recombinant nucleic acid techniques to effect random or targeted mutagenesis.
Isolated esterase proteins of the present invention have the ftrtl -r characteristic of being encoded by nucleic acid molecules that hybridize under stringent hybridization conditions to a gene encoding a Ctenocephalidesfelis protein a C. felis esterase gene). As used herein, stringent hybridization conditions refer to standard hybridization conditions under which nucleic acid molecules, including oligonucleotides, are used to identify similar nucleic acid molecules. Such standard conditions are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratoy Manual, Cold Spring Harbor Labs Press, 1989; Sambrook et al., ibid., is incorporated by reference herein in its entirety. Stringent hybridization conditions typically permit isolation of nucleic acid molecules having at least about 70% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction. Formulae to calculate the appropriate hybridization and wash conditions to achieve hybridization permitting or less mismatch of nucleotides are disclosed, for example, in Meinkoth et al., 1984, Anal. Biochem. 138, 267-284; Meinkoth et al., ibid., is incorporated by reference herein in its entirety.
As used herein, a C.felis esterase gene includes all nucleic acid sequences related to a natural C.felis esterase gene such as regulatory regions that control production of the C. feis esterase protein encoded by that gene (such as, but not limited to, transcription, translation or post-translation control regions) as well as the coding region itself. In one embodiment, a C. felis esterase gene of the present invention includes the nucleic acid sequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76 and/or a nucleic acid molecule encoding a protein comprising amino acid sequence SEQ ID NO:74. Nucleic acid sequence SEQ ID NO:1 represents the deduced sequence of the coding s*ra,.d of a PCR amplified nucleic acid molecule denoted herein as nfE1 401 the production of which is disclosed in the Examples. The complement of SEQ ID NO:1 (represented herein by SEQ ID NO:3) refers to the nucleic acid sequence of the strand complementary to the strand having SEQ ID NO:1, which can easily be determined by those skilled in the art. Likewise, a nucleic acid sequence complement of any nucleic acid sequence of the present invention refers to the nucleic acid sequence of the nucleic acid strand that is complementary to can form a complete double helix with) the strand for which the sequence is cited.
Nucleic acid sequence SEQ ID NO:4 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nfE2 364 the production of which is disclosed in the Examples. The complement of SEQ ID NO:4 is represented herein by SEQ ID NO:6.
Nucleic acid sequence SEQ ID NO:7 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nfE3 421 the production of which is disclosed in the Examples. The complement of SEQ ID NO:7 is represented herein by SEQ ID NO:9.
Nucleic acid sequence SEQ ID NO:10 represents the deduced sequence of the coding strand ofa PCR amplified nucleic acid molecule denoted herein as nfE4 524 the production of which is disclosed in the Examples. The complement of SEQ ID NO:10 is represented herein by SEQ ID NO:12.
Nucleic acid sequence SEQ ID NO:13 represents the deduced sequence of the coding strand of an apparent coding region of a complementary DNA (cDNA) nucleic acid molecule denoted herein as nfE5, 9 82 the production of which is disclosed in the Examples. The complement of SEQ ID NO:13 is represented herein by SEQ ID Nucleic acid sequence SEQ ID NO:18 represents the deduced sequence of the coding strand of an apparent coding region ofa cDNA nucleic acid molecule denoted herein as nfE6 79 2 the production of which is disclosed in the Examples. The complement of SEQ ID NO:18 is represented herein by SEQ ID Nucleic acid sequence SEQ ID NO:24 represents the deduced sequence of the coding strand of an apparent coding region of a cDNA nucleic acid molecule denoted herein as ifE7 836 the production of which is disclosed in the Examples. The complement of SEQ ID NO:24 is represented herein by SEQ ID NO:26.
Nucleic acid sequence SEQ ID NO:30 represents the deduced sequence of the coding strand of an apparent coding region of a cDNA nucleic acid molecule denoted herein as nfE82, 80 the production of which is disclosed in the Examples. The complement of SEQ ID NO:30 is represented herein by SEQ ID NO:32.
-18- Nucleic acid sequence SEQ ID NO:36 represents the deduced sequence of the coding strand of an apparent coding region of a cDNA nucleic acid molecule denoted herein as nfE9 2 007 the production of which is disclosed in the Examples. The complement of SEQ ID NO:36 is represented herein by SEQ ID NO:38.
Nucleic acid sequence SEQ ID NO:57 represents the deduced sequence of the coding strand of an apparent coding region of a cDNA nucleic acid molecule denoted herein as nfE5 21 44 the production of which is disclosed in the Examples. The complement of SEQ ID NO:57 is represented herein by SEQ ID NO:59.
Nucleic acid sequence SEQ ID NO:67 represents the deduced sequence of the coding strand of an apparent coding region of a cDNA nucleic acid molecule denoted herein as nfE10 1 8 7 the production of which is disclosed in the Examples. The complement of SEQ ID NO:67 is represented herein by SEQ ID NO:69.
:It should be noted that since nucleic acid sequencing technology is not entirely error-free, the nucleic acid sequences and amino acid sequences presented herein represent, respectively, apparent nucleic acid sequences of nucleic acid molecules of. the present invention and apparent amino acid sequences of esterase proteins of the present invention.
In another embodiment, a C. felis esterase gene can be an allelic variant that includes a similar but not identical sequence to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59, SEQ ID SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76 and/or a nucleic acid molecule encoding a protein comprising amino acid sequence SEQ ID NO:74. An allelic variant of a C. felis esterase gene is a gene that occurs at essentially the same locus (or loci) in the genome as the gene including SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ -19- ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76 and/or a nucleic acid molecule encoding a protein comprising amino acid sequence SEQ ID NO:74, but which, due to natural variations caused by, for example, mutation or recombination, has a similar but not identical sequence. Allelic variants typically encode proteins having similar activity to that of the protein encoded by the gene to which they are being compared. Allelic variants can also comprise alterations in the 5' or 3' untranslated regions of the gene in regulatory control regions). Allelic variants are well known to those skilled in the art and would be expected to be found within a given arthropod since the genome is diploid and/or among a group of two or more arthropods.
The minimal size of an esterase protein homolog of the present invention is a size sufficient to be encoded by a nucleic acid molecule capable of forming a stable hybrid hybridize under stringent hybridization conditions) with the complementary sequence of a nucleic acid molecule encoding the corresponding natural protein. As such, the size of the nucleic acid molecule encoding such a protein homolog is dependent on nucleic acid composition and percent homology between the nucleic acid molecule and complementary sequence. It should also be noted that the extent of homology required to form a stable hybrid can vary depending on whether the homologous sequences are interspersed throughout the nucleic acid molecules or are clustered localized) in distinct regions on the nucleic acid molecules. The minimal size of such nucleic acid molecules is typically at least about 12 to about 15 nucleotides in length if the nucleic acid molecules are GC-rich and at least about 15 to about 17 bases in length if they are AT-rich. As such, the minimal size of a nucleic acid molecule used to encode an esterase protein homolog of the present invention is from about 12 to about 18 nucleotides in length. Thus, the minimal size of an esterase protein homolog of the present invention is from about 4 to about 6 amino acids in length. There is no limit, other than a practical limit, on the maximal size of such a nucleic acid molecule in that the nucleic acid molecule can include a portion of a gene, an entire gene, multiple genes, or portions thereof The preferred size of a protein encoded by a nucleic acid molecule of the present invention depends on whether a full-length, fusion, multivalent, or functional portion of such a protein is desired.
One embodiment of the present invention includes an arthropod esterase protein having CE enzyme activity. Such a CE protein preferably includes: a catalytic triad of serine histidine glutamic acid as well as the essential amino acids arginine and i aspartic acid at positions similar to those described for juvenile hormone esterase, for example by Ward et al., 1992, Int JBiochem 24: 1933-1941; this reference is incorporated by reference herein in its entirety. Analysis of the apparent full-length protein sequences disclosed herein indicates that each of these amino acid sequences includes these amino acid motifs, as well as surrounding consensus sequences.
i Suitable arthropods from which to isolate esterase proteins having general CE activity of the present invention (including isolation of the natural protein or production of the protein by recombinant or synthetic techniques) preferably include insects and acarines but not Culicidae, Drosophilidae, Calliphoridae, Sphingidae, Lymantriidae, Noctuidae, Fulgoroidae and Aphididae. Preferred arthropods from which to isolate CE proteins having general CE activity include fleas, ticks, black flies, lice, true bugs, sand flies, Psychodidae, tsetse flies, cattle.grub, mites, horn flies, heel flies, deer flies, Culicoides and warble flies. Preferred arthropods from which to isolate an esterase proteins having general CE activity include fleas, midges,, sand flies, black flies, horse flies, snipe flies, louse flies, ho n flies, deer fies, tsetse flies, buffalo flies, blow flies, stable flies, myiasis-causing flies, biting gnats, lice, mites, bee, wasps, ants, true bugs and ticks, preferably fleas, ticks and blow flies, and more preferably fleas. Preferred fleas from which to isolate esterase proteins having general CE activity include Ctenocephalides, Ceratophyllus, Diamanus, Echidnophaga, Nosopsyllus, Pulex, Tunga, Oropsylla, Orchopeus and Xenopsylla. More preferred fleas include Ctenocephalides -21felis, Ctenocephalides canis, Ceratophyllus pulicidae, Pulex irritans, Oropsylla (Thrassis) bacchi, Oropsylla (Diamanus) montana, Orchopeus howardi, Xenopsylla cheopis and Pulex simulans, with C felis being even more preferred.
A preferred arthropod esterase protein of the present invention is a compound that when administered to an animal in an effective manner, is capable of protecting that animal from hematophagous ectoparasite infestation. In accordance with the present invention, the ability of an esterase protein of the present invention to protect an animal from hematophagous ectoparasite infestation refers to the ability of that protein to, for example, treat, ameliorate and/or prevent infestation caused by hematophagous arthropods. In particular, the phrase "to protect an animal from hematophagous i ectoparasite infestation" refers to reducing the potential for hematophagous ectoparasite population expansion on and around the animal reducing the hematophagous ectoparasite burden). Preferably, the hematophagous ectoparasite population size is decreased, optimally to an extent that the animal is no longer bothered by hematophagous ectoparasites. A host animal, as used herein, is an animal from which hematophagous ectoparasites can feed by attaching to and feeding through the skin of the animal. Hematophagous ectoparasites, and other ectoparasites, can live on a host animal for an extended period of time or can attach temporarily to an animal in order to feed. At any given time, a certain percentage of a hematophagous ectoparasite population can be on a host animal whereas the remainder can be in the environment of the animal. Such an environment can include not only adult hematophagous ectoparasites, but also hematophagous ectoparasite eggs and/or hematophagous ectoparasite larvae. The environment can be of any size such that hematophagous ectoparasites in the environment are able to jump onto and off of a host animal. For e.ample, the environment of an animal can include plants, such as crops, from which hematophagous ectoparasites infest an animal. As such, it is desirable not only to reduce the hematophagous ectoparasite burden on an animal per se, but also to reduce the hematophagous ectoparasite burden in the environment of the animal. In one embodiment, an esterase protein of the present invention can elicit an immune response (including a humoral and/or cellular immune response) against a hematophagous ectoparasite.
Suitable hematophagous ectoparasites to target include any hematophagous ectoparasite that is essentially incapable of infesting an animal administered an esterase protein of the present invention. As such, a hematophagous ectoparasite to target includes any hematophagous ectoparasite that produces a protein having one or more epitopes that can be targeted by a humoral and/or cellular immune response against an esterase protein of the present invention and/or that can be targeted by a compound that otherwise inhibits esterase activity a compound that inhibits hydrolysis of anapthyl acetate, hydrolysis of the methyl ester group ofjuvenile hormone, and/or binds to DFP), thereby resulting in the decieased ability of the hematophagous ectoparasite to infest an animal. Preferred hematophagous ectoparasite to target include ectoparasites disclosed herein as being useful in the production of esterase proteins of the present invention.
The present invention also includes mimetopes of esterase proteins of the present invention. As used herein, a mimetope of an esterase protein of the present invention refers to any compound that is able to mimic the activity of such a protein ability to elicit an immune response against an arthropod esterase protein of the present invention and/or ability to inhibit esterase activity), often because the mimetope has a structure that mimics the esterase protein. It is to be noted, however, that the mimetope need not have a structure similar to an esterase protein as long as the mimetope functionally mimics the protein. Mimetopes can be, but are not limited to: peptides that have been modified to decrease their susceptibility to degradation; anti-idiotypic and/or catalytic antibodies, or fragments thereof; non-proteinaceous immunogenic portions of an isolated protein carbohydrate structures); synthetic or natural organic or inorganic molecules, including nucleic acids; and/or any other peptidomimetic compounds.
Mimetopes of the present invention can be designed using computer-generated structures of esterase proteins of the present invention. Mimetopes can also be obtained by generating random samples of molecules, such as oligonucleotides, peptides or other organic molecules, and screening such samples by affinity chromatography techniques -23using the corresponding binding partner, an esterase substrate, an esterase substrate analog, or an anti-esterase antibody). A preferred mimetope is a peptidomimetic compound that is structurally and/or functionally similar to an esterase protein of the present invention, particularly to the active site of the esterase protein.
The present invention also includes mimetopes of esterase proteins of the present invention. As used herein, a mimetope of an esterase protein of the present invention refers to any compound that is able to mimic the activity of such an esterase protein, often because the mimetope has a structure that mimics the esterase protein. Mimetopes can be, but are not limited to: peptides that have been modified to decrease their susceptibility to degradation; anti-idiotypic and/or catalytic antibodies, or fragments thereof; non-proteinaceous immunogenic portions of an isolated protein ~carbohydrate structures); and synthetic or natural organic molecules, including nucleic S acids. Such mimetopes can be designed using computer-generated structures of proteins of the present invention. Mimetopes can also be obtained by generating random samples of molecules, such as oligonucleotides, peptides or other organic molecules, and screening such samples by affinity chromatography techniques using the corresponding binding partner.
One embodiment of an arthropod esterase protein of the present invention is a fusion protein that includes an arthropod esterase protein-containing domain attached to one or more fusion segments. Suitable fusion segments for use with the present invention include, but are not limited to, segments that can: enhance a protein's stability; act as an immunopotentiator to enhance an immune response against an esterase protein; and/or assist purification of an esterase protein by affinity chromatography).
A
suitable fusion segment can be a domain of any size that has the desired function imparts increased stability, imparts increased immnmc genicity to a protein, and/or simplifies purification of a protein). Fusion segments can be joined to amino and/or carboxyl termini of the esterase-containing domain of the protein and can be susceptible to cleavage in order to enable straight-forward recovery of an esterase protein. Fusion proteins are preferably produced by culturing a recombinant cell transformed with a fusion nucleic acid molecule that encodes a protein including the fusion segment -24attached to either the carboxyl and/or amino terminal end of an esterase-containing domain. Preferred fusion segments include a metal binding domain a polyhistidine segment); an immunoglobulin binding domain Protein A; Protein G; T cell; B cell; Fc receptor or complement protein antibody-binding domains); a sugar binding domain a maltose binding domain); and/or a "tag" domain at least a portion of P-galactosidase, a strep tag peptide, other domains that can be purified using compounds that bind to the domain, such as monoclonal antibodies). More preferred fusion segments include metal binding domains, such as a poly-histidine segment; a maltose binding domain; a strep tag peptide, such as that available from Biometra in Tampa, FL; and an S10 peptide. Examples of particularly preferred fusion proteins of the present invention include PHIS-PfE6 5 4 0 PHIS-PfE7 275 PHIS-PfE7 57 0 PHIS-PfE8 570 and PHIS-PfE9 528 production of which are disclosed herein. go In another embodiment, an arthropod esterase protein of the present invention also includes at least one additional protein segment that is capable of protecting an animal from hematophagous ectoparasite infestations. Such a multivalent protective protein can be produced by culturing a cell transformed with a nucleic acid molecule comprising two or more nucleic acid domains joined together in such a manner that the resulting nucleic acid molecule is expressed as a multivalent protective compound containing at least two protective compounds, or portions thereof, capable of protecting an animal from hematophagous ectoparasite infestation by, for example, targeting two V different arthropod proteins.
Examples of multivalent protective compounds include, but are not limited to, an esterase protein of the present invention attached to one or more compounds protective against one or more arthropod compounds. Preferred second compounds are proteinaceous compoui.ds that effect active immunization antigen vaccines), passive immunization antibodies), or that otherwise inhibit a arthropod activity that when inhibited can reduce hematophagous ectoparasite burden on and around an animal.
Examples of second compounds include a compound that inhibits binding between an arthropod protein and its ligand a compound that inhibits flea ATPase activity or a compound that inhibits binding of a peptide or steroid hormone to its receptor), a compound that inhibits hormone (including peptide or steroid hormone) synthesis, a compound that inhibits vitellogenesis (including production of vitellin and/or transport and maturation thereof into a major egg yolk protein), a compound that inhibits fat body function, a compound that inhibits muscle action, a compound that inhibits the nervous system, a compound that inhibits the immune system and/or a compound that inhibits hematophagous ectoparasite feeding. Examples of second compounds also include proteins obtained from different stages of hematophagous ectoparasite development.
Particular examples of second compounds include, but are not limited to, serine proteases, cysteine proteases, aminopeptidases, serine protease inhibitor proteins, calreticulins, larval serum proteins and echdysone receptors, as well as antibodies to and 9 inhibitors of such proteins. In one embodiment, an arthropod esterase protein of the present invention is attached to one or more additional compounds protective against hematophagous ectoparasite infestation. In another embodiment, one or more protective compounds, such as those listed above, can be included in a multivalent vaccine comprising an arthropod esterase protein of the present invention and one or more other protective molecules as separate compounds.
A preferred isolated protein of the present invention is a protein encoded by a O nucleic acid molecule that hybridizes under stringent hybridization conditions with nucleic acid molecules nfEl 40 1 nfE2 3 6 4 nfE3 42 nfE4 524 nfE5 1 s2, nfE5s,,,, nfE5 2 nfE5 1650 nfE6 488 nfE6 1 7 92 nfE6 1 6 5 nfE7 2836 nfE7 1788 nfE7,,to, nfE7 650 nfE8 280 1 fnfEf 1 78 5 nfE8 1 7 10 n9 2 00 7 nfE91, nfE9 1 5 4 0 nfElO,,0 1 and/or nfE 10159. A further preferred isolated protein is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having nucleic acid sequence SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID SEQ TD NO:17, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:52, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:69 and/or SEQ ID NO:71.
Translation of SEQ ID NO:1 suggests that nucleic acid molecule nfE1 4 0 encodes a non-full-length arthropod esterase protein of about 103 amino acids, referred to herein as PfEl103, represented by SEQ ID NO:2, assuming the first codon spans from nucleotide 92 through nucleotide 94 of SEQ ID NO:1.
Comparison of amino acid sequence SEQ ID NO:2 the amino acid sequence ofPfEl103) with amino acid sequences reported in GenBank indicates that SEQ ID NO:2, showed the most homology, about 33% identity, between SEQ ID NO:2 and alpha esterase protein from Drosophila melanogaster.
Translation of SEQ ID NO:4 suggests that nucleic acid molecule nfE2 3 encodes a non-full-length arthropod esterase protein of about 121 amino acids, referred to herein as PfE2 12 1 represented by SEQ ID NO:5, assuming the first codon spans from nucleotide '10 2 through nucleotide 4 of SEQ ID NO:4.
Comparison of amino acid sequence SEQ ID NO:5 the amino acid sequence ofPfE2 1 2 1 with amino acid sequences reported in GenBank indicates that SEQ ID showed the most homology, about 38% identity, between SEQ ID NO:5 and alpha esterase protein from Drosophila melanogaster.
Translation of SEQ ID NO:7 suggests that nucleic acid molecule nfE3 42 1 encodes a non-full-length arthropod esterase protein of about 103 amino acids, referred to herein as PfE32 0 3 represented by.SEQ ID NO:8, assuming the first codon spans from nucleotide 113 through nucleotide 115 of SEQ ID NO:7.
Comparison of amino acid sequence SEQ ID NO:8 the amino acid sequence 20 ofPfE3 0 3 with amino acid sequences reported in GenBank indicates that SEQ ID NO:8, showed the most homology, about 39% identity, between SEQ ID NO:8 and alpha esterase protein from Drosophila melanogaster.
Translation of SEQ ID NO:10 suggests that nucleic acid molecule nfE4 5 24 encodes a non-full-length arthropod esterase protein of about 137 amino acids, referred to herein as PfE4,3,, represented by SEQ ID NO:11, assuming the first rcd )n spans from nucleotide 113 through nucleotide 115 of SEQ ID Comparison of amino acid sequence SEQ ID NO: 11 the amino acid sequence ofPfE4, 37 with amino acid sequences reported in GenBank indicates that SEQ ID NO:11, showed the most homology, about 30% identity, between SEQ ID NO: 11 and Leptinotarsa decemlineata acetylcholinesterase.
-27- Translation of SEQ ID NO:57 suggests that nucleic acid molecule nfE5 2 encodes a full-length arthropod esterase protein of about 550 amino acids, referred to herein as PfE5s 5 o, represented by SEQ ID NO:58, assuming an open reading frame in which the initiation codon spans from nucleotide 30 through nucleotide 32 of SEQ ID NO:57 and the termination (stop) codon spans from nucleotide 1680 through nucleotide 1682 of SEQ ID NO:57. The complement of SEQ ID NO:57 is represented herein by SEQ ID NO:59. The coding region encoding PfE5 5 so is represented by the nucleic acid molecule nfE5 1 6 5s, having a coding strand with the nucleic acid sequence represented by SEQ ID NO:60 and a complementary strand with nucleic acid sequence SEQ ID NO:61.
10 The deduced amino acid sequence ofPfE 55 s 0 SEQ ID NO:58) predicts that PfE555 has an estimated molecular weight of about 61.8 kD and an estimated pi of about Comparison of amino acid sequence SEQ ID NO:58 the amino acid sequence ofPfE5 550 with amino acid sequences reported in GenBank indicates that SEQ ID NO:58 showed the most homology, about 36% identity between SEQ ID NO:58 15 and Drosophila melanogaster alpha esterase protein.
Translation of SEQ ID NO:18 suggests that nucleic acid molecule nfE61 792 encodes a full-length arthropod esterase protein of about 550 amino acids, referred to herein as PfE6 5 50 represented by SEQ ID NO:19, assuming an open reading frame having an initiation codon spanning from nucleotide 49 through nucleotide 51 of SEQ 20 ID NO:18 and a stop codon spanning from nucleotide 1699 through nucleotide 1701 of SEQ ID NO:18. The coding region encoding PfE6 5 5 s, is represented by nucleic acid molecule nfE6Mso, having a coding strand with the nucleic acid sequence represented by SEQ ID NO:21 and a complementary strand with nucleic acid sequence SEQ ID NO:22.
The proposed mature protein, denoted herein as PfE6 53 o, contains about 530 amino acids which is represented herein as SEQ ID NO:53. The nucleic acid molecule encoding PfE6 5 30 is denoted herein as nfE6s 5 and has a coding strand having the nucleic acid sequence SEQ ID NO:23. The deduced amino acid sequence SEQ ID NO:19 suggests a protein having a molecular weight of about 61.8 kD and an estimated pi of about Comparison of amino acid sequence SEQ ID NO:19 the amino acid sequence ofPfE6 5 5 0 with amino acid sequences reported in GenBank indicates that SEQ -28- ID NO:19 showed the most homology, about 28% identity between SEQ ID NO:19 and Drosophila melanogaster alpha esterase protein.
Translation of SEQ ID NO:24 suggests that nucleic acid molecule nfE7 2836 encodes a full-length arthropod esterase protein of about 596 amino acids, referred to herein as PfE7 59 6 represented by SEQ ID NO:25, assuming an open reading frame having an initiation codon spanning from nucleotide 99 through nucleotide 101 of SEQ ID NO:24 and a stop codon spanning from nucleotide 1887 through nucleotide 1889 of SEQ ID NO:24. The coding region encoding PfE7 596 is represented by nucleic acid molecule nfE7 1 ss, having a coding strand with the nucleic acid sequence represented by i' 10 SEQ ID NO:28 and a complementary strand with nucleic acid sequence SEQ ID NO:29.
The proposed mature protein, denoted herein as PfE757 0 contains about 570 amino acids which is represented herein as SEQ ID NO:54. The nucleic acid molecule encoding PfE7 57 0 is denoted herein as nfE7 1 7 10 and has a coding strand having the nucleic acid sequence SEQ ID NO:27. The deduced amino acid sequence SEQ ID N0:25 suggests a protein having a molecular weight of about 68.7 kD and an estimated pi of about 6.1.
Comparison of amino acid sequence SEQ ID NO:25 the amino acid sequence of PfE7 596 with amino acid sequences reported in GenBank indicates that SEQ .ID NO:25 showed the most homology, about 27% identity between SEQ ID and Drosophila melanogaster alpha esterase protein.
Translation of SEQ ID NO:30 suggests that nucleic acid molecule nfE8 28 0 1 encodes a full-length arthropod esterase protein of about 595 amino acids, referred to herein as PfE8 595 represented by SEQ ID NO:31, assuming an open reading frame having an initiation codon spanning from nucleotide 99 through nucleotide 101 of SEQ ID NO:30 and a stop codon spanning from nucleotide 1884 through nucleotide 1886 of SEQ ID NO- 30. The coding region encoding PfE8 595 is represented by nucleic acid molecule nfE8 17 85 having a coding strand with the nucleic acid sequence represented by SEQ ID NO:34 and a complementary strand with nucleic acid sequence SEQ ID The proposed mature protein, denoted herein as PfE8 5 7 o, contains about 570 amino acids which is represented herein as SEQ ID NO:55. The nucleic acid molecule encoding PfE8 5 7 o is denoted herein as nfE8 1 7 1 and has a coding strand having the nucleic acid sequence SEQ ID NO:33. The deduced amino acid sequence SEQ ID NO:31 suggests a protein having a molecular weight of about 68.6 kD and an estimated pi of about 6.1.
Comparison of amino acid sequence SEQ ID NO:31 the amino acid sequence of PfE85 95 with amino acid sequences reported in GenBank indicates that SEQ ID NO:31 showed the most homology, about 28% identity between SEQ ID NO:31 and estalpha-2 esterase of Culexpipiens quinquefasciatus.
Translation of SEQ ID NO:36 suggests that nucleic acid molecule nfE9 200 7 encodes a full-length arthropod esterase protein of about 528 amino acids, referred to herein as PfE9 5 28 represented by SEQ ID NO:37, assuming an open reading frame 10 having an initiation codon spanning from nucleotide 11 through nucleotide 13 of SEQ ID NO:36 and a stop codon spanning from nucleotide 1595 through nucleotide 1597 of S...SEQ ID NO:36. The coding region encoding PfE9 5 28, is represented by nucleic acid molecule nfE9 15 having a coding strand with the nucleic acid sequence represented by SEQ ID NO:51 and a complementary strand with nucleic acid sequence SEQ ID NO:52.
The deduced amino acid sequence SEQ ID NO:37 suggests a protein having a molecular weight of about 60 kD and an estimated pi of about 5.43.
Comparison of amino acid sequence SEQ ID NO:37 the amino acid sequence ofPfE9 5 2s) with amino acid sequences reported in GenBank indicates that SEQ ID NO:37 showed the most homology, about 37% identity between SEQ ID NO:37 20 and alpha esterase protein from Drosophila melanogaster.
Translation of SEQ ID NO:67 suggests that nucleic acid molecule nfE10l 987 encodes a full-length flea esterase protein of about 530 amino acids, referred to herein as PfE10530, having amino acid sequence SEQ ID NO:68, assuming an open reading frame in which the initiation codon spans from nucleotide 231 through nucleotide 233 of SEQ ID NO:67 and a stop codon spanning from nucleotide 1821 through nucleotide 1823 of SEQ ID NO:67. The complement of SEQ ID NO:67 is represented herein by SEQ ID NO:69. The coding region encoding PfE10 3 0 is represented by nucleic acid molecule nfEi01590, having a coding strand with the nucleic acid sequence represented by SEQ ID and a complementary strand with nucleic acid sequence SEQ ID NO:71. The amino acid sequence of P1E10 530 0(ie., SEQ ID NO:68) predicts that PfE IO,, 30 has an estimated molecular weight of about 59.5 kD and an estimated pI of about Comparison of amino acid sequence SEQ ID NO:68 the amino acid sequence of PfE 1053.) with amino acid sequences reported in GenBank indicates that 3 SEQ ID NO:68 showed the most homology, about 30% identity between SEQ ID NO:68 and Culexpipens esterase b I precurser protein (swissprot P 16854).
More preferred arthropod esterase proteins of the present invention include proteins comprising amino acid sequences that are at least about 40%, preferably at least about 45%, more preferably at least about 50%, even more preferably at least about 55%, even more preferably at least about 60%, even more preferably at least about *even more preferably at least about 80%, even more preferably at least about 90%, and even more preferably at least about 95%, identical to amino acid sequence SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: II, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO:25, SEQ ID NO:3 1, SEQ ID NO:3 7, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:68, SEQ ID NO:73 and/or SEQ ID NO:74.
.More preferred arthropod esterase proteins of the present invention include proteins encoded by a nucleic acid molecule comprising at least a portion of nfE 401 nfE2 3 64, nfB3 42 nfE4 524 nfE51 982 nfB5, 515 nfE5 2144 nfE51 65 0 nfE6 1488 nfE6 1 792 nfE-6 1650 nfE7 8 6 nE 1 7 m 8 nfE7 1 710 7 nfE7 650 nfE8 2801 nfES 17 8 5 nfE-8 1710 nfE9 2007 nfE9 1 584 nfB-9 1540 nfE10, 987 and/or nfB1 0 1590 or of allelic variants of such nucleic acid molecules.
More preferred is an esterase protein encoded by nfE 1401, nfE2 3 64, nfE3 4 2 1 nfE4 52 4 1 982 nfE5 1515 nfE5 21 44, nfE5 1650 nfE6 48 nfE-6 1 792 nfE6 165 0 nfE7 2836 nfE7 7 nfE7 171 0 nfE7 650 nfES 28 nfE-8 1 785 nfB8 1710 nfE9 200 7 nfE9 1584 TfF.-9 154 nfEl10 19 7 and/or nfE 10 1590 or by an allelic variant of such nucleic acid molecules. Particularly preferred arthropod esterase proteins are PfEl 103, PfE2 121 PfE31 03 PfE4 1 37 PfB5 505 PfE5 5 PfE6 550 PfB,6 530 PfE7 5 96 PfB7 570 PfE8 595 PfB8 570 PfE952 and PfE1 0 530 In one embodiment, a preferred esterase protein of the present invention is encoded by at least a portion of SEQ ID NOAl, SEQ ID NO:4, SEQ ID NO:7, SEQ ID -31- SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:51, SEQ ID NO:57, SEQ ID and/or SEQ ID NO:67, and, as such, has an amino acid sequence that includes at least a portion of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:58 and/or SEQ ID NO:68. Also preferred is a protein encoded by an allelic variant of a nucleic acid molecule comprising at least a portion of the above-listed nucleic acid sequences.
10 Particularly preferred esterase proteins of the present invention include SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:54,SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:68, SEQ ID NO:73 and/or SEQ ID o" 15 NO:74. (including, but not limited to, the proteins consisting of such sequences, fusion proteins and multivalent proteins) and proteins encoded by allelic variants of SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, SEQ ID S: 20 NO:51, SEQ ID NO:57, SEQ ID NO:60 and/or SEQ ID NO:67.
Another embodiment of the present invention is an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a C. felis esterase gene.
The identifying characteristics of such a gene are heretofore described. A nucleic acid molecule of the present invention can include an isolated natural arthropod esterase gene or a homolog thereof, the latter ofv hich is described in more detail below. A nucleic acid molecule of the present invention can include one or more regulatory regions, fulllength or partial coding regions, or combinations thereof. The minimal size of a nucleic acid molecule of the present invention is the minimal size that can form a stable hybrid with a C. felis esterase gene under stringent hybridization conditions.
In accordance with the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu that has been subject to human manipulation) and can include DNA, RNA, or derivatives of either DNA or RNA. As such, "isolated" does not reflect the extent to which the nucleic acid molecule has been purified. An isolated arthropod esterase nucleic acid molecule of the present invention can be isolated from its natural source or can be produced using recombinant DNA technology polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Isolated esterase nucleic acid molecules can include, for example, natural allelic variants and nucleic acid molecules modified by nucleotide S. 10 insertions, deletions, substitutions, and/or inversions in a manner such that the modifications do not substantially interfere with the nucleic acid molecule's ability to encode an esterase protein of the present invention or to form stable hybrids under stringent conditions with natural gene isolates.
An arthropod esterase nucleic acid molecule homolog can be produced using a 15 number of methods known to those skilled in the art (see, for example, Sambrook et al., ibid.). For example, nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis and recombinant
DNA
techniques site-directed mutagenesis, chemical treatment, restriction enzyme cleavage, ligation of nucleic acid fragments and/or PCR amplification), synthesis of 20 oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof. Nucleic acid molecule homologs can be selected by hybridization with a C.felis esterase gene or by screening for the function of a protein encoded by the nucleic acid molecule ability to elicit an immune response against at least one epitope of an arthropod esterase protein, hydrolyze a-napthyl acetate, hvdr jlyze the methyl ester group of juvenile hormone and/or bind to DFP).
An isolated nucleic acid molecule of the present invention can include a nucleic acid sequence that encodes at least one arthropod esterase protein of the present invention, examples of such proteins being disclosed herein. Although the phrase "nucleic acid molecule" primarily refers to the physical nucleic acid molecule and the phrase "nucleic acid sequence" primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding an arthropod esterase protein.
A preferred nucleic acid molecule of the present invention, when administered to an animal, is capable of protecting that animal from infestation by a hematophagous ectoparasite. As will be disclosed in more detail below, such a nucleic acid molecule can be, or can encode, an antisense RNA, a molecule capable of triple helix formation, a ribozyme, or other nucleic acid-based drug compound. In additional embodiments, a nucleic acid molecule of the present invention can encode a protective esterase protein 10 an esterase protein of the present invention), the nucleic acid molecule being delivered to the animal, for example, by direct injection as a naked nucleic acid) or in a vehicle such as a recombinant virus vaccine or a recombinant cell vaccine.
One embodiment of the present invention is an esterase nucleicacid molecule that hybridizes under stringent hybridization conditions with nucleic acid molecule nfE14 01 and preferably with a nucleic acid molecule having nucleic acid sequence SEQ ID NO:1 and/or SEQ ID NO:3.
Another embodiment of the present invention is an esterase nucleic acid molecule that hybridizes under stringent hybridization conditions with nucleic acid molecule nfE2 3 6 and preferably with a nucleic acid molecule having nucleic acid sequence SEQ ID NO:4 and/or SEQ ID NO:6.
Another embodiment of the present invention is an esterase nucleic acid molecule that hybridizes under stringent hybridization conditions with nucleic acid molecule nfE3 421 and preferably with a nucleic acid molecule having nucleic acid sequence SEQ ID NO:7 and/or SEQ ID NO:9.
Another embodiment of the present invention is an esterase nucleic acid molecule that hybridizes under stringent hybridization conditions with nucleic acid molecule nfE4 5 24 and preferably with a nucleic acid molecule having nucleic acid sequence SEQ ID NO:10 and/or SEQ ID NO:12.
Another embodiment of the present invention is an esterase nucleic acid molecule that hybridizes under stringent hybridization conditions with nucleic acid molecule nfE5 2 and preferably with a nucleic acid molecule having nucleic acid sequence SEQ ID NO:57 and/or SEQ ID NO:59.
Another embodiment of the present invention is an esterase nucleic acid molecule that hybridizes under stringent hybridization conditions with nucleic acid molecule nfE6 792 and preferably with a nucleic acid molecule having nucleic acid sequence SEQ ID NO: 18 and/or SEQ ID Another embodiment of the present invention is an esterase nucleic acid molecule that hybridizes under stringent hybridization conditions with nucleic acid molecule nfE7 2 836 and preferably with a nucleic acid molecule having nucleic acid sequence SEQ ID NO:24 and/or SEQ ID NO:26.
Another embodiment of the present invention is an esterase nucleic acid molecule that hybridizes under stringent hybridization conditions with nucleic acid molecule nfE8 28 0 and preferably with a nucleic acid molecule having nucleic acid sequence SEQ ID NO:30 and/or SEQ ID NO:32.
Another embodiment of the present invention is an esterase nucleic acid molecule that hybridizes under stringent hybridization conditions with nucleic acid molecule nfE9 2 007 and preferably with a nucleic acid molecule having nucleic acid sequence SEQ ID NO:36 and/or SEQ ID NO:38.
Another embodiment of the present invention is an esterase nucleic acid 20 molecule that hybridizes under stringent hybridization conditions with nucleic acid molecule nfEI0, 98 7 and preferably with a nucleic acid molecule having nucleic acid sequence SEQ ID NO:67 and/or SEQ ID NO:69.
Comparison of nucleic acid sequence SEQ ID NO:1 the nucleic acid sequence ofnfE1 40 1 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:1 showed no identifiable identity with any se'quence reported in GenBank.
Comparison of nucleic acid sequence SEQ ID NO:4 the coding strand of nucleic acid sequence ofnfE2 3 6) with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:4 showed the most homolog, about 43% identity, between SEQ ID NO:4 and a H. virescens juvenile hormone esterase gene.
Comparison of nucleic acid sequence SEQ ID NO:7 the coding strand of nucleic acid sequence of nfE3 4 21 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:7 showed the most homolog, about 53% identity, between SEQ ID NO:7 and a Torpedo marmorata acetylcholinesterase gene.
Comparison of nucleic acid sequence SEQ ID NO:10 the coding strand of nucleic acid sequence ofnfE4, 24 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO: 10 showed the most homolog, about 47% identity, between SEQ ID NO: 10 and an Anas platyrhyncos thioesterase B gene.
Comparison of nucleic acid sequence SEQ ID NO:57 the coding strand of 10 nucleic acid sequence ofnfE5 2 1 44 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:57 showed the most homolog, about 41% identity, S between SEQ ID NO:57 and a esterase mRNA from Myzus persicae.
Comparison of nucleic acid sequence SEQ ID NO:18 the coding strand of nucleic acid sequence ofnfE6 1792 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:18 showed the most homolog, about 41% identity, between SEQ ID NO:18 and a esterase gene from Myzus persicae.
Comparison of nucleic acid sequence SEQ ID NO:24 the coding strand of nucleic acid sequence of nfE7 28 36 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:24 showed the most homolog, about 48% identity, between SEQ ID NO:24 and an Anas platychyncos thioesterase B gene.
Comparison of nucleic acid sequence SEQ ID NO:30 the coding strand of nucleic acid sequence of nfE8 28 0 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:30 showed the most homolog, about 46% identity, between SEQ ID NO:30 and a Mus musculus carboxyl ester lipase gene.
Comparison ofnuc:eic acid sequence SEQ ID NO:36 the coding strand of nucleic acid sequence of nfE9207) with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:36 showed the most homolog, about 47% identity, between SEQ ID NO:36 and a hamster mRNA for CE precursor gene.
Comparison of nucleic acid sequence SEQ ID NO:67 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:67 showed the most -36homology, about 48% identity, between SEQ ID NO:67 and a Lucilia cuprina alpha esterase gene (genembl U56636) gene.
Preferred arthropod esterase nucleic acid molecules include nucleic acid molecules having a nucleic acid sequence that is at least about 55%, preferably at least about 60%, more preferably at least about 65%, more preferably at least about more preferably at least about 75%, more preferably at least about 80%, more preferably at least about 90%, and even more preferably at least about 95% identical to nucleic acid sequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ 10 ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID t. NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID 15 NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76 and/or a nucleic acid molecule encoding a protein comprising amino acid sequence SEQ ID NO:74.
Another preferred nucleic acid molecule of the present invention includes at least a portion of nucleic acid sequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID 20 NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76 and/or a nucleic acid molecule encoding a protein comprising amino acid sequence SEQ ID NO:74, that is capable of hybridizing to a C.felis esterase gene of the present invention, as well as allelic variants thereof. A more preferred nucleic acid molecule includes the nucleic acid sequence SEQ ID NO:1, SEQ ID NO:3, -37- SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76 and/or a nucleic acid molecule encoding a protein comprising amino acid sequence SEQ ID NO:74, as well as allelic variants thereof. Such nucleic acid molecules can include nucleotides in addition to those included in the SEQ ID NOs, such as, but not limited to, a full-length gene, a fulllength coding region, a nucleic acid molecule encoding a fusion protein, or a nucleic acid molecule encoding a multivalent protective compound. Particularly preferred nucleic acid molecules include nfE1 401 nfE236, nfE3 4 21 nfE4 5 2 4 nfE5 1 9 s 8 nfE5 15 nfE5 2 44, nfE5 1 6 5 nfE6, 1 48 nfE6, 792 nfE6 6 so, nfE7 28 36 nfE7 1 7 8 nfE7 1 71 nfE7 6 5 0 nfE8 2 801 nfE8 1 7 85 nfE8,7o, nfE9 20 07 nfE9,, 84 nfE9 1 5 0 o, nfE10, 8 7 and nfEl0 1 5 90 The present invention also includes a nucleic acid molecule encoding a protein aving at least a portion of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NQ:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:68, SEQ ID NO:73 and/or SEQ ID NO:74, including nucleic acid molecules that have been modified to accommodate codon usage properties of the cells in which such nucleic acid molecules are to be expressed.
Knowing the nucleic acid sequences of certain arthropod esterase nurle;, acid molecules of the present invention allows one skilled in the art to, for example, make copies of those nucleic acid molecules, obtain nucleic acid molecules including at least a portion of such nucleic acid molecules nucleic acid molecules including full-length genes, full-length coding regions, regulatory control sequences, truncated coding regions), and obtain esterase nucleic acid molecules from other arthropods.
-38- Such nucleic acid molecules can be obtained in a variety of ways including screening appropriate expression libraries with antibodies of the present invention; traditional cloning techniques using oligonucleotide probes of the present invention to screen appropriate libraries or DNA; and PCR amplification of appropriate libraries or DNA using oligonucleotide primers of the present invention. Preferred libraries to screen or from which to amplify nucleic acid molecule include flea pre-pupal, 3rd instar or adult cDNA libraries as well as genomic DNA libraries. Similarly, preferred DNA sources to screen or from which to amplify nucleic acid molecules include flea pre-pupal, 3rd instar or adult cDNA and genomic DNA. Techniques to clone and amplify genes are disclosed, for example, in Sambrook et al., ibid.
SThe present invention also includes nucleic acid molecules that are oligonucleotides capable of hybridizing, under stringent hybridization conditions, with complementary regions of other, preferably longer, nucleic acid molecules of the present invention such as those comprising arthropod esterase genes or other arthropod esterase nucleic acid molecules. Oligonucleotides of the present invention can be RNA, DNA, or derivatives of either. The minimum size of such oligonucleotides is the size required for "formation of a stable hybrid between an oligonucleotide and a complementary sequence on a nucleic acid molecule of the present invention. Minimal size characteristics are disclosed herein. The present invention includes oligonucleotides that can be used as, for example, probes to identify nucleic acid molecules, primers to produce nucleic acid molecules or therapeutic reagents to inhibit esterase protein production or activity as antisense-, triplex formation-, ribozyme- and/or RNA drug-based reagents). The present invention also includes the use of such oligonucleotides to protect animals from disease using one or more of such technologies. Appropriate oligonucleotide-containing therapeutic compositions can be administered to an animal using techniques known to those skilled in the art.
One embodiment of the present invention includes a recombinant vector, which includes at least one isolated nucleic acid molecule of the present invention, inserted into any vector capable of delivering the nucleic acid molecule into a host cell. Such a vector contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not -39naturally found adjacent to nucleic acid molecules of the present invention and that preferably are derived from a species other than the species from which the nucleic acid molecule(s) are derived. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid. Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulation of arthropod esterase nucleic acid molecules of the present invention.
One type of recombinant vector, referred to herein as a recombinant molecule, comprises a nucleic acid molecule of the present invention operatively linked to an expression vector. The phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell. As used herein, an expression vector is a o* DNA or RNA vector that is capable of transforming a host cell and of effecting ooo.
S..expression of a specified nucleic acid molecule. Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids. Expression vectors of the present invention include any vectors that function direct gene expression) in recombinant cells of the present invention, including in bacterial, fungal, endoparasite, insect, other animal, and plant cells. Preferred expression vectors of the present invention can direct gene expression in bacterial, yeast, insect and mammalian cells and more preferably in the cell types disclosed herein.
In particular, expression vectors of the present invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of nucleic acid molecules of the present invention.
In particular, r.ccmbinant molecules of the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art. Preferred transcription control sequences include those which function in bacterial, yeast, insect and mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rmB, bacteriophage lambda(such as lambda PL and lambda PR and fusions that include such promoters), bacteriophage T7, T7lac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis virus subgenomic promoters), antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, 10 other poxvirus, adenovirus, cytomegalovirus (such as intermediate early promoters), simian virus 40, retrovirus, actin, retroviral long terminal repeat, Rous sarcoma virus, heat shock, phosphate and nitrate transcription control sequences as well as other o sequences capable of controlling gene expression in prokaryotic or eukaryotic cells.
Additional suitable transcription control sequences include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters promoters inducible by interferons or interleukins). Transcription control sequences of the present invention can also include naturally occurring transcription control sequences naturally associated with arthropods, such as, C. felis.
Suitable and preferred nucleic acid molecules to include in recombinant vectors of the present invention are as.disclosed herein. Preferred nucleic acid molecules to .include in recombinant vectors, and particularly in recombinant molecules, include nfE1 401 nfE23,, nfE3 4 21 nfE4 524 nfE5, 9 8 2 nfE,5 nfE5 2 nfE5so 6 nfE6,, 4 8 nfE6 1 792 nfE6 165 o, nfE7 2 36 nfE7 1788 nfE7 1 7 1 0 nfE7 650 nfE82So, nfE8 17 s 8 nfE 171 nfE920 7 nfE9 5 84, nfE9 1 5 40 nfEl0 1 98 7 and/or nfEl0 1 59 0 Particularly preferred recombinant molecules of the present invention include pCro-nfE,6 1 8 8 pTrc-nfE7 6 50 pTrc-nfE 1 7 1 0 pTrc-nfE8 1 7 o, pTrc-nfE5 1 65 0 pTrc-nfE9 1 5 40 pFB-nfE6, 1 67 pVL-nfE7 1 8 02 pVL-fE8 1 7 9 and pVL-nfE9 1 60 o, the production of which are described in the Examples section.
Recombinant molecules of the present invention may also contain secretory signals signal segment nucleic acid sequences) to enable an expressed arthropod protein of the present invention to be secreted from the cell that produces the protein -41and/or contain fusion sequences which lead to the expression of nucleic acid molecules of the present invention as fusion proteins. Examples of suitable signal segments include any signal segment capable of directing the secretion of a protein of the present invention. Preferred signal segments include, but are not limited to, tissue plasminogen activator interferon, interleukin, growth hormone, histocompatibility and viral envelope glycoprotein signal segments, as well as natural signal sequences. Suitable fusion segments encoded by fusion segment nucleic acids are disclosed herein. In addition, a nucleic acid molecule of the present invention can be joined to a fusion segment that directs the encoded protein to the proteosome, such as a 10 ubiquitin fusion segment. Recombinant molecules may also include intervening and/or .untranslated sequences surrounding and/or within the nucleic acid sequences of nucleic acid molecules of the present invention.
Another embodiment of the present invention includes a recombinant cell comprising a host cell transformed with one or more recombinant molecules of the present invention. Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed recombinant) cell in such a manner that their ability to be expressed is retained. Preferred nucleic acid molecules with which to transform a cell include arthropod esterase nucleic acid molecules disclosed herein.
Particularly preferred nucleic acid molecules with which to transfo--m a cell includ,: nfEl 401 nfE2 3 64, nfE3 42 nfE4 524 nfE5 982 ,nf5 5 nfE5 21 nfE5 1650 nfE6 148 8, nfE6 1792 nfE6 1 5 nfE7E 2 36 nfE7 88 nE 171 0 nfE7 650 nfE8 280 1 nfE 178 nfES 1710 nfE9 2007 nfE9 1 5 4, nfE9, 5 4o, nfE 10197 and/or nfE 10590.
Suitable host cells to transform include any cell that can be transformed with a nucleic acid molecule of the present invention. Host cells can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule nucleic acid molecules encoding one or more proteins of the present invention and/or other proteins useful in the production of multivalent vaccines). Host cells of the present invention either can be endogenously naturally) capable of producing arthropod esterase proteins of the present invention or can be capable of producing such proteins after being transformed with at least one nucleic acid molecule of the present invention.
Host cells of the present invention can be any cell capable of producing at least one protein of the present invention, and include bacterial, fungal (including yeast), parasite, other insect, other animal and plant cells. Preferred host cells include bacterial, mycobacterial, yeast, insect and mammalian cells. More preferred host cells include Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells (normal dog kidney cell line for canine herpesvirus cultivation), CRFK cells (normal cat kidney cell line for feline herpesvirus cultivation), CV-1 cells (African monkey kidney cell line used, for example, to culture raccoon poxvirus), COS COS-7) cells, and Vero cells.
Particularly preferred host cells are Escherichia coli, including E. coli K-12 derivatives; Salmonella typhi; Salmonella typhimurium, including attenuated strains such as UK-1 x3987 and SR-11 x40 7 2 Spodopterafrugiperda; Trichoplusia ni; BHK cells; MDCK cells; CRFK cells; CV-1 cells; COS cells; Vero cells; and non-tumorigenic mouse myoblast G8 cells ATCC CRL 1246). Additional appropriate mammalian cell hosts include other kidney cell lines, other fibroblast cell lines human, murine or chicken embryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovary cells, mouse NIH/3T3 cells, LMTK 3 cells and/or HeLa cells. In one embodiment, the proteins may be expressed as heterologous proteins in myeloma cell lines employing immunoglobulin promoters.
A recombinant cell is preferably produced by transforming a host cell with one or more recombinant molecules, each comprising one or more nucleic acid molecules of the present invention operatively linked to an expression vector containing one or more transcription control sequences. The phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell.
A recombinant molecule of the present invention is a molecule that can include at least one of any nucleic acid molecule heretofore described operatively linked to at least one of any transcription control sequence capable of effectively regulating expression of the nucleic acid molecule(s) in the cell to be transformed, examples of which are disclosed herein. Particularly preferred recombinant molecules include pCro-nfE6 48 8, pTrc-nfE7so, pTrc-nfE7, 7 1 0 pTrc-nfE8 1 7 o, pTrc-nfE5 1 6 pTrc-nfE9 5 40 pFB-nfE667,,, pVL-nfE7 8 02 pVL-fE 79 ,2 and pVL-nfE9,o.
1 0 A recombinant cell of the present invention includes any cell transformed with at least one of any nucleic acid molecule of the present invention. Suitable and preferred nucleic acid molecules as well as suitable and preferred recombinant molecules with which to transform cells are disclosed herein. Particularly preferred recombinant cells include E. coli:pCro-nfE6 48 3, E. coli:pTrc-nfE7 1 7 1 o, E coli:pTrc-nfE7so, E. coli:pTrcnfE8 1 7 o, E. coli:pTrc-nfE5J 65 o, E. coli:pTrc-nfE9j 4 o, S. frtgiperda:pVL-nfE7 80 o2 S.
frugiperda:pVL-nfE8 79 2 S. frugiperda:pVL-nfE9600o and S. firgiperda:pFB-nfE6,67'.
Details regarding the production of these recombinant cells are disclosed herein.
Recombinant cells of the present invention can also be co-transformed with one or more recombinant molecules including arthropod esterase nucleic acid molecules encoding one or more proteins of the present invention and one or more other nucleic acid molecules encoding other protective compounds, as disclosed herein to produce multivalent vaccines).
Recombinant DNA technologies can be used to improve expression of transformed nucleic acid molecules by manipulating, for example, the number of copies of the nr.cleic acid m )lecules within a host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency ofpost-translational modifications. Recombinant techniques useful for increasing the expression of nucleic acid molecules of the present invention include, but are not limited to, operatively linking nucleic acid molecules to high-copy number plasmids, integration of the nucleic acid molecules into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals promoters, operators, enhancers), substitutions or modifications of translational control signals ribosome binding sites, Shine-Dalgarno sequences), modification of nucleic acid molecules of the present invention to correspond to the codon usage of the host cell, deletion of sequences that destabilize transcripts, and use of control signals that temporally separate recombinant cell growth from recombinant enzyme production during fermentation. The activity of an expressed recombinant protein of the present invention may be improved by fragmenting, modifying, or derivatizing nucleic acid molecules encoding such a protein.
Isolated esterase proteins of the present invention can be produced in a variety of ways, including production and recovery of natural proteins, production and recovery of ~recombinant proteins, and chemical synthesis of the proteins. In one embodiment, an isolated protein of the present invention is produced by culturing a cell capable of :i expressing the protein under conditions effective to produce the protein, and recovering 15 the protein. A preferred cell to culture is a recombinant cell of the present invention.
oooo• Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. An effective medium refers to any medium in which a cell is cultured to produce an arthropod esterase protein of the present invention. Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art. Examples of suitable conditions are included in the Examples section.
Depending on the vector and host system used for production, resultant proteins of the present invention may either remain within the recombinant cell; be secreted into the fermentation medium; be secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or be retained on the outer surface of a cell or viral membrane. The phrase "recovering the protein", as well as similar phrases, refers to collecting the whole fermentation medium containing the protein and need not imply additional steps of separation or purification. Proteins of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization. Proteins of the present invention are preferably retrieved in "substantially pure" form. As used herein, "substantially pure" refers to a purity that allows for the effective use of the protein as a therapeutic composition or diagnostic.
A
:.therapeutic composition for animals, for example, should exhibit no substantial toxicity and preferably should be capable of stimulating the production of antibodies in a treated animal.
The present invention also includes isolated removed from their natural milieu) antibodies that selectively bind to an arthropod esterase protein of the present 9 invention or a mimetope thereof anti-arthropod esterase antibodies). As used herein, the term "selectively binds to" an esterase protein refers to the ability of antibodies of the present invention to preferentially bind to specified proteins and mimetopes thereof of the present invention. Binding can be measured using a variety of methods standard in the art including enzyme immunoassays ELISA), immunoblot assays, etc.; see, for example, Sambrook et al., ibid. An anti-arthropod esterase antibody .999 preferably selectively binds to an arthropod esterase protein in such a way as to reduce the activity of that protein.
Isolated antibodies of the present invention can include antibodies in a bodily fluid (such as, but not limited to, serum), or antibodies tha' hr.ve been purified to varying degrees. Antibodies of the present invention can be polyclonal or monoclonal, functional equivalents such as antibody fragments and genetically-engineered antibodies, including single chain antibodies or chimeric antibodies that can bind to more than one epitope.
A preferred method to produce antibodies of the present invention includes (a) administering to an animal an effective amount of a protein, peptide or mimetope thereof of the present invention to produce the antibodies and recovering the antibodies. In another method, antibodies of the present invention are produced recombinantly using techniques as heretofore disclosed to produce arthropod esterase proteins of the present invention. Antibodies raised against defined proteins or mimetopes canbe advantageous because such antibodies are not substantially contaminated with antibodies against other substances that might otherwise cause interference in a diagnostic assay or side effects if used in a therapeutic composition.
Antibodies of the present invention have a variety of potential uses that are within the scope of the present invention. For example, such antibodies can be used (a) as therapeutic compounds to passively immunize an animal in order to protect the animal from arthropods susceptible to treatment by such antibodies and/or as tools to screen expression libraries and/or to recover desired proteins of the present invention from a mixture of proteins and other contaminants. Furthermore, antibodies of the present invention can be used to target cytotoxic agents to hematophagous ectoparasites o such as those discloses herein, in order to directly kill such hematophagous ectoparasites. Targeting can be accomplished by conjugating stably joining) such antibodies to the cytotoxic agents using techniques known to those skilled in the art.
Suitable cytotoxic agents are known to those skilled in the art.
One embodiment of the present invention is a therapeutic composition that, when administered to an animal in an effective manner, is capable of protecting that animal from infestation by hematophagous ectoparasite. Therapeutic compositions of the present invention include at least one of the following protective compounds: an isolated hematophagous arthropod este:ase protein (including a peptide); a mimetope of such a protein; an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a Ctenocephalidesfelis esterase gene; an isolated antibody that selectively binds to an hematophagous arthropod esterase protein; and inhibitors of hematophagous arthropod esterase activity (including esterase substrate analogs). As used herein, a protective compound refers to a compound that, when administered to an -47animal in an effective manner, is able to treat, ameliorate, and/or prevent disease caused by an arthropod of the present invention. Preferred arthropods to target are heretofore disclosed. Examples of proteins, nucleic acid molecules, antibodies and inhibitors of the present invention are disclosed herein.
A preferred therapeutic composition of the present invention includes at least one of the following protective compounds: an isolated hematophagous ectoparasite carboxylesterase protein (including a peptide); a mimetope of such a protein; an isolated hematophagous ectoparasite carboxylesterase nucleic acid molecule that hybridizes under stringent hybridization conditions with a Ctenocephalidesfelis carboxylesterase 10 gene; an isolated antibody that selectively binds to a hematophagous ectoparasite carboxylesterase protein; and an inhibitor ofcarboxylesterase activity identified by its ability to inhibit the activity of a flea carboxylesterase (including a substrate analog).
Suitable inhibitors of esterase activity are compounds that interact directly with an esterase protein's active site, thereby inhibiting that esterase's activity, usually by S 15 binding to or otherwise interacting with or otherwise modifying the esterase's active site.
Esterase inhibitors can also interact with other regions of the esterase protein to inhibit esterase activity, for example, by allosteric interaction. Inhibitors of esterases are Susually relatively small compounds and as such differ from anti-esterase antibodies.
Preferably, an esterase inhibitor of the present invention is identified by its ability to bind to, or otherwise interact with, a flea esterase protein, thereby inhibiting the activity of the flea esterase.
Esterase inhibitors can be used directly as compounds in compositions of the present invention to treat animals as long as such compounds are not harmful to host animals being treated. Esterase inhibitors can also be used to identify preferred types of arthropod esterases to target using compositions of the present invention, for example by affinity chromatography. Preferred esterase inhibitors of the present invention include, but are not limited to, flea esterase substrate analogs, and other molecules that bind to a flea esterase to an allosteric site) in such a manner that esterase activity of the flea esterase is inhibited; examples include, but are not limited to, juvenile hormone analogs and cholinesterase inhibitors as well as other neural transmission inhibitors. An esterase substrate analog refers to a compound that interacts with binds to, associates with, modifies) the active site of an esterase protein. A preferred esterase substrate analog inhibits esterase activity. Esterase substrate analogs can be of any inorganic or organic composition, and, as such, can be, but are not limited to, peptides, nucleic acids, and peptidomimetic compounds. Esterase substrate analogs can be, but need not be, structurally similar to an esterase's natural substrate as long as they can interact with the active site of that esterase protein. Esterase substrate analogs can be designed using computer-generated structures of esterase proteins of the present invention or computer structures of esterases' natural substrates. Substrate analogs can also be obtained by 10 generating random samples of molecules, such as oligonucleotides, peptides, peptidomimetic compounds, or other inorganic or organic molecules, and screening such samples by affinity chromatography techniques using the corresponding binding partner, a flea esterase). A preferred esterase substrate analog is a peptidomimetic compound a compound that is structurally and/or functionally similar to a natural substrate of an esterase of the present invention, particularly to the region of the substrate that interacts with the esterase active site, but that inhibits esterase activity upon interacting with the esterase active site).
Esterase peptides, mimetopes and substrate analogs, as well as other protective compounds, can be used directly as compounds in compositions of the present invention 20 to treat animals as long as such compounds are not harmful to the animals being treated.
The present invention also includes a therapeutic composition comprising at least one arthropod esterase-based compound of the present invention in combination with at least one additional compound protective against hematophagous ectoparasite infestation. Examples of such compounds are disclosed herein.
In one embodiment, a therapeutic composition of the present invention c"n '.e used to protect an animal from hematophagous ectoparasite infestation by administering such composition to a hematophagous ectoparasite, such as to a flea, in order to prevent infestation. Such administration could be oral, or by application to the environment spraying). Examples of such compositions include, but are not limited to, transgenic vectors capable of producing at least one therapeutic composition of the -49present invention. In another embodiment, a hematophagous ectoparasite, such as a flea, can ingest therapeutic compositions, or products thereof, present in the blood of a host animal that has been administered a therapeutic composition of the present invention.
Compositions of the present invention can be administered to any animal susceptible to hematophagous ectoparasite infestation a host animal), including warm-blooded animals. Preferred animals to treat include mammals and birds, with cats, dogs, humans, cattle, chinchillas, ferrets, goats, mice, minks, rabbits, raccoons, rats, sheep, squirrels, swine, chickens, ostriches, quail and turkeys as well as other furry animals, pets, zoo animals, work animals and/or food animals, being more preferred.
10 Particularly preferred animals to protect are cats and dogs.
In accordance with the present invention, a host animal an animal that is or is capable of being infested with a hematophagous ectoparasite) is treated by administering to the animal a therapeutic composition of the present invention in such a manner that the composition itself an esterase inhibitor, an esterase synthesis suppressor a compound that decreases the production of esterase in the hematophagous ectoparasite), an esterase mimetope, or an anti-esterase antibody) or a product generated by the animal in response to administration of the composition antibodies produced in response to administration of an arthropod esterase protein or *ooo nucleic acid molecule, or conversion of an inactive inhibitor "prodrug" to an active esterase inhibitor) ultimately enters the hematophagous ectoparasite. A host animal is 000. preferably treated in such a way that the compound or product thereof enters the blood stream of the animal. Hematophagous ectoparasites are then exposed to the composition or product when they feed from the animal. For example, flea esterase inhibitors administered to an animal are administered in such a way that the inhibitors enter the blood stream of the animal, where they can be taken ip by feeding fleas. In another embodiment, when a host animal is administered an arthropod esterase protein or nucleic acid molecule, the treated animal mounts an immune response resulting in the production of antibodies against the esterase anti-esterase antibodies) which circulate in the animal's blood stream and are taken up by hematophagous ectoparasites upon feeding. Blood taken up by hematophagous ectoparasites enters the hematophagous ectoparasites where compounds of the present invention, or products thereof, such as anti-esterase antibodies, esterase inhibitors, esterase mimetopes and/or esterase synthesis suppressors, interact with, and reduce esterase activity in the hematophagous ectoparasite.
The present invention also includes the ability to reduce larval hematophagous ectoparasite infestation in that when hematophagous ectoparasites feed from a host animal that has been administered a therapeutic composition of the present invention, at least a portion of compounds of the present invention, or products thereof, in the blood taken up by the hematophagous ectoparasite are excreted by the hematophagous ectoparasite in feces, which is subsequently ingested by hematophagous ectoparasite larvae. In particular, it is of note that flea larvae obtain most, if not all, of their nutrition from flea feces.
In accordance with the present invention, reducing esterase activity in a hematophagous ectoparasite can lead to a number of outcomes that reduce hematophagous ectoparasite burden on treated animals and their surrounding environments. Such outcomes include, but are not limited to, reducing the viability ofhematophagous ectoparasites that feed from the treated animal, reducing the fecundity of female hematophagous ectoparasites that feed from the treated animal, (c) reducing the reproductive capacity of male hematophagous ectoparasites that feed from the treated animal, reducing the viability of eggs laid by female hematophagous ectoparasites that feed from the treated animal, altering the blood feeding behavior of hematophagous ectoparasites that feed from the treated animal hematophagous ectoparasites take up less volume per feeding or feed less frequently), reducing the viability of hematophagous ectoparasite larvae, for example due to the feeding of larvae from feces of hemator hagous ectoparasites that feed from the treated animal and/or (g) altering the development of hematophagous ectoparasite larvae by decreasing feeding behavior, inhibiting growth, inhibiting slowing or blocking) molting, and/or otherwise inhibiting maturation to adults).
Therapeutic compositions of the present invention also include excipients in which protective compounds are formulated. An excipient can be any material that the animal to be treated can tolerate. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal or o-cresol, formalin and benzyl alcohol. Standard formulations can either be 10 liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient can comprise dextrose, human serum albumin, dog serum albumin, cat serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration.
In one embodiment of the present invention, a therapeutic composition can 15 include an adjuvant. Adjuvants are agents that are capable of enhancing the immune response of an animal to a specific antigen. Suitable adjuvants include, but are not limited to, cytokines, chemokines, and compounds that induce the production of cytokines and chemokines granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), colony stimulating factor (CSF), erythropoietin (EPO), interleukin 2 interleukin-3 interleukin 4 interleukin 5 interleukin 6 interleukin 7 interleukin 8 interleukin 10 interleukin 12 (IL-12), interferon gamma, interferon gamma inducing factor I (IGIF), transforming growth factor beta, RANTES (regulated upon activation, normal T cell expressed and presumably secreted), macrophage inflammatory proteins MIP-1 alpha and MIP-1 beta), and Leishmania elongation initiating factor (LEIF); bacterial components endotoxins, in particular superantigens, exotoxins and cell wall components); aluminum-based salts; calcium-based salts; silica; polynucleotides; toxoids; serum proteins, viral coat proteins; block copolymer adjuvants Hunter's TitermaxTM adjuvant (VaxcelTM, Inc. Norcross, GA), Ribi adjuvants (Ribi ImmunoChem Research, Inc., Hamilton, MT); and saponins and their derivatives Quil A (Superfos Biosector A/S, Denmark). Protein adjuvants of the present invention can be delivered in the form of the protein themselves or of nucleic acid molecules encoding such proteins using the methods described herein.
In one embodiment of the present invention, a therapeutic composition can include a carrier. Carriers include compounds that increase the half-life of a therapeutic composition in the treated animal. Suitable carriers include, but are not limited to, polymeric controlled release vehicles, biodegradable implants, liposomes, bacteria, viruses, other cells, oils, esters, and glycols.
10 One embodiment of the present invention is a controlled release formulation that is capable of slowly releasing a composition of the present invention into an animal. As used herein, a controlled release formulation comprises a composition of the present invention in a controlled release vehicle. Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdernal delivery systems. Other controlled release formulations of the present invention include liquids that, upon administration to an animal, form a solid or a gel in situ. Preferred controlled release formulations are biodegradable bioerodible).
A preferred controlled release formulation of the present invention is capable of releasing a composition of the present invention into the blood of an animal at a constant rate sufficient to attain therapeutic dose levels of the composition to protect an animal from hematophagous ectoparasite infestation. The therapeutic composition is preferably released over a period of time ranging from about 1 to about 12 months. A preferred controlled release formulation of the present invention is capable of effr .g a treatment preferably for at least about 1 month, more preferably for at least about 3 months, even more preferably for at least about 6 months, even more preferably for at least about 9 months, and even more preferably for at least about 12 months.
Acceptable protocols to administer therapeutic compositions of the present invention in an effective manner include individual dose size, number of doses, frequency of dose administration, and mode of administration. Determination of such protocols can be accomplished by those skilled in the art. A suitable single dose is a dose that is capable of protecting an animal from disease when administered one or more times over a suitable time period. For example, a preferred single dose of a protein, mimetope or antibody therapeutic composition is from about 1 microgram (Ag) to about milligrams (mg) of the therapeutic composition per kilogram body weight of the animal. Booster vaccinations can be administered from about 2 weeks to several years after the original administration. Booster administrations preferably are administered when the immune response of the animal becomes insufficient to protect the animal from disease. A preferred administration schedule is one in which from about 10 Ag to S. about 1 mg of the therapeutic composition per kg body weight of the animal is administered from about one to about two times over a time period of from about 2 oo o weeks to about 12 months. Modes of administration can include, but are not limited to, subcutaneous, intradermal, intravenous, intranasal, oral, transdermal, intraocular and intramuscular routes.
S00According to one embodiment, a nucleic acid molecule of the present invention can be administered to an animal in a fashion to enable expression of that nucleic acid molecule into a protective protein or protective RNA antisense RNA, ribozyme, *triple helix forms or RNA drug) in the animal. Nucleic acid molecules can be delivered to an animal in a variety of methods including, but not limited to, administering a naked not packaged in a viral coat or cellular membrane) nucleic acid vaccine as naked DNA or RNA molecules, such as is taught, for example in Wolff et al., 1990, Science 247, 1465-1468) or administering a nucleic acid molecule packaged as a recombinant virus vaccine or as a recombinant cell vaccine the nucleic acid molecule is delivered by a viral or cellular ehicle).
A naked nucleic acid vaccine of the present invention includes a nucleic acid molecule of the present invention and preferably includes a recombinant molecule of the present invention that preferably is replication, or otherwise amplification, competent.
A
naked nucleic acid vaccine of the present invention can comprise one or more nucleic acid molecules of the present invention in the form of, for example, a bicistronic -54recombinant molecule having, for example one or more internal ribosome entry sites.
Preferred naked nucleic acid vaccines include at least a portion of a viral genome a viral vector). Preferred viral vectors include those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, and retroviruses, with those based on alphaviruses (such as Sindbis or Semliki virus), species-specific herpesviruses and species-specific poxviruses being particularly preferred. Any suitable transcription control sequence can be used, including those disclosed as suitable for protein production. Particularly preferred transcription control sequence include cytomegalovirus intermediate early (preferably in conjunction with Intron-A), Rous Sarcoma Virus long terminal repeat, and tissue- 10 specific transcription control sequences, as well as transcription control sequences endogenous to viral vectors if viral vectors are used. The incorporation of "strong" poly(A) sequences are also preferred.
Naked nucleic acid vaccines of the present invention can be administered in a variety of ways, with intramuscular, subcutaneous, intradermal, transdermal, intranasal 15 and oral routes of administration being preferred. A preferred single dose of a naked nucleic acid vaccines ranges from about 1 nanogram (ng) to about 100 depending on the route of administration and/or method of delivery, as can be determined by those skilled in the art. Suitable delivery methods include, for example, by injection, as drops, aerosolized and/or topically. Naked DNA of the present invention can be contained in 20 an aqueous excipient phosphate buffered saline) alone or a carrier lipid-based vehicles).
A recombinant virus vaccine of the present invention includes a recombinant molecule of the present invention that is packaged in a viral coat and that can be expressed in an animal after administration. Preferably, the recombinant molecule is packagin'-d ;ficient and/or encodes an attenuated virus. A number of recombinant viruses can be used, including, but not limited to, those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, and retroviruses. Preferred recombinant virus vaccines are those based on alphaviruses (such as Sindbis virus), raccoon poxviruses, species-specific herpesviruses and species-specific poxviruses. An example of methods to produce and use alphavirus recombinant virus vaccines is disclosed in PCT Publication No. WO 94/17813, by Xiong et al., published August 18, 1994, which is incorporated by reference herein in its entirety.
When administered to an animal, a recombinant virus vaccine of the present invention infects cells within the immunized animal and directs the production of a protective protein or RNA nucleic acid molecule that is capable of protecting the animal from hematophagous ectoparasite infestation. For example, a recombinant virus vaccine comprising an arthropod CE nucleic acid molecule of the present invention is administered according to a protocol that results in the animal producing a sufficient immune response to protect itself from hematophagous ectoparasite infestation. A 10 preferred single dose of a recombinant virus vaccine of the present invention is from about 1 x 10 4 to about 1 x 10 7 virus plaque forming units (pfu) per kilogram body weight of the animal. Administration protocols are similar to those described herein for protein- *based vaccines, with subcutaneous, intramuscular, intranasal and oral administration routes being preferred.
15 A recombinant cell vaccine of the present invention includes recombinant cells of the present invention that express at least one protein of the present invention.
Preferred recombinant cells for this embodiment include Salmonella, E. coli, Listeria, S. Mycobacterium, S. frugiperda, yeast, (including Saccharomyces cerevisiae), BHK, CV- 1, myoblast G8, COS COS-7), Vero, MDCK and CRFK recombinant cells.
20 Recombinant cell vaccines of the present invention can be administered in a variety of ways but have the advantage that they can be administered orally, preferably at doses ranging from about 10' to about 10" 2 .cells per kilogram body weight. Administration protocols are similar to those described herein for protein-based vaccines. Recombinant cell vaccines can comprise whole cells, cells stripped of cell walls or cell lysates.
The efficacy of a therapeutic composition of the present invention to protect an animal from hematophagous ectoparasite infestation can be tested in a variety of ways including, but not limited to, detection of anti-arthropod esterase antibodies (using, for example, proteins or mimetopes of the present invention), detection of cellular immunity within the treated animal, or challenge of the treated animal with hematophagous ectoparasites to determine whether, for example, the feeding, fecundity or viability of -56hematophagous ectoparasites feeding from the treated animal is disrupted. Challenge studies can include attachment of chambers containing hematophagous ectoparasites onto the skin of the treated animal. In one embodiment, therapeutic compositions can be tested in animal models such as mice. Such techniques are known to those skilled in the art.
One preferred embodiment of the present invention is the use of arthropod protective compounds, such as proteins, mimetopes, nucleic acid molecules, antibodies and inhibitory compounds of the present invention, to protect an animal from hematophagous ectoparasite, and particularly flea, infestation. Preferred protective 10 compounds of the present invention include, but are not limited to, C.felis esterase nucleic acid molecules, C. felis esterase proteins and mimetopes thereof, anti-C felis ,esterase antibodies, and inhibitors of C felis esterase activity. More preferred protective S" compounds of the present invention include, but are not limited to, CE or JHE formulations of the present invention, C. felis CE nucleic acid molecules, C felis CE 15 proteins and mimetopes thereof, anti-flea CE antibodies, anti-flea JHE antibodies, inhibitors of C. felis CE activity and inhibitors of flea JHE activity. Additional protection may be obtained by administering additional protective compounds, including other proteins, mimetopes, nucleic acid molecules, antibodies and inhibitory compounds, as disclosed herein.
20 One therapeutic composition of the present invention includes an inhibitor of arthropod esterase activity, a compound capable of substantially interfering with the function of an arthropod esterase susceptible to inhibition by an inhibitor of arthropod esterase activity. An inhibitor of esterase activity can be identified using arthropod esterase proteins of the present invention. One embodiment of the present invention is a method to identify a compound capable of inhibiting esterase r ity of an rrthropod.
Such a method includes the steps of contacting combining, mixing) an isolated flea esterase protein, preferably a C. felis esterase protein of the present invention, with a putative inhibitory compound under conditions in which, in the absence of the compound, the protein has esterase activity, and determining if the putative inhibitory compound inhibits the esterase activity. Putative inhibitory compounds to screen include small organic molecules, antibodies (including mimetopes thereof) and substrate analogs. Methods to determine esterase activity are known to those skilled in the art; see, for example, the Examples section of the present application.
The present invention also includes a test kit to identify a compound capable of inhibiting esterase activity of an arthropod. Such a test kit includes an isolated flea esterase protein, preferably a C. felis esterase protein, having esterase activity and a means for determining the extent of inhibition of esterase activity in the presence of effected by) a putative inhibitory compound. Such compounds are also screened to identify those that are substantially not toxic in host animals.
10 Esterase inhibitors isolated by such a method, and/or test kit, can be used to inhibit any esterase that is susceptible to such an inhibitor. Preferred esterase proteins to inhibit are those produced by arthropods. A particularly preferred esterase inhibitor of the present invention is capable of protecting an animal from hematophagous ectoparasite infestation. Effective amounts and dosing regimens can be determined 15 using techniques known to those skilled in the art.
The following examples are provided for the purposes of illustration and are not intended to limit the scope of the present invention.
EXAMPLES
It is to be noted that the Examples include a number of molecular biology, 20 microbiology, immunology and biochemistry techniques considered to be known to those skilled in the art. Disclosure of such techniques can be found, for example, in Sambrook et al., ibid., Borovsky, Arch Insect Biochem. and Phys., 7:187-210, 1988, and related references.
Example 1 This example describes :ling ofproteases and esterases with radiolabeled diisopropylfluorophosphate.
Tissue samples were isolated from unfed or bovine blood-fed 1st instar Ctenocephalidesfelis flea larvae; bovine blood-fed or cat blood-fed 3rd instar Ctenocephalidesfelis flea larvae; bovine blood-fed or cat blood-fed Ctenocephalides felis prepupal flea larvae; bovine blood-fed or cat blood-fed adult Ctenocephalidesfelis -58flea midgut tissue, and whole unfed, bovine blood-fed or cat blood-fed adult Ctenocephalidesfelis fleas. The 1st instar, 3rd instar, prepupal and adult midgut tissues were then homogenized by freeze-fracture and sonicated in a Tris buffer comprising mM Tris, pH 8.0 and 100 mM CaCI 2 The whole adult flea sample was then homogenized by freeze-fracture and ground with a microtube mortar and pestle. The extracts were centrifuged at about 14,000 x g for 20 minutes (min.) and the soluble material recovered. The soluble material was then diluted to a final concentration of about 1 to about 1.2 tissue equivalents per microliter (pl) of Tris buffer. Each sample was labeled with [1 ,3-H]-diisopropylfluorophosphate 3 H-DFP) (available from 10 DuPont-NEN, Wilmington, DE) using the method generally described in Borovsky, ibid.
About 20 tissue equivalents of each tissue sample were mixed with about 1 iCi of H- DFP and incubated for about 18 hours at 4oC. Proteins contained in each sample were then resolved using a 14% Tris-glycine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (available from Novex, San Diego, CA) under reducing 15 conditions. The gel was soaked in Entensify (available from DuPont-NEN) according to manufacturers instructions, and exposed to X-ray film (available from Kodak X-Omat AR, Rochester, NY) for about 3 days at -70 0
C.
Analysis of the resulting autoradiogram (shown in Fig. 1) indicated that tissue samples from 3rd instar, prepupal larvae-and whole adult flea contained proteins that labeled with DFP, having a molecular weight (MW) of about 60 kilodalton No.
.proteins of this MW were labeled in tissue samples from unfed or fed 1st instar larvae and adult midgut. The results indicated preferred tissue distribution and stage-specific expression ofDFP-labeled serine esterases in fleas.
Example 2 This ex'ample describes the identification of general CE activity in flea tissue extracts.
Tissue samples and soluble extracts were prepared as described above in Example 1, except not labelled, from unfed (UF) and bovine blood-fed 1st instar flea larvae, bovine blood-fed 3rd instar flea larvae, bovine blood-fed prepupal flea larvae, unfed whole adult fleas, cat blood-fed adult (ACF) whole fleas, cat blood-fed adult fleas -59that have had their heads and midguts removed (referred to herein as fed adult partial fleas), unfed adult flea midguts and cat blood-fed adult flea midguts. About 5 tissue equivalents of each tissue were assayed for general CE activity using the following method. Tissue samples of about 5 gl were added to separate wells of flat-bottomed microtiter plate (available from Becton Dickinson, Lincoln Park, NJ). A control well was prepared by adding about 5 pi of Tris buffer to an empty well of the plate. About gl of 25 mM Tris-HCI (pH 8.0) was then added to each sample to increase the volume in each well to about 100 gl. About 100 gl of 0.25 mM a-napthyl acetate (available from Sigma, St. Louis, MO) dissolved in 25 mM Tris-HC1 (pH 8.0) was then 10 added to each well. The plate was then incubated for about 15 min. at 37°C. Following the incubation, about 40 ul of 0.3% Fast Blue salt BN (tetrazotized o-dianisidine; available from Sigma) dissolved in 3.3% SDS in water was added to each well.
The microtiter plate was then analyzed using a Cambridge Technology, Inc.
(Watertown, PA) model 7500 Microplate Reader set to 590 nm. The absorbance value 15 for the control sample was subtracted from absorbance values of experimental samples, such that the background value was zero.
The results shown in Fig. 2 indicated that general CE activity was detected in all tissue samples. The level of activity varied, with unfed and fed 1st instar larvae, unfed adult flea midguts, and fed adult flea midguts having relatively lower activity than in the -20 other tissues. Thus, the results indicated preferred tissue distribution and stage-specific expression of general CE activity in fleas.
Example 3 This example describes the determination of general CE activity using isoelectric focusing (IEF)-PAGE and non-reducing SDS-PAGE.
A. Non-reducing SDS-PAGE.
Soluble extracts from unfed and bovine blood-fed 1 st instar flea larvae, bovine blood-fed 3rd instar flea larvae, bovine blood-fed prepupal flea larvae, bovine blood-fed adult (ABF) whole fleas and cat blood-fed adult whole fleas were prepared using the method described in Example 1. Each soluble extract sample was combined with SDS sample buffer (available from Novex) and proteins in the samples were resolved by gel electrophoresis using 14% Tris-glycine SDS electrophoresis gels (available from Novex). The gels were run at room temperature for about 1 hour at 200 volts. After electrophoresis, the gels were soaked for about for 30 minutes in 50 mM Tris, pH containing 2.5% Triton X-100 to renature the proteins. The gels were then soaked in mM Tris, pH 8.0, for about 5 minutes and then stained for about 5 min. in 50 milliliters (ml) of 25 mM Tris, pH 8.0, containing 50 mg Fast blue salt BN and 10 mg a-napthyl acetate (dissolved in 1 ml acetone). Once protein was detected on the stained gels, the gels were rinsed with water and photographed.
B. IEF-PAGE.
10 Soluble extracts from unfed and bovine blood-fed 1st instar flea larvae, bovine blood-fed 3rd instar flea larvae, bovine blood-fed prepupal flea larvae, unfed and cat blood-fed whole fleas, cat blood-fed adult partial fleas and cat blood-fed adult midguts were prepared as described above in Section A. The extracts were each combined with IEF sample buffer pH 3-7 (available from Novex) and loaded onto pH 3-7 IEF electrophoresis gels (available from Novex). The gels were electrophoresed at room temperature first for about 1 hour at about 100 volts, then for about 1 hour at about 200 volts, and then for about 30 min. at about 500 volts. Following electrophoresis, the gels were soaked in 25 mM Tris buffer, pH 8.0, for about 5 min. and then stained for about 1- 5 min. in 50 ml of 25 mM Tris buffer, pH 8.0, containing 50 mg Fast blue salt BN and 20 10 mg a-napthyl acetate (dissolved in 1 ml acetone). Once protein was detected on the stained gels, the gels were rinsed with water and photographed.
C. Results.
The results from gel electrophoresis experiments described above in Sections A and B are shown in Figs. 3 and 4. The results indicated that certain flea tissues contain proteins having MW's of from about 60 to about 70 k7' ad native pi values of from about 4.7 to about 5.2 that have CE activity. In particular, CE activity was identified in prepupal larvae and fed adult flea extracts resolved by non-reduced SDS-PAGE. No CE activity was identified in unfed and fed 1st instar larvae or fed 3rd instar larvae extracts (see Fig. When extracts were resolved by native IEF-PAGE, CE activity was identified in fed 3rd instar larvae, prepupal larvae, unfed and fed whole adult flea, and fed adult partial flea extracts (see Fig. 4, lanes No CE activity was identified in unfed or fed 1st instar larvae, or in fed adult flea midgut extracts (see Fig. 4, lanes 1, 2, and 8).
Example 4 This example describes the purification of CE protein from prepupal flea larvae.
About 15,000 bovine blood-fed prepupal flea larvae were collected and the larvae were homogenized in TBS by sonication in 50 ml Oak Ridge centrifuge tubes (available from Nalgene Co., Rochester, NY) by sonicating 4 times 20 seconds each at a setting of 5 of a model W-380 Sonicator (available from Heat Systems-Ultrasonics, Inc.). The sonicates were clarified by centrifugation at 18,000 RPM for 30 minutes to produce an extract. Soluble protein in the extract was removed by aspiration and diluted to a volume of about 20 ml in TBS (equivalent to about 1 larva per pl TBS). The extract .was then added to a column containing about 5 ml ofp-aminobenzamidine linked to agarose beads (available from Sigma, St. Louis, MO) and incubated overnight at 4oC.
15 The column was then washed with about 30 ml TBS to remove unbound protein. The collected unbound protein was then concentrated to a volume of about 20 ml using a Macrosep 10 centrifugal protein concentrator (Filtron Technology Corp., Northborough, MA) and filtered sequentially through a 1 pm syringe filter and then through a 0.2 gm syringe filter to clarify the sample for chromatography.
S 20 Aliquots of about 0.5 ml were loaded onto a 20 ml Superdex 200 HR gel filtration column (available from Pharmacia, Piscataway, NJ) equilibrated in TBS, operated on a BioLogic liquid chromatography system (available from BioRad, Burlingame, CA). About 1 ml fractions were then collected. Repetitive runs were performed until about 30 ml of each fraction was collected. The fractions were analyzed for CE activity using thb a.say desc ribed above in Example 2. In preparation for cation exchange chromatography, fractions having CE activity (V,=16-18 ml) were combined and dialyzed against about 2 liters of 20 mM MES buffer morpholino)ethanesulfonic acid), pH 6.0, containing 10 mM NaCI, for about 1.5 hours, and then against about 1 liter of the same buffer overnight at 4oC. Prior to loading onto the cation exchange chromatography column, the sample was again filtered through a 0.2 Am syringe filter to remove precipitated proteins. The sample was then applied to a Bio-Scale S2 cation exchange column (available from BioRad) at a rate of about ml/min. The column was washed with MES buffer until all unbound protein was removed. Protein bound to the column was then eluted with a linear gradient from mM to 1 M NaCI in 20 mM MES buffer, pH 6. Fractions were assayed for CE activity using the assay described above in Example 2. The results indicated that CE activity was not retained on the cation exchange column using the above conditions, and all of the activity was found in the flow-through fractions.
Fractions containing CE activity were pooled and adjusted to pH 7 using 0.5 M 10 Tris, pH 8.0, in preparation for anion exchange chromatography. The pooled fractions were then loaded onto a 4.5 mm x 50 mm Poros 10 HQ anion exchange chromatography column (available from PerSeptive Biosystems, Cambridge, MA) equilibrated in mM Tris buffer, pH 6.8. The column was washed with the loading buffer, and bound proteins were eluted with a linear gradient of 0 to 1 M NaCI in 25 mM Tris buffer, pH 15 6.8. Fractions were tested for CE activity using the assay described above in Example 2.
The results indicated that CE activity was eluted at about 170 mM NaCI. Fractions containing CE activity were pooled and diafiltered into TBS.
Example This example describes the determination of N-terminal amino acid sequences of 20 carboxylesterases isolated from prepupal flea larvae.
A. Anion exchange chromatography fractions.
Anion exchange chromatography fractions described above in Example 4 that contained proteins having CE activity were pooled, diafiltered into TBS buffer and concentrated 3-fold in a Speed-Vac Concentrator (available from Savant Instruments, Holbrook, NY). Proteins in the concentrated samples were then resolved on a reducing, SDS-PAGE Tris-glycine gel (available from Novex) for 1 hour at about 200 V.
The proteins on the gel were then blotted onto a polyvinylidene difluoride
(PVDF)
membrane (available from Novex) for about 70 min in 10 mM CAPS buffer (3- [cyclohexylamino]-l-propanesulfonic acid; available from Sigma), pH 11, with 0.5 mM dithiothreitol (DTT). The membrane was then stained for 1 minute in 0.1% Coomassie -63- Blue R-250 dissolved in 40% methanol and 1% acetic acid. The membrane was destained in 50% methanol for about 10 minutes, rinsed with MilliQ water and air dried.
Three stained protein bands were identified having apparent molecular weights of about 64 kD, 65 kD, and 66 kD, respectively. The portion of the membrane containing each band was excised separately. Protein contained in each membrane segment was subjected to N-terminal amino acid sequencing using a 473A Protein Sequencer (available from Applied Biosystems, Foster City, CA) and using standard techniques.
The results indicated that the N-terminal amino acid sequence of the putative 64 kD protein was DPPTVTLPQGEL (denoted SEQ ID NO:39); the N-terminal amino acid sequence of the putative 65 kD protein was DPPTVTLPQGELVGKATNEnxk (denoted SEQ ID NO:40); and the N-terminal amino acid sequence of the putative 66 kD protein was DppTVTLPQGEL (denoted SEQ ID NO:41), in which the lower case letters designate uncertainties and designates an undetermined residue.
B. Proteins Resolved by Native IEF-PAGE.
Proteins isolated by anion exchange chromatography as described above in Section A were further resolved by native IEF-PAGE. Proteins were loaded onto a pH 3-10 IEF gel (available from Novex) and separated in Novex's IEF buffers according to Novex's standard procedure (60 min at 100 V; then 60 min at 200 V; and then 30 min at 500 Following electrophoresis, part of the gel was stained for CE activity using the method described above in Example 2. The remaining portion of the gel was blotted i onto PVDF membrane by reversing the orientation of the gel and membrane so that positively charged proteins migrated to the membrane, electrophoresing the protein for min at 10 V, using 0.7% acetic acid as the transfer buffer. The membrane was stained as described above in Section A. After the membrane was dried, stained protein bands on the membrane were compared to bands on the gel tested for CE ac'iv;Cy to identify corresponding bands. Protein bands on the membrane corresponding to proteins having CE activity were excised and submitted to N-terminal sequencing as described in Section A.
N-terminal amino acid sequence was obtained for protein contained in two bands having pi values of about pi 4.8 and about pI 4.9. N-terminal amino acid sequence of the pi 4.8 band was DPPTVTLPQGELVGKALSNen (denoted SEQ ID NO:42) and Nterminal amino acid sequence of the pi 4.9 band was DPPTVTLP (denoted SEQ ID NO:43). A comparison of the N-terminal amino acid sequences identified here and described in Section A indicates closely related proteins having a consensus sequence of DPPTVTLPQGELVGKALTNEnGk (denoted SEQ ID NO:44).
The amino acid sequences of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:44 are substantially contained within SEQ ID NO:5, SEQ ID NO:19 and SEQ ID NO:53, which are described below in Example 11.
Example 6 This example describes partial purification of CE from 3rd instar flea larvae.
Using the extract preparation methods described in Example 1 without labelling, extracts were prepared from about 50,000 bovine blood-fed 3rd instar flea larvae. The extract was then further purified over a p-aminobenzamidine linked agarose bead 15 column using the method also described in Example 1. Collected unbound protein was concentrated to about 70 ml using a 200 ml stirred cell fitted with a YM-10 membrane (available from Amicon, Beverly, MA). Seven ml (about 5,000 3rd instar flea larval equivalents) of the concentrated extract was used for the remainder of the purification scheme described in Example 4. Resulting fractions from the anion exchange chromatography column were tested for CE activity using the assay described above in Example 2.
The results indicated that CE activity was eluted in two overlapping peaks at about 120 mM and about 210 mM NaCI.
Example 7 This example describes the identificao.. of JHE activity in different flea tissues.
Tissue samples were prepared as described above in Example 1 from unfed anid bovine blood-fed 1st instar flea larvae, bovine blood-fed 3rd instar flea larvae, bovine blood-fed prepupal flea larvae, unfed and cat blood-fed whole adult fleas, cat blood-fed adult partial fleas and cat blood-fed adult flea midguts. About 5 tissue equivalents of each tissue was assayed for JHE activity as follows.
Unlabeled juvenile hormone (JH; available from ICN Biomedicals, Inc., Aurora, OH) was diluted in hexane to concentration of about 0.025 M. Labeled hormone available from Dupont-NEN) was diluted in hexane to concentration of about 80,000 cpm/il. A JH substrate mixture was prepared by mixing about 20 pl of unlabeled JH with about 80 l1 of'H-JH (about 5 iCi) in a 4 ml screw cap vial. The substrate mixture was then covered with nitrogen "blanketed") and the solvent contained in the mixture was evaporated by heating the mixture at 35oC. When just dry, about 1 ml of absolute anhydrous ethanol (final concentration 5 x 10 4 M, or 6400 cpm/pl) was added to the vial. The substrate mixture was then stored at About 5 equivalents of each tissue (about 5 pl of protein) was added into the bottom of a small glass autosampler vial. About 95 gl of Tris-buffered saline (TBS) was added to each vial to bring the final volume in each vial to about 100 pl. Two control samples were also prepared by adding 100 gl TBS to two separate vials. About 1 ul of the substrate mixture described above was added to all of the vials including the control samples. The final JH concentration in each vial was about 5 x 10.6 M. The vials were o then capped and spun in a microfuge to bring all of the liquid to the bottom of each vial.
The vials were then transferred to a heat block and incubated at 35oC for about S. minutes. Following the incubation, enzyme activity was stopped by adding about 50 pl .of methanol buffer (methanol:water:concentrated ammonium hydroxide at a 10:9:1 ratio, respectively) to each vial and removing the vials from the heat block.
To measure labeled juvenile hormone acid, about 250 gl isooctane was added to each vial. Each vial was vortexed for about 15 seconds or until an emulsion formed.
Each vial was then centrifuged in a microfuge for about 1 minute to separate aqueous and organic phases. About 75 pl of the aqueous layer was removed from each vial and added to about 2 nl Eco-lame scintillation fluid (available from ICN). The amount of 3 H-juvenile hormone acid contained in each vial was determined using a Beckman LS- 1801 liquid scintillation counter (available from Beckman, Fullerton, CA).
The results shown in Fig.5 indicated that all flea tissues tested contain active JHE. Referring to Example 2, the level of CE activity differed from JHE activity in various tissue samples. The combined JHE and CE data indicated the differential expression of these two enzymatic activities during the development of a flea.
Example 8 This example describes the purification of JHE protein from cat blood-fed adult midguts.
About 23,000 cat blood-fed adult midguts were collected and prepared using the method described in Example 1. The extract was then added in 4 aliquots to columns containing about 3 to about 5 ml ofp-aminobenzamidine linked agarose beads (available from Sigma), equilibrated in 50 mM Tris (pH 100 mM CaCI 2 400 mM NaCI, and 10 incubated overnight at 4°C. The columns were then washed with about 15 to about 125 ml of the equilibration Tris buffer to remove unbound protein. The collected unbound protein was pooled and then concentrated to a volume of about 5 ml using an Ultrafree- 20 10 kD centrifugal concentrator (available from Millipore, Bedford, MA) and filtered sequentially through a 0.2 gm centrifugal ultrafiltration membrane (available from Lida, Kenosha, WI) to clarify the sample for chromatography.
S
Aliquots of about 0.5 ml were loaded onto a Superdex 200 HR gel filtration column using the method described in Example 4. Repeated runs were performed until about 10 ml of each fraction was collected. The fractions were analyzed for JHE activity using the assay described in Example 7. In preparation for anion exchange chromatography, fractions having JHE activity (V,=17-18 ml) were combined and dialyzed overnight against about 1 L of 20 mM Tris buffer, pH 8.0, containing 10 mM NaC1. The sample was then loaded onto a Poros 10 HQ anion exchange column using the method described in Example 4. Resulting fractions were tested for JHE activity as described in Example 7.
The results indicated that midgut JHE activity was eluted from the anion exchange column in a single peak at about 120 mM NaC1.
Example 9 This example describes partial purification ofJHE from prepupal flea larvae and 3rd instar larvae.
A. JHE Purification from Prepupal Tissue.
Using the extract preparation methods described in Example 1, gel filtration fractions were obtained using a Superdex 200 HR gel filtration column (available from Pharmacia) using the method described in Example 4, from about 15,000 bovine bloodfed prepupal flea larvae. The fractions were analyzed for JHE activity using the assay described above in Example 7. Those fractions containing protein having JHE activity (V,=16-18 ml) were combined and dialyzed using the method described in Example 8.
The fractions were then further purified by passing the fractions over a Bio-Scale S2 cation exchange column (available from BioRad) at a rate of about 0.5 ml/min. The column was washed with MES until all unbound protein was eluted. Bound protein was then eluted with a linear gradient of 20 mM MES buffer, pH 6.0, containing 10 mM NaCI to 1 M NaCI. Resulting fractions were assayed for JHE activity using the method described in Example 7. The results indicated that proteins having JHE activity using prepupal tissue eluted from the column in about 200 to 300 mM NaC1.
The fractions containing JHE activity were combined and the pH adjusted to pH o* 7 using 0.5 M Tris buffer (pH The fractions were then dialyzed twice against about 1 liter of 10 mM phosphate buffer (pH 7.2) containing 10 mM NaCl at 4°C. The resulting dialyzed fractions were then loaded onto a Bio-Scale CHT2-I Hydroxyapatite Column (available from BioRad) at a rate of about 0.5 ml/min. Unbound protein was washed from the column using the dialysis buffer. Bound protein was then eluted with a linear gradient of from 10 mM phosphate buffer, pH 7.2, containing 10 mM NaCI to M phosphate buffer pH 6.5 containing 10 mM NaCl. One ml fractions were collected and each tested for JHE activity by the method-described in Example 7.
The results indicated that JHE eluted in 2 overlapping peaks at about 100 mM and 150 mM phosphate. These two JHE activities were designated PF JHE I and PP JHE II, and were kept separate for the remainder of the purification. Both JhE samples., were dialyzed overnight against 20 mM Tris buffer (pH 8.0) containing 10 mM NaCl.
The two samples were then loaded, separately, onto a 4.5 mm x 50 mm Poros 10 HQ anion exchange chromatography column (available from PerSeptive Biosystems) equilibrated with 20 mM Tris buffer, pH 8.0, containing 10 mM NaCI. Unbound proteins were washed from the column using the same buffer. Bound proteins were eluted with a linear gradient of from 10 mM to 1 M NaCI in 20 mM Tris buffer, pH Resulting fractions were tested for JHE activity using the method described in Example 7.
The results indicated that in both samples, JHE activity was eluted from the column in a single peak at about 100 mM NaCI.
B. JHE Purification from 3 r d Instar Tissue Using the procedure described above in Section A, proteins having JHE activity were obtained using about 5,000 bovine blood-fed 3 rd instar flea larvae. Following purification by cation exchange, proteins having JHE activity using 3rd instar tissue were found to elute in 2 peaks. The first peak having JHE activity was not retained on the colunm and also exhibited CE activity (referred to herein as CE/JHE fractions). The second peak having JHE activity eluted from the column in about 100-200 mM NaCI and did not contain CE activity.
The CE/JHE fractions were pooled and adjusted to about pH 7 using 0.5 M Tris, pH 8.0. The fractions were then loaded onto a4.5 mm x 50 mm Poros 10 HQ anion exchange chromatography column (available from PerSeptive Biosystems) and the column was equilibrated in 25 mM Tris buffer, pH 6.8. The column was washed with the same buffer and bound proteins were eluted with a linear gradient of 0 to I M NaCI in 25 mM Tris buffer, pH 6.8. Fractions were then tested for JHE activity using the method described in Example 7. JHE activity was eluted in two overlapping peaks at about 120 mM and 210 mM NaCI. The fraction containing JHE activity also contained CE activity when tested using the method described in Example 2.
Fractions from the cation exchange column containing only JHE activity were combined, diluted in 20 mM Tris buffe pH 8.0 containing 10 mM NaC1, and concentrated to about 5 ml. The fractions were purified on a Poros 10 HQ anion exchange chromatography column as described immediately above. Fractions were then tested for JHE activity using the method described in Example 7. The JHE activity was elutedin a single peak at about 120 mM. The peak contained no detectable CE activity.
Example -69- This example describes the purification of JHE protein from unfed adult midguts.
About 16,000 unfed adult midguts were collected in 20 mM Tris buffer (pH 7.7), containing 130 mM NaCI, 1 mM sodium EDTA, 1 mM Pefabloc@ (available from Boehringer Mannheim, Indianapolis, IN), 1 microgram/ml (jg/ml) leupeptin and 1 ug/ml pepstatin. The midguts were homogenized by freeze-fracture and sonication, and then centrifuged at about 14,000 x g for 20 min. The soluble material from the centrifugation step was recovered. The soluble material was then concentrated to about 1 ml using an Ultrafree-20 10 kD centrifugal concentrator (available from Millipore) and filtered sequentially through a 0.2 pm centrifugal ultrafiltration membrane to clarify the sample for chromatography. Aliquots of about 0.5 ml were loaded onto a Superdex 200 HR gel filtration column using the method described in Example 4. Repeated column runs were performed until about 2 ml of each fraction was collected. The fractions were analyzed for JHE activity using the assay described in Example 7. In preparation for cation exchange chromatography, fractions having JHE activity (V,=15-17 ml) were S 15 combined and dialyzed overnight against about 1 L of 20 mM MES buffer, pH containing 10 mM NaCI. The sample was then applied to a Bio-Scale S2 cation exchange column using the method described in Example 4. Fractions ofeluate were assayed for JHE activity using the method described in Example 7.
The results indicate that JHE is present in unfed midguts in two forms, one that is not retained on the cation exchange column and one that is bound to the column under low salt conditions at about 100 mM NaC1. The form that was not retained under low salt conditions was shown to have general CE activity using the methods described in Example 2.
Example 11 This example describes the identification of certain esterase nucleic acid molecules of the present invention.
Several flea esterase nucleic acid molecules, representing one or more partial flea esterase genes, were PCR amplified from a flea mixed instar cDNA library or a flea prepupal cDNA library. The flea mixed instar cDNA library was produced using unfed 1st instar, bovine blood-fed 1st instar, bovine blood-fed 2 nd instar and bovine blood-fed 3 r instar flea larvae (this combination of tissues is referred to herein as mixed instar larval tissues for purposes of this example). The flea prepupal cDNA library was produced using prepupal flea larvae. For each library, total RNA was extracted from mixed instar or prepupal tissue, respectfully, using an acid-guanidinium-phenolchloroform method similar to that described by Chomczynski et al., 1987, Anal.
Biochem. 162, p. 156-159. Approximately 5,164 mixed instar larvae or 3,653 prepupal larvae were used in each RNA preparation. Poly A+ selected RNA was separated from each total RNA preparation by oligo-dT cellulose chromatography using Poly(A)Quick® mRNA isolation kits (available from Stratagene Cloning Systems, La Jolla, CA), according to the method recommended by the manufacturer.
0 A mixed instar cDNA expression library and a prepupal cDNA expression library S were constructed in lambda Uni-ZAPTMXR vector (available from Stratagene Cloning Systems) using Stratagene's ZAP-cDNA Synthesis Kit@ protocol. About 6.34 g of mixed instar poly A+ RNA were used to produce the mixed instar library and about 6.72 tg of prepupal poly A+ RNA were used to produce the prepupal library.
The resultant mixed instar library was amplified to a titer of about 2.17 x 1010 pfu/ml with about 97% recombinants. The resultant prepupal library was amplified to a titer of about 3.5 x 1010 pfu/ml with about 97% recombinants.
A pair of primers was used to amplify DNA from the cDNA libraries. A sense vector primer T-3X (corresponding to the vector in which nucleic acid molecules of the o present invention had been ligated), having the nucleic acid sequence AATTAACCCT CACTAAAGGG (available from Gibco BRL, Gaithersburg, MD; denoted SEQ ID was used in combination with a degenerate primer, the design of which was based on a highly conserved esterase amino acid sequence (disclosed in Hanzlik.et al., J.
Biol. Chem. 264:12419-12423, 1989; I Y/H G G G F/L) located in a region downstream from the mature amino terminus in a number of known esterases. The degenerate primer, referred to herein as FCEF, is an anti-sense primer having the nucleic acid sequence ARDCCDCCDC CRTRDAT R indicating an A or G; and D indicating an A, G or T; denoted SEQ ID NO:46). The resultant PCR products from the mixed instar cDNA library, obtained using standard PCR conditions Sambrook et al., ibid.), were about 550 nucleotides. The resultant PCR products from the prepupal cDNA library were from about 500 nucleotides to about 860 nucleotides.
A. PCR Products.
PCR products were gel purified and cloned into the TA Vectorr (available from InVitrogen Corp., San Diego, CA). Approximately 8 clones were identified from the prepupal library and 6 clones were identified from the mixed instar library. These nucleic acid molecules were subjected to nucleic acid sequencing using the Sanger dideoxy chain termination method, as described in Sambrook et al., ibid.
1. Flea esterase clone 1 isolated from the mixed instar cDNA library was determined to comprise nucleic acid molecule nfE14, 0 the nucleic acid sequence of the coding strand which is denoted herein as SEQ ID NO:1. Translation of SEQ ID NO:1 suggests that nucleic acid molecule nfE1401 encodes a non-full-length flea esterase protein of about 103 amino acids, referred to herein as PfEl 03, having amino acid sequence SEQ ID NO:2, assuming an initiation codon spanning from nucleotide 92 through nucleotide 94 of SEQ ID NO: 1. The complement of SEQ ID NO:1 is represented herein by SEQ ID NO:3. Comparison of amino acid sequence SEQ ID NO:2 the amino acid sequence ofPfEl 103) with amino acid sequences reported in GenBank indicates that SEQ ID NO:2, showed the most homology, about 33% identity, between SEQ ID NO:2 and alpha esterase protein from Drosophila melanogaster.
2. Flea esterase clone 2 isolated from the mixed instar cDNA library was determined to comprise nucleic acid molecule nfE2 3 the nucleic acid sequence of the coding strand which is denoted herein as SEQ ID NO:4. Translation of SEQ ID NO:4 suggests that nucleic acid molecule nfE2 3 encodes a non-full-length flea esterase protein of about 121 amino acids, referred to herein as PfE.
12 having amino acid sequence SEQ ID NO:5, assuming the first codon spans from nucleotide 2 through nucleotide 4 of SEQ ID NO:4. The complement of SEQ ID NO:4 is represented herein by SEQ ID NO:6. Comparison of nucleic acid sequence SEQ ID NO:4 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:4 showed the most homology, about 43% identity, between SEQ ID NO:4 and a H. virescens JHE gene.
-72- Comparison of amino acid sequence SEQ ID NO:5 the amino acid sequence of PfE2 1 2 i) with amino acid sequences reported in GenBank indicates that SEQ ID showed the most homology, about 38% identity, between SEQ ID NO:5 and alpha esterase protein from Drosophila melanogaster.
3. Flea esterase clone 3 isolated from the prepupal cDNA library was determined to comprise nucleic acid molecule nfE3 4 2 1 the nucleic acid sequence of the coding strand which is denoted herein as SEQ ID NO:7. Translation of SEQ ID NO:7 suggests that nucleic acid molecule nfE3 42 encodes a non-full-length flea esterase protein of about 103 amino acids, referred to herein as PfE310, having amino acid sequence SEQ ID NO:8, assuming an initiation codon spanning from nucleotide 113 S. through nucleotide 115 of SEQ ID NO:7. The complement of SEQ ID NO:7 is represented herein by SEQ ID NO:9. Comparison of nucleic acid sequence SEQ ID NO:7 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:7 showed the most homology, about 53% identity, between SEQ ID NO:7 and a Torpedo marmorata acetylcholinesterase gene. Comparison of amino acid sequence SEQ ID NO:8 the amino acid sequence of PfE3 1 03 with amino acid sequences reported in GenBank indicates that SEQ ID NO:8, showed the most homology, i.e., about 39% identity, between SEQ ID NO:5 and alpha esterase protein from Drosophila melanogaster.
4. Flea esterase clone 4 isolated from the prepupal cDNA library was determined to comprise nucleic acid molecule nfE4 5 24 the nucleic acid sequence of the coding strand which is denoted herein as SEQ ID NO:10. Translation of SEQ ID suggests that nucleic acid molecule nfE4 524 encodes a non-full-length flea esterase protein of about 137 amino acids, referred to herein as PfE4t37, having amino acid sequence SEQ ID NO:11, ?ssl.ming at initiation codon spanning from nucleotide 113 through nucleotide 115 of SEQ ID NO: 10. The complement of SEQ ID NO:10 is represented herein by SEQ ID NO:12. Comparison of nucleic acid sequence SEQ ID with nucleic acid sequences reported in GenBank indicates that SEQ ID showed the most homology, about 47% identity, between SEQ ID NO:10 and an Anas platyrhyncos thioesterase B gene. Comparison of amino acid sequence SEQ ID -73- NO: 11 the amino acid sequence of PfE4 3 7 with amino acid sequences reported in GenBank indicates that SEQ ID NO: 11, showed the most homology, about identity, between SEQ ID NO:11 and Leptinotarsa decemlineata acetylcholinesterase.
B. cDNA Clones.
Certain amplified PCR fragments were used as probes to identify full-length flea esterase genes in the prepupal cDNA library.
1. Nucleic acid molecule nfE236 was labeled with 3 P and used as a probe to screen the mixed instar cDNA library described in Section A, using standard hybridization techniques. Two clones were isolated. A first clone included about a 2 300-nucleotide insert, referred to herein as nfE5 23 00 Nucleic acid sequence was
S'.
obtained using standard techniques from nfE5 2 00, to yield a flea esterase nucleic acid molecule named nfE51 98 2 having a nucleic acid sequence of the coding strand which is .i denoted herein as SEQ ID NO:13. Translation of SEQ ID NO:13 suggests that nucleic acid molecule nfE5 1 9 82 encodes a non-full-length flea esterase protein of about 505 amino acids, referred to herein as PfE5 5 o 5 having amino acid sequence SEQ ID NO:14, *assuming the first codon spans from nucleotide 1 through nucleotide 3 of SEQ ID NO:13 and the stop codon spans from nucleotide 1518 through nucleotide 1520 of SEQ ID NO:13. The complement of SEQ ID NO:13 is represented herein by SEQ ID S The amino acid sequence of PfE5so, SEQ ID NO:14) predicts that PfE5 50 s has an estimated molecular weight of about 56.8 kD and an estimated pi of about 5.5. The nucleic acid molecule representing the coding region for PfE5s 05 is referred to herein as nfE5sis,; the nucleic acid sequences of the coding strand and the complementary strand are represented by SEQ ID NO:16 and SEQ ID NO:17, respectively.
The nucleic acid sequence ofnfE5, 98 2 was used to design primers to use in -ombination with a vector primer to PCR amplify the 5' terminal fragment of the remainder of the flea esterase coding region from the flea mixed instar cDNA library. A pair of primers was used to amplify DNA from the cDNA library. A sense vector primer T3-X (corresponding to the vector in which nucleic acid molecules of the present invention had been ligated), having the nucleic acid sequence 5' AATTAACCCT CACTAAAGGG 3' (denoted SEQ ID NO:45), was used in combination with an anti- -74sense primer M6/M265', having the nucleic acid sequence 5' GTGCGTACAC GTTTACTACC 3' (denoted SEQ ID NO:56). The resultant PCR product from the mixed instar cDNA library, obtained using standard PCR conditions Sambrook et al., ibid.), were about 354 nucleotides.
The PCR product was subjected to DNA sequencing analysis, and a composite sequence representing a full-length flea esterase coding region was deduced. The nucleic acid sequence of the composite nucleic acid molecule, referred to herein as 2144 is denoted herein as SEQ ID NO:57. Translation of SEQ ID NO:57 suggests that nucleic acid molecule nfE5 21 44 encodes a full-length flea esterase protein of about 550 amino acids, referred to herein as PfE5 55 s, having amino acid sequence SEQ ID NO:58, assuming an open reading frame in which the initiation codon spans from nucleotide 30 through nucleotide 32 of SEQ ID NO:57 and the stop codon spans from nucleotide 1680 through nucleotide 1682 of SEQ ID NO:57. The complement of SEQ ID NO:57 is represented herein by SEQ ID NO:59. The coding region encoding PfE5 55 0 is represented by the nucleic acid molecule nfE5 5 0 having a coding strand with the S* nucleic acid sequence represented by SEQ ID NO:60 and a complementary strand with nucleic acid sequence SEQ ID NO:61. The amino acid sequence ofPfE5 5 50
SEQ
ID NO:58) predicts that PfE5 5 5 ohas an estimated molecular weight of about 61.8 kD and an estimated pI of about Comparison of nucleic acid sequence SEQ ID NO:57 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:57 showed the most homology, about 41% identity, between SEQ ID NO:57 and a M persicae esterase FE4 mRNA sequence. Comparison of amino acid sequence SEQ ID NO:58 the amino acid sequence ofPfE555 0 with amino acid sequences reported in GenBank indicates that SEQ ID NO:58 showed the most homology, about 36% identity between SEQ ID NO:58 and Drosophila melanogster alpha esterase protein.
A second clone included about a 1900 nucleotide insert, referred to herein as nfE6,,90. Nucleic acid sequence was obtained using standard techniques from nfE6 1 9 00 to yield a flea esterase nucleic acid molecule named nfE6 1792 having a nucleic acid sequence of the coding strand which is denoted herein as SEQ ID NO:18. Translation of SEQ ID NO:18 suggests that nucleic acid molecule nfE6 1 7 92 encodes a full-length flea esterase protein of about 550 amino acids, referred to herein as PfE6 55 0 having amino acid sequence SEQ ID NO:19, assuming an open reading frame in which the initiation codon spans from nucleotide 49 through nucleotide 51 of SEQ ID NO:18 and a stop codon spanning from nucleotide 1699 through nucleotide 1701 of SEQ ID NO:18. The complement of SEQ ID NO:18 is represented herein by SEQ ID NO:20. The coding region encoding PfE6ss 0 is represented by nucleic acid molecule nfE6 6 50 having a coding strand with the nucleic acid sequence represented by SEQ ID NO:21 and a complementary strand with nucleic acid sequence SEQ ID NO:22. The proposed mature protein, denoted herein as PfE6s 30 contains about 530 amino acids which is represented herein as SEQ ID NO:53. The nucleic acid molecule encoding PfE6 5 0 is denoted herein 1 as nfE6 5 9 0 and has a coding strand having the nucleic acid sequence SEQ ID NO:23.
The amino acid sequence of PfE6 0 SEQ ID NO:19) predicts that PfE6 5 0 has an .estimated molecular weight of about 61.8 kD and an estimated pi of about Comparison of nucleic acid sequence SEQ ID NO:18 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:18 showed the most a homology, about 41% identity, between SEQ ID NO:18 and a Myzius pericae S..esterase gene. Comparison of amino acid sequence SEQ ID NO:19 the amino acid sequence ofPfE65 5 o) with amino acid sequences reported in GenBank indicates that SEQ ID NO:19 showed the most homology, about 28% identity between SEQ ID NO:19 and Drosophila melanogaster alpha esterase protein.
2. Nucleic acid molecule nfE4 5 24 was labeled with 3 "P and used as a probe to screen the prepupal cDNA library described in Example 11, using standard hybridization techniques Sambrook et al., ibid.). Two clones were isolated. A first clone included about a 3000 nucleotide insert, referred to herein as nfE7 30 00 Nucleic acid sequence was obtained using standard techniques from nfE7 30 0 0 to yield a flea esterase nucleic acid molecule named nfE7 836 having a nucleic acid sequence of the coding strand which is denoted herein as SEQ ID NO:24. Translation of SEQ ID NO:24 suggests that nucleic acid molecule nfE7 283 6 encodes a full-length flea esterase protein of about 596 amino acids, referred to herein as PfE7 5 96 having amino acid sequence SEQ ID NO:25, assuming an open reading frame in which the initiation codon spans from nucleotide 99 through nucleotide 101 of SEQ ID NO:24 and a stop codon spanning from nucleotide 1887 through nucleotide 1889 of SEQ ID NO:25. The complement of SEQ ID NO:24 is represented herein by SEQ ID NO:26. The coding region encoding PfE7,,,, is represented by nucleic acid molecule nfE7, 78 8 having a coding strand with the nucleic acid sequence represented by SEQ ID NO:28 and a complementary strand with nucleic acid sequence SEQ ID NO:29. The proposed mature protein, denoted herein as PfE7 5 s 0 contains about 570 amino acids which is represented herein as SEQ ID NO:54. The nucleic acid molecule encoding PfE7 5 70 is denoted herein as nfE7 1 7 0 and has a coding strand having the nucleic acid sequence SEQ ID NO:27. The amino acid sequence of PfE7s 96 SEQ ID NO:25) predicts that PfE7 59 has an estimated molecular weight of about 68.7 kD and an estimated pi of about 6.1.
Comparison of nucleic acid sequence SEQ ID NO:24 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:24 showed the most homology, about 48% identity, between SEQ ID NO:24 and an Anas platychyncos thioesterase B gene. Comparison of amino acid sequence SEQ ID NO:25 the amino acid sequence ofPfE7 596 with amino acid sequences reported in GenBank indicates that SEQ ID NO:25 showed the most homology, about 27% identity between SEQ ID NO:25 and Drosophila melanogaster alpha esterase protein.
A second clone included about a 3000 nucleotide insert, referred to herein as nfE8 3 000 Nucleic acid sequence was obtained using standard techniques from nfE8 30 oo, to yield a flea esterase nucleic acid molecule named nfE8 2 go having a nucleic acid sequence of the coding strand which is denoted herein as SEQ ID NO:30. Translation of SEQ ID NO:30 suggests that nucleic acid molecule nfE8 28 s 0 encodes a full-length flea esterase protein of about 595 amino acids, referred to herein as PfE895, h -ir g amino acid sequence SEQ ID NO:31, assuming an open reading frame in which the initiation codon spans from nucleotide 99 through nucleotide 101 of SEQ ID NO:30 and a stop codon spanning from nucleotide 1884 through nucleotide 1886 of SEQ ID NO:30. The complement of SEQ ID NO:30 is represented herein by SEQ ID NO:32. The coding region encoding PfES8 59 is represented by nucleic acid molecule nfE8 7 8 s, having a coding strand with the nucleic acid sequence represented by SEQ ID NO:34 and a complementary strand with nucleic acid sequence SEQ ID NO:35. The proposed mature protein, denoted herein as PfE8 57 0 contains about 570 amino acids which is represented herein as SEQ ID NO:55. The nucleic acid molecule encoding PfE8 57 0 is denoted herein as nfE8 1 71 0 and has a coding strand having the nucleic acid sequence SEQ ID NO:33.
The amino acid sequence ofPfE8 5 9 5 SEQ ID NO:3 1) predicts that PfE8 5 95 has an estimated molecular weight of about 68.6 kD and an estimated pi of about 6.1.
Comparison of nucleic acid sequence SEQ ID NO:30 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:30 showed the most homology, about 46% identity, between SEQ ID NO:30 and a Mus musculus carboxyl ester lipase gene. Comparison of amino acid sequence SEQ ID NO:31 the amino acid sequence ofPfE8 5 95 with amino acid sequences reported in GenBank Sindicates that SEQ ID NO:31 showed the most homology, about 28% identity between SEQ ID NO:31 and estalpha-2 esterase of Culex pipiens quinquefasciatus.
3. Nucleic acid molecule nfE3 42 1 was labeled with 3 P and used as a probe to screen the prepupal cDNA library using standard hybridization techniques Sambrook et al., ibid.). Two clones were isolated. One clone included about a 1900 nucleotide insert, referred to herein as nfE9 9 0 oo. Nucleic acid sequence was obtained using standard techniques from nfE9, 0 oo, to yield a flea esterase nucleic acid molecule named nfE9 2 00 7 having nucleic acid sequence of the coding strand which is denoted herein as SEQ ID NO:36. Translation of SEQ ID NO:36 suggests that nucleic acid molecule nfE9 2 0 07 encodes a full-length flea esterase protein of about 528 amino acids, referred to herein as PfE9 528 having amino acid sequence SEQ ID.NO:37, assuming an open reading frame in which the initiation codon spans from nucleotide 11 through nucleotide 13 of SEQ ID NO:36 and a stop co ion spanning from nucleotide 1595 through nucleotide 1597 of SEQ ID NO:36. The complement of SEQ ID NO:36 is represented herein by SEQ ID NO:38. The coding region encoding PfE9 52 8 is represented by nucleic acid molecule nfE9 58 4 having a coding strand with the nucleic acid sequence represented by SEQ ID NO:51 and a complementary strand with nucleic acid sequence SEQ ID NO:52. The amino acid sequence ofPfE9, 5 2 SEQ ID NO:37) predicts that PfE9 5 2 s has an estimated molecular weight of about 60 kD and an estimated pi of about 5.43.
Comparison of nucleic acid sequence SEQ ID NO:36 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:36 showed the most homology, about 47% identity, between SEQ ID NO:36 and a hamster mRNA for carboxylesterase precursor gene. Comparison of amino acid sequence SEQ ID NO:37 the amino acid sequence of PfE9 5 28 with amino acid sequences reported in GenBank indicates that SEQ ID NO:37 showed the most homology, about 37% identity between SEQ ID NO:37 and alpha esterase protein from Drosophila nlelanogaster.
o* As is the case for any of the nucleic acid molecules described in this example, variations between sequences may be due to a number of factors, such as but not limited to, sequencing errors or allelic variation.
:i 4. Nucleic acid molecule nfEl14! was labeled with 32 P and used as a probe to screen the mixed instar cDNA library using standard hybridization techniques Sambrook et al., ibid.). A clone was isolated that included about a 2000 nucleotide insert, referred to herein as nfE1020o. Nucleic acid sequence was obtained using standard techniques from nfE10 2000 to yield a flea esterase nucleic acid molecule named nfE10 9 having nucleic acid sequence of the coding strand which is denoted herein as SEQ ID NO:67. Translation of SEQ ID NO:67 suggests that nucleic acid molecule nfE10,g 8 7 encodes a full-length flea esterase protein of about 530 amino acids, referred to herein as PfE10 5 3 o, having amino acid sequence SEQ ID NO:68, assuming an open reading frame in which the initiation codon spans from nucleotide 231 through nucleotide 233 of SEQ ID NO:67 and a stop codon spanning from nucleotide 1821 through nucO -o:de 1823 of SEQ ID NO:67. The complement of SEQ ID NO:67 is represented herein by SEQ ID NO:69. The coding region encoding PfE10530, is represented by nucleic acid molecule nfE 1015t, having a coding strand with the nucleic acid sequence represented by SEQ ID NO:70 and a complementary strand with nucleic acid sequence SEQ ID NO:71. The amino acid sequence ofPfE10 53 0 SEQ ID -79- NO:68) predicts that PfE10 30 has an estimated molecular weight of about 59.5 kD and an estimated pi of about Comparison of nucleic acid sequence SEQ ID NO:67 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:67 showed the most homology, about 48% identity, between SEQ ID NO:67 and a Lucilia cuprina alpha esterase gene (genembl U56636) gene. Comparison of amino acid sequence SEQ ID NO:68 the amino acid sequence ofPfE10530) with amino acid sequences reported in GenBank indicates that SEQ ID NO:68 showed the most homology, about identity between SEQ ID NO:68 and Culexpipens esterase bl precurser protein (swissprot P16854).
As is the case for any of the nucleic acid molecules described in this example, variations between sequences may be due to a number of factors, such as but not limited to, sequencing errors or allelic variation.
Example 12 This Example demonstrates the production of esterase proteins of the present invention in E. coli cells.
A. Flea esterase protein PHIS-PfE7 57 0 and flea esterase protein PHIS-PfE 5 7 o were produced in the following manner. A pair of primers was used to amplify DNA from flea esterase nucleic acid molecule nfE7 2 836 or nfE8 2801 produced as described in Example 11. A sense primer containing an X7ho site (shown in bold) having the nucleic acid sequence 5' TGTGCTCGAG ATGGGATAAC CTAGATCAGC ATTTGTGC 3' (denoted SEQ ID NO:47), was used in combination with an anti-sense primer containing a KpnI site (shown in bold) having the nucleic acid sequence 5' TTAAGGTACC -TCATCTAATA CTTCCTTCAT TACAG 3' (denoted SEQ ID NO:48). A PCR product was derived from nfE7 2836 and is referred to herein as nfE7 1 7 10 having nucleic acid sequence SEQ ID NO:27. The PCR product was digested with XhoI and KpnI restriction endonucleases, gel purified and subcloned into expression vector pTrcHisB (available from InVitrogen). The resultant recombinant molecule, referred to herein as pTrc-nfE7 1 7 1 0 was transformed into E. coli HB 101 competent cells (available from Gibco BRL) to form recombinant cell E. coli:pTrc-nfE7 1 7 1 o.
The PCR product derived from nfE8 2 8 o, using the primers is referred to herein as nfE8 7 1 o, having nucleic acid sequence SEQ ID NO:33. PCR product nfE8 1 7 t, was digested with Xiho and KpnI restriction endonucleases, gel purified and subcloned into expression vector pTrcHisB. The resultant recombinant molecule, referred to herein as pTrc-nfE8, 7 was transformed into E. coli HB 101 competent cells to form recombinant cell E. coli:pTrc-nfE8 7 1 o.
The recombinant cells were cultured in enriched bacterial growth medium containing 0.1 mg/ml ampicillin and 0.1% glucose at about 32 0 C. When the cells reached an OD,oo of about 0.4-0.5, expression of recombinant protein was induced by the addition of 0.5 mM isopropyl-B-D-thiogalactoside (IPTG), and the cells were cultured for about 2 hours at about 32 0 C. Immunoblot analysis of recombinant cell E. coli:pTrcnfE7ano and E. coli:pTrnfEfE8 7 o lysates using a T7 tag monoclonal antibody (available from Novagen, Inc., Madison, WI) directed against the fusion portion of the recombinant PHIS-PfE7 5 7 o and PHIS-PfE8 57 0 fusion proteins identified proteins of appropriate size, namely an about 65 kD protein for each fusion protein.
B. Flea esterase protein PHIS-PfE6 5 4 0 was produced in the following manner. A pair of primers was used to amplify DNA from flea esterase nucleic acid molecule nfE6 1792 produced as described in Example 11. A sense primer containing an XhoI site having the nucleic acid sequence 5' AAACTCGAGT CCCCCGACTG TAACTTTGC 3' (denoted SEQ ID NO:62; XhAoI site shown in bold), was used in -combination with an anti-sense primer containing a PstI site having the nucleic acid sequence 5' TCATCTGCAG TTATTGACTG TGCAAAGTTT TTGTGG 3' (denoted SEQ ID NO:63; PstI site shown in bold). A PCR product was derived from nfE6 1 7 92 and is referred to herein as nfE6 1 488 having nucleic acid sequence SEQ ID NO:76. The PCR product was digested with XhoI and PstI restriction endontcleases, gel purified and subcloned into expression vector lambdaPR/T
Z
ori/S 10HIS-RSET-A9, that had been digested with XhoI and PstI and dephosphorylated.. The resultant recombinant molecule, referred to herein as pCro-nfE6 4 8 8 was transformed into E. coli HB 101 competent cells (available from Gibco BRL) to form recombinant cell E. coli:pCronfE6, 488 The recombinant cells were cultured using the method generally described in Section A of this example, except that the cells were grown under heat shift conditions rather than in the presence of IPTG. The cells were grown at 32°C for about 2 hours, and then grown at 42°C. Immunoblot analysis of recombinant cell E. coli:pCro-nfE6 4 8 lysate using a T7 tag monoclonal antibody directed against the fusion portion of the recombinant PHIS-PfE6 540 fusion protein identified proteins of appropriate size, namely an about 60 kD protein for each fusion protein.
Expression of the recombinant PHIS-PfE6 5 40 fusion protein was improved by transforming supercoiled plasmid pCro-nfE6 48 8 DNA harvested from E. coli:pCronfE6, 488 cells into the BL-21 strain of E. coli (available from Novagen). The amount of expression PHIS-PfE6 5 was confirmed by immunoblot using the method described immediately above.
c* E. coli cells expressing PHIS-PfE6 5 4o protein were harvested from about 2 liters of media and suspended in about 140 ml of 50 mM Tris, pH 8.0, 50 mM NaC1, 0.1 mM phenylmethylsulfonylfluoride (PMSF) (Solubilization Buffer). The cells were broken by passage through a microfluidizer at 30 psi for 30 cycles. The sample was centrifuged at about 16,000 X g for 30 min at 40C. The supematant (SI) was recovered and the pellet was resuspended in about 80 ml of Solubilization Buffer and centrifuged at about 16,000 X g for 30 min at 40C. The supernatant (S2) was recovered and the pellet was resuspended in about 80 ml of Solubilization Buffer containing 0.1% Triton-X 100 and centrifuged at about 16,000 X g for 30 min at 40C. The supernatant (S3) was recovered and the pellet was resuspended in about 140 mis 50 mM Tris, pH 8.0, 8 M Urea, 0.1 M PMSF and centrifuged at about 16,000 X g. The supernatant (S4) was recovered and the pellet was resuspended in 40 mis 50 mM Tris, 8 M Urea, 0.1 M PMSF. Aliquots of each pellet and supernatant were analyz -d y SDS-PAGE and immunoblot using the T7 tag monoclonal antibody described above. The results indicated that the PHIS-PfE6 54 0 protein was located in the final supernatant The PHIS-PfE6 5 40 protein was loaded onto a 5.0 ml, Metal chelating HiTrap column charged with NiCl 2 (obtained from Pharmacia Biotech Inc., Piscataway, NJ), previously equilibrated with 50 mM Tris, 1 mM PMSF, 1 mM 1-mercaptoethanol (PME), 8 M urea, pH 8.0 (Buffer The column -82was washed with 10 column volumes (cv) of Buffer A and then with 10 cv with 50 mM Tris, 25 mM sodium acetate,1 mM PMSF, 1 mM PME, 8 M urea, pH 6.0 (Buffer B) to remove loosely bound proteins. Bound PHIS-PfE6 40 protein was eluted with 10 cv of mM Tris, 25 mM sodium acetate, 1 mM PMSF, 1 mM PME, 8 M urea, pH (Buffer Column fractions were analyzed for the presence of PHIS-PfE6 5 40 protein by immunoblot using the T7 tag monoclonal antibody as described above. The results indicated that the majority of the PHIS-PfE6s 0 o protein was eluted by Buffer C. The fractions containing the PHIS-PfE6 5 4 0 protein were combined and loaded onto a 5 ml SP- Sepharose HiTrap column (obtained from Pharmacia Biotech Inc.) previously equilibrated with 50 mM Tris, 25 mM Sodium Acetate, 1 mM PMSF, 1 mM PME, 8 M Urea, pH 4.5 (SP-Sepharose Buffer). The column was washed with SP-Sepharose .Buffer until most of the unbound protein was removed. Bound protein was eluted with an increasing salt gradient to 1 M NaCI over 100 ml (20 cv) in SP-sepharose buffer.
Column fractions were analyzed for the presence of PHIS-PfE6 5 40 protein by immunoblot using the T7 tag monoclonal antibody as described above. The results indicated that the PHIS-PfE6 5 40 protein was eluted at about 0.75 M NaC1.
The purified PHIS-PfE6, 48 protein was used to produce an anti-M6 polyclonal antiserum as follows. Rabbits were immunized with PHIS-PfE6,4s 8 protein diluted to a concentration of about 0.1 mg/ml in PBS. One milliliter of the dilution was mixed 1:1 mix with Complete Freunds Adjuvant. In the primary immunization, about 500 pl of the 1:1 mix was injected subcutaneously into 5 different sites (0.1 ml/site) and 500 pl was injected intradermally into 5 different sites (0.1 ml/site) on the rabbit. Booster shots were administered to the rabbit intramuscularly in 4 sites using 250 gl/site of a 1:1 mix of PHIS-PfE6,4g, protein with Incomplete Freunds Adjuvant. The booster shots were adnr ;nstered at days 14 and 35. Serum samples were obtained prior to immunization (pre-bleed), and at day 14 after primary immunization and day 14 after the first and second boost.
C. Flea esterase protein PHIS-PfE9 52 8 was produced in the following manner. A pair of primers was used to amplify DNA from flea esterase nucleic acid molecule nfE9 2007 produced as described in Example 11. A sense primer containing an -83- BamHI site having the nucleic acid sequence 5' -TTC CGG ATC CGG CTG ATC TAC AAG TGA CTT TG 3' (denoted SEQ ID NO:64; BamHI site shown in bold), was used in combination with an anti-sense primer containing a XhoI site having the nucleic acid sequence 5' TGG TAC TCG AGT CAT AAA AAT TTA TTC CAA AAT C 3' (denoted SEQ ID NO:65; XhoI site shown in bold). A PCR product was derived from nfE9 2007 and is referred to herein as nfE9 1 5 4 0 having nucleic acid sequence SEQ ID NO:51. The PCR product was digested with Bamn and XlhoI restriction endonucleases, gel purified and subcloned into expression vector pTrcHisB (available from InVitrogen). The resultant recombinant molecule, referred to herein as pTrc-nfE95, 40 was transformed into E. coli HB101 competent cells (available from Gibco BRL) to form recombinant cell E.
coli:pTrc-nfE9 1 5 4 0 The-recombinant cells were cultured using the method described in Section A of this example. Immunoblot analysis of recombinant cell E. coli:pTrc-nfE9 1540 lysate using a T7 tag monoclonal antibody directed against the fusion portion of the 15 recombinant PHIS-PfE9 52 8 fusion protein identified proteins of appropriate size, namely an about 59 kD protein for each fusion protein.
Expression of the recombinant PHIS-PfE9 5 2 8 fusion protein was improved by transforming supercoiled plasmid pTrc-nfE9 1 5 8 4 DNA harvested from E. coli:pTrcnfE91 5 4 0 cells into the BL-21 strain ofE. coli. The amount of expression PHIS-PfE9 5 20 was confirmed by immunoblot using the method described immediately above.
Two liters of media from cultures of E. coli cells expressing PHIS-PfE9 528 protein were harvested and S4 supernatant was prepared using the method described above in section B. The PHIS-PfE9 5 2 8 protein contained in the S4 supernatant was loaded onto a 5.0 ml, Metal chelating HiTrap column. charged with NiCl 2 (available from Pharmacia Biotech Inc., Piscataway, previously equilibrated with 50 mM Tris, 1 mM PMSF, 1 mM PME, 8 M urea, pH 8.0 (Buffer The column was washed with cv of Buffer A until all unbound protein was removed. Bound protein was eluted with a linear gradient from Buffer A to 50 mM Tris, 1 mM PMSF, 1 mM PME, 8 M urea, 1 M NaC1, pH 4.0. Column fractions were analyzed for the presence of PHIS-PfE9 5 2 s protein by immunoblot using the T7 tag monoclonal antibody as described above. The -84results indicated that the majority of the PHIS-PfE9 528 protein was eluted at about 250 mM NaC1. The fractions containing the PHIS-PfE9s2 protein were combined and loaded onto a C4-reversed phase column (obtained from Vydak, Hesperia,
CA),
previously equilibrated with 0.05% trifluoroacetic acid (TFA). The column was washed with 0.05% TFA until all unbound protein was removed. Bound proteins were eluted with a linear gradient from 0.05% TFA to 0.05% TFA in acetonitrile. Column fractions were analyzed for the presence of PHIS-PfE9 52 s protein by immunoblot using the T7 tag monoclonal antibody as described above. The results indicated that the PHIS-PfE9 52 8 protein was eluted at about 40% acetonitrile. The fractions containing the PHIS-PfE9 528 protein were combined and loaded onto a 5 ml Q-S.epharose HiTrap column previously equilibrated with 50 mM Tris, 25 mM Sodium Acetate, 1 mM PMSF, 1 mM BME, 8 M Urea, pH 8.5 (Q-Sepharose Buffer). The column was washed with Q-Sepharose Buffer until all unbound protein was removed. Bound protein was eluted with an increasing salt gradient to 1 M NaCI over 100 ml (20 cv) in Q-sepharose buffer. Column fractions were analyzed for the presence of PHIS-PfE9 528 protein by immunoblot using the T7 tag monoclonal antibody as described above. The results indicated that the PHIS-PfE9 528 protein was eluted at about 0.3 M NaCI.
The purified PHIS-PfE9 5 28 protein was used to produce an anti-P polyclonal antiserum as follows. Rabbits were immunized with PHIS-PfE9 52 s protein diluted to a concentration of about 0.1 mg/ml in PBS. One milliliter of the dilution was mixed 1:1 mix with Complete Freunds Adjuvant. In the primary immunization, about 500 pl of the 1:1 mix was injected subcutaneously into 5 different sites (0.1 ml/site) and 500 Pl was injected intradermally into 5 different sites (0.1 ml/site) on the rabbit. Booster shots were administered to the rabbit intramuscularly in 4 sites using 250 gl/site of a 1:1 mix ofPHIS-PfE9 528 protein with Incomplete Freunds Adjuvant The booster shots were administered at days 14 and 35. Serum samples were obtained prior to immunization (pre-bleed), and at day 14 after primary immunization and day 14 after the first and second boost.
D. Flea esterase protein PHIS-PfE7 27 5 was produced in the following manner. A 650-bp fragment was produced by digesting nfE7 2836 DNA with the restriction enzymes BamHI and Bg/II. The BamHI and BglII fragment derived from nfE7 2 3 is referred to herein as nfE7, 65 having nucleic acid sequence SEQ ID NO:72 and amino acid SEQ ID NO:73. The fragment was purified using a QiaquickTM Kit (available from Qiagen, Santa Clarita, CA), according to methods provided by the manufacturer. The purified fragment was subcloned into expression vector pTrcHisC which had been digested with BamHI and BgII. The resultant recombinant molecule, referred to herein as pTrc-nfE7 6 5 0 was transformed into E. coli DH-5a competent cells (available from Gibco BRL) to form recombinant cell E. coli:pTrc-nfE7 65 o.
The recombinant cells were cultured using the method described above in section A. Immunoblot analysis of recombinant cell E. coli:pTrc-nfE75o lysate using a T7 tag monoclonal antibody directed against the fusion portion of the recombinant PHIS- PfE7 27 fusion protein identified proteins of appropriate size, namely an about 35 kD o protein for each fusion protein.
Expression of the recombinant fusion protein was improved by transforming supercoiled plasmid pTrc-nfE7 6 5 DNA harvested from E. coli:pTrc-nfE7 65 o cells into the BL-21 strain of E. coli. The amount of expression E. coli:pTrc-nfE7 6 5 0 was confirmed by immunoblot using the method described immediately above.
Example 13.
This Example demonstrates the production of esterase proteins of the present invention in eukaryotic cells.
A. Recombinant molecule pBv-nfE7, 78 containing a flea esterase nucleic acid molecule spanning nucleotides from about 99 through about 1886 of SEQ ID NO:24, and pBv-nfE8, 78 5 containing a flea esterase nucleic acid molecule spanning nucleotides from about 99 through about 1883 of SEQ ID NO:30 each, operatively linked to baculovirus pol he Iron transcription control sequences were produced in the following manner. In order to subclone a flea esterase nucleic acid molecule into baculovirus expression vectors, flea esterase nucleic acid molecule-containing fragments were separately PCR amplified from nfE72s 36 or nfE8 2 8 01 DNA. A PCR fragment of 1858 nucleotides, named nfE7, 18 s, was amplified from nfE7 2 8 3 6 using a sense primer E1113 FWD having the nucleic acid sequence AAAACTGCAG
TATAAATATG
TTACCTCACA GTAGTG 3' (SEQ ID NO:49; PstI site shown in bold) and an antisense primer E 1113/2212 REV having the nucleic acid sequence TGCTCTAGAT TATCTAATAC TTCCTTCATT ACAG (SEQ ID NO:50; XbaI site shown in bold). A PCR fragment of 1858 nucleotides, named nfE8 18 5 8 was amplified from nfE8 280 using a sense primer E2212 FWD having the nucleic acid sequence AAAACTGCAG TATAAATATG TTACCTCACA GTGCATTAG (SEQ ID NO:66; PstI site shown in bold), and the antisense primer E .1113/2212 REV. The Nterminal primer was designed from the pol h sequence ofbaculovirus with modifications to enhance expression in the baculovirus system.
i' 10 In order to produce a baculovirus recombinant molecule capable of directing the production of PfE7 5 96 the about 1,802 base pair PCR product (referred to as Bv-nfE7,so 2 was digested with PstI and Xbal and subcloned into unique PstI and XbaI sites of pVL1392 baculovirus shuttle plasmid (available from Pharmingen, San Diego, CA) to produce the recombinant molecule referred to herein as pVL-nfE7 80 2,.
15 In order to produce a baculovirus recombinant molecule capable of directing the production of PfE8 5 95 the about 1,792 base pair PCR product (referred to as Bv-nfE8, 7 92 was digested with PstI and XbaI and subcloned into PstI and XbaI digested to produce the recombinant molecule referred to herein as pVL-nfE8 7 9 2 The resultant recombinant molecules, pVL-nfE7 8 0 2 and pVL-nfE8, 79 were 20 verified for proper insert orientation by restriction mapping. Such a recombinant molecule can be co-transfected with a linear Baculogold baculovirus DNA (available from Pharmingen) into S.frugiperda Sf9 cells (available from InVitrogen) to form the recombinant cells denoted S. frugiperda:pVL-nfE7 102 and S. fiugiperda:pVL-fE8 1 7 2. S.
friugiperda:pVL-nfE7to2 can be cultured in order to produce a flea esterase protein PfE7 596 S. frugiperda:pVL-nfE8 1 79 2 can be cultured in order to produce a flea esterase protein PfE8 595 B. Recombinant molecule pBv-PfE9 5 2 g, containing a flea esterase nucleic acid molecule spanning nucleotides from 14 through 1595 of SEQ ID NO:36, operatively linked to baculovirus polyhedron transcription control sequences were produced in the following manner. In order to subclone a flea esterase nucleic acid molecule into baculovirus expression vectors, a flea esterase nucleic acid moleculecontaining fragment was PCR amplified from nfE920o7 DNA. A PCR fragment of about 1600 nucleotides, named nfE9 1 6 0, was amplified from nfE9 2007 using a sense primer P121B1 Sense having the nucleic acid sequence CGC GGA TCC GCT GAT CTA CAA GTG ACT TTG C 3' (SEQ ID NO:75; BamHI site shown in bold) and an antisense primer P121B Anti having the nucleic acid sequence CCG AGC GGC CGC ATA AAA ATT TAT TCC AAA ATC TAA GTC G-3' (SEQ ID NO:76; NotI site shown in bold). The N-terminal primer was designed from the pol h sequence of baculovirus with modifications to enhance expression in the baculovirus system.
10 In order to produce a baculovirus recombinant molecule capable of directing the production of PfE9 52 8 the about 1,600 base pair PCR product (referred to as Bv-nfE9 1 00 was digested with BamHI and NotI and subcloned into unique BamHI and NotI sites of pVL1393 baculovirus shuttle plasmid (available from Pharmingen, San Diego, CA) to produce the recombinant molecule referred to herein as pVL-nfE9,.
15 The resultant recombinant molecule, pVL-nfE9,,6o, was verified for proper insert orientation by restriction mapping. Such a recombinant molecule can be co-transfected with a linear Baculogold baculovirus DNA into S. frugiperda Sf9 cells to form the recombinant cells denoted S. fiugiperda:pVL-nfE9 ,oo. S. frugiperda:pVL-nfE9 1 6 oo can be cultured in order to produce a flea esterase protein PfE9 52 8 20 An immunoblot of supernatant from cultures of S. firugiperda:pVL-nfE9 0 oo cells producing the flea esterase protein PfE9 52 8 was performed using the anti-P 1 polyclonal antiserum described in detail in Example 12. Blots were incubated using serum samples from the pre-bleed or from serum collected 14 days after the first boost of the rabbit.
Analysis of the supernatent from cultures of S. frugiperda:pVL-nfE9 6 oo cells identified an about 66 kD protein C. Recombinant molecule pBv-PfE6 53 o, containing a flea esterase nucleic acid molecule spanning nucleotides from 50 through 1701 of SEQ ID NO:18, operatively linked to baculovirus polyhedron transcription control sequences were produced in the following manner. In order to subclone a flea esterase nucleic acid molecule into baculovirus expression vectors, a flea esterase nucleic acid molecule- -88containing fragment was PCR amplified from nfE6 1792 DNA. A PCR fragment of about 1679 nucleotides, named nfE10,679, was amplified from nfE6 1 79 using a sense primer M6M32 Sense having the nucleic acid sequence GCG AGG CCT TAT AAA TAT GTC TCG TGT TAT TTT TTT AAG TTG 3' (SEQ ID NO:75; StuI site shown in bold) and an antisense primer M6M32 Anti having the nucleic acid sequence
GCA
CTG CAG TTA TTG ACT GTG CAA AGT TTT TGT GG-3' (SEQ ID NO:76; PstI site shown in bold). The N-terminal primer was designed from the pol h sequence of baculovirus with modifications to enhance expression in the baculovirus system.
In order to produce a baculovirus recombinant molecule capable of directing the 10 production ofPfE6 5 30 the about 1,679 base pair PCR product (referred to as Bv-nfE6, 7 was digested with Stil and PstI and subcloned into unique StuI and PstI sites of FAST BACT' baculovirus shuttle plasmid (obtained from Gibco-BRL) to produce the recombinant molecule referred to herein as pFB-nfE6, 6 7 9 The resultant recombinant molecule, pFB-nfE6 1 6 7 9 was verified for proper insert 15 orientation by restriction mapping. Such a recombinant molecule can be transformed into E. coli strain DH10 (obtained from Gibco-BRL) according to the manufacturer's instructions. The pFB-nfE6 1 6 79 isolated from the transformed DHIO cells can then be cotransfected with a linear Baculogold baculovirus DNA into S.frugiperda Sf9 cells to form the recombinant cells denoted S. flrugiperda:pFB-nfE6, 1 7 9 S. firugiperda:pFB- 20 nfE6 1 679 can be cultured in order to produce a flea esterase protein PfE6 5 An immunoblot of supernatant from cultures of S. frugiperda:pFB-nfE6, 67 9 cells producing the flea esterase protein PfE6 53 0 was performed using the anti-M6 polyclonal antiserum described in detail in Example 12. Blots were incubated using serum samples from the pre-bleed or from serum collected 14 days after the first boost of the rabbit.
Analysis of the supernatent fron cultures of ;iperda:pFB-nfE6 1 6 7 9 cells identified an about 66 kD protein.
N-terminal amino acid sequence was obtained using standard methods for the about 66 kD protein identified using the anti-M6 polyclonal antiserum. The N-terminal amino acid sequence was determined to be identical to the N-terminal amino acid sequence of SEQ ID NO:44.
-89- Example 14 This example describes the purification of carboxylesterase protein from fed flea midguts.
About 43,000 cat blood-fed adult flea midguts were collected and prepared as previously described in Example 1. The extract was then added in 2 aliquots to columns containing about 1 to about 2 ml ofp-aminobenzamidine linked agarose beads (available from Sigma), equilibrated in 50 mM Tris (pH 400 mM NaCI, and incubated overnight at 4 0 C. The columns were then drained to remove unbound protein and the two aliquots of unbound protein were combined. The collected unbound protein was S* 10 then concentrated and diafiltered into a total volume of about 16 ml of 25 mM Tris (pH 10 mM NaCI using an Ultrafree-20 10 kD centrifugal concentrator (available from Millipore, Bedford, MA).
Aliquots of about 8 ml were loaded onto an Uno Q6 anion exchange column (available from Bio-Rad, Hercules, CA) equilibrated in 25 mM Tris (pH 10 mM 15 NaC1, operated on a BioLogic liquid chromatography system (available from Bio-Rad).
The column was washed with 25 mM Tris (pH 10 mM NaCl until all unbound protein was removed. Protein bound to the column was then eluted with a linear gradient from 10 mM to 1 M NaCI in 25 mM Tris, pH 8. Fractions were assayed for CE activity using the assay described previously. The results indicated that CE activity was 20 eluted at about 220 mM NaC1.
Fractions containing CE activity were pooled and diafiltered into a total volume of about 3 ml of 20 mM MES buffer (2-(N-morpholino)ethanesulfonic acid), pH containing 10 mM NaC1, in preparation for cation exchange chromatography. The sample was then applied to an Uno S1 cation exchange column (available from Bio-Rad) squilibrated in IS buffer. The column was washed with MES buffer until all unbound protein was removed. Protein bound to the column was then eluted with a linear gradient from 10 mM to 1 M NaCI in 20 mM MES buffer, pH 6. Fractions were assayed for CE activity using the assay described previously. The results indicated that CE activity was not retained on the cation exchange column using the above conditions, and all of the activity was found in the flow-through fractions.
Fractions containing CE activity were pooled and diafiltered into a total volume of about 3 ml of 25 mM Tris (pH 10 mM NaCI, in preparation for an additional anion exchange chromatography step. The sample was then applied to a Bio-Scale Q2 anion exchange column (available from Bio-Rad). The column was washed with 25 mM Tris (pH 10 mM NaCI until all unbound protein was removed. Protein bound to the column was then eluted with a linear gradient from 10 mM to I M NaCl in 25 mM Tris, pH 8. Fractions were assayed for CE activity using the assay described previously. The results indicated that CE activity was eluted at about 130 mM NaCI.
A fraction containing CE activity was diluted into a total volume of about 4 ml of S 10 10 mM phosphate buffer, pH 7.2 containing 10 mM NaC1, in preparation for hydroxyapatite chromatography. The sample was then applied to a Bio-Scale CHT2-I column (available from Bio-Rad) at a flow rate of about 0.5 ml/min. The column was washed with 10 mM-phosphate buffer, pH 7.2 containing 10 mM NaCI until all unbound protein was removed. Protein bound to the column was then eluted with a linear 15 gradient from 10 mM phosphate buffer, pH 7.2 containing 10 mM NaCl to 0.5 M mM phosphate buffer, pH 6.5 containing 10 mM NaCl. Fractions were assayed for CE activity using the assay described previously. The results indicated that CE activity was eluted at about 200 mM phosphate.
Example 20 This example describes the purification of a carboxylesterase protein from wandering flea larvae.
About 120,000 bovine blood-fed adult wandering flea larvae were homogenized in 3 batches of about 40,000 wandering larvae in each batch, in Tris buffered saline (TBS), pH 8.0 as previously described, except that about 1.2 mg ofphenylthiourea was added to each ml of TBS during the extraction procedure to inhibit cross linking reactions. The extracts were dialyzed against 2 changes of about 2 L of 10 mM phosphate buffer, pH 7.2 containing 10 mM NaCl in preparation for hydroxyapatite batch chromatography. The samples were then filtered through glass Acrodiscs (available from Gelman Sciences, Ann Arbor, MI) and added to 14 g of Macro-Prep Ceramic Hydroxyapatite, Type I, 40 pm beads (available from Bio-Rad), previously equilibrated in 10 mM phosphate buffer, pH 7.2 containing 10 mM NaCI. The extracts and beads were rocked at room temperature for about 30 minutes. Following incubation, the beads were centrifuged for about 5 minutes at 500 x g and the supematants removed.
The beads were washed with about 40 ml 10 mM phosphate buffer, pH 7.2 containing 10 mM NaCI, centrifuged as above, and washed and centrifuged again to eliminate all unbound protein. Bound proteins were eluted by washing the beads with about 40 ml of each of 100 mM, 200 mM, 300 mM, and 400 mM phosphate buffer, pH 6.5 containing mM NaCI. Following elution, the supernatants from each concentration of phosphate buffer were tested for juvenile hormone esterase activity as described previously in 10 Example 7. Thejuvenile hormone esterase activity eluted at different phosphate concentrations in each batch, but the activity was generally found in the 200 mM to 300 mM phosphate fractions.
The fractions that contained the highest juvenile hormone esterase activity were combined and diafiltered into a total volume of about 50 ml of 10 mM phosphate buffer, 15 pH 7.2 containing 10 mM NaCI using a stirred cell concentrator fitted with a ultrafiltration membrane (available from Amicon, Beverly, MA). Aliquots of about 5 ml to 10 ml were applied to a chromatography column containing about 10 ml of Macro- Prep Ceramic Hydroxyapatite, Type I, 20 gm beads, previously equilibrated with 10 mM phosphate buffer, pH 7.2 containing 10 mM NaCI. The column was washed with 20 mM phosphate buffer, pH 7.2 containing 10 mM NaCI until all unbound protein was removed. Protein bound to the column was then eluted with a linear gradient from mM phosphate buffer, pH 7.2 containing 10 mM NaCI to 0.5 M 10 mM phosphate buffer, pH 6.5 containing 10 mM NaCl: Fractions were assayed for carboxylesterase activity using the assay described previously. The results indicated that carboxylesterase activity was eluted at about 160 mM phosphate.
The fractions that contained the highest carboxylesterase activity were combined and diafiltered into a total volume of about 15 ml of 20 mM sodium acetate buffer, pH in preparation for cation exchange chromatography. Aliquots of about 3 ml were applied to a PolyCat A cation exchange column (available from PolyLC, Columbia, MD) equilibrated in 20 mM sodium acetate buffer, pH 6.0, operated on a Waters high -92performance liquid chromatography system (available from Waters Corporation, Milford, MA). The column was washed with 20 mM sodium acetate buffer, pH 6.0 until all unbound protein was removed. Protein bound to the column was then eluted with a linear gradient from 20 mM sodium acetate buffer, pH 6.0 to 20 mM sodium acetate buffer, pH 6.0 containing 1 M NaCI. Fractions were assayed for CE activity using the assay described previously. The results indicated that there were two pools of CE activity. The first pool was not retained on the cation exchange column, and the second pool was eluted at about 170 mM NaCI.
The fractions from the second pool that contained the highest carboxylesterase 10 activity were combined and diafiltered into a total volume of about 10 ml of 25 mM Tris (pH 10 mM NaCI, in preparation for anion exchange chromatography. The sample was then applied to a Bio-Scale Q2 anion exchange column (available from Bio-Rad).
The column was washed with 25 mM Tris (pH 10 mM NaCl until all unbound protein was removed. Protein bound to the column was then eluted with a linear 15 gradient from 10 mM to 1 M NaCI in 25 mM Tris, pH 8. Fractions were assayed for carboxylesterase activity using the assay described previously. The results indicated that carboxylesterase activity was eluted at about 350 mM NaC1.
Fractions containing carboxylesterase activity were combined and concentrated to about 175 pl using a Centricon 10 centrifugal concentrator (available from Amicon, 20 Beverly, MA) in preparation for size exclusion chromatography. The sample was applied to a Bio-Select SEC 125-5 size exclusion chromatography column (available from Bio-Rad), previously equilibrated in TBS, pH 7.2. About 250 ul fractions were then collected. The fractions were assayed for carboxylesterase activity using the assay described previously. The results indicated that the carboxylesterase activity was eluted in about 5.5 to 6 ml of buffer, corresr- r ling to a molecular weight of about 40 to 100 kDa based on the elution volumes of gel filtration molecular weight standard proteins (available from Sigma, St. Louis, MO).
Example 16 This example describes the purification of juvenile hormone esterase activity from unfed adult flea midguts by affinity chromatography.
-93- About 16,000 unfed adult flea midguts were collected in 20 mM Tris buffer (pH containing 130 mM NaC1, 1 mM sodium EDTA, 1 mM Pefabloc (available from Boehringer Mannheim, Indianapolis, IN), 1 microgram/ml (pg/ml) leupeptin and 1 gg/ml pepstatin. The midguts were homogenized by freeze-fracture and sonication, and then centrifuged at about 14,000 x g for 20 min. The soluble material from the centrifugation step was recovered, diafiltered into Tris buffered saline (TBS), and applied to a disposable plastic column containing about 1 ml of mercapto)butylthio]- 1,1,1 -trifluoropropan-2-one linked Sepharose 6B beads, prepared similarly to the method described by Venkatesh et al. Biol. Chem., Vol. 265, No. 10 21727-21732, 1990) (the 3 4 '-mercapto)butylthio]-l,l,l-trifluoropropan-2-one was a gift from Novartis Corp., Basel, Switzerland; and the Epoxy-activated Sepharose 6B is available from Pharmacia Biotech Inc., Piscataway, NJ). After overnight incubation at 4 the column was drained and the beads were washed with about 10 ml TBS, then about 10 ml TBS containing 0.1% n-octylglucoside (OG; available from 15 Boehringer Mannheim). The pre-column, flow-through, and wash fractions were tested forjuvenile hormone esterase activity by the method previously described above in Example 7. The results indicate that the flow-through fraction contained approximately less juvenile hormone esterase activity than the pre-column material, and that the washes contained very little activity.
20 Bound protein was eluted from the beads by adding about 10 ml of TBS containing 0.1 OG and 1 mM 3 -octylthio-l,l,l-trifluoropropan-2-one (OTFP; a gift from Novartis Corp.). After a 2 hour incubation at 4 0 C, about 5 ml of the eluate was collected, and the remaining 5 ml was incubated with the beads overnight at 4°C. The following day, the beads were drained, the-eluate collected, and an additional 10 ml of TBS cr nt .ining 0.1 OG and 1 mM OTFP was added to the beads. After an overnight incubation at 4 0 C, the beads were drained and the eluate collected. The final ml elution step was repeated 3 additional times so that we had 6 eluted fractions. The first elution fraction was dialyzed overnight twice against 1 liter of fresh TBS to remove excess OTFP. The second elution fraction was also dialyzed overnight against 1 liter of fresh TBS to remove OTFP. The third through sixth elution fractions were not dialyzed.
All six eluted fractions were tested for juvenile hormone esterase activity by the method previously described above in Example 7. The results indicate that only the third elution fraction contained detectable juvenile hormone esterase activity. Analysis of the eluted fractions by silver-stained SDS-PAGE indicated that several proteins were specifically bound to the affinity beads and were eluted by OTFP. The apparent molecular weights of these proteins, as determined by SDS-PAGE, were about 66 kDa, 55 kDa, and 33 kDa. About 3.5 ml of each elution fraction were combined and concentrated to about 110 .il using a Centriplus 10 centrifugal concentrator (available from Amicon, Beverly, MA). This pool was separated by SDS-PAGE and blotted onto a polyvinylidene 10 -difluoride (PVDF) membrane as described previously in Example 5. The stained protein band at about 66 kDa was excised and subjected to N-terminal sequence analysis as described previously.
The results indicated that the N-terminal amino acid sequence of the putative 66 kDa juvenile hormone esterase protein was DL y/g V k/y/g v/q/n LQGTLKGKE 15 (denoted herein as SEQ ID NO:74), in which the lower case letters designate uncertainties. Below is shown a comparison between different esterase amino acid sequences of the present invention.
*00.
SEQ ID NO:74: DL V
LQGTLKGKE
SEQ ID NO:37: DL Q V T L LQGTLKGKE 20 (Residues 3-17) Example 17 This example describes the purification of an active recombinant juvenile hormone esterase protein from baculovirus supernatants.
About 1 liter of supernatant from cultures of S. frugiperda:pVL-nfE9 6 oo cells producing the flea esterase protein PfE9 528 was brought to'about 50% satu ation with ammonium sulfate and centrifuged at about 20000 x g for about 30 minutes at 4°C to pellet the precipitated material. After centrifugation, the pellet was retained and the supernatant was brought to about 100% saturation with ammonium sulfate and centrifuged as above. The material in both pellets were resuspended separately in about 35 ml of Tris buffered saline (TBS), pH 8.0. The resuspended pellets were assayed for the presence of flea esterase protein PfE9s 28 using standard Western blot techniques and a polyclonal antiserum that binds specifically to PfE952 8 protein. Briefly, a rabbit was immunized with PHIS-PfE9 528 protein purified from E. coli:pTrc-nfE9s4 cells (described above in Example 12C) and boosted using standard procedures. The results indicated that the flea esterase protein PfE952 8 was present in the S.frugiperda:pVLnfE9,6o supernatants and the protein was precipitated by adjusting the ammonium sulfate concentration from about 50% saturation to about 100% saturation.
The resuspended flea protein PfE9 528 was diafiltered into about 10 ml of 25 mM Tris (pH 10 mM NaCI using an Ultrafree-20 10 kD centrifugal concentrator in 10 preparation for anion exchange chromatography. Aliquots of about 5 ml were loaded onto an Uno Q6 anion exchange column equilibrated in 25 mM Tris (pH 10 mM NaCI. The column was washed with 25 mM Tris (pH 10 mM NaCI until most of the unbound protein was removed. Protein bound to the column was then eluted with a linear gradient from 10 mM to 1 M NaCI in 25 mM Tris buffer (pH Fractions were 15 assayed for the presence of flea esterase protein PfE9 2 8 by the immunoblot method described above. The results indicated that the flea esterase protein PfE9 52 8 was eluted at about 200 mM NaCI.
Fractions containing the flea esterase protein PfE9 52 1 were pooled and concentrated to about 440 ul using a Centricon 10 kD centrifugal concentrator in 20 preparation for size exclusion chromatography. The sample was applied in 3 aliquots to S. a Bio-Select SEC 125-5 size exclusion chromatography column (available from Bio- Rad), previously equilibrated in TBS, pH 7.2. The column was eluted with TBS, pH 7.2 at a flow rate of about 0.5 ml/min, and fractions of about 250 pl were collected. The fractions were assayed for the presence of flea esterase protein PfE9 5 28 by the immunoblot method described above. The results indicate t' at the flea esterase protein PfE9 5 28 was eluted with about 6 ml of buffer, corresponding to a molecular weight of about 40 to 100 kDa based on the elution volumes of gel filtration molecular weight standard proteins (available from Sigma, St. Louis, MO).
Fractions containing flea esterase protein PfE9 5 2 8 were then assayed forjuvenile hormone esterase activity as described in Example 7 and carboxylesterase activity as described in Example 2. The results indicated that the purified flea esterase protein P1E9 528 had both juvenile hormone esterase activity and carboxylesterase activity.
S S
S
5S S S
S
*SSS
S. S S S 55 555.
5.55 S. 55
S.
5 9
S
S
*555
*S
S
S
*S.S
S
212 22L Page(s) are claims pages they appear after the sequence listing SEQUENCE LISTING GENERAL INFORMATION:
APPLICANT:
NAME: Heska Corporation STREET: 1825 Sharp Point Drive CITY: Fort Collins STATE: CO COUNTRY: US POSTAL CODE (ZIP): 80525 1 0 TELEPHONE: (970) 493-7272 TELEFAX: (970) 484-9505 (ii) TITLE OF INVENTION: Novel Carboxylesterase Nucleic Acid Molecules, Proteins and Uses Thereof (iii) NUMBER OF SEQUENCES: 76 15 (iv) CORRESPONDENCE
ADDRESS:
ADDRESSEE: LAHIVE COCKFIELD,
LLP
STREET: 28 STATE STREET CITY: BOSTON STATE: MA COUNTRY:
US
ZIP: 02109 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk 02.5 COMPUTER: IBM PC compatible OPERATING SYSTEM: Windows SOFTWARE: ASCII DOS TEXT (vi) CURRENT APPLICATION DATA: APPLICATION
NUMBER:
FILING DATE:
CLASSIFICATION:
o (vii) PRIOR APPLICATION
DATA:
APPLICATION NUMBER: 08/747,221 FILING DATE: November 12, 1996 (viii) ATTORNEY/AGENT
INFORMATION:
NAME: Rothenberger, Scott D.
REGISTRATION NUMBER: 41,277 REFERENCE/DOCKET NUMBER: HKV-010PC (FC-1-C1-PCT) (ix) TELECOMMUNICATION
INFORMATION:
TELEPHONE: (617) 227-7400 TELEFAX: (617) 742-4214 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 401 nucleotides TYPE: nucleic acid -98- STRANDEDN'ESS: single TOPOLOGY: linear (ii) MOLECULE TYPE:
FEATURE:
NAME/KEY:
LOCATION:
(iv) FEATURE:
NAME/KEY:
LOCATION:
v) FEATURE:
NAME/KEY:
LOCATION:
FEATURE:
NAME/KEY:
LOCATION:
cDNA
CDS
92. .400 Xaa Ile, 218 Xaa Lys, 275, 329 Xaa Asn, 332 Thr Lys. or Arg Glu or Gln Tyr or Asp 00 OS 0 S 9 5@ S 0 0OS@ r oO~
S
00p0 *9 9 .5
S.
(vii) SEQUENCE DESCRIPTION: SEQ ID NO:1: TTTACATCAT TAATAAACAT AAATCTAATA AATCTTGTGG ATCAAGATCA AGTTTATTAG TGAGAOTGTT GGATTTGTGA AATATTTCAA A ATG AAT Met Asn *00 S...0 TCT TTA ATT GTA AAA ATT TCT CAA GGA OCT ATT GAG Ser Leu Ile Val Lys Ile Ser Gin Gly Ala Ile Glu GGG AAG Gly Lys is 97 139 181 223 GAA ATG ATT Giu Met Ile OAT AAT GGA AAO Asp Asn Gly Lys TTT AGA GGA TTT Phe Arg Oly Phe GOT ATA CCT TAT OCT AAA CCO CCT ATA Gly Ile Pro Tyr Ala Lys Pro Pro Ile AAT CTT ANA TTT Asn Leu Xaa Phe
AAG
Lys CCT CCT CAA AAO Pro Pro Gin Lys OAT OAT TOG AAT Asp Asp Trp Asn OTT COA CCA Val Arg.Pro CAT ATO CTO His Met Leu 265 307 OCT ACT Ala Thr OAA NAA OCA AAT Oiu Xaa Ala Asn TOT AGA TCG AAA Cys Arg Ser Lys CAG CAT CAT Gin His His ATT ATT OGA GAC Ile Ile Oly Asp
NAA
Xaa 80 NAT TOT CTA TAC Xaa Cys Leu Tyr CTA AAC Leu Asn GTN TAT OTT Vai Tyr Val TTO ACT TCC AAA TTO GAO AAA CTA CCA Leu Thr Ser Lys Leu Giu Lys Leu Pro -99-
ATG
Met (2) TTC TGG G Phe Trp INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 103 amino acids TYPE: amino acid TOPOLOGY: linear
S.
S*
S
*SS**e
S
*5*S *5 (ii) MOLECULE TYPE: protein (iii) FEATURE NAME/KEY: Xaa le, Thr, LOCATION: 43 (iv) FEATURE NAME/KEY: Xaa =Lys, Glu LOCATION: 62, 15
FEATURE
NAME/KEY: Xaa Asn, Tyr (B3) LOCATION: 81 (vi) SEQUENCE DESCRIPTION: SEQ ID NO Met Asn Ser Leu Ile Val Lys Ile Ser Gin Gly 1 5 10 Gly Lys Giu Met Ile Asn Asp Asn Gly Lys Ser 1520 Phe Leu Gly Ile Pro Tyr Ala Lys Pro Pro Ile 35 25 Xaa Phe Lys Pro Pro Gin Lys Pro Asp Asp Trp 45 50 Arg Pro Ala Thr Giu Xaa Ala Asn Gly Cys Arg 65 Met Leu Gin His His Ile Ile Gly Asp Xaa Xaa 75 80 Leu Asn Vai Tyr Val Pro Leu Thr Ser Lys Leu 90 Pro Vai Met Phe Trp 100 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 401 nucleotides TYPE: nucleic acid STRANDEDNESS: single Lys or Arg or Gin or Asp :2: Ala I Phe Aj Gly AE Asn As Ser L) Cys Le Giu
L\
le Glu Gly Leu Val His Tyr Leu -100- OPOLOGY: linear (ii) MOLECULE
TYPE:-
cDNA (iii) SEQUENCE DESCRIPTION: SEQ ID NO:3:
CCCAGAACAT
ACGTTTAGGT
ATGTTTCGAT
TCCAATCATC
GGTTTAGCAT
ATTAATCATT
AAGAATTCAT
TTGATCTTGA
A
TACTGGTAGT
ATAGACAATN
CTACAACCAT
AGGCTTTTGA
AAGGTATACC
TCCTTCCCCT
TTTGAAATAT
TCCACAAGAT
TTCTCCAATT
TTNGTCTCCA
TTGCTTNTTC
GGAGGCTTAA
CAAAAATCCT
CAATAGCTCC
TTCACAAATC
TTATTAGATT
TOGAAGTCAA
TGGAACATN
ATAATATGAT
GCTGCAGCAT
AGTAGCTGGT
CGAACATCAT
ATNTAAGATT
TCCTATAGGC
CTAAACGACT
TTCCATTATC
TTGAGAAATT
TTTACAATTA
CAACACTCTC
ACTAATAAAC
TATGTTTATT AATGATGTAA 100 200 250 300 350 400 401 9 9 9* 9* 9. 9* 9 INFORMATION FOR SEQ ID NO:4: C) SEQUENCE CHARACTERISTICS: LENGTH: 364 nucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE
TYPE:
(iii) FEATURE:
NAME/KEY:
LOCATION:
cDNA
CDS.
2. .364 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: G TCT COT GTT ATT TTT TTA AGT TGT ATT TTT TTG TTT AGT 25 Ser Arg Val Ile Phe Leu Ser Cys Ile Phe Le~u Phe Ser TTT AAT TTT Phe Asn Phe is CAA GOC OAA Gin Gly Glu ATA AAC TGT OAT TCC CCG ACT GTA ACT TTO CCC Ile Asn Cys Asp Ser Pro Thr Val Thr Leu Pro TTO OTT GOA AAA OCT TTG ACG AAC OAA Leu Val Oly Lys Ala Leu Thr Asn Giu AAT OGA Asn Oly AAA GAG TAT TTT AG. TAC ACA GOT OTA CCT TAT GCT AAA Lys Giu Tyr Phe Ser Tyr Thr Oly Val Pro Tyr Ala Lys CCT OTT OGA GAA Pro Val Oly Glu
CTT
Leu AGA TTT AAO CCT CCA CAG AAA OCT GAG Arg Phe Lys Pro Pro Gin Lys Aia Glu
CCA
Pro TOG CAA GOT OTT TTC A.AC 0CC ACA TTA TAC OGA AAT OTG Trp Gin Gly Val Phe Asn Ala Thr Leu Tyr Oiy Asn Val TGT AAA TCT TTA AAT TTC TTC TTG AAG AAA ATT GAA GGA GAC 292 Cys Lys Ser Leu Asn Phe Phe Leu Lys Lys Ile Glu Gly Asp 90 GAA GAO TGC TTG GTA GTA AAC GTG TAC GCA CCA AAA ACA ACT 334 Glu Asp Cys Leu Val Val Asn Val Tyr Ala Pro Lys Thr Thr 100 105 110 TOT GAT AAA AAA.CTT CCA GTA TTT TTC TGG 364 Ser Asp Lys Lys Leu Pro Val Phe Phe Ti-p 115 120 INFORMATION FOR SEQ ID WI SEQUENCE
CHARACTERISTICS:
LENGTH: 121 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) SEQUENCE DESCRIPTION: SEQ ID Ser Arg Val Ile Phe Leu Ser Cys Ile Phe Leu Phe Ser Phe 1 5 Asn Phe Ile Asn Cys Asp Ser Pro Thr Val Thi- Leu Pro Gin 15. 20 000:Gly Giu Leu Val Gly Lys Ala Leu Thr Asn Glu Asn Gly Lys 35 Giu Tyr Phe Ser Tyr Thi- Gly Val Pro Tyr Ala Lys Pro Pro 50 0::0.425 Val Giy Glu Leu Arg Phe Lys Pro Pro Gin Lys Ala Glu Pro '0.060 65 Ti-p Gin Gly Val Phe Asn Ala Thi- Leu Tyr Gly Asn Val Cys *75 000000LysSer Leu Asn Phe Phe Leu Lys Lys Ile Glu Gly Asp Giu 85 90 Asp Cys Leu Val Val Asn Val Tyr Ala Pro Lys Thi- Thi- Ser 100 10s 110 Asp Lys Lys Leu Pro Val Phe Phe Ti-p 115 120 INFORMATION FOR SEQ ID NO:6: SEQUENCE
CHARACTERISTICS:
LENGTH: 364 nucleotides TYPE: nucieic acid STRANDEDNESS: singie TOPOLOGY: linear -102- (ii) MOLECULE TYPE: cDNA (iii) SEQUENCE DESCRIPTION: SEQ ID NO:6:
CCAGAAAAAT
CGTTTACTAC
TTTAAAGATT
CCATGGCTCA
GTTTAGCATA
TTCGTCAAAG
GGAATCACAG
AAATAACACG
ACTGGAAGTT
CAAGCAGTCT
TACACACATT
GCTTTCTGTG
AGGTACACCT
CTTTTCCAAC
TTTATAAAAT
AGAC
TTTTATCAGA
TCGTCTCCTT
TCCGTATAAT
GAGGCTTAAA
GTGTAGCTAA
CAATTCGCCT
TAAAACTAAA
AGTTGTTTTT
CAATTTTCTT
GTGGCGTTGA
TCTAAGTTCT
AATACTCTTT
TGGGGCAAAG
CAAAAAAATA
GGTGCGTACA
CAAGAAGA
AAACACCTTG
CCAACAGGAG
TCCATTTTCG
TTACAGTCGG
CAACTTAA
so 100 150 200 250 300 350 364 INFORMATION FOR SEQ ID NO:7: i) SEQUENCE CHARACTERISTICS: LENGTH: 421 nucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: (i)FEATURE:
NAME/KEY:
LOCATION:
cDNA
CDS
113. .421 (iv) SEQUENCE DESCRIPTION: SEQ ID NO:7: TTTACATTAC ATCAAATCAT ATTTTTATTA GTATATTTTT
TAGAAGAACC
TAGCCAAAp.A ATATOGACTT TAGACTGTGA TTAATTTATT
TTACCTGAGA
TTTTCCTTTA
CA ATG GGT GAT CTT, CAA GTG ACT TTG TTA CAA Met Gly Asp Leu Gin Val Thr Leu Leu Gin GOT TCT TTO AGA Oly Ser Leu Arg
OGA
Gly AAA GAG CAA ATT AAT GAA AAG OGA AAT Lys Olu Gin Ile Asn Glu Lys Gly Asn TTT TAT AOT TAT Phe Tyr Ser Tyr
TCT
Ser OGA ATT CCA TAT GCC AAA CCT CCA Oly Ilie Pro Tyr Ala Lys Pro Pro GTT GOT Val Gly OAT CTA AGA TTC Asp Leu Arg Phe CCA CCT CAA CCT Pro Pro Gin Pro OCA GAA CCT Ala Oiu Pro TOO TCA GOT Trp Ser Gly GTC CTT OAT OCT Val Leu Asp Ala
ACT
Thr AAA OAA 000 AAT Lys 0Th Oly Asn AOT TOT Ser Cys AGA TCT OTA CAT TTT ATT AAA AAO ATT AAA OTA 000 OCT Arg Ser Val His Phe Ile Lys Lys Ile Lys Val Oly Ala -103- GAT TGT CTA TAC CTC MAT GTC TAT GTA CCA AMA ACA TCA GAG 394 Asp Cys Leu Tyr Leu Asn Val Tyr Val. Pro Lys Thr Ser Glu AAA TCC CTT CTT CCA GTA ATG GTA TGG 421 Lys Ser Leu Leu Pro Val Met Val Trp 100 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 103 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) SEQUENCE DESCRIPTION: SEQ ID NO:8: Met Gly Asp Leu Gin Val Thr Leu Leu Gin Gly Ser Leu Arg 1 5 *Gly Lys Giu Gin Ile Asn Glu Lys Gly Asn Val Phe Tyr Ser is 1 20 Tyr Ser Gly Ile Pro Tyr Ala Lys Pro Pro Val Gly Asp Leu 35 Arg Phe Lys Pro Pro Gin Pro Ala Giu Pro Trp Ser Gly Val 50 Leu Asp Ala Thr Lys Giu Gly Asn Ser Cys Arg Ser Val His 65 Phe Ile Lys Lys Ile Lys Val Gly Ala Giu Asp Cys Leu Tyr 75 .*.Leu Asn Vai Tyr Val Pro Lys Thr Ser Giu Lys Ser Leu Leu 90 Pro Val Met Val. Trp 100 INFORMATION FOR SEQ ID NO:9: SEQUENCE
CHARACTERISTICS:
LENGTH: 421 nucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULhE TYPE: cDNA (iii) SEQUENCE DESCRIPTION: SEQ ID NO:9: CCATACCATT ACTGGAAGAA GGGATTTCTC TGATGTTTTT GGTACATAGA CATTGAGGTA TAGACAATCT TCAGCCCCTA CTTTAATCTT TTTAATAA 100 -104-
TGTACAGATC
CCAAGGTTCT
GTTTGGCATA
TTAATTTGCT
ATCACCCAT'r
AAAGTCCATA
TATGATTTGA
TACAACTATT CCCTTCTTTA GTAGCATCAA
GGACACCTGA
GCAGGTTGAG GTGGCTTGA. TCTTAGATCA
CCAACTGGAG
TGGAATTCCA GAATAACTAT AAAACACATT
TCCCTTTTCA
CTTTTCCTCT CAAAGAACCTTGTAACAAAG
TCACTTGAAG
GTAAAGGAAA ATCTCAGGTA AAATAAATTA
ATCACAGTCT
TTTTTTGGCT AGGTTCTTCT AAAAAATATA
CTAATAAAAA
TGTAATGTA
A
IS0 200 250 300 350 400 421 INFORMATION FOR SEQ ID NO:iO: SEQUENCE CHARACTERISTICS: LENGTH: 524 nucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: (iii) FEATURE:
NAME/KEY:
LOCATION:
cDNA
CDS
113. .523 (iv) SEQUENCE DESCRIPTION: SEQ ID NO:i0: GAACGTTGAT ACGATAGACA TGTCGTCTTC AAAACGTCTA
TTTTATCATA
AACAAAACGA GATAAATAAT AACAATTAAG CAACCAAAAT
GCATTAAAAA
ACACAATAAA
S
S
AA ATG TTA CCT CAC AOT AGT GCA TTA OTT TTA Met Leu Pro His Ser Ser Ala Leu Val Leu TTT TTA TTT TTT TTA TTT TTC TTA TTT 25 Phe Leu Phe Phe Leu Phe Phe Leu Phe
ACA
Thr CCT ATC TTG TGC Pro Ile Leu Cys ATA CTA TGG OAT AAC Ile Leu Trp Asp Asn OAT CAG CAT TTO TOC AGA OTT CAA Asp Gin His Leu Cys Arg Val Gin 184 226 268 TTT AAC Phe Asn AGO ATC ACO GAA OGA AAA CCG TTC CGA Arg Ile Thr Glu Gly Lys Pro Phe Arg 45 TAT AAA OAT Tyr Lys Asp CAT AGO *"A7 His Arg Asn .55 OAT GTA TAT TOT TCT TAT TTG OGA ATT Asp Val Tyr Cys Ser Tyr Leu Oly Ile 60 CCT TAT Pro Tyr 0CC GAA CCG Ala Giu Pro
CCT
Pro ATT OGA CCA TTA Ile Gly Pro Leu TTT CAG TCT CCA Phe Gin Ser Pro CCA ATA TCA AAT Pro Ile Ser Asn AAA ACA GGA TTC OTA CAG OCT CGA ACT Lys Thr Giy Phe Val Gin Aia Arg Thr -105- TTG GGA GAC AAA TGT TTC CAG GAA AGT CTA ATA TAT TCT TAT Leu Gly Asp Lys Cys Phe Gin Glu Ser Leu Ile Tyr Ser Tyr 100 105 GCA GGA AGC GAA GAT TGC TTA TAT CTG AAT ATA TTC ACG CCA Ala Gly Ser Giu Asp Cys Leu Tyr Leu Asn Ile Phe Thr Pro 110 115 120 GAG ACT GTT AAT TCT GCG AAC AAT ACA AAA TAT CCT GTA ATG Giu Thr Val Asn Ser Ala Asn Asn Thr Lys Tyr Pro Val Met 125 130 135 TTC T Phe INFORMATION~ FOR SEQ ID NO:li: 436 478 520 a a a.
S
a.
a a a i) SEQUENCE CHARACTERISTICS: LENGTH: 137 amino acids TYPE: amino acid TOPOLOGY: linear Met 1 Leu 15 Asn Thr Val Ile Pro Cys Asp Ser Leu Pro Phe Phe Leu Asp 30 Giu Gly Tyr Cys Gly Pro Lys Thr Phe Gin 100 Cys Leu Ala Asn MOLECULE TYPE: protein SEQUENCE DESCRIPTION: SEQ ID NO:11: His Ser Ser Ala Leu Val Leu Phe Leu 5 Leu Phe Thr Pro Ile Led Cy ,s Ile Leu 20 Gin His Leu Cys Arg Val Gin Phe Asn 35 Lys Pro Phe Arg Tyr Lys Asp His Arg 50 Ser Tyr Leu Gly Ile Pro Tyr Ala Giu 60 65 Leu Arg Phe Gin Ser Pro Lys Pro Ile 75 Gly Phe Val Gin Ala Arg Thr Leu Gly 90 Glu Ser Leu Ile Tyr Ser Tyr Ala Gly l0S 110 Tyr Leu Asn Ile Phe Thr Pro Glu Thr 115 120 Asn Thr Lys Tyr Pro Val Met Phe Phe Phe rrp Asp krg Ile ksn Asp Pro Pro 3er Asn k.sp Lys 'er Glu lai Asn 125 INFORMATION FOR SEQ ID NO:12: -106- Wi SEQUENCE CHARACTERISTICS: LENGTH: 524 nucleotides TYPE: nucleic acid STRANDEIDNESS: single D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
AGAACATTAC
GTGAATATAT
TAGACTTTCC
CTGTTTTTGG
ATAGOCOOTT
CCTATOATCT
GAACTCTGCA
GGTGTAAATA
GTGAGGTAAC
TGTTATTATT
GACATGTCTA
AGGATATTTT
TCAGATATAA
TGGAAACATT
ATTTGATAT-T
CGGCATAAGG
TTATATCGGA
CAAATGCTGA
AGAAAAATAA
ATTTTTTATT
TATCTCGTTT
TCGTATCAAC
OTATTGTTCG
GCAATCTTCG
TGTCTCCCAA
OGTTTTGGAG
AATTCCCAAA
ACGGTTTTCC
TCTAGOTTAT
AAAAAATAAA
GTGTTTTTTA
TGTTTATGAT
GTTC
CAGAATTAAC
CTTCCTGCA.T
AGTTCGAGCC
ACTGAAATCG
TAAGAACAAT
TTCCGTGATC
CCCATAGTAT
AATAAAACTA
ATGCATTTTG
AAAATAGACG
AOTCTCTOGC
AAGAATATAT
TGTACGAATC
TAATGGTCCA'
ATACATCATT
CTGTTAAATT
GCACAAGATA
ATGCACTACT
GTTGCTTAAT
TTTTGAAGAC
100 150 200 250 ~300 350 400 450 500 524 INFORMATION FOR SEQ ID NO:13: i) SEQUENCE CHARiACTERISTICS: LENGTH: 1982 nucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: (iii) FEATURE:
NAME/KEY:
LOCATION:
(iv) FEATURE:
NAME/KEY:
LOCATION:
cDNA CDs 31. .1517 Asx Asn or Asp 300 SEQUENCE DESCRIPTIO'N: SEQ ID NO:13: AT TTT AOC TAC ACA OGT GTA CCT TAT GCT AAA CCT CCT GTT Phe Ser Tyr Thr Gly Val Pro Tyr Ala Lys Pro Pro Vr 1 5 OGA OAA CTT AGA TTT AAO CCT CCA CAG AAA OCT GAG CCA TG Gly Olu Leu Arg Phe Lys Pro Pro Gin Lys Ala Olu Pro Trp 20 CAA GOT OTT TTC AAC 0CC ACA TTA TAC OGA AAT OTO TOT AAA Gin Oly Val Phe Asn Ala Thr Leu Tyr Oly Asn Vai Cys .Lys 35 -107- TCT TTA AAT TTC TTC TTG AAG AAA ATT Ser Leu Asn Phe Phe Leu Lys Lys Ile 50 TGC TTG GTA GTA AAC GTG TAC GCA CCA GAA GGA GAC GAA GAC Giu Giy Asp Giu Asp AAA ACA ACT TCT GAT Lys Thr Thr Ser Asp 167 209 Cys Leu Val Val Asn Val.
Tyr Ala Pro
AAA
Lys AAA CTT CCA GTA Lys Leu Pro Val
TTT
Phe 75 TTC TGG GTT CAT GGT GGT GGT TTT Phe Trp Val His Gly Gly GJly Phe GTG ACT Val Thr GGA TCC GGA AAT Gly Ser Gly Asn GAA TTC CAA AGC Glu Phe Gin Ser CCA GAT TAT Pro Asp Tyr TTA GTA RAT Leu Val Asx 100 TTT GAT GTT ATT Phe Asp Val. Ile
TTC
Phe 105 GTA ACT TTC AAT Val Thr Phe Asn TAC CGA Tyr Arg 110 a TTG GGA CCT Leu Gly.Pro CCA GGA AAT 20 Pro Gly Asn
CTC
Leu 115 GGA TTT CTG AAT Gly Phe Leu Asn
TTG
Leu 120 GAG TTG GAG GGT GCT Glu Leu Glu Gly Ala GTA GGA Val Gly 130 TTA TTG GAT CAG Leu Leu Asp Gin GCA GCT CTG AAA Ala Ala Leu Lys
TGG
Trp 140 ACC AAA GAA AAC Thr Lys Giu Asn
ATT
Ile 145 GAG AAA TTT GGT Glu Lys. Phe Gly
GGA
Gly 150 GAT CCA GAA Asp Pro Glu GCA AGT GTT Ala Ser Val 165 25 AAT ATT ACA Asn Ile Thr 155 CAT TAT CTT His Tyr Leu 30 170 AGG GCA ATT Arg Ala Ile ATT GGT GGT GTT TCT GCT GGT GGA Ile Gly Gly Val Ser Ala Gly Gly 419 461 503 545 587 TTG TTA TCT CAT Leu Leu Ser His ACC ACT GGA CTT TAC AAA Thr Thr Gly Leu Tyr Lys CAA AGT GGA AGT GCT TTT AAT CCA TGG GCC Gin Ser Gly Ser Ala Phe Asn Pro Trp Ala TTC CAA AGA CAT Phe Gin Arg His
CCA
Pro 200 GTA AP G 2'GT AGT Val Lys Arg Ser CAA CTT GCT GAG Gin Leu Ala Glu
ATA
Ile 210 TTG GGT CAT CCC Leu Gly His Pro
ACA
Thr 215 AAC AAT ACT CAA GAT GCT TTA GAA Asn Asn Thr Gin Asp Ala Leu Giu 220 TTC TTA Phe Leu 225 CAA AAA GCC CCC Gin Lys Ala Pro
GTA
Val 230 GAC AGT CTC CTG Asp Ser Leu Leu AAG AAA ATG Lys Lys Met 235 -108- CCA GCT GAA Pro Ala Glu 240 ACA GAA GGT GAA ATA ATA GAA GAG TTT GTC TTC Thr Giu Giy Giu Ile Ile Giu Giu Phe Val Phe 245 250 755 797 GTA CCA TCA ATT GAA AAA GTT TTC CCA DVal Pro Ser Giu Lys Val Phe TCC CAC CAA CCT TTC Ser His Gin Pro Phe 265 TTG GAA GAA TCA Leu Glu Giu Ser AAC AAA GTA CCT Asn Lys Val. Pro 280
CCA
Pro 270
TTA
Leu TTG GCC AGA ATG AAA TCC GGA TCC TTT Leu Aia Arg Met Lys Ser Gly Ser Phe
TI'A
Leu 285 GTT GGA TTT Val Giy Phe 275
AAC
Asn
AGT
Ser 290 GCA GAA GGA Aia Giu Gly GAG ATG CTG Giu Met Leu' 305
S
S S
S
*5*S
*SSS
S
CTT TTG TTC Leu Leu Phe 295 AAC CAA GCT Asn Gin Aia 310 TTT GAA TTA 20 Phe Giu Leu AAA TTC TTC Lys Phe Phe AAA GAA AAA CCA Lys Glu Lys Pro GAA GCA GAT TTT Glu Ala Asp Phe
GAA
Giu 315 AGA CTC GTA CCA Arg Leu Vai Pro GCC GAA Ala Glu 320 923 965 1007 CAT GGA TCA GAG GAA TCG AAA AAA CTT His Gly Ser Giu Giu Ser Lys Lys Leu 330
GCA
A-1a 335 GAA AAA ATC AGG Glu Lys Ile Arg
AAG
Lys 340 TTT TAC TTT GAC Phe Tyr Phe Asp
GAT
Asp 345 AAA CCC GTT CCA Lys Pro Val Pro
GAA
Giu 350 AAT GAA CAG AAA Asn Giu Gin Lys
TTT
Phe 355 ATT GAC TTG ATA Ile Asp Leu Ile
GGA
Gly 360 GAT ATT TGG Asp Ile Trp TCT GTG GAG Ser Vai Glu 375 1049 1091 1133 1175 TTT ACT Phe Thr 30 365 AGA GGT GTT GAC Arg Gly Vai Asp
AAG
Lys 370 CAT GTC AAG TTG His Val Lys Leu AAA CAA GAC Lys Gin Asp 380 GAA CCA GTT TAT Giu Pro Val Tyr TAT GAA TAT TCC Tyr Giu Tyr Ser TTC TCG Phe Ser 390 GAA AGT CAT CCT GCA AAA GGA ACA TTT GGT GAT CAT PAT Giu Ser His Pro Ala Lys Giy Thr Phe Gly Asp His Asn 395 400
CTG
Leu 405 1217 1259 ACT GGT GCA TGC CAT GGA GAA GAA CTT GTG PAT TTA TTC AAA Thr Gly Ala Cys His Gly Giu Giu Leu Val Asn Leu Phe Lys
GTC
Val1 420' GAG ATG ATG PAG, CTG GA AAA GAT AAA Glu Met Met Lys Leu Giu Lys Asp Lys CCT PAT GTT CTA Pro Asn Val Leu 430 1301 -109- TTA ACA AAA GAT AGA GTA CTT GCC ATG TbG ACT AAC TTC ATC Leu Thr Lys Asp Arg Val Leu Ala Met Trp Thr Asn Phe Ile 1343 435 440 AAA AAT GGA Lys Asn Gly 450 GTT AAA TG Val Lys Trp, AAT CCT ACT Asn Pro Thr GAA CCT GCC Glu Pro Ala 465 CCT GAA GTA Pro Giu Val 455 ACA AAA GAC Thr Lys Asp 470 ACA GAA TTA TTG CCA Thr Glu Leu Leu Pro 460 1385 1427 AAG TTG AAT TAT Lys Leu Asn Tyr
TTG
Leu 475 AAC ATT GAT GCC ACC TTA Asn Ile Asp Ala Thr Leu 480 ACT TTG GGA ACA AAT CCT GAG GCA Thr Leu Gly Thr Asn Pro Glu Ala 485 GAA GAC GCC ACA AAA TCT TTG CAC Giu Asp Ala Thr Lys Ser Leu His 500 1469 AAC CGA Asn Arg 15 4-90 GTC AAA TTT TGG Val Lys Phe Trp 0 0 GGT CAA TAA TAATTTATGA AAATTGTTTT AAATACTTTA GGTAATATAT Gly Gin
TAGGTAAATA
TTATGTGTAT
TGAAATATCA
AAAATTTCCA
ATACACTAAA
AATAATTTTT
AATTTTTGTT
TTAGTATTTC
TATATTTAAA
AAAATTAAAA
CAGTTCTAAT
TGGTTTTAAT
ATAGATATGT
ATCAGTAAAA
TTTAAAGAAA
TAACTTAAAA
TGATATCATT
ATAAAATTAT
AATAACAATT
TTTATTTATT
TTTCAAAACA
TATATTAAGT
ATACATTAAC
ATACCAAAAA
ATAAAATTAA
AGTGAAAATT
GT
TTTATGTT'TT
TATTCTTGTT
CAACGTCGTT
ACTCTGAAGT
TAAAAATATA
TAAAGTAAAA
CTCTTCAATA
ATATTTTGAT
ATGTATTGGC
TTGCTTGTTT
TGTTTTTAGC
ATTTTTATAT
AGATATTTTC
TTCCAAACGG
ATTTTGATAA
AATACGTATT
1511 1560 1610 1660 1710 1760 1810 1860 1910 1960 1982 INFORMATION FOR SEQ ID NO:14: Wi SEQUENCE CHARACTERISTICS: LENGTH: 505 amino acids TYPE: amino acid TOPOLOGY: linear 0 0 00..
0 *000 (ii) MOLECULE TYPE: protein (iii) SEQUENCE DESCRIPTION: SEQ ID NO:14: Phe Ser Tyr Thr Gly Val Pro Tyr Ala Lys Pro Pro Val Gly 1 510 Giu Leu Arg Phe Lys Pro Pro Gin Lys Ala Giu Pro Trp Gin 20 Gly Val Phe Asn Ala Thr Leu Tyr Gly Asn Val Cys Lys Ser 35 Leu Asn Phe Phe Leu Lys Lys Ile Giu Gly Asp Giu Asp Cys Leu Val Val Asn Val. Tyr Ala Pro Lys Thr Thr Ser Asp Lys 65 Lys Leu Pro Val Phe Phe Trp Val His Gly Gay Gly Phe Val Thr Gay Ser Giy Asn Leu Glu Phe Gin Ser Pro Asp Tyr Leu 90 Val Asx Phe Asp Val Ile Phe Val Thr Phe Asn Tyr Arq Leu 100 105 110 Gly Pro Leu Gay Phe Leu Asn Leu Glu Leu Giu Gly Ala Pro 115 120 125 Gly Asn Val Gay Leu Leu Asp Gin Val Ala Ala Leu Lys Trp 130 135 140 Thr Lys Giu Asn Ile Giu Lys Phe Gly Gly Asp Pro Giu Asn 145 150 Ile Thr Ile Gly Gay Val Ser Ala Gay Gly Ala Ser Vai His *155 160 165 Tyr Leu Leu Leu Ser His Thr Thr Thr Gly Leu Tyr Lys Arg 170 175 180 Ala Ile Ala Gin Ser Gly Ser Ala Phe Asn Pro Trp Ala Phe 185 190 195 VGin Arg His Pro Val Lys Arg Ser Leu Gi'2n Leu Ala Giu Ile 200 205 210 LeuG- yHi Pro TrAsn Asn Thr Gin Asp Aia Leu Giu Phe .215 220 Leu Gin Lys Ala Pro Vai Asp Ser Leu Leu Lys Lys Met Pro 225 230 235 Ala Glu Thr Giu Giy Giu Ile Ile Glu Giu Phe Val Phe Val 240 245 250 Pro Ser Ile Giu Lys Val Phe Pro Ser His Gin Pro Leu Leu 255 260 265 Glu Giu Ser Pro Leu Ala Arg Met Lys Ser Gay Ser Phe Asn 270 275 280 Lys Val Pro Leu Leu Val Gly Phe Asn Ser Ala Giu Giy Leu 285 290 Leu Phe Lys Phe Phe Met Lys Giu Lys Pro Giu Met Leu Asn 295 300 305 Gin Ala Glu Ala Asp The Glu Arg Leu Val Pro Ala Giu Phe 310 Giu Leu Lys Ile Asn Giu Val 325 Arg Gin His Lys 340 Lys Gly Ser -Giu Phe Tyr Phe Phe Ile Asp 355 Giu Ser Lys Lys 330 Asp Asp Lys Pro 345 Leu Ile Gly Asp Leu Vai Aia 335 Pro Giu Giu 350 Phe 360 Thr Arg Giy Vai Asp 365 Gin Asp Giu Pro Vai .4 4* .4 4 '4 4* 4 4 *4*4 4**4 4 4**4 4* .4 4 4 444.
*444*4 4 ,.44.
4 4 *44* .4 4 4.44 4* 44 4 4 4 *444 4 4444 380 Ser His Giy Aia 15 Giu Met Thr Lys 435 Asn Giy 20 450 Lys Trp Ile Asp Pro 395 Cys Met Asp Asn Giu 465 Aila Aila His 410 Lys Arg Pro Pro Thr Lys Giy Leu 425 Vai Thr Aia Leu Lys His Vai 370 Tyr Tyr Tyr 385 Giy Thr Phe 400 Giu Giu Leu Giu Lys Asp Leu Aia Met 440 Pro Giu Val 455 Thr Lys Asp Lys Giu Giy Val 415 Lys Trp Thr Leu Tyr Asp Asn Pro 430 Thr Giu Ser 375 Ser His Leu Asn Asn 445 Leu Vai Phe 390 As n Phe Vai Phe Leu 460 Giu Ser Leu 405 Lys Leu Ile Pro Lys Giu Thr Vai 420 Leu Lys Val 470 Lys Leu Asn Tyr Leu Asn 475 Thr Leu Giy Thr Asn Pro Giu Aia Asn 485 490 480 Arg Vai Lys Phe Trp Giu Asp Ala Thr Lys Ser Leu His Gi' 495 500 Gin 505 INFORMATION FOR SEQ ID Ci) SEQUENCE CHARACTERISTICS: LENGCH: 1982 nucieotides TYPE: nucleic acid STRANDEDNESS: singie TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) SEQUENCE DESCRIPTION: SEQ ID NO:iS: ACATAATTTT ATTTTAAATA TAAATACGTA TTATCAAAT
ATAATTTTCA
CTAATGATAT CAGAAATACT AATTATCAAA ATTATTGAAG
AGTTAATTTT
ATTTTTAAGT TAAACAAAAA TTCCGTTTGG AATTTTACTT
TATTTTTGGT
y s0 100 150 -112- ATTTTCTTTA
AAAAAAATT
ATTTTTACTG
ATTTTAGTG
TAACATATCT
ATTGGAT
AAATTAAAAC
CATGATATT
AAATTAGAAC
TGATACACA'
TT TTT TAATT TTTATTTAC( TTTCATAAAT
TATTATTGA(
ATTTGACTCG
GTTTGCCTC;
ATGTTCAAAT
AATTCAACTJ
CAATAATTCT
GTTACTTCAC
TCCACATGGC
AAGTACTCTA
TTTTCCAGCT
TCATCATCTC
ATGGCATGCA
CCAGTCAGAT
*-GACTTTCCGA.
GAAGGAATAT
TCCACAGACA
ACTTGACATG
*TCCTATCAAG
TCAATAAATT
CAAAGTAAA
CTTCCTGATT
CCATGGACTA
ATTCAAATTC
AGCTTGGTTC
AGCATCTCTG
20 GTCCTTCTGC
ACTGTTAAAT
CCGGATTTCA
TTCTGGCCA
TGGGAAAACT
TTTTCAATTG
CACCTTCTGT
TTCAGCTGGC
TTTTGTAAGA
ATTCTAAAGC
25 TATCTCAGCA
AGTTGAAGAC
ATGGATTA
AGCACTTCCA
GTGGTTGTAT
GAGATAACAA
AACACCACCA
ATTGTAATAT
TTTCTTTGGT
CCATTTCAGA
30 CCTGGAGCAC
CCTCCAACTC
GTAATTGAAA
GTTACGAAAA
TTTGGAATTC
TAAATTTCCG
CAGAAAAATA
CTGGAAGTTT
GTTTACTACC
AAGCAGTCTT
35 TTAAAGATTT
ACACACATTT
CATGGCTCAG
CTTTCTGTGG
TTTAGCATAA
GGTACACCTG
ATTGAAAATA'.
T ATATATAAAJU T TTGCTAAAAJ T CAAAACAAGC T' AAGCCAATAc
CTAATATATT;
7CGTGCAAAGA
~GGATTTGTTC
GTCTTTTGTG
GAGTAGGATT
TCTTTTGTTA
GACTTTGAAT
*TATGATCACC
*TCATAATAAT
CTTGTCAACA
TCTGTTCATT
TTTTCTGCAA
GGCTGGTACG
GTTTTTCTTT
CCAACTAATA
TGGTGATTCT
ATGGTACGAA
ATTTTCTTCA.
ATCTTGAGTA
TACGCTTTAC
CTTTGAGCAA
AAGATAATGA
TTTCTGGATC
GCTGCCACCT
CAAATTCAGA
TAACATCAAA
GATCCAGTCA
TTTATCAGA
CGTCTCCTTC
CCGTATAATG
AGGCTTA.AAT
TGTAGCTAAA
9 9 99 9 *99* *99* .999 .9 9* *4 99** *9*9 9**9 9 r CTTATATTTT k ATACTTCAG.
k. CAAACGACGT
'AAAACAAGAA
7ATAAAACATA
LCCTAAAGTAT
TTTTGTGGCG
*CCAAAGTTAA
GCAGGTTCCC
*TCCATTTTTG
ATAGAACATTr
AAATTCACAA
AAATGTTCCT
AAACTGGTTC
CCTCTAGTAA2
TTCTGGAACG
GTTTTTTCGA
I
AGTCTTTCAA
CATGAAGAAT
I
AAGGTACTTT
G
TCCAAGAAAG
G
GACAAACTCT
T
GGAGACTGTC
TI
TTGTTTGTGG
G
TGGATGTCTT
T
TTGCCCTTTT 0 ACACTTGCTC
C
TCCACCAAAT
T
GATCCAATAA
T
AATCCGAGAG
G
ATYTACTAAA
T
CAAAACCACC
A
GTTGTTTTTG,
G
AATTTTCTTC
A
TGGCGTTGAA
A
CTAAGTTCTC
C.
AT
TAGTTAATGT
GTACTTAATA
TGTGTTTTGA
TAAATAAATA
AAAATTGTTA
TTAAAACAAT
TCTTCCCAA
GGTGGCATCA
ATTTAACTGG
ATGAAGTTAG
PGGTTTATCT
GTTCTTCTCC
TTTGCAGGAT
3TCTTGTTTC k.CCAAATATC
'GTTTATCGT
~TCCTCTGAT
LATCTGCTTC
~TGAACAAAA
TTAAAGGAT
;TTGGTGGGA
CTATTATTT
ACGGGGGCT
ATGACCCAA
'GGAAGGCCC
TAAAGTCCA
ACCAGCAGA
TCTCAATGT
CCTACATTT
TCCCAATCG
AATCTGGGC
CCATGAACC
TGCGTACAC
PGAAGAAAT
ALCACCTTGC
1AACAGGAGG 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 1982 INFORMATION FOR SEQ ID NO:16: Wi SEQUENCE
CHARACTERISTICS:
LENGTH: 1515 nucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: ii) FEATURE:
NAME/KEY:
LOCATION:
FEATURE:
NAME/ICEY:
LOCATION:
cDNA CDs 1. .1515 Asx Asn or Asp 298 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: -113- TTT AGC TAC ACA GGT GTA. CCT TAT GCT AAA CCT CCT GTT Phe Ser Tyr Thr Gay Val Pro Tyr Ala Lys Pro Pro Val GGA GAA CTT Gly Glu Leu AGA TTT AAG Arg Phe Lys
CCT
Pro 20 CCA CAG AAA GCT Pro Gin Lys Ala GAG CCA TGG Giu Pro Trp CAA GGT GTT Gin Gly Val TTC AAC GCC ACA TTA TAC GGA AAT GTG Phe Asn Ala Thr Leu Tyr Gly Asn Val 35 TCT TTA AAT Ser Leu Asn TGT AAA Cys Lys GAA GAC Giu Asp
TTC
Phe TTC TTG AAG AAA Phe Leu Lys Lys ATT GAA GGA GAC Ile Giu Gly Asp TGC TTG GTA GTA Cys Leu Val Val GTG TAC GCA CCA Val Tyr Ala Pro ACA ACT TCT GAT Thr Thr Ser Asp 9.
*9 9 *9 9 .9.9 **99 9 9**9 S. 4 9*
S.
*599 9 5999 0 *99* 9 909 9 9*95 9 9 9*99 .9 9.
9 9
S
165 207 249 291
AAA
Lys 70 AAA CTT CCA GTA Lys Leu Pro Val TTC TGG GTT CAT Phe Trp Val His
GGT
Gly GGT GGT TTT Gly Gly Phe GTG ACT 20 Val Thr GGA TCC GGA AAT Gly Ser Gly Asn
TTA
Leu 90 GAA TTC CAA AGC CCA GAT TAT Giu Phe Gin Ser Pro Asp Tyr TTA OTA PAT Leu Vai Asx 100 TTG GGA CCT Leu Gay Pro TTT GAT GTT ATT TTC GTA ACT TTC AAT Phe Asp Val Ile Phe Val Thr Phe Asn 105 TAC CGA Tyr Arg 110
CTC
Leu 115 GGA TTT CTG AAT Gly Phe Leu Asn
TTG
Leu 120 GAG TTG GAG GGT Glu Leu Glu Gly
GCT
Al a 125 CCA GGA AAT GTA Pro Gly Asn Val
GGA
Gly 130 TTA TTG GAT CAG GTG GCA GCT CTG AAA Leu Leu Asp Gin Val Ala Ala Leu Lys TGG ACC Trp Thr 140 AAA GAA AAC ATT GAG AAA TTT GGT GGA GAT CCA GAA Lys Glu Asn Ile Glu Lys Phe Gly Gly Asp Pro Giu 145 1 CA AAT ATT Asn Ile 155 ACA ATT GGT GGT Thr Ile Gly Giy
GTT
Val 160 TCT GCT GGT GGA Ser Ala Gly Gly GCA AGT GTT Ala Ser Val 165 CAT TAT CTT His Tyr Leu 170 AGG GCA ATT Arg Ala Ile TTG TTA TCT CAT Leu Leu Ser His ACC ACT GGA CTT Thr Thr Gly Leu TAC AAA Tyr Lys 180 CAA AGT GGA AGT Gin Ser Gly Ser
GCT
Al a 190 TTT AAT CCA TGG Phe Asn Pro Trp -114- TTC CAA AGA CAT CCA GTA AAG CGT AGT Phe Gin Arg His Pro Val Lys Arg Ser 200
CTT
Leu 205 CAA CTT GCT GAG Gin Leu Ala Glu
ATA
Ile 210 TTG GGT CAT CCC Leu Gly His Pro
ACA
Thr 215 AAC AAT ACT CAA Asn Asn Thr Gin
GAT
Asp 220 GCT TTA GAA Ala Leu Glu AAG AAA ATG Lys Lys Met 235 669 711 TTC TTA Phe Leu 225 CAA AAA GCC CCC Gin Lys Ala Pro GAC AGT CTC CTG Asp Ser Leu Leu CCA GCT GAA Pro Ala Glu 240 ACA GAA GGT GAA Thr Giu Gly Glu
ATA
Ile 245 ATA GAA GAG TTT Ile Giu Giu Phe GTC TTC Val Phe 250 GTA CCA TCA Val Pro Ser
ATT
lie 255 GAA AAA GTT TTC Glu Lys Val Phe TCC CAC CAA CCT Ser His Gin Pro
TTC
Leu 265 6O 00
S
SS
S @565 0@
S.
5
ES
S
S
0
S
TTG GAA GAA TCA Leu Giu Giu Ser
CCA
Pro 270 TTG GCC AGA ATG Leu Aia Arg Met
AAA
Lys 275 TCC GGA TCC TTT Ser Gly Ser Phe 753 795 837 879 921
AAC
20 Asn 280 AAA GTA CCT TTA Lys Val. Pro Leu
TTA
Leu 285 GTT GGA TTT AAC Val Gly Phe Asn
AGT
Ser 290 GCA GAA GGA Ala Gu Gly GAG ATG CTG Glu Met Leu 305 CTT TTG Leu Leu 295 TTC AAA TTC TTC Phe Lys Phe Phe AAA GAA AAA CCA Lys Giu Lys Pro AAC CAA GCT Asn Gin Ala 310 TTT GAA TTA Phe Giu Leu GAA GCA GAT TTT Glu Ala Asp Phe
GAA
Glu 315 AGA CTC GTA CCA Arg Leu Val Pro GCC GAA Ala Glu 320
GTC
Val 325 CAT GGA TCA GAG His Gly Ser Glu TCG AAA AAA CTT Ser Lys Lys Leu
GCA
Ala 335 GAA AAA ATC AGG Glu Lys Ile Arg
AAG
Lys 340
AAA
Lys TTT TAC TTT GAC Phe Tyr Phe Asp TTT ATT GAC TTG Phe Ile Asp Leu
GAT
Asp 345
ATA
112 AAA CCC GTT CCA Lys Pro Vai Pro 963 1005 1047 1089 1131 GAA AAT Glu Asn 350 TTT ACT Phe Thr 365 GAA CAG Glu Gin 355 AGA GGT GTT GAC Arg Giy Val Asp GAT ATT TGG Asp Ile Trp TCT GTG GAG Ser Val Glu 375 CAT GTC AAG TTG His Val Lys Leu AAA CAA GAC Lys Gin Asp 380 GAA CCA GTT TAT Glu Pro Val Tyr
TAT
Tyr 385 TAT GAA TAT TCC Tyr Giu Tyr Ser TTC TCG Phe Ser 390 1173 -115- GAA AGT CAT CCT GCA AAA GGA ACA TTT GGT GAT CAT AAT CTG Giu Ser His Pro Ala Lys Gly Thr Phe Gly Asp His Asn Leu 395 400 405 1215 ACT GGT GCA TGC CAT GGA GAA GAA Thr Gly Ala Cys His Gly Giu Glu 410 CTT GTG AAT TTA TTC AAA~ Leu Val Asn Leu Phe Lys 415 GAT AAA CCT AAT GTT CTA Asp Lys Pro Asn Val Leu 430 1257 GTC GAG ATG ATG AAG Val Giu Met Met Lys CTG GAA AAA Leu Glu Lys 425 1299 TTA ACA Leu Thr 435 AAA GAT AGA Lys Asp Arg GTA CTT Val Leu 440 GCC ATG TGG ACT Ala Met Trp Thr AAC TTC ATC Asn Phe Ile 445 TTA TTG CCA Leu Leu Pro 460 1341 1383 AAA AAT GGA Lys Asn Gly 450 AAT CCT ACT, CCT Asn Pro Thr Pro GTA ACA GAA Val Thr Glu 0000 0 0000 GTT AAA TGG Val Lys Trp
GAA
Giu 465 CCT GCC ACA AAA Pro Ala Thr Lys
GAC
Asp 470 AAG TTG AAT TAT Lys Leu Asn Tyr AAC ATT GAT GCC ACC TTA ACT TTG GGA ACA Asn Ile Asp Ala Thr Leu Thr Leu Gly Thr 480 485 AAT CCT GAG GCA Asn Pro Glu Ala AAA TCT TTG CAC Lys Ser Leu His 500 1425 1467 1509
AAC
Asn 490 CGA GTC AAA TTT Arg Val Lys Phe TGG GAA GAC GCC ACA Trp Giu Asp Ala Thr 495 GGT CAA Gly Gin INFORMATION FOR SEQ ID NO:17: i) SEQUENCE CHARACTERISTICS: LENGTH: 1515 nucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear I515 (ii) MOLECULE TYPE: cDNA (iii) SEQUENCE DESCRIPTION: SEQ ID NO:17:
TTGACCGTGC
CCTCAGGATT
AACTTGTCTT
TTCAGGAGTA
CTCTATCTTT
ATCTCGACTT
CAGATTATGA
AATATTCATA
ACATGCTTGT
AAATTTCTGT
AAAGATTTTG
TGTTCCCAAA
TTGTGGCAGG
GGATTTCCAT
TGTTAATAGA
TGAATAAATT
TCACCAAATG
ATAATAAACT
CAACACCTCT
TCATTTTCTG
TGGCGTCTTC
GTTAAGGTGG
TTCCCAT'rTA
TTTTGATGAA
ACATTAGGTT
CACAAGTTCT
TTCCTTTTGC
GGTTCGTCTT
AGTAAACCAA
GAACGGGTTT
CCAAAATTTG
CATCAATGTT
ACTGGCAATA
GTTAGTCCAC
TATCTTTTTC
TCTCCATGGC
AGGATGAT
GTTTCTCCAC!
ATATCTCCTA
ATCGTCAAAG
ACTCGGTTTG
CAAATAATTC
ATTCTGTTAC
ATGGCAAGTA
CAGCTTCATC
ATGCACCAGT
TCCGAGAAGG
AGACAACTTG
TCAAGTCAAT
TAAAACTTCC
100 150 200 250 300 350 400 450 500 -116- TGATTTTTTC TGCAAGTTT7 AATTCGGCTG
GTACGAGTCT
CTCTGGTTTT
TCTTTCATGA
TAAATCCAAC
TAATAAAGGT
GCCAATGGTG
ATTCTTCCA
AATTGATGGT
ACGAAGACA
CTGGCATTTT
CTTCAGGAGA
AAAGCATCTT GAGTATTGTT AAGACTACGC TTTACTGGAT TTCCACTTTG
AGCAATTGCC
AACAAAAGAT
AATGAACACT
AATATTTTCT
GGATCTCCAC
TCAGAGCTGC
CACCTGATCC
AACTCCAAAT TCAGAAATCC GAAAATAACA
TCAAAATYTA
TTCCGGATCC AGTCACAAAA AGTTTTTTAT CAGAAGTTGT GTCTTCGTCT
CCTTCAATTT
*CATTTCCGTA
TAATGTGGCG
TGTGGAGGCT TAAATCTAAG ACCTGTGTAG
CTAAA
*TTCGATTCC7I
TTCAAAATCT
AGAATTTGAA
ACTTTGTTAA
GAAAGGTTGG
ACTCTTCTAT
CTGTCTACGG
TGTGGGATGA
GTCTTTGGAA.
CTTTTGTAAA
TGCTCCACCA
CAAATTTCTC
AATAATCCTA
GAGAGGTCCC
CTAAATAATC
CCACCACCAT
TTTTGGTGCG
TCTTCAAGAA
TTGAAAACAC
TTCTCCAACA
CTGATCCATG
GCTTCAGCTT
CAAAAGTCCT
AGGATCCGGA
TGGGATGGGA
TATTTCACCT
GGGCTTTTTG
CCCAATATCT
GGCCCATGGA
GTCCAGTGGT
GCAGAAACAC
AATGTTTTCT
CATTTCCTGG
AATCGGTAAT
TGGGCTTTGG
GAACCCAGAA
TACACGTTTA
GAAATTTAAA
CTTGCCATGG
GGAGGTTTAG
GACTAATTCA
GGTTCAGCAT
TCTGCACTGT
TTTCATTCTG
AAACTTTTTC
TCTGTTTCAG
TAAGAATTCT
CAGCAAGTTG
TTAAAAGCAC
TGTATGAGAT
CACCAATTGT
TTGGTCCATT
AGCACCCTCC
TGAAAGTTAC
AATTCTAAAT
AAATACTGGA
CTACCAAGCA
GATTTACACA
CTCAGCTTTC
CATAAGGTAC
550 G00 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1j50 1400 1450 1500 1515 INFORMATION FOR SEQ ID NO:18: i) SEQUENCE CHARACTERISTICS: LENGTH:' 1792 nucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: ii) FEATURE:
NAM~E/KEY:
LOCATION:
cDNA
CDS
49. .1701 (iv) SEQUENCE DESCRIPTION: SEQ ID NO:18: ACTGTGTGCT AATAATTCAG TACACACAGT CAATAGTCTA GATCCAAG ATG 'TCT CGT GTT ATT TTT TTA AGT TGT ATT TTT TTG TTT AGT Met Ser Arg Val Ile Phe Leu Ser Cys Ile Phe Leu Phe Ser
TTT
Phe AAT TTT ATA AAA Asn Phe Ile Lys GAT CCC CCG ACT GTA ACT TTG CCC Asp Pro Pro Thr Val Thr Leu Pro CAG GGC Gin Gly GAA TTG GTT GGA AAA GCT TTG ACG AAC GAA AAT GGA Glu Leu Val Gly Lys Ala Leu Thr Asn Glu Asn Gly AAA GAG TAT TTT AGC Lys Glu Tyr Phe Ser TAC ACA GGT GTG Tyr Thr Gly Val TAT GCT AAA CCT Tyr Ala Lys Pro -50 -117- CCA GTT GGA GAA CTT AGA TTT AAG CCT CCA CAG AAA GCT GAG Pro Val Gly Glu Leu Arg Phe Lys Pro Pro Gin Lys Ala Glu CCA TGG AAT Pro Trp, Asn GGT GTT TTC AAC GCC Gly Val Phe Asn Ala ACA TCA CAT Thr Ser His GGA PAT GTG Gly Asn Val
TGC
Cys AAA GCT TTG AAT Lys Ala Leu Asn TTC TTC TTG AAA Phe Phe Leu Lys 90 AAA ATT GPA GGA GAC Lys Ile Glu Giy Asp GCA CCA AAA ACA ACT Ala Pro Lys Thr Thr 110 342 384 GAA GAC Glu Asp 100 TGC TTG TTG GTG Cys Leu Leu Val
PAT
Asn 105 GTG TAC Val Tyr
S
TCT GAC AA Ser Asp Lys 15 115 GGT TTT GTG Gly Phe Val AAA CTT CCA GTA Lys Leu Pro Val *TTC TGG GTT CAT Phe Trp Val His GGT GGC Gly Gly 125
ACT
Thr 130 GGA TCC GGA AAT Gly Ser Gly Asn
TTA
Leu 135 GAA TTT CAA AGC Giu Phe Gin Ser 426 468 510 552 GAT TAT TTA GTA Asp Tyr Leu Val TAT GAT GTT ATT TTT Tyr Asp Val Ile Phe 150 GTA ACT TTC PAT Val Thr Phe Asn CGA TTG GGA CCA Arg Leu Gly Pro GGA TTT TTG PAT Gly Phe Leu Asn
TTG
Leu 165 GAG TTG GA Giu Leu Giu GTA GCA GCT Vai Ala Ala 180 25 GGT GCT Gly Ala 170 CCT GGA PAT GTA Pro Gly Asn Val
GGA
Gly 175 TTA TTG GAT CAG Leu Leu Asp Gin TTG A.AA TGG Leu Lys Trp 30 185 CCA GPA PAT Pro Giu Asn ACC AAA GPA PAT Thr Lys Giu Asn
ATT
Ile 190 GAG AAA TTT GGT Glu Lys Phe Gly GGA GAT Gly Asp 195
ATT
Ile 200 ACA ATT GGT GOT Thr Ile Gly Gly
GTT
Val 205 TCT GCT GGT GGA Ser Ala Gly Giy
GCA
Al a 210 636 678 720 762 AGT GTT CAT TAT Ser Vai His Tyr
CTT
Leu 215 TTA TTG TCA CAT Leu Leu Ser His ACC ACT GGA CTT Thr Thr Gly Leu
TAC
Tyr 225 APA AGG GCA ATT Lys Arg Ala Ile CAA AGT GGA AGT Gin Ser Gly Ser TTA P-AT CCA Leu Asn Pro TOG GCC Trp Ala 240 TTC CPA AGA CAT Phe Gin Arg His
CCA
Pro 245 GTA PAG CGT AGT CTT CPA CTT Val Lys Arg Ser Leu Gin Leu -118- GCT GAG ATA TTA GGT CAT CCC ACA AAC Ala Giu Ile Leu Gly His Pro Thr Asn 255 260 AAC ACT CAA GAT GCT Asn Thr Gin Asp Ala 265 TTA GAA TTC Leu Glu Phe
TTA
Leu 270 CAA AAA GCC Gin Lys Ala CCA GTA Pro Val 275 GAC AGT CTC CTG Asp Ser Leu Leu Lys AAA ATG CCA GCT Lys Met Pro Ala GAA ACA Giu Thr 285 GAA GGT GAA Glu Gly Giu
ATA
Ile 290 ATA GAA GAG TTC Ile Giu Giu Phe
GTC
Val 295 TTC GTA CCA TCA Phe Val Pro Ser
ATT
Ile 300 GAA AAA GTT TTC Giu Lys Val Phe TCC CAC CAA Ser His Gin AAA TCT GGA Lys Ser Gly 320 CCT TTC TTG GAA GAA 0.0.
*.0.0 Pro Phe 310 L eu Glu Giu TCA CCA Ser Pro 315 TTG GCC AGA ATG Leu Ala Arg Met 888 930 972 1014 1056 1098 1140 1182 TCC TTT AAC Ser Phe Asn 325 GAA GGA CTT 20 Giu. Giy Leu AAA GTA CCT Lys Val Pro TTA GTT*GGA TTC AAC AGC GCA Leu 330 Vai Giy Phe Asn Ser Ala
TTG
Leu 340 TAC AAA TTC TTT Tyr Lys Phe Phe
ATG
Met 34 5 AAA GAA AAA CCA Lys Giu Lys Pro
GAG
Giu ATG CTG AAC CAA Met Leu Asn Gin
GCT
Ala 355 GAA GCA GAT TTC Giu Ala Asp Phe AGA CTC GTA CCA Arg Leu Val Pro GAA .TCG AAA AAA Glu Ser Lys Lys 375
GCC
Al a 365 GAA TTT GAA TTA Giu Phe Giu Leu
GCC
Al a 370 CAT GGA TCA GAA His Gly Ser Giu CTT GCA Leu Ala 380 GAA AAA ATC AGG Giu Lys Ile Arg TTT TAC TTT GAC Phe Tyr Phe Asp GAT AAA CCC Asp Lys Pro 390 1224 GTT CCT GAA Val Pro Giu 395 AAT GAG CAG AAA Asn Giu Gin Lys TTT ATT Phe Ile 400 GAC TTG ATA Asp Leu Ile GGA GAT Gly Asp' 1266 ATT TGG TTT Ile Trp Phe ACT AGA Thr Arg 410 GGC ATT GAC AAG CAT GTC AAG TTG Giy Ile Asp Lys His Val Lys Leu 415
TCT
Ser GTA GAA AAA CAA Val Glu Lys Gin
GAC
Asp 425 GAG CCA GTA TAT TAT TAT GAA TAT TCT Giu Pro Val Tyr Tyr Tyr Glu Tyr Ser 430 1308 1350 1392
TTC
Phe 435 TCT GAA AGT CAT CCT GCA AAA GGA ACA Ser Glu Ser His Pro Ala Lys Gly Thr
TTT
Phe 445 GGT GAC CAT Gly Asp His -119- AAC TTG ACT GGA GCA TGT CAT GGT GAA GAA CTT GTG AAT TTA Asn Leu Thr Gly Ala Cys His Gly Glu Giu Leu Val Asn ILeu 450. 455 460 1434 TTC AAA GTC Phe Lys Val 465 GAG ATG ATG AAG Glu Met Met Lys
CTG
Leu 470 GAA AAA GAT AAA Giu Lys Asp Lys CCG AAT Pro Asn 475 1476 GTT TTA TTA ACA AAA GAT AGG GTA CTT GCT Val Leu Leu Thr 480 Lys Asp Arg Val Leu 485 Ala ATG TGG ACG Met Trp Thr
AAC
Asn 490 1560 TTC ATC AAA AAT Phe Ile Lys Asn
GGA
Gly 495 AAT CCT ACT CCT Asn Pro Thr Pro GAA GTA ACT GAA TTA Glu Val Thr Giu Leu 500
TTG
Leu 15 s5 CCA GTT AAA TGG Pro Val Lys Trp
GAA
Glu 510 CCT GCC ACA AAA GAC AAG TTG AAT Pro Ala Thr Lys Asp Lys Leu Asn TAT TTG Tyr Leu 520 AAC ATT GAT GCC Asn Ile Asp Ala TTA ACT TTG Leu Thr Leu GGA ACA AAT CCA Gly Thr Asn Pro 530 GCC ACA AAA ACT Ala Thr Lys Thr 545 1602 1644 1686 1731 GAA GAA ACC CGA Glu Glu Thr Arg 535 GTC AAA TTY TGG GAA GAT Val Lys Phe Trp Glu Asp TTG CAC AGT CAA TAA AAATGTATGA AAATTGTTTT
AA'ITATTTTA
Leu His Ser Gin 550 25 GGTAATACAT TAGGTAAATA AAAATTNAAA AATAACNAAA ~AAAAAAAA AAAAAA
A
1781 1792 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 550 amino acids (1B) TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: protein ii) SEQUENCE DESCRIPTION: SEQ ID NO:19: Ser Arg Val Ile Phe Leu Ser Cys Ile Phe Leu Phe Ser 5 Met 1 Phe Asn Phe Gin Gly Glu Ile Lys Cys Asp Pro Pro Thr Val Thr Leu Pro 20 Leu Val Gly Lsys Ala Leu Thr Asn Giu Asn Gly Lys Glu Tyr Phe Ser Tyr Thr Gly Val Pro Tyr Ala Lys Pro 50
SS
-120- Pro Val Gly Glu Leu Arg Phe Lys Pro Pro Gin Lys Ala Glu 65 Pro Trp Asn Gly Val Phe Asn Ala Thr Ser His Gly Asn Val Cys Lys Ala Leu Asn Phe Phe Leu Lys Lys Ile Glu Gly Asp 90 Glu Asp Cys Leu. Leu Val Asn Val Tyr Ala Pro Lys Thr Thr 100 105 110 Ser Asp Lys Lys Leu Pro Val Phe Phe Trp Val His Gly Gly 115 120 125 Gly Phe Val Thr Gly Ser Gly Asn Leu Glu Phe Gin Ser Pro 130 135 140 As y LuVlAsn TrAsp Va Il nPe Va Tr PeAsn *.145 150 Tyr Arg Leu Gly Pro Leu Gly Phe Leu Asn Leu Giu Leu Giu 0**155 160 165 Gly Ala Pro Gly Asn Val Gly Leu Leu Asp Gin Val Ala Ala 170 175 180 Leu Lys Trp Thr Lys Giu Asn Ile Giu Lys Phe Gly Gly Asp 185 190 195 Pro Glu Asn Ile Thr Ile Gly Gly Val Ser Ala Gly Gly Ala .200 205 210 Ser Vai His Tyr Leu Leu Leu Ser His Thr Thr Thr Gly Leu 215 220 Tyr Lys Arg Ala Ile Ala Gin Ser Gly Ser Ala Leu Asn Pro *225 230 235 Trp Ala Phe Gin Arg His Pro Val Lys Arg Ser Leu Gin Leu 240 245 250 Ala Giu Ile Leu Gly His Pro Thr Asn Asn Thr Gin Asp Ala 255 260 265 Leu Giu Phe Leu Gin Lys Ala Pro Val Asp Ser Leu Leu Lys 270 275 280 Lys Met Pro Ala Giu Thr Giu Gly Giu Ile Ile Glu Giu Phe 285 290 Vai Phe Val Pro Ser Ile Glu Lys Val Phe Pro Ser His Gin 295 300 305 Pro Phe Leu Giu Glu Ser Pro Leu Ala Arg Met Lys Ser Gly 310 i -Ser Phe Asn Lys Val Pro Leu Leu Val Gly Phe Asn Ser Ala 325 330 335 Giu Gly Leu Leu Tyr Lys Phe Phe Met Lys Glu Lys Pro Giu 340 345 350 Met Leu Asn Gin Ala Glu Ala Asp Phe Glu Arg Leu Val Pro 355 360 Ala Giu Phe Giu Leu Ala His Gly Ser Giu Glu Ser Lys Lys 365 370 375 Leu Ala Giu Lys Ile Arg Lys Phe Tyr Phe Asp Asp Lys Pro 380 385 390 Val Pro Glu Asn Glu Gin Lys Phe Ile Asp Leu Ile Gly Asp 395 400 405 I..le Trp Phe Thr Arg Gly Ile Asp Lys His Val Lys Leu Ser *..410 415 420 Val Giu Lys Gin Asp Giu Pro Val Tyr Tyr Tyr Giu Tyr Ser 425 430 Phe Ser Giu Ser His Pro Ala Lys Gly Thr Phe Gly Asp His 435 440 445 Asn Leu Thr Giy Aia Cys His Gly Giu Glu Leu Val Asn Leu 450 455 460 Phe Lys Val Giu Met Met Lys Leu Giu Lys Asp Lys Pro Asn 465 470 475 *Val Leu Leu Thr Lys Asp Arg Val Leu Ala Met Trp, Thr Asn 480 485 490 Phe Ile Lys Asn Gly Asn Pro Thr Pro Glu Vai Thr Glu Leu *..49550 Leu Pro Val Lys Trp Giu Pro Ala Thr Lys Asp Lys Leu Asn 505 510 515 Tyr Leu Asn Ile Asp Ala Thr Leu Thr Leu Gly Thr Asn Pro 520 525 530 Giu Giu Thr Arg Vai Lys Phe Trp Giu Asp Ala Thr L~ys Thr 535 540 545 Leu His Ser Gin 550 INFORMATION FOR SEQ ID SEQUENCE
CHARACTERISTICS:
LENGTH: 1792 nucleotides TYPE: nucleic acid STRANDEDNESS: single -122- TOPOLOGY: linear (ii) MOLECULE
TYPE:
cDNA (iii) SEQUENCE DESCRIPTION: SEQ ID S S. S S S
S
*SS*
S
*5 S S S. S.
S
*SSS
TTTTTTTTTT
TTTTTTTTTI
AATGTATTAC CTAAAATAA7 GTGCAAAGTT
TTTGTGGCAI
GATTTGTTCC
CAAAGTTAAG
TCTTTTGTGG
CAGGTTCCCA
AGTAGGATTT
CCATTTTTGA
CTTTTGTTAA
TAAAACATTC
ACTTTGAATA
AATTCACAAG
ATGGTCACCA
AATGTTCCTT
CATAATAATA
TACTGGCTCG
TTGTCAATGC
CTCTAGTAAA
CTGCTCATTT
TCAGGAACGG
TTTCTGCAAG
TTTTTTCGAT
GCTGGTACGA
GTCTTTCGA
TTTTTCTTTC
ATAAAGAATT
CAACTAATAA
AGGTACTTTG
20 GGTGATTCTT
CCAAGAAAGG
TGGTACGAAG
ACGAACTCTT
TTTTTTTCAG
GAGACTGTCT
TCTTGAGTGT
TGTTTGTGGG
ACGCTTTACT
GGATGTCTTT
25 TTTGAGCAAT
TGCCCTTTTG
AGATAATGAA
CACTTGCTCC
TTCTGGATCT
CCACCAAATT
CTGCTACCTG
ATCCAATAAT
AAATTCAAAA
ATCCGAGTGG
30 AACATCATAA TTTACTAAAT ATCCAGTCAC
AAAACCGCCA
TTGTCAGAAG
TTGTTTTTGG
GTCTCCTTCA
ATTTTTTTCA
CATGTGATGT
GGCGTTGAAJA
35 GGCTTAAATC
TAAGTTCTCC
GTAGCTAAA
TACTCTTTTC
ATTCGCCCTG
GGGCAAAGTT
AAACTAAACA
AAAAAATACA
TCTAGACTAT
TGACTGTGTG
TTTTNGTTAI
TAAAACAAT'i
CTTCCCARAA
GTGGCATCAA
TGAAGTTCGT
GGTTTATCTT
TTCTTCACCA
TTGCAGGATG
TCTTGTTTTT
CCAAATATCT
GTTTATCGTC
TCTTCTGATC
ATCTGCTTCA
TGTACAAAAG
TTAAAGGATC
TTGGTGGGAT
CTATTATTTC
ACTGGGGCTT
ATGACCTAAT
GGAAGGCCCA
TAAAGTCCAG
ACCAGCAGAA
TCTCAATATT
CCTACATTTC
TCCCAATCGG
AATCTGGGCT
CCATGAACCC
TGCGTACACA
AGAAGAAATT
ACACCATTCC
AACTGGAGGT
CATTTTCGTT
ACAGTCGGGG
ACTTAAAAAA
TACTGAATTA
TTTTNAATT'
TTCATACAT)
TTTGACTCGC
TGTTCAATz
AATAATTCAC
CCACATAGC.A
TTTCCAGCTT
TGACATGCTC
ACTTTCAGAG
CTACAGACA
CCTATCAAGT
AAAGTAAAAC
CATGGGCTAA
GCTTGGTTCA
TCCTTCTGCG
CAGATTTCAT
GGGAAAACTT
ACCTTCTGTT
TTTGTAAGAA
ATCTCAGCA
TGGATTTAAA
TGGWTGTATG
ACACCACCAA
TTCTTTGGTC
CAGGAGCACC
TAATTGAAAG
TTGAAATTCT
AGAAAAATAC
TTCACCAACA
CAAAGCTTTG
ATGGCTCAGC
TTAGCATAAG
CGTCAAAGCT
GATCACATTT
ATAACACGAG
TTAGCACACA
"r TTTATTTGACT
;GTTTCTTCTG
LATTCAACTTG
TTACTTCAGG
AGTACCCTAT
CATCATCTCG
CAGTCAAGTT
AAAGAATATT
CTTGACATGC
CAATAAATTT
TTCCTGATTT
TTCAAATTCG
GCATCTCTGG
CTGTTGAATC
TCTGGCCAAT
TTTCAATTGA
TCAGCTGGCA
TTCTAAAGCA
GTTGAAGACT
GCACTTCCAC
TGACAATAAA
TTGTAATATT
CATTTCAAAG
TTCCAACTcC
TTACAAAAT
AAATTTCCGG
TGGAAGTTTT
AGCAGTCTTC
CACACATTTC
TTTCTGTGGA
GCACACCTGT
TTTCCAACCA
TATAAAATTA
ACATCTTGGA
GT
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1 250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1792 INFORMATION FOR SEQ ID NO-21: SEQUENCE
CHARACTERISTICS:
LENGTH: 1650 nucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) SEQUENCE DESCRIPTION: SEQ ID NO:21: -123- ATG TCT CGT GTT ATT TTT TTA AGT TGT ATT TTT TTG TTT AGT Met Ser Arg Val Ile Phe Leu Ser Cys Ile Phe LeuPhe Ser 1 5 TTT AAT TTT ATA AAA TGT GAT CCC CCG ACT GTA ACT TTG CCC Phe Asn Phe Ile Lys Cys Asp Pro Pro Thr Val Thr Leu Pro 20 CAG GGC Gin Giy GAA TTG GTT Giu Leu Val.
GGA A.AA Gly Lys 35 GCT TTG ACG AAC Ala Leu Thr Asn GAA AAT GGA Giu Asn Gly AAA GAG TAT TTT AGC TAC ACA Lys Giu Tyr Phe Ser Tyr Thr
GGT
Gly so GTG CCT TAT GCT Val Pro Tyr Ala AAA CCT Lys Pro CCA GTT GGA Pro Val Gly
GAA
Glu 60 CTT AGA TTT AAG Leu Arg Phe Lys
CCT
Pro 65 CCA CAG AAA GCT Pro Gin Lys Ala
GAG
Glu CCA TGG AAT GGT Pro Trp Asn Gly
GTT
Val 75 TTC AAC GCC ACA Phe Asn Ala Thr
TCA
Ser CAT GGA AAT GTG His Gly Asn Val 210 252 294 336
TGC
Cys AAA GCT TTG AAT Lys Ala Leu Asn
TTC
Phe 90 TTC TTG AAA AAA Phe Leu Lys Lys GAA GGA GAC Giu Gly Asp AAA ACA ACT Lys Thr Thr 110 GAA GAC Giu Asp 100 TGC TTG TTG GTG Cys Leu Leu Val
AAT
Asn 105 GTG TAC GCA CCA Val. Tyr Ala Pro TCT GAC AAA Ser Asp Lys 115 AAA CTT CCA GTA Lys Leu Pro Val TTC TGG GTT CAT Phe Trp Va]. His GGT GGC Gly Gly 125 GGT TTT GTG ACT GGA TCC GGA AAT Gly Phe Val Thr Gly Ser Giy Asn
TTA
Leu 135 GAA TTT CAA AGC Glu Phe Gin Ser
CCA
Pro 140 420 462 GAT TAT TTA OTA AAT TAT GAT GTT ATT Asp Tyr Leu Val Asn Tyr Asp Val Ile 145 GTA ACT TTC AAT Val Thr Phe Asn
TAC
Tyr 155 CGA TTG GGA CCA Arg Leu Gly Pro GGA TTT TTG AAT Gly Phe Leu Asn GAG TTG GAA Giu Leu Glu GGT GCT Gly Ala 170 CCT GGA AAT GTA Pro Gly Asn Vai
GGA
Gly 175 TTA TTG GAT CAG OTA OCA GCT Leu Leu Asp Gin Val Ala Ala 180 TTG AAA TGG Leu Lys Trp 185 ACC AAA GAA AAT Thr Lys Glu Asn GAG AAA TTT GGT Glu Lys Phe Gly GGA GAT Gly Asp 195 -124- CCA GAA AAT ATT Pro Giu Asn Ile 200 AGT GTT CAT TAT Ser Val Hi s Tyr ACA ATT GGT GGT GTT Thr Ile Gly Giy Val 205 TCT GCT GGT GGA GCA Ser Ala Gly Gly Ala 210 630 672 CTT TTA TTG Leu Leu Leu 215 TCA CAT ACA ACC ACT GGA Cr1' Ser His Thr Thr Thr Gly Leu 220 TAC AAA AGG GCA ATT GCT CAA AGT GGA Lys Arg Ala Ile Ala 230 Gin Ser Gly AGT GCT Ser Ala 235 TTA AAT CCA Leu Asn Pro CTT CAA CTT Leu Gin Leu 250 TGG GCC Trp Ala 240 TTC CAA AGA CAT Phe Gin Arg His
CCA
Pro 245 GTA AAG CGT AGT Val Lys Arg Ser GCT GAG ATA Ala Giu Ile 255 TTA GAA TTC Leu Giu Phe TTA GGT CAT CCC Leu Gly His Pro
ACA
Thr 260 AAC AAC ACT CAA Asn Asn Thr Gin GAT GCT Asp Ala 265
TTA
Leu 270 CAA AAA GCC CCA Gin Lys Ala Pro GAC AGT CTC CTG Asp Ser Leu Leu 798 840 882 AAA ATG CCA GCT Lys Met Pro Ala
GAA
Giu 285 ACA GAA GGT GAA Thr Giu Gly Glu
ATA
Ile 290 ATA GAA GAG TTC Ile Giu Giu Phe
GTC
Vai 295 TTC GTA CCA TCA ATT GAA AAA GTT TTC CCA TCC CAC CAA Phe Val Pro Ser Ile Giu Lys Val Phe Pro Ser His Gin 25 CCT TTC Pro Phe 310 TTG GAA GAA TCA Leu Giu Giu Ser
CCA
Pro 315 TTG GCC AGA ATG Leu Ala Arg Met AAA TCT GGA Lys.Ser Giy 320 966 TCC TTT AAC Ser Phe Asn 325 GAA GGA CTT Giu Gly Leu AAA GTA CCT TTA Lys Val Pro Leu
TTA
Leu 330 GTT GGA TTC AAC Val Gly Phe Asn AGC GCA Ser Ala 335
TTG
Leu 340 TAC AAA TTC TTT Tyr Lys Phe Phe AAA GAA AAA CCA Lys Giu Lys Pro ATG CTG AAC CAA Met Leu Asn Gin GAA GCA GAT TTC Glu Ala Asp Phe AGA CTC GTA CCA Arg Leu Val Pro 1008 1050 109') 1134 1176
GCC
Al a 365 GAA TTT GAA TTA Giu Phe Giu Leu
GCC
Al a 370 CAT GGA TCA GAA His Gly Ser Giu
GAA
Giu 375 TCG AAA AAA Ser Lys Lys GAT AAA CCC Asp Lys Pro 390 CTT GCA Leu Ala 380 GAA AAA ATC AGG Giu Lys Ile Arg
AAG
Ly s 385 TTT TAC TTT GAC Phe Tyr Phe Asp -125- GTT CCT GAA AAT GAG CAG AAA TTT ATT GAC TTG ATA GGA GAT Val Pro Giu 'Asn Giu Gin Lys Phe Ile Asp Leu Ile Gly Asp 395 400 405 ATT TGG TTT ACT AGA GGC ATT GAC AAG CAT GTC AAG TTG TCT Ile Trp Phe Thr Arg Gly Ile Asp Lys His Vai Lys Leu Ser 410 415 420 GTA GAA AAA CAA GAC GAG CCA GTA TAT TAT TAT GAA TAT TCT Val Giu Lys Gin Asp Giu Pro Val Tyr Tyr Tyr Giu Tyr Ser 425 430 1218 1260 1302
TTC
Phe 435 TCT GAA AGT CAT Ser Giu Ser His
CCT
Pro 440 GCA AAA GGA ACA Aia Lys Giy Thr GGT GAC CAT Gly Asp His GTG AAT TTA Val Asn Leu 460 1344 AAC TTG Asn Leu 15 450 ACT GGA GCA TGT Thr Giy Aia Cys GGT GAA GAA CTT Gly Giu Glu Leu a. a.
a a.
a a a 9*ea a. a a a a a a. a.
a TTC AAA GTC GAG Phe Lys Val Giu 465 GTT TTA TTA ACA Vai Leu Leu .Thr 480 ATG ATG AAG Met Met Lys
CTG
Leu 470 GAA AAA GAT AAA Giu Lys Asp Lys CCG AAT Pro Asn 475 AAA GAT AGG GTA Lys Asp Arg Vai
CTT
Leu 485 GCT ATG TGG ACG Aia Met Trp5 Thr
AAC
Asn 490 1386 1428 1470 1512 1554 TTC ATC AAA AAT GGA AAT CCT ACT CCT Phe Ile Lys Asn Gly Asn Pro Thr Pro GTA ACT GAA TTA Val Thr Giu Leu
TTG
Leu 505 CCA GTT AAA TGG Pro Vai Lys Trp
GAA
Giu 510 CCT GCC ACA AAA GAC AAG TTG AAT Pro Ala Thr Lys Asp Lys Leu Asn 515 TAT TTG Tyr Leu 30 520 AAC ATT GAT GCC Asn Ile Asp Ala
ACC
Thr 525 TTA ACT TTG GGA Leu Thr Leu Gly ACA AAT CCA Thr Asn Pro 530 1596 GAA GAA ACC Giu Giu Thr 535 CGA GTC AAA TTY Arg Val Lys Phe
TGG
Trp 540 GAA GAT GCC ACA Glu Asp Ala Thr AAA ACT Lys Thr 545 1638 TTG CAC AGT CAA Leu His Ser Gin INFORM4ATION FOR SEQ ID NO:22:- SEQUENCE CHARACTERISTICS: LENGTH: 1650 nlucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: i~near 1650 (ii) MOLECULE TYPE: cDNA -126- (iii) SEQUENCE DESCRIPTION: SEQ ID NO:22: TTGACTGTGC AAAGTTTT'rc CTTCTGGATr TGTTCCCA.
AACTTGTCTT
TTGTGGCAGG
TTCAGGAGTA GGATTTCCAT CCCTATCTTT TGTTAATAAA ATCTCGACTT TGAATAAATT CAAGTTATGG TCACCAAATG AATATTCATA ATAATATACT ACATGCTTGT CAATGCCTCT AAATTTCTGC TCATTTTCAG TGAT TTT TTC TGCAAGTTTT AATTCGGCTG
GTACGAGTCT
CTCTGGI'TT TCTTTCATAA TGAATCCAAC TAATAAAGGT GCCAATGGTG ATTCTTCCAA AATTGATGGT ACGAAGACGA *CTGGCATTTT
TTTCAGGAGA
a *uAAAGCATCTT
GAGTGTTGTT
AAGACTACGC TTTACTGGAT *TTCCACTTTG
AGCAATTGCC
:AATAAAAGAT
AATGAACACT
TCAAAGCTGC TACCTGATCC AACTCCAAAT TCAAAAATCC AAAAA.TAACA TCATAATTTA TTCCGGATCC AGTCACAAAA AGTTTTTTGT CAGAAGTTGT GTCTTCGTCT CCTTCAATTT CATTTCCATG TGATGTGGCG TGTGGAGGCT
TAAATCTAAG
:ACCTGTGTAG
CTAAAATACT
CAACCAATTC GCCCTGGGGC AAATTAAAAC TAAACAAAAA
TGGCATC'ITC
GTTAAGGTGC
TTCCCATTT.A
TTTTGATGAA
ACATTCGGTT
CACAAGTTCT
TTCtTTTTGC
GGCTCGTCTT
AGTA.AACCAA
GAACGGGTTT
TTCGATTCTT
TTCGAAATCT
AGAATTTGTA
ACTTTGTTAA
GAAAGGTTGG
ACTCTTCTAT
CTGTCTACTG
TGTGGGATGA
GTCTTTGGAA
'CTTTTGTAAA
TGCTCCACCA
CAAATTTCTC
AATAATCCTA
GAGTGGTCCC
CTAAATAATC
CCGCCACCAT
TTTTGGTGCG
TTTTCAAGAA
TTGAAAACAC
TTCTCCAACT
CTTTTCC.ATT
AAAGTTACAG
AATACAACTT
*CCAR.AATTTC
CATCAATGTI
ACTGGCAATPA
*GTTCGTCCAC
TATCTTTTTC
TCACCATGAC
AGGATGACTT
GTTTTTCTAC
ATATCTCCTA
ATCGTCAAAG
CTGATCCATG
GCTTCAGCTT
CAAAAGTCCT
AGGATCCAGA
TGGGATGGGA
TATTTCACCT
GGGCTTTTTG
CCTAATATCT
GGCCCATGGA
GTCCAGTGGT
GCAGAAACAC
AATATTTTCT
CATTTCCAGG
AATCGGTAAT
TGGGCTTTGA
GAACCCAGAA
TACACATTCA
GAAATTCAAA
CATTCCATGG
GGAGGTTTAG
TTCGTTCGTC
TCGGGGGATC
AAAAAAATAA
ACTCGGGTT
*CAJAATAATTC
6ATTCAGTTAC
ATAGCAAGTA
*CAGCTTCATC
ATGCTCCAGT
TCAGAGAAAG
AGACAACTTG
TCAAGTCAAT
TAAAACTTCC
GGCTAATTCA
GGTTCAGCAT
TCTGCGCTGT
TTTCATTCTG
AAACTTTTTC
TCTGTTTCAG
TAAGAATTCT
CAGCAAGTTG
TTTAAAGCAC
TGTATGTGAC
CACCAATTGT
TTGGTCCATT
AGCACCTTCC
TGAAAGTTAC
AATTCTAAAT
AAATACTGGA
CCAACAAGCA
GCTTTGCACA
CTCAGCTTTC
CATAAGGCAC
AAAGCTTTTC
ACATTTTATA
CACGAGACAT
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 .1300 1350 1400 1450 1500 1550 1600 1650 9 0 35 INFORMATION FOR SEQ ID NO:23: i) SEQUENCE CHARACTERISTICS: LENGTH: 1590 nucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MO
T
E(.ULE TYPE: (iii) FEATURE:
NAME/KEY:
LOCATION:
cDNA
CDS
1.-.1590
GAT
Asp
I
(iv) SEQUENCE DESCRIPTION: SEQ ID NO:23: CCC CCG ACT GTA ACT TTG CCC CAG GGC GAA TTG GTT GGA Pro Pro Thr Val Thr Leu Pro Gln Gly Glu Leu Val Gly AAA GCT Lys Ala is ACA GGT Thr Gly TTT AAG TTG ACG Leu Thr GTG CCT Val Pro CCT CCA
AAC
Asn
TAT
Tyr
CAG
-127- GAA AAT GGA AAA GAG TAT Glu Asn Gly Lys Glu Tyr 20 GCT AAA CCT CCA GTT GGA Ala Lys Pro Pro Val Gly 35 AAA GCT GAG CCA TGG AAT TTT AGC TAC Phe Ser Tyr GAA CTT AGA GIlu Leu Arg 84 126 168 iPne L~ys Pro Pro Gin Lys Ala Glu Pro Trp, Asn Gly Val Phe 0 0*
AAC
Asn
TTC
Phe
AAT
Asn
GTA
Val
GGA
Gly 25 GAT Asp
GGA
Gly 30
GGA
Gly 155
AAT
Asn
GGT
Gly
GCC
Al a
TTG
Leu
GTG
Val
TTT
Phe 100
AAT
Asn
GTT
Val
TTT
Phe
TTA
Leu PiTT Ile 170
GGT
Gly
ACA
Thr
AAA
Lys
TAC
Tyr
TTC
Phe
TTA
Leu 115
ATT
Ile
TTG
Leu
TTG
Leu
GAG
Glu
GTT
Val 185
TCA
Ser
AAA
Lys
GCA
Ala
TGG
Trp
GAA
Glu
TTT
Phe 130
AAT
Asn
GAT
Asp
PAA
Lys
TCT
Ser His
ATT
Ile 75
CCA
Pro
GTT
Val
TTT
Phe
GTA
Val1
TTG
Leu 145
CAG
Gln
TTT
Phe
GCT
Al a Gly
GAA
Glu
AAA
Lys 90
CAT
His
CAA
Gln
ACT
Thr
GAG
Glu
GTA
Val 160
GGT
Gly
GGT
Gly Asn
GGA
Gly
ACA
Thr
GGT
Gly 105
AGC
Ser
TTC
Plie
TTG
Leu
GCA
Ala
GGA
Gly 175
GGA
Gly Val
GAC
Asp
ACT
Thr
GGC
Gly
CCA
Pro 120
AAT
Asn
GAA
Glu
GCT,
Al a
GAT
Asp
GCA
Al a 190 Cys 65
GAA
Glu
TCT
Ser
GGT
Gly
GAT
Asp
TAC
Tyr 135
GGT
Gly
TTG,
Leu
CCA
Pro
AGT
Ser Lys
GAC
Asp
GAC
Asp Phe
TAT
Tyr
CGA
Arg
GCT
Ala 150
AAA
Lys
GAA
Glu
GTT
Val Ala
TGC
Cys
AAA
Lys
GTG
Val
TTA
Leu
TTG
Leu
CCT
Pro
TGG
Trp 165
AAT
Asn
CAT
His Leu
TTG
Leu
AAA
Lys
ACT
Thr 110
GTA
Val1
GGA
Gly
GGA
Gly
ACC
Thr
ATT
I le 180
TAT
Tyr CAT GGA AAT GTG TGC AAA GCT TTG AAiT Asn
TTG
Leu
CTT
Leu
GGA
Gly
AAT
Asn 125
CCA
Pro
AAT
Asn'
AAA
Lys
ACA
Thr
CTT
Leu 195
TTC
Phe
GTG
Val1
CCA
Pro
TCC
Ser
TAT
Tyr
CTC
Lieu 140
GTA
Val
GAA
G1u
ATT
Ile
TTA
Leu 210 252 294 336 378 420 462 504 546 588 630 TTG TCA CAT ACA ACC ACT GGA CTT TAC AAA AGG GCA Leu Ser His Thr Thr Thr Gly Leu Tyr Lys Arg Ala 200 205 ATT GCT Ile Ala -128- CAA AGT GGA AGT Gin Ser Gly Ser
GCT
Al a 215 TTA AAT CCA TGG GCC TTC CAA AGA CAT Leu Asn Pro Trp Ala Phe Gln A-rg His 220 672 CCA GTA AAG Pro Val Lys 225 CGT AGT CTT CAA CTT Arg Ser Leu Gin Leu 230 GCT GAG ATA TTA GGT CAT Ala Giu Ile Leu Gly His 235 CCC ACA AAC AAC ACT CA-A GAT GCT Pro Thr 240 Asn Asn Thr Gin Asp 245 Al a
AAA
Lys 260 TTA GAA Leu Giu TTC TTA CAA AAA Phe Leu Gin Lys 250 GCC CCA GTA Ala Pro Vai 255 GA-A GGT GAA Glu Gly Glu GAC AGT CTC CTG Asp Ser Leu Leu AAA ATG CCA GCT Lys Met Pro Ala GAA ACA Glu Thr 265
A-TA
Ile 270 ATA GAA GAG TTC Ile Giu Giu Phe
GTC
Val 275 TTC GTA CCA TCA Phe Val Pro Ser GAA AAA GTT TTC Giu Lys Vai Phe
CCA
Pro 285 TCC CAC CAA CCT Ser His Gin Pro TTG GAA GA-A TCA Leu Giu Giu Ser 798 840 882 924 966
CCA
Pro 295 TTG GCC AGA ATG Leu Aia Arg Met
AAA
Lys 300 TCT GGA TCC TTT Ser Giy Ser Phe AAA GTA CCT Lys Vai Pro TTG TAC AAA Leu Tyr Lys 320 TTA TTA Leu Leu 310 GTT GGA TTC AAC Val Gly Phe Asn
AGC
Ser 315 GCA GA-A GGA CTT Ala Glu Giy Leu 25 TTC TTT ATG Phe Phe Met 325 GCA GAT TTC Ala Asp Phe 30 CAT .GGA TCA His Gly Ser AAA GAA AAA CCA Lys Giu Lys Pro ATG CTG A-AC CAA Met Leu Asn'Gin GCT GAA A-ia Giu 335
GAA
Glu 340 AGA CTC GTA CCA A-rg Leu Val Pro
GCC
Ala 345 GAA TTT GAA TTA Giu Phe Giu Leu 1008 1050 1092 1134 GA-A GAA Giu Glu 355 TCG A-AA AAA CTT Ser Lys Lys Leu GAA AAA ATC AGG Glu Lys Ile A-rg
A-AG
Lys 365 TTT TA-C TTT GA-C Phe Tyr Phe Asp
GAT
Asp 370 AAA CCC GTT CCT Lys Pro Val Pro A-AT GAG CAG A-sn Giu Gin AAA TTT Lys Phe 380 ATT GAC TTG ATA Ile Asp Leu Ile
GGA
Gly 385 GAT ATT TGG TTT ACT A-GA GGC Asp Ile Trp Phe Thr A-rg Gly 390 1176 ATT GAC A-AG CAT GTC Ile Asp Lys His Val 3 95 A-AG TTG TCT Lys Leu Ser 400 GTA GAA AA-A CAA Vai Giu Lys Gin GA-C GAG Asp Glu 405 1218 -129- CCA GTA TAT TAT TAT Pro Val. Tyr Tyr Tyr 410 GCA AAA GGA ACA TTT Ala Lys Gly Thr Phe 425 GAA TAT TCT TTC TCT GAA AGT CAT CCT Glu Tyr Ser Phe Ser Glu Ser His Pro 1260 GGT GAC CAT Gly Asp His AAC TTG ACT GGA GCA TGT Asn Leu Thr Gly Ala Cys 430 1302 CAT GGT GAA GAA CTT GTG AAT TTA His Gly 435 AAG CTG Lys Leu 450 Glu Glu Leu GAA AAA GAT Glu Lys Asp Val Asn Leu 440 AAA CCG AAT Lys Pro Asn 455 TTC AAA GTC GAG ATG ATG Phe Lys Val Glu Met Met 445 GTT TTA TTA ACA AAA GAT 1344 1386 Val. Leu Leu Thr Lys Asp 460 AGG GTA CTT Arg Val Leu 465 CCT ACT CCT Pro Thr Pro GCT ATG TGG ACG Ala Met T-p, Thi- TTC ATC AAA AAT Phe Ile Lys Asn GGA AAT Gly Asn 475
GAA
Glu 480 GTA ACT GAA TTA Val Thi- Glu Leu CCA GTT AAA TGG Pro Val Lys Ti-p 1428 1470 1512 1554 CCT GCC ACA AAA Pro Ala Thi- Lys AAG TTG AAT TAT Lys Leu Asn Tyr
TTG
Leu 500 AAC AITr GAT GCC Asn Ile Asp Ala
ACC
Thr 505 TTA ACT TTG GGA Leu Thi- Leu Gly
ACA
Thi- 510 AAT CCA GAA GAA Asn Pro Glu Glu
ACC
Thi- 515 CGA GTC AAA Arg Val Lys 25 TTY TGG Phe Ti-p 520 GAA GAT GCC ACA Glu Asp Ala Thi-
AAA
Lys 525 ACT TTG CAC AGT Thr Leu His Ser 1590 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 2836 nucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: (iii) FEATURE:
NAME/KEY:
LOCATION:
cDNjk CDs 99. .1889 (iv) SEQUENCE DESCRIPTION: SEQ ID NO:24: TAGACATGTC GTCITCAAAA CGTCTATTTT ATCATAAACA
AAACGAGATA
AATAATAACA ATTAAGCAAC CAAAATG CAT TAAAAAACAC
AATAAAAA
-130- ATG TTA CCT CAC AGT AGT GCA TTA GTT TTA TTT TTA TTT TTT Met Leu Pro His Ser Ser Ala Leu Val Leu Phe Leu Phe Phe TTA TTT Leu Phe TTC TTA TTT Phe Leu Phe
ACA
Thr 20 CCT ATC TTG TGC Pro Ile Leu Cys ATA CTA TGG GAT Ile Leu Trp Asp AAC CTA GAT CAG CAT TTG Asn Leu Asp Gin His Leu AGA GTT CAA TTT Arg Val. Gin Phe AAC GGG ATC Asn Gly Ile ACG GAA GGA Thr Giu Gly AAA CCG TTC Lys Pro Phe CGA TAT Arg Tyr 50 AAA GAt CAT AGG Lys ASP His Arg AAT GAT Asn Asp GTA TAT TGT Val Tyr Cys TAT TTG GGA ATT Tyr Leu Gly Ile TAT GCC GAA CCG Tyr Ala Giu Pro TTT GGA CCA TTA Phe Giy Pro Leu
CGA
Arg TTT CAG TCT CCA Phe Gin Ser Pro CCA ATA TCA AAT Pro Ile Ser Asn 266 308 350 392 434
CCA
Pro AAA ACA GGA TTC Lys Thr Gly Phe
GTA
Val CAG GCT CGA ACT Gin Ala Arg Thr GGA GAC AAA Gly Asp Lys GGA AGC GAA Gly Ser Giu 110 TGT TTC Cys Phe 100 CAG GAA AGT CTA Gin Glu Ser Leu
ATA
Ile 105 TAT TCT TAT GCA Tyr Ser Tyr Ala GAT TGC TTA Asp Cys Leu 115 TCT GCG AAC Ser Aia Asn TAT CTG AAT ATA Tyr Leu Asn Ile ACG CCA GAG ACT Thr Pro Giu Thr GTT AAT Val Asn 125 ACA AAA TAT CCT Thr Lys Tyr Pro
GTA
Val 135 ATG TTC TGG ATC Met Phe Trp Ile GGA GGC GCA ITC Gly Gly Ala Phe
AAC
Asn 145 CAA GGA TCA GGA Gin Gly Ser Gly TAT AAT TTT TTT Tyr Asn Phe Phe 476 518 560 602 644
GGA
Gly 155 CCT G'iT TAT TTG Pro Asp Tyr Leu AGG GAA GGA ATT Arg Giu Gly Ile
ATT
Ile 165 TTG GTC ACT Leu Val. Thr TCA GCG CCG Ser Ala Pro 180 ATC AAC Ile Asn 170 TAT AGA TTA GGA Tyr Arg Leu Gly TTC GGT TTT CTA Phe Gly Phe Leu GAA TGG GAT ATC CAT GGA AAT Giu Trp Asp Ile His Gly Asn 185 GGT CTA AAA GAC Gly Leu Lys Asp CAG AGA Gin Arg 195 686 -13 1- TTG GCA OTA AAA Leu Ala Leu Lys 200 GGA GAO AGA GAA Gly Asp Arg Giu TGG GTT TAC GAO AAO ATO GAA AAG Trp, Val Tyr Asp Asn Ile Glu Lys 205 TTT GGT Phe Gly 210 AAA ATT Lys Ile 215 ACA ATT GOT Thr Ile Ala
GGA
Gly 220 GAA TOT GOT GGA Giu Ser Ala Giy 770 812 GCA GCA AGT GTC OAT TTT Ala 225 Ala Ser Val His CTG ATG ATG GAO Leu Met Met Asp Phe 230
AAC
Asn 235 TOG ACT AGA Ser Thr Arg AAA TAO Lys Tyr 240 TAO CAA AGG Tyr Gin Arg GOO ATT .TTG OAG AGT GGG ACA TTA OTA Ala Ile 245 Leu Gin Ser Gly Thr Leu Leu 250 S S S S.
S
*5 S S
S
*5
S
S
55*5 AAT OOG ACT Asn Pro Thr 255 AAA OTO AAA Lys Leu Lys GOT AAT CAA ATT CAA OTT OTG OAT AGA Ala Asn Gin Ile Gin Leu Leu His Arg TTT GAA Phe Giu 265
CAA
Gin 270 GTG OTA AAC ATO Vai Leu Asn Ile
AOG
Thr 275 CAA AAA CAA GAA Gin Lys Gin Giu
OTO
Leu 280 896 9 38 980 1022 OTA AAO OTG GAT Leu Asn Leu Asp
AAA
Lys 285 AAO CTA ATT Asn Leu Ile TTA OGA Leu Arg 290 GOA GOO TTA AAO Ala Ala Leu Asn
AGA
Arg 295 GTT COT GAT AGO Val Pro Asp Ser
AAO
Asn 300 GAO OAT GAO OGA Asp His Asp Arg
GAO
As p 305 ACA GTA OOA Thr Val Pro OCA GATOCCA Pro Asp Pro.
320 25 GTA TTT Vai Phe 310 AAT OCA GTO TTA Asn Pro Val Leu
GAA
Giu 315 TOA OOA GAA TOT Ser Pro Giu Ser 1064 ATA ACA TTT Ile Thr Phe 325 OCA TOT GOO TTG Pro Ser Ala Leu AGA ATG AGA AAT Arg Met Arg Asn GGT GAA Gly Giu 335 1106 TTT OCT GAT GTO GAT GTO ATO ATT Phe Pro Asp Val Asp Vai Ile Ile 340
GGT
Gly 345 TTO AAT AGT GOT Phe Asn Ser Ala 1148 GGT TTA AGA TOT Gly Leu Arg Ser GOA AGA GTA ACC Ala Arg Val Thr
AGA
Arg 360 GGA AAO ATG GAA Gly Asn Met Giu 1190 1232 GTT CAC Val His 365 AAG ACT TTG Lys Thr Leu
ACA
Thr 370 AAT ATA GAA AGG Asn Ile Giu Arg ATA COT AGA Ile Pro Arg -13 2- GAT GCT AAT ATT TGG AAA AAT CCA AAT GGT ATT GAG GAG AAA Asp Ala Asn Ile .Trp Lys Asn Pro Asn Gly Ile Giu Giu Lys 380 385 390 AAA CTA ATA AAA ATG CTT ACA GAG TTT TAT GAC CAA GTG AAA Lys Leu Ile Lys Met Leu Thr Giu Phe Tyr Asp Gin Val Lys 1274 1316 GAA CAA AAC Glu Gin Asn GAT GAC Asp Asp 410 ATT GAA GCC Ile Glu Ala
TAC
Tyr 415 GTC CAA CTA AAA Vai Gin Leu Lys
GGC
Gly 420 GAT GCT GOT TAC Asp Ala Gly Tyr
CTC
Leu 425 CAA GGA ATC TAC Gin Gay Ile Tyr
CGT
Arg 430 ACC TTG AAA GCC Thr Leu Lys Ala 1358 1400 1442
ATA
Ile 435 TTT TTC AAT GAA Phe Phe Asn Glu AGA AGO PAT TCC Arg Arg Asn Ser
AAT
As n 445 TTG TAT TTG Leu Tyr Leu TAC AG Tyr Arg 450 TTA TCA GAC OAT ACG TAT AGT GTA TAT Leu Ser Asp Asp Thr Tyr Ser Val Tyr AAA AGT TAT Lys Ser Tyr 460 1484 1526 ATC TTG CCC TAT CGA TOG GGT Ile Leu Pro Tyr Arg Trp Gly 465
TCC
Ser 470 TTG CCA GGA GTT Leu Pro Gly Val AGT CAT Ser His 475 GGT GAT GAT Gly Asp Asp
TTA
Leu 480 GGA TAT CTT TTT Gly Tyr Leu Phe GCA PAC Ala Asn 485 TCG TTG GAT Ser Leu Asp CCT ATT TTG GGA Pro Ile Leu Gly
ACA
Thr 495 ACG CAC ATT TCT Thr His Ile Ser CCG CAA GAT GCT Pro Gin Asp Ala 1568 1610 1652 1694
ATG
Met 30 505 CAG ACT CTG GAA AGG ATO GTC AGO ATC TGG ACC PAT TTT Gin Thr Leu Glu Arg Met Vai Arg Ile Trp Thr Asn Phe GTA PAG Val Lys 520 PAT OGA AAA CCT Asn Gly Lys Pro
ACA
Thr 525 TCA PAC ACT GA Ser Asn Thr Glu GAT GCA TCA Asp Ala Ser 530 TOT GAT ACA AAA AGA CAT TTA Cys Asp Thr Lys Arg His Leu 535
AAC
Asn 540 GAC ATT TTT TGG Asp Ile Phe Trp GPA CCA r It. Pro 545 1736 TAC PAC GAC Tyr Asn Asp
GPA
Glu 550 GAA CCA AAA TAT 01u Pro Lys Tyr
TTG
Leu 555 GAC ATG GGA AAA Asp Met Gly Lys
A
Giu 560 1778 1820 AAT TTT GAA ATG Asn Phe Giu Met
APA
Lys 565 P-AT ATT TTG GA Asn Ile Leu Glu
CTA
Leu 570 AAA CGC ATG ATG Lys Arg Met Met -133- CTT TGG Leu Trp, 575 GAT GAA GTT Asp Glu Val TAT AGA AAT GCG Tyr Arg Asn Ala 580 AGT ATT AGA TGA Ser Ile Arg 595 AAT TTG CGG TTT AGA Asn Leu Arg Phe Arg 585
GTTTTTTTA.A
1862 1899 GTC TGT AAT GAA GAA Val Cys Asn Glu Glu 590 TTTTACATAC
AGCCGAGAGG
GAAAAAGAAA
AATCACATGG
TCAAGTGTAT CAAGAAAACT AAATTCTTGG AAAAACACTT
TTTTTCTTCT.TTTACCAAAT
TTTATGACAT GGCAATTAAT GGTAGTATTT TCTAATAAGA GATTTGCTCG GCTATATATA TTTGATGGTA AAAATATGTT TTAGATTAAA
TTAAAATATG
ATGTACTATA TTTACCAGTA TACAATATTT ATTTCTCAAA GAGTTAATGT TGTAAATTAA 20 TGGTGCAAAG AAAAATTTTA TACGAATTGT AAACTCACAA CATACATGAC TTTTCTATAT TCAACATGTA
AAATACAAAT
TGTTATATAA AATCTGAATA AAAAAAAAA
AAACATGACT
ACCATAGTAA
TATTGCATCA
TTTATGACTG
CGCCAAATTT
GTGTTAAACA
AGGTTATATA
AAAACTTANC
GATTTTCCTA.
ATATTTTCAA
CTATGAAACT
AATGTTTAGT
ACTATTTTTT
CTGTGATAAT
TTATGTGAAT
CATTTTATAT
GCGGTTGTTA
AATGTACTTT
AAAATTGGAA
CTTTATTACA
AAGAAAATAT
ACATGGCCCA
TGTAGCGTCA
TTCAACTCTA
AAAAGACTTG
GTCTCGTTAT
ATAATCTAAG
TTAATTAATT
ATTTTAAATA
GTAACAAGAC
ATCTATCACA
ATTTGACP.TT
ATTGTTTTTT
TACGGTGATA
AAAATAATCT
TAAGTAAAAA
AGAAAAATCA
TGATTTAGT
TATTTTGCCA
TAATTGAAGC
GACACATTTA
TATTAAAAAT
AAAATAATAA
GCTAAACTTT
ATATTATATT
TTAGTTTTAA
TATTTTTTAT
CATTAAATTA
ACCGCTTAAT
TACACAATAT
GTTAAAAAAA
TGGATTAATG
GTATTAAAAT
1949 1999 2049 2099 2149 2199 2249 2299 2349 2399 2449 2499 2549 2599 2649 2699 2749 2799 2836 S
S
SS
S
S
*5SS
S
S
S
AAAAAA AAAAAAA INFORMATION FOR SEQ ID i) SEQUENCE CHARACTERISTICS: LENGTH: 596 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein *5 S
S
S
(iii) SEQUENCE DESCRIPTION: SEQ ID Met Leu Pro His Ser Ser Ala Leu Val Leu Phe Leu Phe Phe Leu Phe Phe Leu Phe Thr Pro Ile Leu Cys Leu Trp Asp Asn Leu Asp Thr G1*u Gly Val Tyr Cys Gin His Leu Cys Arg Val Gin Phe Asn Gly Ile Lys Pro Phe Arg Lys Asp His Arg Asn Asp Tyr Leu Gly Ile TyAl uPr Tyr Ala Glu Pro Phe Pro Gly Pro Leu Lys Thr Gly Arg Phe Phe Val -134- Ser Pro Lys Ala Arg Thr Pro Leu Ser Asn Asp Lys 90 Cys Phe Gin Giu Ser Leu Ile Tyr Ser Tyr Ala Gi\ a. a a a a a a a. a a a a Asp Ser Gly 155 Ile Giu Leu 20 Gly Ala 225 25 Lys Asn Lys Arg 295 100 Cys Ala Gly Pro Asn 170 Trp Al a Asp Al a Tyr 240 Pro Leu Asn Val Leu 115 Asn Ala Asp Tyr Asp 185 Leu Arg Ser Tyr Thr 255 Lys Leu Pro Tyr Asn 130 Phe Tyr Arg Ile Lys 200 Giu Val Gin Ala Gin 270 Asp Asp *Leu Thr As n 145 Leu Leu His Trp Lys 215 His Arg As n Val Lys 285 Ser Asn Lys Gin Ile 160 Gly Gly Val Ile Phe 230 Al a Gin Leu Asn Asn 300 105 Ile Tyr Gly Arg Val 175 Asn Tyr Thr Leu Ile 245 Ile Asn Leu Asp Phe 120 Pro Ser Giu Phe Met 190 Asp Ile Met Leu Gin 260 Ile Ile His Thr Val1 135 Gly Giy Gly Giy Asn 205 Al a Met Gin Leu Thr 275 Leu Asp *Prc Met Ser 150 Ile Phe Leu Ile Gly 220 Asp Ser Leu Giln Arg 290 Arg Giu Phe Tyr Ile 165 Leu Lys Giu Giu Asn 235 Giy His Lys Ala Asp 305 110 Thr Trp Asn Leu S er 180 Asp Lys Ser S er rhr 250 Arg Gln Ala rhr Ser Val 125 Ile Phe Val Ala Gin 195 Phe Ala Thr Leu I Phe C 265 Giu I Leu Val E Giu Asn His 140 Phe Thr Pro k.rg 'iy 210 .rg jeu 1u ~eu ~sn ~ro Val Phe 310 Asn Pro Val Leu Ser Pro Glu Ser Pro Asp Pro 320 Ile Thr Phe Pro Ser Ala 325 Phe Pro Asp Val Asp Val 340 Gly Leu Arg Ser Met Ala 355 Val His Lys Thr Leu Thr 365 370 Asp Ala Asn Ile Trp, Lys 380 Leu Ile Arg Asn -135- Glu Arg met Arg 330 Ile Gly Phe Asn 345 Val Thr Arg Gly 360 Ile Giu Arg Ala 375 Asn Gly Glu 335 Ser Ala Giu 350 Asn Met Giu Ile Pro Arg Glu Glu Lys 385 Lys Leu Ile Lys Met Leu Thr Giu Phe Tyr Asp Gin Val L too.*: 0 *009.
C' 6006o Glu Asp Ile 435 Tyr Ile Gly Pro Met 505 Val Cys Tyr~ Gin Al a Phe Arg 450 Leu Asp Ile Gin Lys 520 A-sn 395 Asn Gly Phe Leu Pro 465 Asp Leu Thr Asn rhr 535
!LSP
Asl 410 Asn Ser Tyr Leu 480 Giy Leu Gly Lys Giu 550 Asp Leu 425 Giu Asp Arg Giy Thr 495 Giu Lys Arg Giu Lys 565 400 405 Ile Giu Ala Gin Phe 440 Asp Trp Tyr Thr Arg 510 Pro His Pro Gly Arg Thr 455 Gly Leu His Met Thr 525 Leu Lys Ile Arg Tyr Ser 470 Phe Ile Vali Ser Asn S40 T'yr Tyi 415 Tyr Asn Ser Leu Aia 485 Ser Arg Asn Asp Leu 555
I
Val *Arg 430 *Ser Val Pro Asn Ile 500 Ile Thr Ile Asp Gin Thr Asn 445 Tyr Giy Ser Pro Trp 515 Giu Phe Met Leu Leu Leu Lys 460 Val Leu Gin Thr Asp 530 Trp GiyI Lys Lys Tyr Ser Ser 475 Asp Asp Asn klia 3iu .sys Giy 420 Al a Leu Tyr His Val1 490 Al a Phe Ser Pro Giu 560 Asn Phe Glu Met Asn Ile Leu Glu Leu Lys Arg Met Met 570 -136..
Leu Trp Asp Glu Val Tyr Arg Asn Ala Asn Leu Arg Phe Arg 575 580 585 Val Cys Asn Glu Glu Ser Ile Arg 590 595 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 2836 nucleotides TYPE: nucleic acid STRANDEDNESS: single 0 TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) SEQUENCE DESCRIPTION: SEQ ID NO:26:
TTTTTTTTTT
TTACTTAAAA
TTATTTTTAA
CACCGTAATA
AAACAATATT
GTCAAATATT
GATAGATAAA
TTGTTACACT
TTAAAATAGT
TAATTAATTG
AGATTATTAG
ACGAGACGNT
2>5 GTCTTTTTAT
AGTTGAATGT
CGCTACAAAA
GCCATGTCAG
TTTTCTTTGA.
30 AATAAAGTTA
CAATTTTAGT
TCTAATACTT
AAACTTCATC
TCAAAATTTT
TGGTTCCCAA
CTTCAGTGTT
CTGACCATCC
CGTTGTTCCC
AATCATCACC
ATATAACTTT
CAAATTGGAA
GGTAGATTCC
TCAATGTCAT
TTTTATTAGT
CATCTCTAGG
ATGTTTCCTC
GAAACCAATG
CCAAGGCAGA
TTTTTTTTTT
GTACATTTAT
CAACCGCATT
TAAAATGATA
CACATAATTG
ATCACAGTAA
AAATAGTTTA
AAACATTTTT
TTCATAGTAC
AAAATATCAT
GAAAATCAAC
AAGTTTTTAT
ATAACCTTCT
TTAACACATT
TTTGGCGATT
TCATAAAAAG
TGCAATAAGT
CTATGGTCCA
CATGTTTCCT
TCTTCATTAC
CCAAAGCATC
CTTTTCCCAT
AAAATGTCGT
TGATGTAGGT
ITTCCAGAGT
AAAATAGGAA
ATGACTAACT
TATATACACT
TTCCTTCTGA.
TTGGAGGTA.A
CGTTTTGTTC
TTTTTCTCCT
TATAGCCCTT
TGGTTACTCT
ATGACATCGA
TGGAAATGTT
TTTTTTTTTT
TCAGATTTTA
TGTATTTTAC
TAGAAAAGTC
TGAGTTTACA
AATTTTTCTT
ATTTACAACA
GAGAAATAAA
TGGTAAATAT
ATTTTAATTT
ATATTTTTAC
ATATAGCCGA
TATTAGAAAA
AATTGCCATG
TGGTAAAAGA
TGTTTTTCCA
TTTCTTGATA
TGTGATTTTT
CTCGGCTGTA
AGACTCTAAA
ATGCGTTTTA
GTCCAAATAT
TTAAATGTCT
TTTCCATTCT
CTGCATAGCA
CATCCAACGA
CCTGGCAAGG
ATACGTATCG
ATTCATTGAA
CCAGCATCGC
TTTCACTTGG
CAATA CCATT
TCTATATTTG
TGCCATAGAT
CATCAGGAAA.
ATTGGATCTG
TTTTTTTT
TATAACAATT
ATGTTGACAT
ATGTATGTTT
ATTCGTAATA
TGCACCAATT
TTAACTCTAA
TATTGTAATA
AGTACATTTA
AATCTAAAAT
CATCAAAAAA
GCAAATCTTA
TACTACCATT
TCATAAATAA
AGAAAAAGCT
AGAATTTTGG
CACTTGAAAC
CTTTTTCTGA.
TGTAAAATTA
CCGCAAATTC
GTTCCAAAAT
TTTGGTTCTT
TTTTGTATCA
TTACAAAATT.
TCTTGCGGTA
GTTTGCAAAA
AACCCCATCG
TCTGATAAtLA2
AAATATGGCT
CTTTTAGTTG
TCATAAAACT
TGGATTTTTC
TCAAAGTCTT
CTTAAACCTT
TTCACCATTT
GAGATTCTGGj
TTTTTTTTTT
TTAATACAGA
TAATCCATAT
TTTTAACAAA
TTGTGTAAAT
AAGCGGTTGT
TTTAATGGTC
AAAAATATAT
AAACTAAAAT
ATAATATCTT
GTTTAGCATA
TTATTTTCAA
TTTAATATAG
ATGTGTCTGA
TCAATTATGG
CAAAATAATA
TAAATCATGT
TTTTTCTTTC
AAAAAACTCA.
GCATTTCTAT
ATTTTTCATT
CGTCGTTGTA
CATGATGCAT
GGTCCAGATC
TAGAAATGTG
AGATATCCTA
'kTAGGGCAAG
TGTACAAATA
TTCAAGGTAC
GACGTAGGCT
CTGTAAGCAT
CAAATATTAG
GTGAACTTCC
CAGCACTATT
CTCATTCTTT
TGATTCTAAG
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 -137- ACTGGATTAA
ATACTGGTAC
AACTCTGTTT
AAGGCTGCTC
GTTCTTGI-FT
TTGCGTGATG
TGCAGAAGTT
GAATTTGATT
CAAAATGGCC
CTTTGGTAGT
AATGGACACT
TGCTGCTCCA
CTGTCTCCAC
CAAACTTTTC
TCTCTGGTCT
TTTAGACCCA
ATAGAAAACC
GAAAACTCCT
CCTTCCCTGA
TCAAATA.ATC
TTGGTTGAAT
GC-GCCTCCAT
TGTTCGCAGA
ATTAACAGTC
TCTTCGCTTC
CTGCATAAGA
TCCCAAAGTT
CGAGCCTGTAC
TTGGAGACTG AAATCGTAAT
C
CCCAAATAAG
AACAATATAC
TTTTCCT.TCC
GTGATCCCGTI
GGTTATCCCA TAGTATGCAC
A
AATAAAAATA AAACTAATGC
A
20 TTTTTAATGC ATTTTGGTTG
C
TATGATAA TAGACGTTITT
G.
TGTGTCTCGG TCATGGTCGT
GTAAAATTAG
TTTAGCACTT
AGCAGTCGGA
ATTTTCTAGT
GCAGATTCTC
GATGTTGTCG
TATTTCCATG
AATCTATAGT
AGGTCCAAA
GGATCCAGAA
rCTGGCGTGA
W.ATATTAGA
~GAATCCTGT
;GTCCAAAAGC
LTCATTCCTA 'AAATTGAAC
TI
AGATAGGTG
CTACTGTGA
G
TTAATTGTT
A
GTTTTTATCC
GTTTGAGTTT
TTTAGTAATG
CGAGTTGTCC
CAGCAATTGT
TAAACcCATT GATATccCAT
TGATAGTGAC
AAATTATAAG
CATTACAGGA
ATATATTCAG
CTTTCCTGGA E'TTTGGATTT
C
CGGTTCGGC
A
'GATCTTTAT
A
CTGCACAAA
T
'AAATAAGAAA
GTAACATTT
T
.TTATTTATC
T
TGCTATCAGG
AGGTTTAGGA
TTCAAATCTA
TCCCACTCTG
ATCATcAGAA
AATTTTTTCT
TTAGTGCCAA
TCCGGCGCTG
CAAAATAATT
ATCCTGATCC
TATTrTGTAT kTATAAGCA
ACATTTGTC
ATATTGGTT
TAAGGAATT
.TCGGAACGG
GCTGATCTA
AATAAAAAA
TTATTGTGT
CGTTTTGTT
1850 1900 1950 2000 2050 2100 2150 2200 2250 2300 2350 2400 2450 2500 2550 2600 2650 2700 2750 2800 2836 *9 INFORMATION FOR SEQ ID NO:27: i) SEQUENCE
CHARACTERISTICS:
LENGTH: 1710 nucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE
TYPE:
(iii) FEATURE:
NAME/KEY:
LOCATION:
cDNA
CDS
(iv) SEQUENCE DESCRIPTION: SEQ ID NO:27: TGG .GAT AAC CTA GAT CAG CAT TTG TOC AGA GTT CAA TTT AAC Trp Asp Asn Leu Asp Gin His Leu Cys Arg Val Gin Phe Asn 1
GGG
Gly ATC ACG GAA GGA Ile Thr Glu Gly AAA CCG TTC CGA TAT AAA GAT CAT AGG Lys Pro Phe Arg Tyr Lys Asp His Arg AAT GAT GTA TAT TGT TCT TAT TTG GGA ATT CCT Asn Asp Val Tyr Cys Ser Tyr Leu Gly Ile Pro TAT GCC GAA Tyr Ala Glu CCG CCT TTT GGA CCA TTA Pro Pro Phe Gly Pro Leu CGA TTT CAG TCT CCA AAA Arg Phe Gin Ser Pro Lys CCA ATA Pro Ile -138- TCA AAT CCA Ser Asn Pro ACA GGA TTC GTA Thr Gly Phe Val GCT CGA ACT TTG Ala Arg Thr Leu 210 GAC AAA TGT TTC CAG GAA AGT CTA ATA TAT TCT TAT GCA GGA Asp Lys Cys Pile Gin Glu Ser Leu Ile Tyr Ser Tyr Ala Gly
AGC
Ser GAA GAT TGC TTA Giu Asp Cys Leu
TAT
Tyr 90 CTG AAT ATA TTC ACG CCA GAG ACT Leu Asn Ile Phe Thr Pro Glu Thr GTT AAT Val Asn 100 TCT GCG AAC AAT ACA AAA TAT CCT GTA Ser Ala Asn Asn Thr Lys Tyr Pro Vai ATG TTC TGG Met Phe Trp 110 ATC CAT GGA Ile His Gly 115 TTT TTT GGA Pile Pile Gly GGC GCA TTC AAC Gly Ala Pile Asn
CAA
Gin 120 GGA TCA GGA TCT Gly Ser Gly Ser TAT AAT Tyr Asn 125 GAT TAT TTG ATC Asp Tyr Leu Ile GAA GGA ATT ATT Giu Gly Ile Ile GTC ACT ATC AAC Val Thr Ile Asn
TAT
Tyr 145 AGA TTA GGA GTT Arg Leu Gly Vai GGT TTT CTA TCA Gly Phe Leu Ser 336 378 420 462 504 546 588
GCG
Ala 155 CCG GAA TGG GAT Pro'Glu Trp Asp
ATC
Ile 160 CAT GGA A.AT ATG His Gly Asn Met CTA AAA GAC Leu Lys Asp ATC GAA AAG Ile Giu Lys 180 25 CAG AGA Gin Arg 170 TTG GCA CTA AAA Leu Ala Leu Lys GTT TAC GAC AAC Val Tyr Asp Asn TTT GGT GGA Phe Giy Gly 185 GAC AGA GAA AAA Asp Arg Giu Lys
ATT
Ile 190 ACA ATT GCT GGA Thr Ile Ala Gly GAA TCT Glu Ser 195 GCT GGA GCA GCA AGT GTC 6AT TTT CTG ATG ATG GAC AAC Ala Gly Ala Ala Ser Val His Phe Leu Met Met Asp Asn 200 205
ACT
Thr AGA AAA TAC TAC Arg Lys Tyr Tyr 215 CTA AAT CCG ACT Leu Asn Pro Thr CAA AGG GCC ATT Gin Arg Ala Ile CAG AGT GGG ACA Gin Ser Gly Thr 672 714
TTA
Leu 225
GCT
Aia 230 AAT CAA ATT CA Asn Gin Ile Gin CTG CAT AGA Leu His Arg TTT GAA AAA CTC AAA CAA GTG CTA AAC ATC ACG CAA AAA CAA Phe Giu Lys Leu Lys Gin Val Leu Asn Ile Thr Gin Lys Gin 240 245 250 756 -13 9- GAA CTC CTA AAC Glu Leu Leu Asn 255 CTG GAT AAA AAC Leu Asp Lys Asn 260 CCT GAT AGC AAC Pro Asp Ser Asn CTA ATT TTA CGA Leu Ile Leu Arg GCA GCC Ala Ala 265 798 840 TTA AAC AGA Leu Asn Arg
GTT
Val 270 CAT GAC CGA GAC His Asp Arg Asp
ACA
Thr 280 GTA CCA GTA TTWT AAT CCA GTC TTA GAA TCA CCA GAA Val Pro Val Phe Asn 285 Pro Val Leu Glu TCT CCA Ser Pro 882 Ser Pro Glu 290
GAT
Asp 295 CCA ATA, ACA TTT Pro Ile Thr Phe
CCA
Pro 300 TCT GCC TTG GAA AGA ATG AGA AAT Ser Ala Leu Glu 'Arg Met ArgAsn 305.
GGT GAA TTT Gly Giu Phe 15 310 GCT GAA GGT Ala Giu Gly 325 ATG GAA GTT Met Giu Val CCT AGA GAT Pro Arg Asp CCT GAT GTC Pro Asp Val
GAT
Asp 315 GTC ATC ATT GGT Val Ile Ile Gly TTC AAT AGT Phe Asn Ser 320 TTA AGA TCT ATG Leu Arg Ser Met
GCA
Al a 330 AGA GTA'ACC AGA Arg Val Thr Arg GGA AAC Gly Asn 335 AAG ACT TTG ACA Lys Thr Leu Thr 966 1008 1050 1092 1134 ATA GAA AGG GCT Ile Giu Arg Ala
ATA
Ile 350
GAG
Glu ATT TGG AAA Ile Trp Lys
CCA
Pro 360 AAT GGT ATT Asn Gly Ile 25 GAG Glu 365 AAA AAA CTA ATA Lys Lys Leu Ile
AAA
Lys 370 ATG CTT ACA GAG Met Leu Thr Giu TAT GAC CAA Tyr Asp Gin GTC CAA CTA Val Gin Leu 390 GTG AAA GAA Val Lys Giu 30 380 AAA GGC GAT Lys Gly Asp 395 AAA GCC ATA Lys Aia Ile CAA AAC GAT Gin Asn Asp
GAC
Asp 385 ATT GAA GCC TAC Ile Glu Ala Tyr GCT GGT TAC CTC CAA GGA ATC TAC CGT Ala Gly Tyr Leu Gin Gly Ile Tyr Arg ACC TTG Thr Leu 405 1176 1218 1260
TTT
Phe 410 TTC AAT GAA TTC AGA AGG AAT TCC AAT Phe Asn Giu Ph-e Arg Arg Asn Ser Asn 415 TAT TTG TAC AGG Tyr Leu Tyr Arg TCA GAC GAT ACG Ser Asp Asp Thr
TAT
Tyr 430 AGT GTA TAT AAA Ser Val Tyr Lys 1302
AGT
Ser 435 TAT ATC TTG CCC TAT CGA TGG GGT TCC TTG CCA GGA GTT Tyr Ile Leu Pro Tyr Arg Trp Gly Ser Leu Pro Giy Val 1344 -140- AGT CAT GGT GAT GAT TTA GGA TAT CTT TTT GCA AAC TCG TTG Ser His Gly Asp Asp Leu Gly Tyr Leu Phe Ala Asn Ser Leu 450 455 460 1386 GAT GTT CCT ATT TTO GGA ACA ACG CAC Asp Val Pro Ile Leu Gly Thr Thr His 465 470 AT? TCT ATA Ile Ser Ile CCG CAA Pro Gin 475 1428 GAT GCT ATG Asp Ala Met ACT CTG GAA Thr Leu Giu AGG ATG Arg Met 485 GTC AGG ATC TGG Val Arg Ile Trp,
ACC
Thr 490 1470 AAT TTT GTA AAG Asn Phe Vai Lys AAT GGA AAA CCT ACA TCA AAC ACT GAA GAT Asn Gly Lys Pro Thr Ser Asn Thr Glu Asp 495 Soo 1512
GCA
Al a 15 sos TCA TGT GAT ACA Ser Cys Asp Thr S.
S
*S
S
S
*5 *5
AAA
Lys 510 AGA CAT TTA AAC Arg His Leu Asn
GAC
Asp 515 AT? TTT TGG Ile Phe Trp GAC ATG GGA Asp Met Gly 530 1554 159G GAA CCA Giu Pro 520 TAC AAC GAC GAA Tyr Asn Asp Glu
GAA
Glu 525 CCA AAA TAT TTG Pro Lys Tyr Leu
S
*5
S
S
5*5* AAA GAA AAT Lys Glu Asn 535 ATG ATG CTT Met Met Leu TTT GAA ATG AAA Phe Giu Met Lys
AAT
As n 540 ATT TTG GAA CTA Ile Leu Giu Leu AAA CGC Lys Arg 545
TGG
Trp 550 GAT GAA GTT Asp Giu Val TAT AGA Tyr Arg 555 AAT GCG AAT TTG Asn Ala Asn Leu
CGG
Arg 560 1638 1680' 1710 25 TTT AGA GTC TGT AAT GAA GAA AGT ATT AGA Phe Arg Val Cys Asn Giu Giu Ser Ile Arg 565 570 INFORMATION FOP. SEQ ID NO:28: SEQUENCE CHARACTERISTICS: 30 LENGTH: 1788 nucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE:
FEATURE:
NAME/KEY:
LOCATION:
cDNA
CDS
1. .1788
ATG
Met 1 (iv) SEQUENCE DESCRIPTION: SEQ ID NO:28: TTA CC? CAC AGT AG? GCA TTA GTT TTA TTT TTA TTT TTT Leu Pro His Ser Ser Ala Leu Val Leu Phe Leu Phe Phe S -141-
TTA
Leu TTT TTC TTA TTT ACA CCT Phe Phe Leu Phe Thr Pro 20 ATC. TTG TGC ATA CTA TGG GAT Ile Leu Cys Ile Leu Trp Asp AAC CTA GAT CAG CAT TTG TGC AGA OTT CAA TTT AAC GGG ATC Asn Leu Asp Gin His Leu Cys Arg Val. Gin Phe Asn. Gly Ile 35 ACG GAA GGA AAA CCG Thr Giu Oly Lys Pro TTC COA TAT Phe Arg Tyr 50 AAA OAt CAT AGO Lys Asp His Arg AAT OAT Asn Asp GTA TAT TOT TCT Vai Tyr Cys Ser TAT TTG OGA ATT CCT Tyr Leu Gly Ile Pro TAT 0CC GAA CCO Tyr Ala OJlu Pro TTT OGA CCA TTA Phe Giy Pro Leu
COA
Arg TTT CAG TCT CCA Phe Gin Ser Pro CCA ATA TCA AAT Pro Ile Ser Asn .00.
*.0 *.0 0 0
CCA
Pro AAA ACA OGA TTC Lys Thr Oly Phe CAG OCT COA ACT Gin Ala Arg Thr OGA GAC A.AA Gly Asp Lys OGA AGC OAA Gly Ser Glu 110 210 252 294 336 378 420 TOT TTC *Cys Phe 100 CAG OAA AGT CTA Gin Giu Ser Leu
ATA
Ile 105 TAT TCT TAT OCA Tyr Ser Tyr-Ala OAT TOC TTA Asp Cys Leu 115 TCT OCO AAC Ser Ala Asn TAT CTO AAT ATA TTC ACO CCA GAO ACT Tyr Leu Asn Ile Phe Thr Pro Oiu Thr OTT AAT Vai Asn 125
AAT
Asn 130 ACA AAA TAT CCT OTA ATG TTC TOO ATC Thr Lys Tyr Pro Val Met Phe Trp Ile 135
CAT
His 140 OGA GOC OCA TTC Oly Oly Ala Phe CAA OGA TCA OGA Gin Oly Ser Oly TAT AAT TTT TTT Tyr Asn Phe Phe
OGA
Gly 155 CCT OAT TAT TTG Pro Asp Tyr Leu AOG OAA GGA ATT Arg 0Th Oly Ile
ATT
Ile 165 TTO GTC ACT Leu Val Thr TCA GCO CCO Ser Ala Pro 180 ATC AAC Ile Asn 170 TAT AGA TTA OGA Tyr Arg Leu Oly
OTT
Val1 175 TTC GOT TTT CTA Phe Oly Phe Leu OAA TOG OAT Oiu Trp Asp 185 TTO GCA CTA Leu Ala Leu ATC CAT OGA AAT Ile His Gly Asn GOT CTA AAA GAC Oly Leu Lys Asp CAG AGA Gin Arg 195 TOG OTT TAC GAC Trp Val Tyr Asp
AAC
Asn 205 ATC OAA AAG TTT Ile Olu Lys Phe
GT
Oiy 210 -142- GGA GAC AGA GAA AAA ATT ACA ATT GCT GGA Gly Asp Arg Glu Lys Ile Thr Ile Ala Gly 215 220 GAA TCT GCT GGA Giu Ser Ala Gly
GCA
Ala 225 GCA AGT GTC CAT Ala Ser Val His
TTT
Phe 230 CTG ATG ATG GAC Leu Met Met Asp
AAC
Asn 235 TCG ACT AGA Ser Thr Arg ACA TTA CTA Thr Leu Leu 250 714 756 AAA TAC Lys Tyr 240 TAC CAA AGG Tyr Gin Arg GCC ATT Ala Ile 245 TTG CAG AGT GGG Leu Gin Ser Gly AAT CCG ACT Asn. Pro Thr 255 AAA CTC AAA Lys Leu Lys CTA AAC CTG Leu Asn Leu GCT AAT CAA ATT CAA CTT CTG CAT AGA TTT Ala Asn Gin Ile Gin 260 Leu Leu His Arg
CAA
Gin 270 GTG CTA AAC ATC Vai Leu Asn Ile
GAA
Giu
CTC
Leu 280
ACG
Thr 275 CAA =A CAA GAA Gin Ls Gin Giu GAT AAA Asp Lys 285 AAC CTA ATT TTA CGA Asn Leu Ile Leu Arg 290 GCA GCC TTA AAC Ala Ala Leu Asn
AGA
Arg 295 GTT CCT GAT AGC Val Pro Asp Ser
AAC
As n 300 GAC CAT GAC CGA Asp His Asp Arg
GAC
Asp 305 ACA GTA CCA Thr Val Pro CCA GAT CCA Pro Asp Pro 320 798 840 882 924 966 1008 1050 GTA TTT Val Phe 310 AAT CCA GTC TTA Asn Pro Val Leu
GAA
Giu 315 TCA CCA GAA TCT Ser Pro Glu Ser ATA ACA TTT Ile Thr Phe 325 CCA TCT GCC TTG Pro Ser Ala Leu
GAA
Glu 330 AGA ATG AGA AAT Arg Met Arg Asn GGT GAA Giy Giu 335 TTT CCT GAT GTC GAT GTC ATC ATT Phe Pro Asp Vai Asp Vai Ile Ile 340
GGT
Gly 345 TTC AAT AGT.GCT Phe Asn Ser Ala
GAA
Glu 350 GGT TTA AGA TCT Gly Leu Arg Ser
ATG
Met 355 GCA AGA GTA ACC Ala Arg Val Thr
AGA
Arg 360 GGA AAC ATG GAA Giy Asn. Met Giu 1092 1134
GTT
Val 365 CAC AAG ACT TTG His Lys Thr Leu
ACA
Thr 370 AAT ATA GAA AGG, Asn Ile Glu Arg
GCT
Al a 375 GAT GCT Asp Ala 380 AAA CTA Lys Leu AAT ATT TGG AAA Asn Ile Trp Lys
AAT
Asn 385
ACA
Thr CCA AAT GGT ATT Pro Asn Gly Ile ATA CC? AGA Ile ezi~ Arg GAG GAG AAA Giu Glu Lys 390 CAA GTG AAA Gin Val Lys 405 1176 1218
ATA
Ile 395 AAA ATG CTT Lys Met Leu
GAG
Giu 400 TTT TAT GAC Phe Tyr Asp -143- GAA CAA AAC GAT GAC ATT GAA GCC TAC GTC CAA CTA AAA GGC Glu Gin Asn Asp Asp Ile Giu Ala Tyr Val Gin Leu Lys Gly 410 .415 420 1260 GAT GCT GGT Asp Ala Gly TAC CTC Tyr Leu 425 CAA GGA ATC TAC CGT ACC TTG AAA GCC Gin Giy Ile Tyr Arg Thr Leu Lys Ala 430 1302
ATA
Ile 435 TTT TTC AAT Phe Phe Asn GAA TTC Glu Phe 440 AGA AGG AAT TCC Arg Arg Asn Ser
AAT
Asn 445 TTG TAT TTG Leu Tyr Leu AAA AGT TAT Lys Ser Tyr 460 TAC AGG Tyr Arg 450 TTA TCA GAC GAT Leu Ser Asp Asp
ACG
Thr 455 TAT AGT GTA TAT Tyr Ser Val Tyr ATC TTG CCC TAT lie Leu Pro Tyr 465 0* CGA TGG GGT TCC Arg Trp Gly Ser 470 GGA TAT CTT TTT Gly Tyr Leu Phe TTG CCA GGA GTT Leu Pro Gly Vai AGT CAT Ser His 475 GGT GAT GAT Gly Asp Asp
TTA
Leu 480
GCA
Ala 485 AAC TCG TTG GAT Asn Ser Leu Asp
GTT
Val 490- CCT ATT TTG GGA ACA ACG Pro Ile Leu Gly Thr Thr 495 CAC ATT TCT His Ile Ser
ATA
Ile 500 CCG CAA GAT GCT ProGln Asp Ala 1344 1386 1428 1470 1512 1554 1596 1638 1680 1722
ATG
Met 505 CAG ACT CTG GAA Gin Thr Leu Glu
AGG
Arg 510 ATG GTC AGG ATC TGG ACC AAT TTT Met Val Arg Ile Trp Thr Asn Phe GTA AAG AAT Val Lys Asn 520 TGT GAT ACA Cys Asp Thr 535 TAC AAC GAC Tyr Asn Asp GGA AAA CCT Gly Lys Pro
ACA
Thr 525 TCA AAC ACT GAA Ser Asn Thr Glu GAT GCA TCA Asp Ala Ser 530 AAA AGA CAT TTA Lys Arg His Leu
AAC
Asn 540 GAC ATT TTT TGG Asp Ile Phe Trp GAA CCA Glu Pro 545
GAA
Glu 550 GAA CCA AAA TAT Glu Pro Lys Tyr
TTG
Leu 555 GAC ATG GGA AAA Asp Met Giy Lys
GAA
Glu 560 AAT TTT GAA ATG Asn Phe Giu Met
AAA
Lys 565 AAT AT'! TTG GAA Asn ie Leu Glu
CTA
Leu 570 AAA CGC ATG ATG Lys Arg Met Met
CTT
Leu 575 TGG GAT GAA GTT *Trp Asp Glu Val
TAT
Tyr 580 AGA AAT GCG AAT TTG CGG TTT AGA Arg Asn Ala Asn Leu Arg Phe Arg 585 1764 GTC TGT*AAT GAA GAA AGT Val Cys Asn Giu Glu Ser 590 ATT AGA Ile Arg 595 1788 -144- INFORMATION FOR SEQ ID NO:29: SEQUENCE
CHARACTERISTICS:
LENGTH: 1788 nucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) SEQUENCE DESCRIPTION: SEQ ID NO:29: TCTAATACTT TCTTCATTAC AGACTCTAA CCGCAAATTC GCATTTe'Tnl
C.
C
C
I AAACTTCATC
TCAAAATTTT
TGGTTCCCAA
CTTCAGTGTT
CTGACCATCC
15 CGTTGTTCCC
AATCATCACC
ATATAACTTT
CAAATTGGAA
GGTAGATTCC
TCAATGTCAT
TTTTATTAGT
CATCTCTAGG
ATGTTTCCTC
GAAACCAATG
25 CCAAGGCAGA
ACTGGATTAA.
AACTCTGTTT
GTTCTTGTTT
TGCAGAAGTT
CAAAATGGCC
AATGGACACT
CTGTCTCCAC
TCTCTGGTCT
ATAGAAAACC
CCTTCCCTGA
TTGGTTGAAT
TGTTCGCAGA
TCTTCGCTTC
TCCCAAAGTT
TTGGAGACTG2
CCCAAATAAG
T-.TTCCTTCC
GGTTATCCCA
AATAAAA.ATA
CCAAAGCATC
CTTTTCCCAT
AAAATGTCGT
TGATGTAGGT
TTTCCAGAGT
AAAATAGGAA
ATGACTAACT
TATATACACT
TTCCTTCTGA
TTGGAGGTAA
CGTTTTGTTC
TTTtTCTCCT
TATAGCCCTT
TGGTTACTCT
ATGACATCGA
TGGAAATGTT
PLTACTGGTAC
AAGGCTGCTC
rTGCGTGATG
GAATTTGATT
CTTTGGTAGT
rGCTGCTCCA
CAAACTTTTC
rTTAGACCCA 3AAAACTCCT rCAAATAATC 3CGCCTCCAT kTTAACAGTC
:TGCATAAGA
:GAGCCTGTA
%.AATCGTAAT
kACAATATAC 7TGATCCCGT
L'AGTATGCAC
k.AACTAATGC
ATGCGTTTTA
GTCCAAATAT
TTAAATGTCT
TTTCCATTCT
CTGCATAGCA
CATCCAACGA
CCTGGCAAGG
ATACGTATCG
ATTCATTGAA
CCAGCATCGC
TTTCACTTGG
CAATACCATT
TCTATATTTG
TGCCATAGAT
CATCAGGAAA
ATTGGATCTG
TGTGTCTCGG
GTAAAATTAG
TTTAGCACTT
AGCAGTCGGA
ATTTTCTAGT
GCAGATTCTC
GATGTTGTCG
TATTTCCATG
AATCTATAGT
AGGTCCAAAA
GGATCCAGAA
TCTGGCGTGA
ATATATTAGA
CGAATCCTGT
GGTCCAAAAG
ATCATTCCTA
TAAATTGAAC
A.AGATAGGTG
ACTACTGTGA
GTTCCAAAAT
TTTGGTTCTT
TTTTGTATCA
TTACAAAATT
TCTTGCGGTA
GTTTGCAAAA
AACCCCATCG
TCTGATAACC
AAATATGGCT
CTTTTAGTTG
TCATAAAACT
TGGATTTTTC
TCAAAGTCTT
CTTAAACCTT
TTCACCATTT
GAGATTCTGG
TCATGGTCGT
GTTTTTATCC
GTTTGAGTTT
TTTAGTAATG
CGAGTTGTCC
CAGCAATTGT
TAAACCCATT
GATATCCCAT
TGATAGTGAC
AAATTATAAG
CATTACAGGA
PLTATATTCAG2
CTTTCCTGGA
TTTTGGATTT
GCGGTTCGGC
TGATCTTTAT
rCTGCACAAA9
TAAATAAGAA
GGTAACAT
ATTTTTCATT
CGTCGTTGTA
CATGATGCAT
GGTCCAGATC
TAGA.AATGTG
AGATATCCTA
ATAGGGCAAG
TGTACAAATA
TTCAAGGTAC
GACGTAGGCT
CTGTAAGCAT
CAAATATTAG
GTGAACTTCC
CAGCACTATT
CTCATTCTTT
rGATTCTAAG rGCTATCAGG
AGGTTTAGGA-
rTCAAATCTA rCCCACTCTG k.TCATCAGAA
%ATTTTTTCT
ETAGTGCCAA
ECCGGCGCTG
:AAAATAATT
kTCCTGATCC
LATTTTGTAT
kTATAAGCAAk.ACATTTGTC
ATATTGGTT
kTAAGGAATT kTCGGAACGG
~GCTGATCTA
AATAAAAAA
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 11.00 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1788 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 2801 nucleotides TYPE: nucleic acid STRANDEDNESS: single -145- TOPOLOGY: linear (ii) MOLECULE
TYPE:
(iii) FEATURE:
NAME/KEY:
LOCATION:
cDNA
CDS
99. .1886 (iv) SEQUENCE DESCRIPTION: SEQ ID GACATGTCGT CTTCAAAACG TCTATTTTAT CATAAACAAA
ACGAGATAA
TAATAACAAT TAAGCATCCA AAATGCATTA AAAAAAACAT
CATAAAAA
ATG TTA CCT CAC AGT GCA TTA GTT TTA TTT TTA TTT TTT TTA Met Leu Pro His Ser Ala Leu Val Leu Phe Leu Phe Phe Leu s0 98 140 TTT TTC TTA TTT ACA CCT GTC TTG TGC ATA CTA 0 0 *0*0 0* 0 0* *0 0*0**0 *000 0 0 0 000 0 00** *0*0 0* 00 0 0 0**0 Phe Plhe 15 CTA GAT Leu Asp 30 Leu Phe Thr Pro Val Leu Cys Ile TGG GAT AAC Trp, Asp Asn GGG ATC ACG Gly Ile Thr CAG CAT TTG TGC Gin His Leu Cys
AGA
Arg 35 GTT CAA TTT AAC Val Gin Phe Asn GAA GGA AAA Glu Gly Lys 45 TAT TGT TCC Tyr Cys Ser CCG TTC CGA TAT Pro Phe. Arg Tyr
AAA
Lys 50 GAT CAT AAA AAT Asp His Lys Asn GAT GTA Asp Val 182 224 266 308 350 392
TAT
Tyr 60 TTG GGA ATT CCT Leu Gly Ile Pro
TAT
Tyr GCA GAA CCG CCT Ala Glu Pro Pro GGA CCA TTG CGA Gly Pro Leu Arg
TTT
Phe 75 CAG TCT CCA AAA Gin Ser Pro Lys
CCA
Pro ATA TCA AAT CCA Ile Ser Asn Pro
AAA
Lys ACA GGA TTC GTT Thr Gly Phe Val
CAG
Gin 90 GCT CGG TCT TTA Ala Arg Ser Leu
GGA
Gly GAC AAA TGT Asp Lys Cys TTC CAG GAA Phe Gin Giu 100 TGC TTA TAT Cys Leu Tyr 115 AGT CTA ATA Ser Leu Ile
TAT
Tyr 105 TCT TAT GCA GGA AGC GAA GAT Ser Tyr Ala Gly Ser Giu Asp 434 476 CTG AAT ATA TTC Leu Asn Ile Phe CCA GAG ACT GTT Pro Glu Thr Val AAT TCT Asn Ser 125 GCG AAC AAT ACA AAA Ala Asn Asn Thr Lys 130 GGC GCA TTC AAC CAA Gly Ala Phe Asn Gin 145 TAT CCT GTA ATG TTC TGG ATC CAT GGA Tyr Pro Val Met Phe Trp Ile His Gly 135 140 GGA TCA GGA TCT TAT AAT TTT TTT GGA Gly Ser Gly Ser Tyr Asn Phe Phe Gly 150 -146- CCT GAT TAT TTG ATC AGG GAA GGA ATT ATT TTG GTC ACT ATC Pro Asp Tyr Leu Ile Arg Giu Gly Ile Ile Leu Val Thr Ile 155 1gn
I
AAC TAT AGA Asn Tyr Arg 170 TTA GGA GTT Leu Gly Val
TTC
Phe 17S GGT TTT CTA TCA Gly Phe Leu Ser GCG CCG GAA Ala Pro Giu 180 TGG GAT ATC Trp Asp Ile 185 GCA CTA AAA Ala Leu Lys CAT GGA AAT ATG His Gly Asn Met
GGT
Gly 190 CTA AAA GAC CAG Leu Lys Asp Gin AGA TTG Arg Leu 195
TGG
Trp 200 GTT TAT GAC AAC Val Tyr Asp Asn
ATC
I le 205 *GAA AAA TTT GGT Giu Lys Phe Giy
GGA
Gly 210 686 728 770 812 GAC AGA GAT AAA Asp Arg Asp Lys
ATC
Ile 215 ACT ATA GCT GGA Thr Ile Ala Gly 9 9* 9*
GAA
Glu 220 TCT GCT GGA GCA Ser Ala Gly Ala
GCA
Al a 225 AGT GTT CAT TTT Ser Val His Phe
CTG
Leu.
230 ATG ATG GAC AAT Met Met Asp Asn
TCT
Ser 235 ACT AGA AAA Thr Arg Lys TTA CTC AAT Leu Leu Asn 250 TAC TAC Tyr Tyr 240 CAA AGG GCA ATT Gin Arg Ala Ile
TTG
Leu 245 CAG AGT GGG ACA Gin Ser Gly Thr 9 9**9 9 0 9* 09 9 0 9909 CCG ACT GCT Pro Thr Ala 255 CTA AAA CAA Leu Lys Gin AAT CAA ATT CAA Asn Gin Ile Gin CTG CAT AGA TTT Leu His Arg Phe GAA AAA Giu Lys 265
GTG
Val 270 CTG AAC ATC ACG Leu Asn Ile Thr
CAA
Gin 275 AAA CAA GAA CTC Lys Gin Giu Leu
CTA
Leu 280 854 896 938 980 1022 AAT CTG GAC AAA Asn Leu Asp Lys
AAT
Asn 285 CAA ATT TTG CGA Gin Ile Leu Arg
GCA
Aia 290 GCC TTA AAC AGA Ala Leu Asn Arg
GTC
Val1 295 CCA GAT AAC AAC Pro Asp Asn Asn
GAC
Asp 300 CAC GAA AGG GAC His Glu Arg Asp
ACA
Thr 305 GTA CCA GTA Val Pro Val 13AC CCA ATA Asp Pro Ile 320 TTT AAT Phe Asn 310 CCA GTC CTA GAA Pro Val Leu Glu
TCA
Ser 315 CCA GAA TCT CCA Pro Giu Ser P-) 1064 ACA TTT CCA Thr Phe Pro 325 CCT GAC GTT Pro Asp Val TCT GCT TTA GAA Ser Ala Leu Glu
AGA
Arg 330 ATG AGA AAT GGT Met Arg Asn Gly GAA TTT Glu Phe 335 1106 1148
GAT
Asp 340 GTC ATC ATT GGA Vai Ile Ile Gly
TTC
Phe 345 AAT AGT GCT GAA Asn Ser Ala Giu -147- TTA AGA TCT ATG CCA AGA GTA ACC Leu Arg Ser Met Pro Arg Val Thr 355 AGA GGA Arg Gly 360 AAC ATG GAA GTT Asn Met Glu Val 1190
TAC
Tyr 365 AAG ACT TTG ACA Lys Thr Leu Thr
AAT
Asn 370 ATA GAG AGA GCT Ile Glu Arg Ala
ATA
Ile 375 CCT AGA GAT Pro Arg Asp GAG AAA AAA Glu Lys Lys 390 1232 1274 GCT AAT Ala Asn 380 ATT TGG AAA AAT Ile Trp Lys Asn
CCT
Pro 385 AAT GGC ATT GAG Asn Gly Ile Glu CTT ATA AAA Leu Ile Lys 395 ATG CTT ACA GAG Met Leu Thr Glu
TTT
Phe 400 TAT GAC CAA GTT Tyr Asp Gin Val AAA GAA Lys Glu 405 1316 CAA AAC GAT GAC Gin Asn Asp 0 *0*0 0**0 Asp 410 ATC GAA GCC TAT Ile Glu Ala Tyr
GTC
Val 415 CAA CTA AAA GGC Gin Leu Lys Gly
GAT
Asp 420 GCT GGT TAT CTC Ala Gly Tyr Leu
CAA
Gin 425 GGA ATT TAC CGT Gly Ile Tyr Arg
ACC
Thr 430 TTG AAA GCC ATA Leu Lys Ala Ile
TTT
20 Phe 435 TTC AAT GAA ATC Phe Asn Glu Ile
AAA
Lys 440 AGA AAT TCC AAC TTG TAT TTG TAT Arg Asn Ser Asn Leu Tyr Leu Tyr 445 AGG TTA TCA Arg Leu Ser 450 25 TTG CCC TAT Leu Pro Tyr 465 GAT GAT ACG Asp Asp Thr
TAT
Tyr 455 AGT GTA TAT AAA Ser Val Tyr Lys AGT TAT ATC Ser Tyr Ile 460 CGA TGG GGT TCC Arg Trp Gly Ser
TTG
Leu 470 CCA GGA GTT AGT Pro Gly Val Ser CAT GGT His Gly 475 1358 1400 1442 1484 1526 1568 1610 1652 1694 GAT GAT TTA Asp Asp Leu
GGA
Gly 480 TAT CTT TTT GCA Tyr Leu Phe Ala
AAC
Asn 485 TCT TTG GAT GTT Ser Leu Asp Val
CCT
Pro 490 ATT TTG GGA ACA lie Leu Gly Thr
ACG
Thr 495 CAC ATT TCT ATA His Ile Ser Ile
CCG
Pro 500 CAA GAT GCT ATG Gin Asp Ala Met
CAG
Gin 505 ACT CTG GAA AGG Thr Leu Glu Arc, GTC AGG ATC TGG Val Arg Ile Trp
ACC
Thr 515 AAT TTT GTA Asn Phe Val GCA TCA TGT Ala Ser Cys 530 AAG AAT Lys Asn 520 GGA AAA CCT ACA Gly Lys Pro Thr
TCA
Ser 525 AAC ACT GAA GAT Asn Thr Glu Asp GAT ACA AAA Asp Thr Lys 535 AGA CAT TTA AAC Arg His Leu Asn
GAC
Asp 540 ATT TTT TGG GAA Ile Phe Trp Glu CCA TAC Pro Tyr 545 1736 -148- AAC GAC GAA GAA CCA AAA TAT TTG GAC ATG GGA Asn Asp Glu Gin Pro Lys Tyr Leu Asp Met Gly 550 C zC AAA GAA CAT Lys Gin His 1778 1820 TTT GAA ATG Phe Gin Met AAA AAT ATT TTG GAA CTA AAA Lys Asn Ile Leu Glu Len Lys CGG) ATG ATG CTT Arg Met Met Leu GAT GAA GTT TAT AGA AAT GCG AAT TTG CGG Asp Glu Val Tyr Arg Asn Ala Asn Len Arg 580 585 TT AGA GTC Phe Arg Val 1862 TOT AAT GAA GAA AGT ATT AGA TGA GTTTTTTTAA
TTTTACATAC
Cys Asn Glu Gin Ser Ile Arg 590 Cq 1906 .4 .9 4 9 *4 4 9 .4.9 49..
9. 4* 99 9 4 494499 9 9 4 9 AGCCGAGAGG
AAACATGACT
AATCACATGG
ACCATAGTAA
15 CAAGAAAACT
TATTGCATCA
AAAAACACTT
TTTATGACTG
TTTACCWAT
CGCCAAATTT
GGCAATTAT
GTGTTAAACA
TCTAATAAGA
AGGTTATATA
20 GCTATATATA
AAAACTTANJC
AAAATATGTT
GATTTTCCTA
TTAAAATATG
ATATTTTCAA
TTTACCAGTA
CTATGAAACT
ATTTCTCAA
AATGTTTAGT
25 TGTAAATTA
ACTATTTTTT.
AAAAATTTTA
CTGTGATAAT
AAACTCACAA
TTATGTGAAT
TTTTCTATAT
CATTTTATAT
AAATACAAAT
GCGGTTGTTA
30 AATCTGAATA
AATGTACTTT
AAAATTGGAA
CTTTATTACA
AAGAAAATAT
ACATGGCCCA
TGTAGCGTCA
TTCAACTCTA
AAAAGACTTG
GTCTCGTTAT
ATAATCTAAG
TTAATTAATT
ATTTTAAATA
GTAACAAGAC
ATCTATCACA
ATTTGACATT
ATTGTTTTTT
TACGGTGATA
AAAATAATCT
TAAGTAAAAA
AGAAAAATCA
TGATTTAGTT
TATTTTGCCA
TAATTGAAGC
GACACATTTA
TATTAAAAAT
AAAATAATAA
GCTAAACTTT
ATATTATATT
TTAGTTTTAA
TATTTTTTAT
CATTAAATTA
ACCGCTTAAT
TACACAATAT
GTTAAAAAAA
TGGATTAATG
GAAAAAGAAA
TCAAGTGTAT
AAATTCTTGG
TTTTTCTTCT
TTTATGACAT
GGTAGTATTT
GATTTGCTCG
TTTGATGGTA
TTAGATTAAA
ATGTACTATA
TACAATATTT
GAGTTAATGT
TGGTGCAAAG
TACGAATTGT
CATACATGAC
TCAACATGTA
TGTTATATAA
1956 2006 2056 2106 2156 2206 2256 2306 2356 2406 2456 2506 2556 2606 2656 2706 2756 2801 AAAnAAnA AAAAA *4 4 4 4 *494 9 *49* INFORMATION FOR SEQ ID NO:3.1: SEQUENCE
CHARACTERISTICS:
LENGTH: 595 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) SEQUENCE DESCRIPTION: SEQ ID NO:31: Met Leu Pro His Ser Ala Len Val Len Phe Leu Phe Phe Len Phe Phe Leu Phe Thr Pro Val Leu Cys Ile Leu Trp Asp Asn 20 Len Asp Gin His Leu Cys Arg Val Gin Phe Asn Gly Ile Thr 35 Gin Gly Lys Pro Phe Arg Tyr LYS Asp His Lys Asn Asp Val 50 Tyr Cys Gly Pro Lys Thr Phe Gin 100 Ser Leu Gly Glu Tyr Leu Gly Arg Phe Gin Phe Val Gin 90 Ser Leu Ile Ile Ser Ala Tyr Pro Pro Arg Ser -149- Tyr Ala 65 Lys Pro Ser Leu Tyr Ala Glu Pro Pro Ile Ile Ser Asn Pro Gly Asp Lys Cys Gly Ser Giu Asp 110 Cys Leu Tyr Leu Asr 115 Ile 00 @0 0
S
.99.
SS S @0 @0 0000 0
S
00
S
0 Ala Gly Pro 155 Asn Trp 20 Ala Asp 25 Ala 225 Tyr Pro Leu Asn Vai 295 Phe Asn Ala Asp Tyr 170 Asp Leu Arg Ser Tyr 240 Thr Lys Leu Pro Asn 310 Asn Phe Tyr Arg Ile 185 Lys Asp Val Gin Ala 255 Gin Asp Asp Pro Thr 130 Asn Leu Leu His Trp 200 Lys His Arg Asn Va1 270 Lys Asn Va1 Lys Gin 145 Ile Gly Gly Va1 Ile 215 Phe Ala Gin Leu Asn 285 Asn Leu Tyr Gly Arg 160 Val Asn Tyr Thr Leu 230 Ile Ile Asn Gln Asp 300 Glu Phe Thr 120 Pro Val Ser Gly Glu Gly Phe Gly 175 Met Gly 190 Asp Asn Ile Ala Met Met Leu Gin 245 Gin Pro 260 Ile Thr Ile Leu 2 His Giu 2 Ser Pro C 315 Prc Met 135 Ser lie Phe Leu Iie.
205 Gly Asp 3er Leu 31n a75 krg %xg liu Glu Phe Tyr 150 Ile Leu Lys Glu Glu 220 Asn Gly His Lys Ala 2 290 Asp Ser I Thl Trp Asn Leu 165 Ser Asp Lys Ser Ser 235 rhr Arg Aln kla thr 305 Pro Val Asn 125 Ile His Phe Phe Val Thr Ala Pro 180 Gin Arg 195 Phe Gly Ala Gly Thr Arg Leu Leu 2 250 Phe Giu I 265 Glu Leu I Leu Asn Vai Pro A Asp Pro I 320 Ser Gly 140 Gly Ile Glu Leu Gly 210 kla ys ksn ys .eu rg tal le Thr Pro Phe Asp Pro 325 Val Ser Asp Al a Val1 Giu Arg 330 Ile Gly -150- Met Arg Phe Asn Gly Ala Phe Gly 350 340 345 Le Ty2 .1 0%.
o o.
.0.0 0 Goa* 1U Ala Leu Gin Al a Phe 435 Arg Leu Asp Ile Gin 505 Lys Asp Asn Phe Arg Lys As n 380 Ile Asn Gly Phe Leu 450 Pro Asp Leu Thr Asn 520 Thr Asp Glu Ser Thr Ile Lys 395 Asp Tyr Asn Ser Tyr 465 Leu Gly Leu Gly Lys 535 G1u Me t Met *Leu Trp, Met Asp 410 Leu Giu Asp Arg Gly 480 Thr Glu Lys Arg Giu 550 Lys Pro 355 Thr Lys Leu Ile Gin 425 Ile Asp Trp T'yr rhr 495 krg Pro 5li Pro ksn 565 Arg Val Thr Asn Ile Giu 370 Asn Pro Asn 385 Thr Glu Phe 400 Giu Aia Tyr Gly Ile Tyr Lys Arg Asn 44b Thr Tyr Ser 455 Gly Ser Leu 470 Leu Phe Ala His Ile Ser Met Vai Arg 510 Thr Ser Asn 525 Leu Asn Asp 545 Lys Tyr Leu Ile Leu Giu Arg Arg Gly 360 Al a 375 Met Pro Gly Tyr Val1 415 Arg Ser Val1 Pro Asn 485 Ile Ile Thr Ile Asp 555 Lieu Ile Asp Gin Thr 430 As n Tyr Gly Ser Pro 500 Trp Giu he Met Lys 570 Glu Gin Leu Leu Leu 445 Lys Vali Leu Gin Thr Asp Trp Gly Arg Glu 390 Val1 Lys Lys Tyr Ser 460 Ser Asp Asp As n Al a 530 Giu Lys Met Lys Lys 405 Gly Al a Leu Tyr His 475 Val1 Ala Phe Ser Pro 545 Glu MIet Gil Arg Val Asp Lys Giu Asp 420 Ile Tyr le Gly Pro 490 Met Val Cys Tyr His 560 Leu Asp Giu Vai Tyr Arg Asn Ala Asn Leu Arg Phe Arg Val -151- Cys Asn Glu Glu Ser Ile Arg 590 ,q; INFORMATION FOR SEQ ID N'O:32: Wi SEQUENCE CHARACTERISTICS: LENGTH: 2801 nucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) SEQUENCE DESCRIPTION: SEQ ID NO:32;
S
TTTTTTTTTT
TTTTTTTTTT
TAACAATTTT
AATACAGATT
GTTGACATTA
ATCCATATCA
GTATGTTTTT
TTAACAAAAA
TCGTAATATT
GTGTAAATGT
CACCAATTAA
GCGGTTGTGA
AACTCTAATT
TAATGGTCTT
TTGTAATAA.A
AAATATATTT
TACATTTAAA
ACTAAAATTA
20 TCTAAAATAT
AATATCTTAG
TCAAAAAAGT
TTAGCATAAC
AAATCTTATT
ATTTTCAAGT
CTACCATTTT
TAATATAGAG
ATAAATAAAT
GTGTCTGACG
AAAAAGCTTC
AATTATGGGC
AATTTTGGCA
AAATAATATT
CTTGAAACTA
AATCATGTAA
TTTTCTGATT
TTTCTTTCCA
TAAAATTAA AAAACTCATC 30 GCAAATTCGC
ATTTCTATAA
TCCAAAATAT
TTTTCATTTC
TGGTTCTTCG
TCGTTGTATG
TTGTATCACA
TGATGCATCT
ACAAATGG
TCCAGATCCT
35 TTGCGGTATA
GAAATGTGCG
TTGCAAAAAG
ATATCCTAAA
CCCCATCGAT
AGGGCAAGAT
TGATAACCTA
TACAAATACA
ATATGGCTTT
CAAGGTACGG
ITTAGTTGGA C\ TAGGCTTC ATAAAACTC, .L I'AAGCATTT GATTTTTCCA
AATATTAGCA
AAAGTCTTGT
AAACTTCCAT
TAAACCTTCA
GCACTATTGA
CACCATTTCT.
CATTCTTTCT
GATTCTGGTG
ATTCTAGGAC
GTGGTCGTTG
TTATCTGGGA
TTTTGTCCAG
ATTTAGGAGT
TTTAGTTTT
CAAATCTATG
GAGTAATGTC
CCACTCTGCA
AATTGTCCAT
CATCAGAAAA
GCTATAGTGA
TTTTATCTCT
ACTTAAAAGT
ATTTTTAACA
CCGTAATATA
ACAATATTCA
CAAATATTAT
TAGATAAAAA
GTTACACTAA
AAAATAGTTT
ATTAATTGAA
ATTATTAGGA
GAGACGNTAA
CTTTTTATAT
TTGAATGTTT
CTACAAAATT
CATGTCAGTC
TTCTTTGATG
TAAAGTTACT
ATTTTAGTCA
TAATACTTTC
ACTTCATCCC
AAAATGTTCT
GTTCCCAAAA
TCAGTGTTTG
GACCATCCTT
TTGTTCCCAA
TCATCACCAT
ATAACTTTTA
AGTTGGAATT
TAAATTCCTT
GATGTCATCG
TTATAAGTTT
TCTCTAGGTA
GTTTCCTCTG
ATCCAATGAT
AAAGCAGATG
TGGATTAAAT
CTCTGTTTAA
TCTTGTTTTT
CAGAGGTTGA
AAATTGCCCT
TGAACACTTG
GTCTCCACCA
ACATTTATTC
ACCGCATTTG
AAATGATATA
CATAATTGTG
CACAGTAAAA
ATAGTTTAAT
ACATTTTTGA
CATAGTACTG
AATATCATAT
AAATCAACAT
GTTTTTATAT
AACCTTCTTA
AACACATTAA
TGGCGATTTG
ATAAAAAGTG
CAATAAGTTT
ATGGTCCATG
TGTTTCCTCT
TTCATTACAG
AAAGCATCAT
TTTCCCATGT
AATGTCGTTT
ATGTAGGTTT
TCCAGAGTCT
AATAGGAACA
GACTAACTCC
TATACACTAT
TCTTTTGATT
GGAGATAACC
TTTTGTTCTT
TTTCTCCTCA
TAGCTCTCTC
GTTACTCTTG
GACATCAACG
GAAATGTTAT
ACTGGTACTG
GGCTGCTCGC
GCGTGATGTT
ATTTGATTAG
TTGGTAGTAT
CTGCTCCAGC
AATTTTTCGA
AGATTTTATA
TATTTTACAT
GAAAAGTCAT
AGTTTACAAT
TTTTTCTTTG
TTACAACATT
GAAATAAATA
GTAAATATAG
TTTAATTTAA
ATTTTTACCA
ATAGCCGAGC
TTAGAAAATA
TTGCCATGTC
GTAAAAGAAG
TTTTTCCA.AG
TCTTGATACA
TGATTTTTCT
CGGCTGTATG
ACTCTAAACC
GCGTTTTAGT
CCAAATATTT
AAATGTCTTT
TCCATTCTTT
GCATAGCATC
TCCAAAGAGT
TGGCAAGGAA
ACGTATCATC
TCATTGAAAA
AGCATCGCCT
TAACTTGGTC
ATGCCATTAG
TATATTTGTC
GCATAGATCT
TCAGGAAATT
TGGGTCTGGA
TGTCCCTTTC
AAAATTTGAT
CAGCACTTGT
CAGTCGGATT
TTTCTAGTAG
AGATTCTCCA
TGTTGTCATA
s0 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 11so 1200 1250 1300 1350 1400.
1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000 2050 2100 -152-
AACCCATTTT
TATCCCATTC
ATAGTGACCA
ATTATAAGAT
TTACAGGATA
ATATTCAGAT
TTCCTGGAAA
TTGGATTTGA
GGTTCTGCAT
ATCTTTATAT
TGCACAAATG
AATAAGAAAA
CATTTTTTAT
ATTTATCI'CG
C
AGTGCCAATC
CGGCrOCTGAT
AAATAATTCC
CCTGATCCTT
TTTTGTATTG
ATAAGCAATC
CATTTGTCTC
TATTGGTTTT
AAGGAATTCC
CGGAACGGTT'
CTGATCTAGG
ATAAAAAA
GATGTTTTTT
rTTTGTTTAT TCTGGTCTTT
TAGACCCATA
AGAAAACCGA AAACTCCTAA
TTCCCTGATC
GGTTGAATGC
TTCGCAGAAT
TTCGCTTCCT
CTAAAGACCG
GGAGACTGAA
CAAATAGGAA
TTCCTTCCGT
TTATCCCATA
AAATAATCAG
GCCTCCATGG
TAACAGTCTC
GCATAAGAT
AGCCTGAACG
ATCGCAATGG
CAATATACAT
GATCCCGTTA
GTATGjCACAA
TTTCCATGGA
TCTATAGTTG
GTCCAAAAA
ATCCAGAACA
TGGCGTGAAT
ATATTAGACT
AATCCTGTTT
TCCAATAGGC
CATTTTTATG
AATTGAACTC
GACAGGTGTA
TGTGAGGTAA
AATTGTTATT
GACGACATGT
2150 2200 2250 2300 2350 2400 2450 2500 2550 2600 2650 2700 2750 2800 2801 TTAATGCATT
TTGGATGCTT
GATAAAATAG ACGTTTTGAA INFORMATION FOR SEQ 11) NO:33: (4 SEQUENCE
CHARACTERISTICS:
LENGTH: 1710 nlucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE
TYPE:
FEATURE:
NME/KEY:
LOCATION:
cDNA
CDS
1.-1710
TG
Trp 1 (iv) SEQUENCE DESCRIPTION: .SEQ ID NO:33: GAT AAC CTA GAT CAG CAT TTG TGC AGA GTT CAA TTT AAC Asp Asn Leu Asp Gln His Leu Cys Arg Val Gin Phe Asn 30 GOG ATC ACG GAA GGA AAA CCG TTC CGA TAT AAA OAT CAT A-AA Gly Ile Thr Glu Oly Lys Pro Phe Arg Tyr Lys Asp His Lys AAT OAT Asn Asp GTA TAT TGT TCC Val Tyr Cys Ser
TAT
Tyr 35.
TTG GGA ATT CCT Leu Gly Ile Pro TAT GCA GAA Tyr Ala Olu CCG CCT ATT Pro Pro Ile TCA AAT CCA Ser Asn Pro GGA CCA TTG CGA Oly Pro Leu Arg
TTT
Phe so CAG TCT CCA AAA Gln Ser Pro Lys CCA ATA Pro Ile
AAA
Lys ACA OGA TTC GTT Thr Gly Phe Val
CAG
Gin 65 OCT COG TCT TTA GGA Ala Arg Ser Leu Gly 168 210 252 GAC AAA TGT TTC ASP Lys Cys Phe
CAG
Gin GAA AGT CTA ATA Glu Ser Leu Ile TCT TAT GCA GGA Ser Tyr Ala Gly -153- AGC GAA GAT TGC TTA TAT CTG AAT ATA TTC ACG CCA GAG ACT Ser Giu Asp Cys Leu Tyr Leu Asn Ile Phe Thr Pro Giu Thr 90 294 OTT AAT Val Asn' 100 TCT GCG AAC AAT Ser Ala Asn Asn
ACA
Thr 105 AAA TAT CCT GTA ATG TTC TOO Lys Tyr Pro Vai Met Phe Trp, 110 ATC CAT GGA GGC GCA TTC AAC Ile His Gly Gly Ala Phe Asn 115
CAA
Gin 12 0 GGA TCA GGA TCT Giy Ser Gly Ser TAT AAT Tyr Asn 125 378 TTT TTT GGA CCT Phe Phe Gly Pro 130 GTC ACT ATC AAC Val Thr Ile Asn GAT TAT TTG ATC AGG Asp Tyr Leu Ile Arg 135 GAA GGA ATT ATT Glu Gly Ile Ile
TTG
Leu 140
TAT
Tyr 145 AGA.TTA GGA OTT Arg Leu Giy Vai
TTC
Phe
ISO
GOT TTT CTA TCA Giy Phe Leu Ser 420 462 504 546 9*
GCG
Ala 155 CCG GAA TGG GAT Pro Giu Trp Asp ATC CAT GGA AAT ATG His Gly Asn Met
GOT
Gly 165 CTA A.AA GAC Leu Lys Asp ATC GAA AAA Ile Giu Lys 180 CAG AGA 20 Gin Arg 170 TTG GCA CTA AAA Leu Ala Leu Lys GTT TAT GAC A.AC Val Tyr Asp Asn TTT GOT GGA GAC Phe Gly Gly Asp 185 GCT GGA GCA GCA Ala Gly Ala Ala 200 ACT AGA AAA TAC Thr Arg Lys Tyr AGA GAT AAA Arg Asp Lys ACT ATA GCT GGA Thr Ile Ala Gly GAA TCT Giu Ser 195 AGT OTT CAT TTT CTG Ser Val His Phe Leu 205 ATG ATG GAC AAT Met Met Asp Asn 0 0 *0 0 588 630 672 714
TAC
Tyr 215 CAA AGO GCA ATT Gin Arg Ala Ile
TTG
Leu 220 CAG AGT GGG ACA Gin Ser Gly. Thr
TTA
Leu 225 CTC AAT CCG ACT Leu Asn Pro Thr
GCT
Al a 230 AAT CAA ATT CAA Asn Gin Ile Gin
CCT
Pro 235 TTT GAA Phe Giu 240 GAA CTC GJlu Leu AAA CTA AAA CAA Lys Leu Lys Gin
GTG
Val1 245
AAA
Lys CTG AAC ATC ACG Leu Asn Ile Thr CTG CAT AGA Leu His Arg CAA AAA CA.
Gin Lys 250 CGA GCA GCC Arg Ala Ala 265 756 798
CTA
Leu 255 AAT CTG GAC Asn Leu Asp
AAT
Asn 260 CAA ATT TTG Gin Ile Leu TTA AAC AGA Leu Asn Arg
GTC
Val 270 CCA GAT AAC AAC Pro Asp Asn Asn
GAC
Asp 275 CAC GAA AGG GAC His Giu Arg Asp -154- GTA COA GTA TTT AAT CCA GTC CTA GAA TCA CCA GAA TOT OCA Val Pro Val Phe Asn Pro Val Lau Giu. Ser Pro Giu Ser Pro GAO CCA ATA ACA TTT OCA TCT GCT TTA GAA AGA ATG 295 Pro Ile Thr Phe Pro 300 Ser Ala Leu Giu Met AGA AAT Arg Asn 882 924 9"6 GGT GAA Gly Giu 310 TTT OCT GAO GTT Phe Pro Asp Val
GAT
Asp 315 GTC ATO ATT Val Ile Ile GGA TTO AAT AGT Gly Phe Asn Ser 320 GOT GAA GGT TTA AGA TOT ATG Ala Glu Gly Leu Arg Ser Met 325
OCA
Pro 330 AGA GTA ACC AGA Arg Val Thr Arg GGA AAO Gly Asn 335 1008 ATG GAA GTT Met Giu Val
TAO
Tyr 340 AAG ACT TTG ACA Lys Thr Leu Thr ATA GAG AGA GOT Ile Giu Arg Ala a a 4 4 a 4 a 4 OCT AGA GAT GOT AAT ATT TGG AAA AAT Pro Arg Asp Ala Asn Ile Trp Lys Asn
OCT
Pro 360 AAT GGO ATT GAG Asn Gly Ile Glu 1050 1092 1134 1176
GAG
Glu 365 AAA AAA OTT ATA Lys Lys Leu Ile
AAA
Lys 370 ATG CTT ACA GAG Met Leu. Thr Glu TAT GAO CAA Tyr Asp Gin GTO CAA OTA Val Gin Leu 390 GTT AAA Val Lys 380 GAA CAA AAO GAT Glu Gin Asn Asp
GAO
Asp 385 ATO GAA GOC TAT Ile Glu Ala Tyr AAA GGO GAT Lys Gly Asp 395 AAA GCC ATA Lys Ala Ile GOT GGT TAT OTO Ala Gly Tyr Leu
CAA
Gln 400 GGA ATT TAO CGT Gly Ile Tyr Arg AAA AGA AAT TOO Lys Arg Asn Ser 415 ACC TTG Thr Leu 405 AAC TTG Asn Leu 420
TTT
Phe 410 TTO AAT GAA ATO Phe Asn Giu Ile 1218 1260 1302 TAT .TTG TAT AGG Tyr Leu Tyr Arg
TTA
Leu 425 TCA GAT GAT ACG Ser Asp Asp Thr
TAT
Tyr 430 AGT GTA TAT AAA Ser Vai Tyr Lys
AGT
Ser 435 TAT ATO TTG CCC Tyr'lie Lau Pro
TAT
Tyr 440 OGA T.;G GGT TOO TTG OCA GGA GTT Arg Trp Gly Ser Leu Pro Gly Val 445 1344 AGT OAT Ser His 450 GGT GAT GAT TTA Gly Asp Asp Leu
GGA
Gly 455 TAT OTT TTT GCA Tyr Leu Phe Ala AAC TOT TTG Asn Ser Leu 460 1386 GAT GTT COT Asp Vai Pro 465 ATT TTG GGA ACA Ile Leu Gly Thr CAC ATT TCT ATA His Ile Ser Ile COG CAA Pro Gin 475 1428 -155- GAT GCT ATG CAG ACT CTG GAA AGO ATG GTC AGG ATC TGG ACC Asp Ala Met Gin Thr Leu Glu Arg Met Val. Arg Ile Tr)b Thr 480 485 490 1470 AAT TTT GTa Asn Phe Val.
AAG AAT GGA Lys Asn Gly 495.
AAA CCT ACA Lys Pro Thr
TCA
Ser S00 AAC ACT GAA GAT Asn Thr Glu Asp 1512
GCA
Al a 505
GAA
Glu 520 TCA TGT GAT ACA Ser Cys Asp Thr CCA TAC AAC GAC Pro Tyr Asn Asp
AAA
Lys 510
GAA
Glu 525 AGA CAT TTA AAC Arg His Leu Asn GAA CCA AAA TAT Glu Pro Lys Tyr
GAC
Asp 515 aTT TTT TG Ile Phe Trp 1554 1596 TTG GAC ATG GGA Leu Asp Met Gly 530 GAA CTA AAA CGC Glu Leu Lys Arg 545 AAA GAA Lys Glu 535 CAT TTT GAA ATG His Phe Glu Met
AAA
Lys 540 A-AT ATT TTG Asn Ile Leu 1638 S
S
S
S.
ATG ATG CTT Met Met Leu 550 TTT AGA GTC 20 Piae Arg Val TGG GAT GAA OTT Trp Asp Glu Val TAT AGA AAT GCG AAT Tyr Arg A-sn Ala Asn 555 AGT ATT A-GA Ser Ile Arg 570 TTG CG Leu Arg 560 1680 1710
TOT
Cys 565 A-AT GAA GAA Asn. Glu Glu INFORMATION FOR SEQ ID NO:34: i) SEQUENCE CHARACTERISTICS: LENGTH: 1785 nucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear
S.
S
S
S
S
(ii) MOLECULE TYPE: cDNA
ATG
Met ~1
TTT
Phe (iii) FEATURE: NAME/KEY:
CDS
LOCATION: 1. .1785 (iv) -SEQUENCE DESCRIPTION: SEQ ID NO:34: T'2A CCT CAC AGT OCA TTA OTT TTA TTT TTA TTT TTT TTA Leu Pro His Ser Ala Leu Val Leu Phe Leu Phe Phe Leu 5 TTC TTA TTT A-CA CCT GTC TTG TGC ATA CTA TGO OAT A-AC Phe Leu Phe Thr Pro Val Leu Cys Ile Leu Trp Asp A-sn 20 CTA OAT CAG Leu Asp Gin CAT TTG TOC AGA OTT CAA. TTT AAC 000 ATC ACG His Leu Cys Arg Val Gin Phe Asn Oly Ile Thr 35 -156- GAA GGA AAA CCG TTC CGA TAT Glu GJly Lys Pro Phe Arg Tyr GAT CAT AAA AAT GAT GTA Asp His Lys Asn Asp Val TAT TGT TCC TAT TTG Cys Ser Tyr Leu GGA ATT CCT GlY Ile Pro CAG TCT CC.A Gin Ser Pro TAT GCA Tyr Ala AAA CCA Lys Pro GAA CCG CCT Glu P-ro Pro GGA CCA TTG CGA Gly Pro Leu Arg ATA TCA AAT CCA Ile Ser Asn Pro GGA GAC AAA TGT Gly Asp Lys Cys 210 252 294
AAA
Lys ACA GGA TTC GTT Thr Gly Phe Vai
CAG
Gin 90 GCT CGG TCT TTA Ala Arg Ser Leu TTC CAG Phe Gin 100 GAA AGT CTA ATA Giu Ser Leu Ile
TAT
Tyr 105 TCT TAT GCA GGA AGC GAA GAT Ser Tyr Aia Gly Ser Glu Asp
C
C. TGC TTA TAT CTG AAT ATA TTC ACG CCA GAG ACT GTT Cys Leu Tyr Leu Asn Ile Phe Thr Pro Giu Thr Val 115 120n AAT TCT Asn Ser 125 GCG AAC AAT 20 Ala Asn Asn AAA TAT CCT GTA ATG TTC TGG ATC CAT Lys Tyr Pro Val Met Phe Trp Ile His GGC GCA TTC AAC Gly Ala Phe-Asn C
CAA
Gin 145 GGA TCA GGA TCT TAT AAT TTT TTT GGA Gly Ser Gly Ser Tyr Asn Phe Phe Gly 378 420 462 504 546 588 GAT TAT TTG ATC Asp Tyr Leu Ile
AGG
Arg 160 GAA GGA ATT ATT Glu Gly Ile Ile
TTG
Leu 165 GTC ACT ATC Val Thr Ile AAC TAT Asn Tyr 170 AGA TTA GGA GTT Arg Leu Gly Val
TTC
Phe 175 GGT TTT CTA Giy Phe Leu TCA GCG CCG GAA Ser Ala Pro Giu 180 GAC CAG AGA TTG Asp Gin Arg Leu 195 TGG GAT ATt Trp Asp Ile 18S CAT GGA AAT ATG GGT His Giy Asn Met Gly 190 TGG GTT TAT GAC AAC Trp Vai Tyr Asp Asn CTA AAA Leu Lys GCA CTA A.AA Ala Leu Lys
ATC
Ile 205 GAA AAA TTT GGT Giu Lys Phe Giy
GGA
Gly 210 GAC AGA GAT AAA Asp Arg Asp Lys
ATC
Ile 215 ACT ATA GCT GGA Thr Ile Ala Giy
GAA
Giu 220 TCT GCT GGA GCA Ser Ala Giy Ala GCA ACT GTT CAT TTT Ala Ser Val His Phe 225
CTG
Leu 230 ATG ATG GAC AAT Met Met Asp Asn
TCT
Ser 235 ACT AGA AAA Thr Arg Lys 7- TAO TAO Tyr Tyr 240 CAA AGG GCA ATT TTG Gin Arg Ala Ile Leu 245 CAG AGT 000 ACA TTA CTC AAT Gin Ser Gly Thr Leu Leu Asn 250 CCG ACT GCT Pro Thr Ala 255 AAT CAA ATT CAA Asn Gin Ile Gin CTO CAT AGA TTT Leu His Arg Phe GAA AAA Oiu Lys CTA AAA CAA GTG OTO AAC ATC ACG CAA Leu Lys Gin Leu Asi Ile Thr Gin 275 AAA CAA GAA CTC CTA Lys Gin Giu Leu Leu 280 AAT CTG GAC AAA Asn Leu Asp Lys
AAT
Asn 285 CAA ATT TTO Gin Ile Leu CGA GCA Arg Ala 290
GTC
Val 295 OCA GAT AAC AAC Pro Asp Asn Asn
GAC
Asp 300 CAC GAA AGO GAO His Giu Arg Asp GCC TTA AAC AGA Ala Leu Asn Arg ACA OTA CCA GTA Thr Val Pro Val 3059 OCA GAC COA ATA Pro Asp Pro Ile 320
S
TTT AAT CCA GTC OTA GAA TCA CCA Phe Asn Pro Val Leu Giu Ser Pro 31.0 315 OAA TCT Glu Ser ACA TTT CCA 20 Thr Phe Pro 325 CCT GAO OTT Pro Asp Val TOT OCT TTA GAA Ser Ala Leu Oiu ATO AGA AAT GOT Met Arg Asn Oly GAA TTT Giu Phe 335 840 882 924 966 1008 1050 1092 1.134 1.176
GAT
Asp 340 OTO ATO ATT OGA Val Ile Ile Gly
TTC
Phe 345 AAT AGT OCT GAA Asn Ser Ala Giu TTA AGA TOT ATG Leu Arg Ser Met
CCA
Pro 355 AGA GTA ACC AGA Arg Val Thr Arg
OGA
Gly 360 AAC ATO GAA OTT Asn Met Giu Val
TAO
-Tyr 30 365 AAO ACT TTO. ACA Lys Thr Leu Thr
AAT
Asn 370 ATA GAO AGA GOT Ile Oiu Arg Ala COT AGA OAT Pro Arg Asp GAG A.AA AAA Oiu Lys Lys 390 GOT AAT Ala Asn 380 ATT TOO AAA AAT Ile Trp Lys Asn
COT
Pro 385 AAT 000 ATT GAG Asn Gly Ile Oiu OTT ATA AAA Leu Ile Lys 395 CAA AAO OAT Gin Asn Asp ATO OTT ACA GAG Met Leu Thr Oiu
TTT
Phe 400 TAT GAO CAA OTT Tyr Asp Gin AAA GAA Lys Olu 405 1.218 1260
GAO
Asp 410 ATO GAA 000 TAT Ile Giu Ala Tyr
OTO
Val1 42.5 CAA OTA AAA 000 OAT Gin Leu Lys Gly Asp 420 GOT GOT TAT OTO Ala Gly Tyr Leu CAA OGA ATT Gin Oly Ile 425 TAO COT ACC Tyr Arg Thr 430 TTO AAA 000 ATA Leu Lys Ala Ile 1.302 -158- TTT TTC Phe Phe 435 AAT GAA ATC AAA AGA PAT TCC AAC TTG Asn Glu Ile Lys Arg Asn Ser Asn Leu 440 445 TAT TTG TAT Tyr Leu Tyr ACT TAT ATC Ser Tyr Ile 460 1344 AGG TTA Arg Leu 450 TCA GAT GAT ACC Ser Asp Asp Thr TAT AGT Tyr Ser 455 GTA TAT AAA Val Tyr Lys 1386 TTG CCC TAT Leu Pro Tyr 465 CAT CAT TTA Asp Asp Leu CCA TGC CCT TCC Arg Trp Cly Ser
TTC
Leu 470 CCA CCA GTT ACT Pro Cly Val Ser CAT CCT His Cly 475 CGA TAT CJly Tyr 480 CTT TTT CCA Leu Phe Ala TCT TTC CAT OTT Ser Leu Asp Val
CCT
Pro 490 ATT TTC CCA ACA Ile Leu Cly Thr CAC ATT TCT ATA His Ile Ser Ile CtC Pro 500 CAA CAT CCT ATC Gin Asp Ala Met 1428 1470 1512 1554 1596
S.
S S
S*
S
S
*5*S
S
S
S
55S 55..
*SSS
S
CAC
Gln 505 ACT CTC CPA ACC Thr Leu Clu Arg
ATO
Met 510 CTC AGG ATC TG Val Arg Ile Trp PAT TTT CTA Asn Phe Val CCA TCA TCT Ala Ser Cys 530 AC PAT Lys Asn 520 CCA AAA CCT ACA Cly Lys Pro Thr
TCA
Ser 525 PAC ACT CPA CAT Asn Thr Clu Asp CAT ACA AA Asp Thr Lys 535 PAC CAC CPA Asn Asp Clu AGA CAT TTA PAC Arg His Leu Asri
CAC
Asp 540 ATT TTT TGC CPA Ile Phe Trp Clu CCA TAC Pro Tyr 545 CPA CCA Clu Pro 550* AAA TAT TTG Lys Tyr Leu ATG CCA PAA CPA Met Cly Lys Clu 1638 1680 1722 1764 TTT CPA ATC AA Phe Clu Met Lys
PAT
Asn 565 ATT TTC CPA CTA Ile Leu Glu Leu
AAA
Lys 570 CGC ATC ATC CTT Arg Met Met Leu
TG
Trp 575 CAT CPA CTT TAT Asp Clu Val Tyr
AGA
Arg 580 PAT CCG PAT TTC Asn Ala Asn Leu TTT AGA CTC Phe Arg Val TCT P.AT Cys Asn 590 CPA CA ACT ATT Clii Ciu Ser -1- 1785 INFORMATION FOR SEQ ID i) SEQUENCE CHARACTERISTICS: LENCTH: 1785 nucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOCY: linear (ii) MOLECULE TYPE:
CDNA
-159- (iii) SEQUENCE DESCRIPTION: SEQ ID TCTAATACTT
TCTTCATTAC
AAACTTCATC
CCAAAGCATC
TCAAAATGTT CTTTTCCCA1 TGGTTCCCAA AAAATGTCG I CTTCAGTGTT TGATGTAGG I CTGACCATCC
TTTCCAGAGT
CGTTGTTCCC
AAAATAGGAA
AATCATCACC
ATGACTAACT
ATATAACTTT
TATATACACT
CAAGTTGGAA
TTTCTTTTGA
GGTAAATTCC
TTGGAGATAA
TCGATGTCAT
CGTTTTGTTC
TTTTATAAGT
TTTTTCTCCT
CATCTCTAGG
TATAGCTCTC
ATGTTTCCTC
TGGTTACTCT
GAATCCAATG
ATGACATCAA
CTAAAGCAGA
TGGAAATGTT
ACTGGATTAA
ATACTGGTAC
20 GACTCTOTTT
AAGGCTGCTC
GTTCTTGTTT
TTGCGTGATG
TGCAGAGGTT
GAATTTGATT
CAAAATTGCC
CTTTGGTAGT
AATGAACACT
TGCTGCTCCA
CTGTCTCCAC
CAAATTTTTC
TCTCTGGTCT
TTTAGACCCA
ATAGAAAACC GAAAACTCCT CCTTCCCTGA
TCAAATAATC
TTGGTTGAAT GCGCCTCCAT 30 TGTTCGCAGA
ATTAACAGTC
TCTTCG CTT C CTGCATAAGA TCCTAAAGAC
CGAGCCTGAA
TTGGAGACTG
AAATCGCAAT
CCCAAATAGG
AACAATATAC
35 TTTTCCTTCC
GTGATCCCGT
GGTTATCCCA
TAGTATGCAC
AATAAAAATA
AAACTAATGC
C
C**
C
C
C
C
C
AGACTCTAAJ
ATGCGTTTTI;
GTCCAAATA-J
TTAAATGTC'I
T=CCATTC71
CTGCATAGCA
CATCCAAAGA
CCT'GGCAAGG
ATACGTATCA
TTTCATTGA.A
CCAGCATCGC
TTTAACTTGG
CAATGCCATT
TCTATATTTG
TGGCATAGAT
CGTCAGGAAA
ATTGGGTCTG
TGTGTCCCTT
GCAAAATTTG
TTCAGCACTT
AGCAGTCGGA
ATTTTCTAGT
GCAGATTCTC
GATGTTGTCA
TATTTCCATG
AATCTATAGT
AGGTCCAAAA
GGATCCAGAA
TCTGGCGTGA
ATATATTAGA
CGAATCCTGT
GGTCCAATAG
ATCATTTTTA
TAAATTGAAC
AAGACAGGTG
ACTGTGAGGT
k. CCGCAAATTC kGTTCCAAANI
TTTGGTTCT'I
TTTTGTATcA
*TTACAAAATT
TCTTGCGGTA
GTTTGCAAAA
AACCCCATCG
TCTGATAACC
AAATATGGCT
CTTTTAGTTG
TCATAAAACT
AGGATTTTTC
TCAAAGTCTT
CTTAAACCTT
TTCACCATTT
GAGATTCTGG
TCGTGGTCGT
ATTTTTGTCC
GTTTTAGTTT
TTGAGTAATG
AGAATTGTCC
CAGCTATAGT
TAAACCCATT
GATATCCCAT
TGATAGTGAC
AAATTATAAG
CATTACAGGA
ATATATTCAG
CTTTCCTGGA
TTTTGGATTT
GCGGTTCTGC
TGATCTTTAT
TCTGCACAAA
TAAATAAGAA
AACAT
GCATTTCTAT
ATTTTTCATT
CGTCGTTGTA
CATGATGCAT
GGTCCAGATC
TAGAAATGTG
AGATATCCTA
ATAGGGCAAG
TATACAAATA
TTCAAGGTAC
GACATAGGCT
CTGTAAGCAT
CAAATATTAG
GTAAACTTCC
CAGCACTATT
CTCATTCTTT
TGATTCTAGG
TGTTATCTGG
AGATTTAGGA
TTCAAATCTA
TCCCACTCTG
ATCATCAGAA
GATTTTATCT
TTAGTGCCAA
TCCGGCGCTG
CAAAATAATT
ATCCTGATCC
TATTTTGTAT
ATATAAGCAA
AACATTTGTC
GATATTGGTT
ATAAGGAATT
ATCGGAACGG
TGCTGATCTA
AAATAAAAAA
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1785 INFORMATION FOR SEQ ID NO:36: i) SEQUENCE CHARACTERISTICS: LENGTH: 2007 nucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: (iii) FEATURE:
NAME/KEY:
LOCATION:
cDNA CDs 11. .1594 (iv) SEQUENCE DESCRIPTION: SEQ, ID NO:36: -160- AGTTCCAACG ATG GCT GAT CTA CAA GTG ACT TTG CTI' CAA GGT Met Ala Asp Leu Gin Val T1±r Leu Leu Gin Gly
I
ACT TTA AAA GGA AAA GAG CAA ATT AGT Thr Leu Lys Gly Lys Giu Gin Ile Ser GAA AAA GGA AAT GTG Giu Lys Gly Asn Val TTC CAT AGT TAT TOT GGA ATT CCA TAT GCC AAA CCT OCT GTA Phe His Ser Tyr Ser Gly Ile Pro Tyr Ala Lys Pro Pro Val GGT GAT CTA AGA TTT AAG CCA CCT CAA CCT Asp Leu Arg Phe Lys Pro Pro Gin Pro
GCA
Ala s0 GAA CCT TGG Glu Pro Trp AGT TGT AGA Ser Cys Arg TCA GGT Ser Gly GTT CTT GAT GCT Val Leu Asp Ala
AGT
Ser 60 AAA GAA GGG AAT Lys dlu Gly Asn
S.
S
.5 5
S
TCA GTA CAT Ser Val His 70 TTT ATT AAA AAA Phe Ile Lys Lys ATT AAA GTA GGG GCT Ile Lys Val Gly Ala GAA GAT Glu Asp TGT TTA TAC CTC AAT GTC TAT GTA 20 Cys Leu Tyr Leu Asn Val Tyr Val CCA AAA ACA Pro Lys Thr TCA GAG AAA Ser diu Lys 169 211 253 295 337 379 421.
TCA CTT OTT CCA Ser Leu Leu Pro
GTA
Val 100 ATG GTA TGG ATA Met Val Trp Ile
CAT
His 105 GGA GGA GGC TTC Gly Gly Gly Phe
S
S
*5S*
S
*5
S
TFC
Phe 110 ATG GGA TCT GGA Met Giy Ser Gly
AAT
Asn 115 AGT GAT ATG TAT Ser Asp Met Tyr CCT GAA TAT Pro Glu Tyr AAT TAT CGA Asn Tyr Arg 135 TTG ATG Leu Met 30 125 GAT TAT GGA ATT Asp Tyr Gly Ile
GTT
Val 130 CTG GTT ACT TTC Leu Val Thr Phe TTA GGT GTT Leu Gly Val 140 CCT GGO AAT Pro Gly Asn TTG OGA TTT TTG Leu Gly Phe Leu CTG GGA ATA GAA Leu Gly Ile Giu GAA GCG Glu Ala 150
GTT
Val1 155 GGT TTG, ATO GAC Gly Leu Met Asp
CAG
Gin 160 GTT GAA GOT CTA Val Glu Ala Leu TGG GTA AAA AAC Trp Val Lys Asn
AAT
Asn 170 ATT GCA TCC Ile Ala Ser TTT GGT Phe Gly 175 GGT GAC CCC A.AC Gly Asp Pro Asn AAT GTG ACT ATT TTT GGA GAA TCA GCA GGT Asn Val Thr Ile Phe Gly Giu Ser Ala Gly
GGT
d ly 190 GCA AGT GTT Ala Ser Val 589 -161- CAT TAT TTG His Tyr Leu 195 ATG TTA TCA Met Leu Ser GAT CTT TCC Asp Leu Ser 200* AAA GCA CTT TTT CAT Lys Cly Leu Phe His 205 AAA GCG ATC Lys Ala Ile 210 CTT CAA CAT Leu Gin His TCA CAA ACT GGA Ser Gin Ser Gly GAT AAT AAT AAA Asp Asn Asn Lys
AGT
Ser 215 GCT TTT AAT OCT Ala Phe Asn Pro TGG CCA Trp Ala 220 CAA AAT Giu Asn 230 GCA TTC CCC CTC Ala Phe Arg Leu
TGC
Cys 235 673 715 757 799 AAA CTT CTC GCT Lys Leu Leu Cly
CAT
His 240 CCT CTC CAT AAC Pro Vai Asp Asn
GAC
Giu 245 ACA CAA CCT CTA Thr Ciu Ala Leu
AAA
Lys 250 ATC CTT CCT CAA Ile Leu Arg Gin
CC
Ala 255 CCC ATA CAT GAT Pro Ilie Asp Asp ATA CAC AAC Ile Asp Asn ATA CAC TAT Ile Asp Tyr 275 0 0 0e 0 *000
S
*0*0
S
AGA ATA AAA Arg Ile Lys 265 CCT TTT CTA 20 Pro Phe Leu 280 CCA TTC TTC Pro Phe Leu CCA AAA GAC Pro Lys Asp
AAA
Lys 270 CCC CAA CTT ATT Cly Gin Leu Ile CCA ACA ATA GAA Pro Thr Ile Ciu
AAA
Lys 285 CT TAT CAA AAT Arg Tyr Gin Asn T'rr CAA Phe Ciu 290
GAC
Asp 295 CAC TCT CCA TTA Gin Ser Pro Leu
TCA
Ser 300 AAA ATC CAA TCA Lys Met Gin Ser
GCC
Cly 305 841 883 925 967 1009 AAT TTC ACA AAA Asn Phe Thr Lys CCA TTT ATA TCT Pro Phe Ile Cys
GA
Giy 315 TAC AAC ACT CCT Tyr Asn Ser Ala
S*
S 055 .5 0
CAA
Ciu 30 320 GGA ATT TTA GCT Cly lie Leu Cly
TTA
Leu 325 ATG CAC TTC AAG Met Asp Phe Lys
GAT
Asp 330 CAC CCA AAT Asp Pro Asn ATA TTT Ile Phe 335 GAG AAC TTT CAA Giu Lys Phe Giu
GCT
Ala 340 CAT TTT GAA AGA TTT CTA CCA Asp Phe Giu Arg Phe Val Pro 345 1051 GTA CAT TTG Vai Asp Leu 350 TTC GCT CAA Leu Ala Giu AAT CTA ACT TTA A sn Leu Thr Leu TCT AAG CrAA TCT Ser L 7 3u Ser AAA AAA Lys Lys 360
GAA
Ciu 365 ATC AGA AAG TTT Met Arg Lys Phe TAC CAA CAC GAA Tyr Gin Asp Glu
CCT
Pro 375 1093 1135 1177 CTT TCT TCA GAO Vai Ser Ser Asp
AAC
Asn 380 AAA CAA AAA TTT Lys Ciu Lys Phe
GTC
Val 385 ACT CTT ATT ACT Ser Val Ile Ser -162-
GAT
Asp 390 ACT TGG TTT Thr Trp Phe TTG AGA Leu Arg 395 GGG ATT AAA AAT Gly Ilie Lys Asn ACT GCA AGA TAT Thr Al a Arg Tyr 400 1219 ATA ATT Ile Ile 405 GAA CAT TCC TCA Glu Hi-s Ser Ser
GAA
Glu 410 CCG TTA TAT TTA Pro Leu Tyr Leu TAT GTT TAT Tyr Val. Tyr 415 1261 AGT TTT GAT GAT TTT GGT Ser Phe Asp 420 CCT AAT ATT Pro Asn Ile Asp Phe Gly TTT TTG Phe Leu 425 AAG AAA CTT GTA Lys Lys Leu Val TTA GAT Leu Asp 430
GAA
Glu 435 GGA GCA GCT CAT Gly Ala Ala His GAT GAG CTG GGA TAT Asp Glu Leu Gly Tyr CTT TTC AAG ATG Leu Phe Lys Met
AGT
Ser 450 TTT ACA GAA TTT Phe Thr Glu Phe
CCA
Pro 455 AAA GAT TTA CCA Lys Asp Leu Pro 0 0 000 a .04 6
AGT
Ser 460 GCA GTG GTG AAT Ala Val. Val Asn
AGG
Arg 465 GAA CGA TTG TTG Glu Arg Leu Leu
CAA
Gin 470 CTT TGG ACA Leu Trp Thr -AAT TTT GCA Asn Phe Ala 475 GTT ATA ACA Val Ile Thr 490 25 GAT CAT ATG Asp His Met AAA ACA GGA Lys Thr Gly ACA AAA TG Thr Lys Trp
AAT
As n 480 CCC ACT CCT GAA ATC AAT GAT Pro Thr Pro Glu Ile Asn Asp GAT AAA Asp Lys 495 GCT ACT GAG GAA Ala Thr Glu Glu AAA TCA Lys Ser 500 CCA GAT Pro Asp .515 1303 1345 1387 1429 1471 1513 1555 1597 1640 1690 1740 1790 1840 1890 1940 1990 2007
GAT
Asp ATC GAT AAT ACT Ile Asp Asn Thr AGA ATG ATT Arg Met Ile CCT GAT GCA AAA Pro Asp Ala Lys
CGA
Arg 520 CTT AGA TTT TGG Leu Arg Phe Trp
AAT
Asn 525 AAA TTT TTA TGA Lys Phe Leu TAA ATATACCAAT TATCGATTTT ATTATAGAGT TTCTGT1TTA
GTATAATTAT
TATATTTCGA
GTTGTAATAA
GTGATTTGCG
TCTTGAAGAA
ATTTGATATT
TTTTTTTTAT
CACGTTTAGA
TTTCAAATTT
TCAAGAAGTA
CATTCAACAG
CT%...X.CTAAA
ATTACCATCT
TTCAAATATA
AA A A
TGTACGAGAT
ACTCTGATTA
GGTGGTAAAT
ATGGTGTACT
AATGTGATTA
TTGTATCATA
TTGTTTTTTT
TCAATTGGCT CTAATTGAAG TTGGAAAAA
AGCTTTTACA.
TTAGAACA
TTCTGTTTTA
GTGCCTAAAT
TTGTCGCTCT
ATGGACGCCA
CATTATTTAT
TTTGCTTTTA
TTTTTTCATT
ATAAAAAAAA AA~AhAAA INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 528 amino acids TYPE: amino acid TOPOLOGY: linear -163- (ii) MOLECULE TYPE: Protein (iii) SEQUENCE DESCRIPTION: SEQ ID NO:37: Met Ala Asp Leu Gin Val Thr Lau Leu Gin Giy Thr Leu Lys 1 5 Gly Lys Giu Gin Ile Ser Giu Lys Giy Asn Val Phe His Ser 20 Tyr Ser Giy Ile Pro Tyr Ala Lys Pro Pro Val. Gly Asp Leu 35 Arg Phe Lys Pro Pro Gin. Pro Ala Glu Pro Trp Ser Gly Val 45 50 Leu Asp Ala Ser Lys Giu Gly Asn Ser Cys Arg Ser Vai His 6S Phe Ile Lys Lys Ile Lys Vai Gly Ala Glu.Asp Cys Leu Tyr *75 Leu Asn Vai Tyr Val Pro Lys Thr Ser Giu Lys Ser Leu Leu 90 *Pro Val Met Val. Trp Ile His Gly Gly Gly Phe Phe Met Giy 100 105 110 Ser Gly Asn Ser.Asp Met Tyr Giy Pro Giu Tyr Leu Met Asp 115 120 125 Tyr Gly Ile Val Leu Val. Thr Phe Asn Tyr Arg Leu Gly Val 130 135 140 Giy Phe Leu Asn Leu Giy Ile Glu Giu Ala Pro Gly Asn 145 Val. Gly Leu Met Asp Gin Val Giu Ala Leu Lys Trp Val. Lys 155 160 165 Asn Ile Ala Ser Phe Gly Gly Asp Pro Asn Asn Val Thr .170 175 180 Sle Phe Gly Giu Ser Ala Giy Giy Ala Ser Val. His Tyr Leu 185 190 195 Met Leu Ser Asp Leu Ser Lys Gly Leu Phe His Lys Ala Ile 200 205 210 Ser Gin Ser Gly Ser Ala Phe Asn. Pro Trp Ala Leu Gin His 215 220 Asp Asn Asn Lys-Giu Asn Ala Phe Arg Leu Cys Lys Leu Leu 225 230 235 Gly His Pro Val Asp Asn. Glu Thr Giu Ala Leu Lys Ile Leu 240 245 250 Arg Gin Pro Lys Al a 255 Asp Pro Ile Lys Gly Asp Gin -164- Asp Leu Ile Asp Asn 260 Leu Ile Ile Asp Tyr Arg Pro Pro Thr Ile Giu Lys Arg Tyr Gli Asn Phe Giu Prc
S
S
.5 S S
S
5555 5*
S
S S
S
S. Asp 295 Lys Leu Lys 15 Asn Glu 365 Asp Phe His Asp Glu 435 Met Vai L~ys Thr Asp 505 Gir Val 310 Giy Phe Leu Met Asn 380 Leu Ser Phe Gly Ser 450 As n rhr Lie ISer *Pro *Leu 325 Giu Thr Arg Lys Arg 395 SerC Gly I Ala Phe I* Arg G 465 Gly P~ 4 Trp, P Asp A 285 Pro Leu Ser Lys Met 300 Phe Ile Cys Giy Tyr 315 Met Asp Phe Lys Asp 330 Ala Asp Phe Giu Arg 340 Leu Arg Ser Lys Glu 355 Lys Phe Tyr Tyr Gin 370 31u Lys Phe Val Ser 385 31y Ile Lys Asn Thr 400 flu Pro Leu Tyr Leu ~10 ?he Leu Lys Lys Leu 425 lIa His Gly Asp Giu 440 :hr Giu Phe Pro Lys 455 1u Arg Leu Leu Gin 470 Lsn Pro Thr Pro Giu 80 Lsp Lys Ala Thr Glu 495 ~sn Thr Leu Arg Met 510 Gir As V Asp Phe 345 Ser Asp Val1 Al a Tyr -415 Val1 Leu Asp Leu Ile 485 G1u Ile 290.
1Ser Gly 305 Ser Ala Pro Asn Val Pro Lys Lys 360 Glu Pro 375 Ile Ser Arg Tyr Val Tyr Leu Asp 430 Gly Tyr I 445 Leu Pro S Trp Thr I Asn Asp I Lys Ser S00 pro Asp P 515 Asr Glu 320 Ile Val1 Leu Val1 Asp 390 Ile 3er ?rO .eu er ~sn al1 ~sp ~ro Phe IPhe Gly Phe 335 Asp Ala Ser Thr Ile 405 Phe Asri Phe I Ala Phe Ile I 4 His P~ Asp
P
Lys Leu 280 Leu Thr Ile Giu Leu 350 Glu Ser Trp .lu ksp 120 Ile .ys Pal lia 'hr e t la -165- Lys Arg Leu Arg Phe Trp Asn Lys Phe Leu 520 525 INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 2007 nucleoti~des TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) SEQUENCE DESCRIPTION: SEQ ID NO:38: *4 40 0 0 0 0 000* 000* 0**0 0* 0 ~0 0* 000.
0 0000 0 0 0 *00 *0*0 *0*0 0* .0 *0 S 0 *00* 0
TTTTTTTTTT
ATTTGAAATA
TGGTAATAAT
AGTTCAGTTC
15 TTGAATGCGC
TTCTTGATTA
TTTGAAATCG
AAACGTGATA
GGTATATTTA
20 GGATCTGGAA
TTCCTCAGTA
GAGTGGGATT
TCCCTATTCA
CATCTTGAAA
25 TAGGATCTAA
ACATATAAAT
ATTTTTAATC
TTTCTTTGTT
ATTTCTTCAG
30 ATCTACTGGT
TTGGGTCATC
TATCCACATA
TAATGGAGAC
TTGTTGGTAG
35 TTTATTCTGT
TAGAGCTTCT
GGAATGCATT
GCACTTCCAC
TGATAACATC
TAGTCACATT
CATTTTAGAG
TTCTATTCCC
TAACCAGAAC
CTATTTCCAG
TGGAAGAAGT
AACAATCTTC
CAACTATTCC
AGGTTGAGGT
GAATTCCAGA
TTTCCTTTTA
TGGAACT
AAAAAAAAAT
ATCAAATATA
TTCAAGAAGA
AAATCACTAA
TTACAACTGT
AAATATACTT
ATTATACTAA
TCATAAAAAT
TCATTCTCAA
GCTTTATCCC
TCCTGTTTTT
CCACTGCACT
AGATATCCCA
TACAAGTTTC
ATAACGGTTC
CCTCTCAAAA
GTCTGAAGAA
CCAATTTTTT
ACAAATCTTT
CTTGAAGTCC
TAAATGGGAC
TGGTCCAAGA
AAAAGGATAG
TGTCTATAAG
GTCTCGTTAT
TTCTTTATTA
TTTGTGAGAT
AAATAATGAA
GTTGGGGTCA
CTTCAACCTG
AGGTTCAAAA
AATTCCATAA
ATCCCATGAA
GATTTCTCTG
AGCCCCTACT
CTTCTTTACT
GGCTTAAATC
ATAACTATGG
AAGTACC'rrG
GAAAAAATAA
AATAATGTGG
GCGACAAATT
AACAGAATTT
AAAAGCTTTT
CAATTAGAGC
TACAGAAACT
TTATTCCAAA
AGTATTATCG
ATTTTGTTGT
GCAA.AATTTG
TGGTAAATCT
GCTCATCTCC
TTCAAAAAAC
TGAGGAATGT
ACCAAGTATC
ACAGGTTCGT
AGATTCCTTA
CAAAATCAGC
ATTAAACCTA
TTTTGTGAAA
ATGGTTCAAA
TCTATAATAA
ATCATCTATG
CGACAGGATG
TTATCATGTT
CGCTTTATGA
CACTTGCACC
CCACCAAAGG
GTCCATCA; 1,
ATCCCAAAAC
TCCATCAAAT
GAAGCCTCCT
ATGTTTTTGG
TTAATTTTTT
AGCATCAAGA
TTAGATCACC
AACACATTTC
AAGCAAAGTC
TTTTTTTTTT TTTTTTT TrT
TTTTTATAAA
AAGCAAATAT
CGTCCATTAA
TAGGCACAGT
GTTCTAA.ATT
TTTCCAATAA
CAATTGAATC
CTATAATAAA
ATCT.AAGTCG
ATATCCATAT
TATAACATCA
TCCA.AAGTTG
TTTGGAAATT
ATGAGCTGCT
CAAAATCATC
TCAATTATAT
ACTAATAACA
CTTGGTAATA
GACCTTAAAG
TTCAAACTTC
AAATTCCTTC
TTGCCTGATT
ATTTTGATAA
GTTGGCCTTT
GGGGCTTGAC
ACCCAGAAGT
GAAGTGCCCA
AAAAGTCCTT
ACCTGCTGAT
ATGCA.ATATT
:CAACATTGC
ACCTAATCGA
ATTCAGGACC
CCATGTATCC
TACATAGACA
TAATAAAATG
ACACCTGACC
TACAGGAGGT
CTTTTTCACT
ACTTGTAGAT
AAAACAATAT
GATACAAAGA
TCACATTTTT
ACACCATCTG
TACCACCTAC
TCAGAGTAAA
TCGTACATCT
ATCGATAATT
TTTTGCATCA
GATCTGATTT
TTGATTTCAG
CAACAATCGT
CTGTAAAACT
CCTTCAATAT
AAAACTATAA
ATCTTGCAGT
CTGACAAATT
AAACTTTCTC
TTAGATTCAA
TCAAATATAT
AGCACTGTTG
GCATTTTTGA
CGTTTTTCTA
GTCTTTTGGT
GAAGGATTTT
TTGCAGAGGC
AGGATTAAAA
TGGAAAGATC
TCTCCAAAAA
GTTTTTTACC
CAGGCGCTTC
TAATTGAAAG
ATACATATCA
ATACCATTAC
TTGAGGTATA
TACTGATCTA
AAGGTTCTGC
TTGGCATATG
AATTTGCTCT
CAGCCATCGT
100 IS0 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 11.00 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000 2007 -166- INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: peptide (iii) SEQUENCE DESCRIPTION: SEQ ID NO:39: Asp Pro Pro Thr Val Thr Leu Pro Gin Gly Glu Leu 1 5 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 22 amino acids TYPE: amino acid c(C) TOPOLOGY: linear 15 (ii) MOLECULE TYPE: peptide (iii) FEATURE: NAME/KEY: Xaa any amino acid LOCATION: 21 (iv) SEQUENCE DESCRIPTION: SEQ ID 0' 20 Asp Pro Pro Thr Val Thr Leu Pro Gln Gly Glu Leu Val Gly 1 5 Lys Ala Thr Asn Glu Asn Xaa Lys INFORMATION FOR SEQ ID NO:41: 25 SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid TOPOLOGY: linear (ii' MOLECULE TYPE: peptide (iii) SEQUENCE DESCRIPTION: SEQ ID NO:41: Asp Pro Pro Thr Val Thr Leu Pro Gin Gly Glu Leu 1 5 INFORMATION FOR SEQ ID NO:42: SEQUENCE CHARACTERISTICS: LENGTH: 21 amino acids TYPE: amino acid -167- TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) SEQUENCE DESCRIPTION: SEQ ID NO:42: Asp Pro Pro Thr Val Thr Leu Pro Gin Gly Glu Leu Val Gly 1 5 Lys Ala Leu Ser Asn Glu Asn INFORMATION FOR SEQ ID NO:43: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide ~(iii) SEQUENCE DESCRIPTION: SEQ ID NO:43: Asp Pro Pro Thr Val Thr Leu Pro 1 *s -INFORMATION FOR SEQ ID NO:44: S0 SEQUENCE CHARACTERISTICS: eo* LENGTH: 23 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) SEQUENCE DESCRIPTION: SEQ ID NO:44: 2 Asp Pro Pro Thr Val Thr Leu.Pro Gin Gly Glu Leu Val Gly 25 1 5 Lys Ala Leu Thr Asn Glu Asn Gly Lys INFORMATION FOR SEQ ID SEQUENCE
CHARACTERISTICS:
LENGTH: 20 bases TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: primer (xi) SEQUENCE DESCRIPTION: SEQ ID -168- AATTAACCCT
CACTAAAGGG
0000 0*1.
o: o ooso :6.09 0..00 00000 INFORMATION FOR SEQ ID NO:46: SEQUENCE CHARACTERISTICS: LENGTH: 17 bases TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: primer (iii) FEATURE: NAME/KEY: R A or G LOCATION: 2, 12, 14 (iv) FEATURE: NAME/KEY: D A, G or T LOCATION: 3, 6, 9, SEQUENCE DESCRIPTION: SEQ ID NO:46: ARDCCDCCDC CRTRDAT INFORMATION FOR SEQ ID NO:47: SEQUENCE CHARACTERISTICS: LENGTH: 38 bases 20 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: primer (iii) SEQUENCE DESCRIPTION: SEQ ID NO:47: TGTGCTCGAG ATGGGATAAC CTAGATCAGC
ATTTGTGC
(2T INFORMATION FOR SEQ ID N0:48: SEQUENCE CHARACTERISTICS: LENGTH: 35 bases TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: primer (iii) SEQUENCE DESCRIPTION: SEQ ID NO:48: TTAAGGTACC TCATCTAATA CTTCCTTCAT
TACAG
INFORMATION FOR SEQ ID NO:49: -169- SEQUENCE CHARACTERISTICS: LENGTH: 36 bases TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: primer (iii) SEQUENCE DESCRIPTION: SEQ ID NO:49: AAAACTGCAG TATAAATATG TTACCTCACA GTAGTG 36 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 34 bases TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: primer (iii) SEQUENCE DESCRIPTION: SEQ ID TGCTCTAGAT TATCTAATAC TTCCTTCATT ACAG 34 INFORMATION FOR SEQ ID NO:51: SEQUENCE CHARACTERISTICS: LENGTH: 1540 nucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear S* (ii) MOLECULE TYPE: cDNA 25 (iii) FEATURE: NAME/KEY: CDS LOCATION: 1..1540 (iv) SEQUENCE DESCRIPTION: SEQ ID NO:51: CTT CAA GGT ACT TTA AAA GG1 AAA GAG CAA ATT AGT GAA AAA 42 Leu Gin Gly Thr Leu Lys 3-y Lys Glu Gin Ile Ser Glu Lys 1 5 GGA AAT GTG TTC CAT AGT TAT TCT GGA ATT CCA TAT GCC AAA 84 Gly Asn Val Phe His Ser Tyr Ser Gly Ile Pro Tyr Ala Lys 20 CCT CCT GTA GGT GAT CTA AGA TTT AAG CCA CCT CAA CCT GCA 126 Pro Pro Val Gly Asp Leu Arg Phe Lys Pro Pro Gin Pro Ala 35 -170- GAA CCT TGG TCA GGT GTT CTT GAT GJlu Pro Trp Ser Gly Val Leu Asp so GCT AG? AAA GAA GGG AAT Ala Ser Lys Glu Gly Asn 168 210 AG? TGT AGA TCA GTA CAT TTT ATT AAA AAA Ser Cys Arg Val His Phe Ile Lys AT? AAA GTA Ile Lys Val GCT GAA GAT TGT TTA TAC CTC AAT Leu Asn GTC TAT OTA CCA AAA ACA Ala Glu Asp Cys Leu Tyr Val Tyr Val Pro Lys Thr
TCA
Ser GAG AAA TCA CT? Glu Lys Ser Leu
CTT
Leu 90 CCA OTA ATG GTA Pro Val Met Val
TGG
Trp ATA CAT GGA (Ile His Gly ATG TAT GGT Met Tyr Gly 110 294 GGA GGC Gly Gly 100 TTC TTC ATG GGA Phe Phe Met Gly
TCT
Se r 105 GGA MAT AG? GAT Gly Asn Ser Asp CCT GAA TAT Pro Glu Tyr 115 AAT TAT CGA Asn Tyr Arg TTG ATG GA? TAT Leu Met Asp Tyr AT? GT? CTG GT? Ile Val Leu Val ACT TTC Thr Phe 125
TTA
Leu 130 OCT GT? TTG GGA Gly Val Leu Gly TTG MAC CTG GGA Leu Asn Leu Gly
ATA
I le 140 378 420 462 504 GAA GAA GCG CC? Glu Glu Ala Pro
GGC
Gly 145 AAT GTT GGT TTG Asn Val Gly Leu
ATG
Met
ISO
GAC CAG GTT GAA Asp Gin Val Glu OcT Ala 155 CTA AAA TGG GTA Leu Lys Trp Val
AAA
Lys 160 AAC MAT AT? GCA Asn Asn Ile Ala TTT GOT GGT Phe Gly Gly GCA GGT GGT Ala Oly Gly 180 GAC CCC Asp Pro 170 AAC AAT GTG ACT Asn Asn Val Thr TTT OGA GAA TCA Phe Gly Glu Ser GCA AG? OTT CAT TAT TTO ATG Ala Ser Val His Tyr Leu Met 185 TCA GA? CTT TCC Ser Asp Leu Ser AMA GGA Lys Gly 195 C'"T TTT CAT I 3u Phe His GCG ATC TCA CAA Ala Ile Ser Gin GGA AGT GCT TTT Gly Ser Ala Phe
AAT
As n 210 CC? TGG GCA CTT Pro Trp Ala Leu
CAA
Gin 215 CAT GA? MAT MAT His Asp Asri Asn
MA
Lys 220 GMA M.T GCA TTC Glu Asn Ala Phe 630 672 714
CGC
Arg 225 CTC TGC AMA CT? Leu Cys Lys Leu
CTG
Leu 230 GGT CAT CC? GTC Gly His Pro Val
GA?
Asp 235 MAC GAG ACA Asn Glu Thr -171- GAA GCT Glu Ala 240 CTA AAA ATC CTT Leu Lys Ile Leu
CGT
Arg 245 CAA GCC CCC ATA Gin Ala Pro Ile GAT GAT CTT Asp Asp Leu 250 ATA GAC AAC lie Asp Asn 255 AGA ATA AAA CCA Arg Ile Lys Pro
AAA
Lys 260 GAC AAA GGC CAA Asp Lys Gly Gin CTT ATT Leu Ile 265 ATA GAC TAT CCT Ile Asp Tyr Pro TTT CTA CCA ACA Phe Leu Pro Thr
ATA
Ile 275 GAA AAA CGT TAT Glu Lys Arg Tyr
CAA
Gin 280 798 840 882 924 AAT TTT GAA CCA Asn Phe G1u Pro
TTC
Phe 285 TTG GAC CAG TCT Leu Asp Gin Ser
CCA
Pro 290 TTA TCA AAA ATG Leu Ser Lys Met
CAA
Gin 295 TCA GGC AAT TTC Ser Gly Asn Phe AAA GTC CCA TTT Lys Val Pro Phe
ATA
Ile 305 TGT GGA TAC Cys Gly Tyr AAC AGT Asn Ser 310 GCT GAA GGA ATT Ala Giu Gly Ile
TTA
Leu 315 GGT TTA ATG GAC Gly Leu- Met Asp TTC AAG GAT Phe Lys Asp 320 .0 0 0 0.
00 0 GAC CCA AAT ATA 20 Asp Pro Asn Ile 325 TTT GTA CCA GTA Phe Vai Pro.Vai 340 25 TCT AAA AAA TTG Ser Lys Lys Leu TTT GAG AAG Phe Giu Lys GAA GCT GAT TTT Glu Ala Asp Phe GAA AGA Glu Arg 335 GAT TTG AAT CTA Asp Leu Asn Leu
ACT
Thr 345 TTA AGG TCT AAG Leu Arg Ser Lys
GAA
Glu 350 966 1008 1050 1092 1134 1176
GCT
Ala 355 GAA GAA AT.G AGA Glu Giu Met Arg
AAG
Lys 360 TTT TAT TAC CAA Phe Tyr Tyr Gin
GAC
Asp 365 GAA CCT GTT TCT Glu Pro Vai Ser
TCA
Ser 370 GAC AAC AAA GAA Asp Asn Lys Glu
AAA
Lys 375 TTT GTC AGT Phe Val Ser AAA AAT ACT Lys Asn Thr 390 GTT ATT Val Ile 380 AGT GAT ACT TGG Ser Asp Thr Trp
TTT
Phe 385 TTG AGA GGG ATT Leu Arg Gly Ile GCA AGA TAT Ala Arg Tyr 395 TAT GTT TAT Tyr Vai Tyr ATA ATT GAA CAT Ile Ile Giu His
TCC
Ser 400 TCA GAA CCG TTA Ser Giu Pro Leu TAT TTA Tyr Leu 405 1218 1260 AGT TTT Ser Phe 410 GAT GAT TTT GGT TTT Asp Asp Phe Gly Phe TTG AAG AAA CTT Leu Lys Lys Leu 420 CAT GGA GAT GAG His Gly Asp Glu GTA TTA GAT CCT Val Leu Asp Pro
AAT
Asn 425 ATT GAA GGA GCA Ile Giu Gly Ala
GCT
Ala 430 1302 -172- CTG GGA TAT Leu Giy Tyr 435 CTT TTC AAG ATO AGT TTT ACA GAA TTT CC-A AAA Leu Phe Lys Met Ser Phe Thr Giu Phe Pro Lys 1344 GAT TTA CCA AGT GCA GTG Leu 450 Pro Ser Ala Val GTG AAT Val Asn 455 AGG GAA CGA Arg Giu Arg TTG TTG CAA Leu Leu Gin 460 1386 CTT TGG ACA Leu Trp Thr 465 ATC AAT GAT Ile Asn Asp AAT TTT GCA AAA Asn Phe Ala Lys
ACA
Thr 470 GGA AAT CCC ACT Giy Asn Pro Thr CCT GAA Pro Giu 475
GTT
Val 480 ATA ACA ACA AAA Ile Thr Thr Lys GAT AAA GCT ACT Asp Lys Ala Thr
GAG
Glu 490 1428 1470 1512 GAA AAA TCA GAT Glu Lys Ser Asp ATG GAT ATC GAT Met Asp Ile Asp
AAT
As n S00 ACT TTG AGA ATG Thr Leu Arg Met
ATT
Ile CCA GAT CCT GAT Pro Asp Pro Asp
GCA
Ala 510 AAA CGA CIT A Lys Arg Leu 1540 INFORMATION FOR SEQ. ID NO:52: Wi SEQUENCE CHARACTERISTICS: LENGTH: 1584 nucleotides TYPE: nucleic acid STRA.NDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) SEQUENCE DESCRIPTION: SEQ ID NO:52: TAAAAATTTA TTCCAAAATC TAAGTCGTTT TGCATCAGGA
TCTGGAATCA
TTCTCAAAGT ATTATCGATA TCCATATGAT CTGATTTTTC
CTCAGTAGCT
-173- TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) SEQUENCE DESCRIPTION: SEQ ID NO: 52: TAAAAATTTA
TTCCAAAATC
TTCTCAAAGT
ATTATCGATA
TTATCCCATT
TTGTTGTTAT
TGTTTTTGCA
AAATTTGTCC
CTGCACTTGG
TAAATCTTTT
TATCCCAGCT
CATCTCCATG
AAGTTTCTTC AAAAAACCAA ACGGTTCTGA
GGAATGTTCA
CTCAAAAACC AAGTATCACT TGAAGAAACA
GGTTCGTCTT
ATTTTTTAGA FrCCTTAGAC AATCTTTCAA
AATCAGCTTC
GAAGTCCATr
AAACCTAAA
ATGGGACTTT
TGTGAAATTG
TCCAAGAATG
GTTCAAAATT
AGGATAGTCT
ATAATAAGTT
CTATAAGATC
ATCTATGGGG
TCGTTATCGA
CAGGATGACC
TTTATTATTA
TCATGTTGAA
GTGAGATCGC
TTTATGAAAA
TAATGAACAC
TTGCACCACC
GGGGTCACCA
CCAAAGGATG
CAACCTGGTC
CATCAAACCA
TTCAAAAATC
CCAAAACACC
30 TCCATAATCC
ATCAAATATT
CCATGAAGAA
GCCTCCTCCA
TTCTCTGATG
TTTTTGGTAC
CCCTACTTTA
ATTTTTTTAA
CTTTACTAGC ATCAAGAACA TTAAATCTTA
GATCACCTAC
ACTATGGAAC
ACATTTCCTT
TACCTTGAAG
CAAAGTCACT
TAAGTCGTTT
TCCATATGAT
AACATCATTG
AAAGTTGCAA
GGAAATTCTG
AGCTGCTCCT
AATCATCAAA
ATTATATATC
AATAACACTG
GGTAATAAAA
CTTAAAGTTA
AAACTTCTCA
TTCCTTCAGC
CCTGATTGCA
TTGATAACGT
GGCCTTTGTC
GCTTGACGAA
CAGAAGTTTG
GTGCCCAAGG
PGTCCTTTGG
I'GCTGATTCT
CAATATTGTT
I
A.CATTGCCAG C3 rAATCGATAA I1 CAGGACCATA C TGTATCCATA C kTAGACATTG rAAAATGTAC .I :CTGACCAAG
G
kGGAGGTTTG
G
ETTCACTAAT TI E'GTAGATCAG
C
TGCATCAGGA
CTGATTTTTC
ATTTCAGGAG
CAATCGTTcC
TAAAACTCAT
TCAATATTAG
ACTATAAACA
TTGCAGTATT
ACAAATTTTT
CTTTCTCATT
GATTCAAATC
AATATATTTG
ACTGTTGTAT
rTTTTGATAA rTTTCTATTGI
TTTTGGTTTTI.
GGATTTTTAG
:AGAGGCGGA
kTTA-AAAGCA
C
UkAGATCTGA CA.AAAATAG
TI
~TTTACCCAT
TI
;CGCTTCTTc
T
~TGAAAGTAA
C
ATATCACTAT
CATTACTGGA
LGGTATAAACA
'GATCTACAA
C
LTCTGCAGGT
ICATATGGAA
T
TGCTCTTTT
C
CAT
TCTGGAATCA
CTCAGTAGCT
TGGGATTTCC
CTATTCACCA
CTTGAAAAGA
GATCTAATAC
TATAAATATA
TTTAATCCCT
TCTTTGTTGTC
rCTTCAGCCA rACTGGTACA
GGTCATCCTT
CCACATATA
rGGAGACTGG
ETGGTAGAAA
.TTCTGTTGT
.GCTTCTGTC
~TGCATTTTC
TTCCACTTT
'AACATCAAA
'CACATTGTT
TTAGAGCTT
ATTCCCAGG
CAGAACAAT
'TTCCAGATC
AGAAGTGAT
ATCTTCAGC
TATTCCCTT
TGAGGTGGC
TCCAGAATA
CTTTTAAAG
s0 100 150 200 250 300 350 400 450 500 550 600 650 700 750 S00 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1584 INFORMATION FOR SEQ ID NO:53: SEQUENCE
CHARACTERISTICS:
LE TGTH: 530 amino acids TPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) SEQUENCE DESCRIPTION: SEQ ID N0:53: Asp Pro Pro Thr Val Thr Leu Pro Gln Gly Glu Leu Val Gly Lys Ala Leu Thr Asn Glu Asn Gly Lys Glu Tyr Phe Ser Tyr -174- Thr Phe Asn Gly Lys Ala Val Pro Thr Pro Tyr Ala Pro Gin Lys Ser His Gly Lys 35 Al a Asn Pro Giu so Val Pro Pro Cys Val1 Trp Lys Gly Giu Leu Asn Giy Val Ala Leu Asn Arg Phe Phe Phe Asn 85 Val1 Gly 15 Asp Gly Gly 155 Asn Gly 25 Leu Leu Val1 Phe 100 As n Val Phe Leu Ile 170 Gly Ser Ser Val1 Thr 240 Pro
G
1 y Lys Tyr Phe Leu 115 Ile Leu Leu Giu Val 185 His Gly Lys Asn Val 255 Glu Lys Ala Trp Glu Phe 130 Asn Asp Lys Ser Thr 200 Ser Arg Asn Asp Ile 270 Ile Pro Val Phe Val1 Leu 145 Gin Phe Al a Thr Ala 215 Ser Thr Ser Ile Lys 90 His Gin Thr Giu Val 160 Gly Gly Thr Leu Leu 230 Gin Leu Glu Thr Gly 105 Ser Phe Leu Ala.
Gly 175 Gly Gly Asn Gin Asp 245 Leu Glu Thr Gi y Pro 120 Asn Glu Al a Asp Al a 190 Leu Pro Leu ka Lays 260 Phe *Ser Gly Asp Tyr 135 Gly Leu Pro Ser Tyr 205 Trp Al a Leu Lys Val 27S Giu Gly Asp Gi1L Asp Asp Phe Tyr Arg Al a 150 Lys Giu Val1 Lys Al a 220 Glu Giu Met Phe Cys Lys Vai Leu Leu Pro Trp 165 As n His Arg Phe Ile 235 Phe Pro Leu Lys Thr 110 Val1 Gly Gly Thr Ile 180 Tyr Al a Gin Leu Leu 250 Ala Pro Leu Leu Gly As n 125 Pro As n Lys Thr Leu 195 Ile Arg Gly 31n .lu 265 3er Val Pro Ser Tyr Leu 140 Val Giu Ile Leu Al a 210 His His Lys Thr Ile 280 Gin Pro :22 5 Pro Al a Giu -175- Glu Lys Val Phe Pro Ser Hi~s Gin Pro Phe Leu Giu Glu Ser 285 290 Pro Leu Ala Arg Met Lys Ser Gly Ser Phe Asn Lys Val Pro 295 300 305 Leu Leu Val Gly Phe Asn Ser Ala Giu Gly Leu Leu Tyr Lys 310 315 320 Phe Phe Met Lys Giu Lys Pro Glu Met Leu Asn Gin Ala Giu 325 330 335 Ala Asp Phe Giu Arg Leu Val Pro Ala Giu Phe Giu Leu Ala 340 345 350 His Gly Ser Giu Giu Ser Lys Lys Leu Ala Giu Lys Ile Arg 355 360 Lys Phe Tyr Phe Asp Asp Lys Pro Val Pro Glu Asn Giu Gin 365 370 375 Lys Phe Ile Asp Leu Ile Gly Asp Ile Trp Phe Thr Arg Gly .380 385 390 Ile Asp Lys His Vai Lys Leu Ser Val Glu Lys Gin Asp Giu 395 400 .405 Val Tyr Tyr Tyr Giu Tyr Ser Phe Ser Giu Ser His Pro 410 415 420 Ala Lys Gly Thr Phe Gly Asp His Asn Leu Thr Gly Ala Cys 425 430 His Gly Giu Giu Leu Val Asn Leu Phe Lys Vai Giu Met Met 435 440 445 Lys Leu Giu Lys Asp Lys Pro Asn Val Leu Leu Thr-Lys Asp 450 455 460 ***Arg Val Leu Ala met Trp Thr Asn Phe Ile Lys Asn Giy Asn 465 470 475 Pro Thr Pro Giu Vai Thr Giu Leu Leu Pro Vai Lys Trp Giu 480 485 490 Pro Ala Thr Lys Asp Lys Leu Asn Tyr Leu Asn Ile Asp Ala 495 500 Thr Leu Thr Leu Gly Thr Asn Pro Giu Gilt Thr Arg Val Lys 505 510 515 Phe Trp Giu Asp Ala Thr Lys Thr Leu His Ser Gin 520 525 530 -1 76- INFORMATION FOR SEQ ID NO:54: i) SEQUENCE CHARACTERISTICS: LENGTH: 570 amino acids TYPE: amino acid
TOPOLOGY:
linear protein o.
*O.
900.
Trp 1 Gly Asri Pro Ser Asp 20 Ser 85 Val Ile Phe Val Ala 155 Gin Phe Ala (ii) MOLECUL.
(iii) SEQUENCI Asp Asn Leu Asp Ile Thr Giu Gly Asp Val Tyr Cys Pro Phe Gly Pro 45 Asn Pro Lys Thr 60 Lys Cys Phe Gin Glu Asp Cys Leu Asn Ser Ala Asn 100 His Gly Gly Ala 115 Phe Gly Pro Asp 130 Thr Ile Asn Tyr 145 Pro Glu Trp Asp Arg Leu Ala Leu 170 Gly Gly Asp Arg 185 Gly Ala Ala Ser 200 E TYPE: Lys 20 Ser Leu Gly Glu Tyr 90 Asn Phe Tyr Arg Ile 160 Lys Glu Val Pro Tyr 35 Arg Phe Ser Leu Thr 105 As n Leu Leu His Trp? 175 Lys His Phe Leu Phe 50 Val1 Leu Asn Lys Gli 120 Ile Gly Gly Val Ile 190 Phe Arg Giy Gin Gin Ile Ile Tyr Gly Arg 135 Val1 Asn.
Tyr Thr Leu 205 Tyr Ile Ser Ala Tyr Phe Pro Ser Glu Phe 150 M ~t Asp Ile Met Lys Pro Pro Arg Ser Thr Vali Gly Gly Gly Gly 165 As n Al a Met Asp His Tyr Ala Lys Pro Thr Leu Tyr Ala Pro Glu Met Phe 110 Ser Tyr 125 Ile Ile Phe Leu Leu Lys Ile Giu 180 Gly Glu 195 Asp Asn Arg 01 u Ile Gly Gly Thr Trp Asn Leu 140 Ser Asp Lys Ser Ser 210 EDESCRIPTION: SEQ ID NO:54: Gin His LeU CyS Arg Val Gin Phe Asn -1 77- Thr Arg Leu Leu 225 Phe Glu 240 Giu Leu Lys Asn Lys Leu Tyr Tyr Gin 215 Pro Thr Ala 230 Leu Ly s Gin Asn Leu Asp Arg Asn Val1 245 Lys Al a Gin Leu Asn Ile Ile Asn Leu Leu 220 Gin Ile Ile Gin Ser Leu Leu 235 Thr Gin 250 Leu Arg Gly Thr His Arg Lys Gin Ala Ala 265 0* a.
Leu Val1 Asp 295 15 Gly Ala Met Pro Giu 365 25 Val: Lys Lys Tyr Ser 435 Ser 1 Asr2 Pro Pro Giu 310 Glu Giu Arg Lys Lys 380
G
1 y kla eu L'yr ~is so0 Arg Val Ile Phe Gly 325 Val1 Asp Lys Giu Asp 395 Ile Tyr Ile Gly Val Pro Asp Ser Asn Asp His 270 Phe Thr Pro Leu His 340 Ala Leu Gin Al a Phe 410 Arg Leu Asp 275 Asn Pro Val Leu Glu 285 Phe Pro Ser Ala Leu 300 Asp Val Asp Val Ile 315 Arg Ser met Ala Arg 330 Lys Thr Leu Thr Asn 345 Asn Ile Trp Lys Asn 355 Ile Lys Met Leu Thr 370 Asn Asp Asp Ile Glu 385 Gly Tyr Leu Gin Gly 400 Phe Asn Giu Phe Arg 415 Leu Ser Asp Asp Thr 425 Pro Tyr Arg Trp Gly 440 Asp Leu Gly Tyr Leu 455 Ser 290 Giu Ile Val le Pro 360 Glu Ala Ile Arg Tyr 430 Ser Phe Asi Prc Arg 305 Gly Thr Glu Asn Phe 3 Tyr Tyr Asn Ser Lieu 450 Ala Arg Glu Met Phe 320 Arg Arg Gly Tyr Val 390 Arg Ser Val Pro C Asn S 460 Asp Ser Arg Asn Gly 335 Al a Ile Asp Glm rhr 405 ksn ryr fly ~er Thr 280 Pro Asn Ser As n Ile 350 Glu Gin Leu Leu Leu 420 Lys Val Leu Asj Asr Asn Ala 505 Glu Lys Met Phe (2) .00.
sees "to* -178- SVai Pro Ile Leu Gly Thr Thr His Ile Ser Ile 465 470 Ala Met Gin Thr Leu Glu Arg Met Val Arg Ile 480 485.
Phe Val Lys Asn Gly Lys Pro Thr Ser Asn Thr 495 500 Ser Cys Asp Thr Lys Arg His Leu Ass Asp Ile 510 515 Pro Tyr Asn Asp Giu Giu Pro Lys Tyr Leu Asp 520 525 530 Giu Asn Phe Giu Met Lys Asn Ilie Leu Giu Leu 535 540 Met Leu Trp Asp Giu Vai Tyr Arg Asn Ala Asn 550 555 Arg Val Cys Asn Giu Gly Ser Ilie Arg 565 570 INFORMATION FOR SEQ ID Ci) SEQUENCE CHARACTERISTICS: LENGTH: 570 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) SEQUENCE DESCRIPTION: SEQ ID Asp Asn Leu Asp Gin His Leu Cys Arg Vai Gin 5 Ile Thr Giu Giy Lys Pro Phe Arg Tyr Lys Asp Asp Val Tyr Cys Ser Tyr Leu Giy Ile Pro Tyr 35 Pro Ile Gly Pro Leu Arg Phe Gin Ser Pro Lys s0 Ass Pro Lys Thr Giy Phe Val Gin Aia Arg Ser 65 Lys Cys Phe Gin Giu Ser Leu Ilie Tyr Ser Tyr 75 80 Giu Asp Cys Leu Tyr Leu Asn Ilie Phe Thr Pro 90 95 *Pr 47~ Tr Gil Phe Met Lys 545 Leu Phe Hi s Ala Pro Leu Ala Glu 2Thr 490 IAsp Trp Gly Arg Arg 560 Asn Lys Giu Ile Gly Gly Thr Trp 1 Giy Ass Pro Ser Asp Ser -179- Val Asn Ser Ala Asn Asn Thr Lys Tyr Pro Val. Met Phe Trp 100 105 110 Ile His Gly Gly Ala Phe Asn Gin Gly Ser Gly Ser Tyr Asn 115 120 125 Phe Phe dly Pro Asp Tyr Leu Ile Arg Giu Gly Ile Ile Leu 130 135 140 Val Thr Ile Asn Tyr Arg Leu Gly Val Phe Gly Phe Leu Ser 145 150 Ala Pro Giu Trp Asp Ile His Gly Asn met Gly Leu Lys Asp 155 .160 165 Gin Arg Leu Ala Leu Lys Trp Val Tyr Asp Asn Ile Giu Lys 170 175 180 Phe Gly Gly Asp Arg Asp Lys Ile Thr Ile Ala Gly Glu Ser .185 190 195 Ala Gly Ala Ala Ser Val His Phe Leu Met Met Asp Asn Ser 200 205 210 Thr Arg Lys Tyr Tyr Gin Arg Ala Ilie Leu Gin Ser Gly Thr 215 220 Leu Leu Asn Pro Thr Ala Asn Gin Ilie Gln Pro Leu His: Arg 225 230 235 Phe Giu Lys Leu Lys Gin Val Leu Asn Ilie Thr Gin Lys Gin 240 245 250 Giu Leu Leu Asn Leu Asp Lys Asn Gin Ile Leu Arg A *la Ala 255 260 265 .25 Leu Asn Arg Val Pro Asp Asn Asn Asp His Glu Arg Asp Thr 270 275 280 Val Pro Val Phe Asn Pro Val Leu Giu Ser Pro Glu Ser Pro 285 290 Asp Pro Ile Thr Phe Pro Ser Ala Leu Giu Arg Met Arg Asn 295 300 305 Gly Glu Phe Pro Asp Vai Asp Val Ile Ile Gly Phe Asn Ser 310 315 320 Ala Giu Gly Leu Arg Ser Met Pro Arg Val Thr Arg Gly Asn 325 330 335 Met Giu Val Tyr Lys Thr Leu Thr As Ilie Giu Arg Ala Ile 340 34S 350 Pro Glu Arg Asp Lys Lys Al a Leu As n 355 Ile Ile Lys Trp Met -180- LYS Asn Pro 360 Leu Thr Giu Asn Gly Ile Phe Tyr Asp 375 365 370 Val Lys Giu Gin Asn Asp 380 Lys Gly Asp Ala Gly Tyr Asp Ile Giu Ala 385 Leu Gin Giy Ile Tyr Val 390 Tyr Arg Gin Leu Thr Leu 405 Lys Tyr Ser 435 Ser Asp Asp Asn Ala 505 25 Glu Lys Met Phe (2) 395 400 Ala Ile Phe Phe Asn Giu Ile L' 410 4: Leu Tyr Arg Leu Ser Asp Asp Ti 425 Tyr Ile Leu Pro Tyr Arg Trp G] 440 His Gly Asp Asp Leu Giy Tyr Le 450 455 Vai Pro Ile Leu Gly Thr Thr Hi 465 470 Ala Met. Gin Thr Leu Giu Arg Me 480 48 Phe Val Lys Asn Gly Lys Pro Th 495 Ser Cys Asp Thr Lys Arg His Le 510 Pro Tyr Asn Asp Giu Giu Pro Ly 520 525 Giu His Phe Giu Met Lys Asn 11 535* 540 Met Leu Trp Asp Giu Val Tyr Ar 550 55 Arg Vai Cys Asn Giu Gly Sf.r Ii 565 INFORMATION FOR SEQ ID NO:56: SEQUENCE CHAP.ACTERITI~' /s hr
U
5 e Arg Tyr 430 Ser Phe Ile Vai Ser 500 As n Tyr Leu Asn Arg 570 Asn Ser Leu 445 Ala Ser Arg Asn Asp 515 Leu Giu Al a Ser Asn Val Tyr Pro Gly Asn Ser 460 Ile Pro 475 Ile Trp Thr Giu Ile Phe Asp Met 530 Leu Lys 545 Asn Leu Leu 420 Lys Val Leu Gin Thr 490 Asp Trp Gly Arg Arg 560
(-Q
LENGTH: 20 bases TYPE: nucleic acid STRANDEDNESS: snl single -181- TOPOLOGY: linear (ii) MOLECULE TYPE: primer (iii) SEQUENCE DESCRIPTION: SEQ ID N'O:56: GTGCGTACAC G TTT ACTACC INFORMATION FOR SEQ ID NO:57: Wi SEQUENCE CHARACTERISTICS: LENGTH: 2144 nucleotides TYPE: nucleic acid STRANDEDNESS: single 0 TOPOLOGY: linear (ii) MOLECULE TYPE: (iii) FEATURE:
NAME/KEY:
LOCATION:
(iv) FEATURE:
NAME/KEY:
LOCATION:
cDNA CDs 30. .1682 Asx Asn or Asp 462 SEQUENCE DESCRIPTION: SEQ ID NO:57: GTACACATAG TCAATAGTCT AGATCCAAG ATG TCT CGT GTT ATT TTT Met Ser Arg Val Ile Phe TTA AGT TGT ATT TTT TTG TTT AGT TTT AAT TTT ATA AAA Leu Ser Cys Ile Phe Leu Phe Ser Phe Asn Phe Ile Lys **.25 GAT TCC CCG ACT GTA ACT TTG CCC CAA Asp Ser Pro Thr Vai Thr Leu Pro Gin GAA TTG GTT GGA Glu Leu Val Gly
AAA
Lys 35 GCT TTG ACG AAC Ala Leu Thr Asn AAT GGA AAA GAG Asn Gly Lys Glu
TAT
Tyr 'rIT AGC TAC Phe Ser Tyr GAA CTT AGA Glu Leu Arg ACA G';T Tht. ly so GTA CCT TAT GCT Val 'Pro Tyr Ala CCT CCT GTT GGA Pro Pro Val Gly TTT AAG CCT Phe Lys Pro CCA CAG AAA GCT Pro Gin Lys Ala CCA TGG CAA GGT Pro Trp Gin Gly OTT TTC Val Phe -182- AAC GCC ACA TTA TAC GGA AAT GTG TGT AAA TCT TTA AAT TTC Asn Ala Thr Leu Tyr Gly Asn Val Cys Lys Ser Leu Asn Phe 85 TTC TTG AAG AAA ATT GAA GGA GAC GAA GAC TGC TTG GTA GTA Phe Leu Lys Lys Ile Glu Gly Asp Giu Asp Cys Leu Val Val 299 341
AAC
Asn 105 GTG TAC GCA CCA Val. Tyr Ala Pro AAA ACA ACT TCT GAT AAA Lys Thr Thr Ser Asp Lys 110 115 AAA CTT CCA Lys Leu Pro ACT GGA TCC Thr Gly Ser 130 GTA TTT Val Phe 120 TTC TGG GTT CAT Phe Trp Val His
GGT
Gly 125 GGT GGT TTT GTG Gly Gly Phe Val 425 GGA AAT TTA Gly Asn Leu 135 GAT GTT ATT Asp Val Ile GAA TTC CAA AGC Giu Phe Gin Ser
CCA
Pro 140 GAT TAT TTA GTA RAT TTT Asp Tyr Leu Val Asx Phe
TTC
Phe 150 GTA ACT TTC AAT Val Thr Phe Asn CGA TTG GGA CCT Arg Leu Gly Pro
CTC
Leu 160 GGA TrT CTG AAT Gly Phe Leu Asn
TTG
Leu 165 GAG TTG GAG GGT Giu ILeu Glu Gly CCA GGA AAT GTA Pro Gly Asn Val 509 551 593
GGA
Gly 175 TTA TTG GAT CAG Leu Leu Asp Gin
GTG
Val 180 GCA GCT CTG AAA Ala Ala Leu Lys ACC AAA GAA Thr Lys Glu ATT ACA ATT Ile Thr Ile 200 AAC ATT Asn Ile 190 GAG AAA TTT GGT Giu Lys Phe Gly
GGA
Gly 195 GAT CCA GAA AAT Asp Pro Glu Asn GGT GGT GTT Gly Gly Val 205 TTA TCT CAT Leu Ser His TCT GCT GGT GGA Ser Ala Gly Gly
GCA
Ala 210 AGT GTT CAT TAT CTT TTG Ser Val His Tyr Leu Leu ACC ACT GGA CTT Thr Thr Gly Leu AAA AGG GCA ATT Lys Arg Ala Ile CAA AGT GGA AGT GCT TTT AAT CCA TGG Gin Ser Gly Ser Ala Phe Asn Pro Trp 235
GCC
Al a 240 TTC CAA AGA CAT Phe Gin Arg His
CCA
Pro 245 GTA AAG CGT AGT Val Lys Arg. Ser CTT CAA Leui Gin 250 CTT GCT GAG Leu Ala Glu TTG GGT CAT Leu Gly His -183- CCC ACA Pro Thr 260 MAC AAT ACT Asn Asn Thr CAA GAT Gin Asp 265 GCT TTA GAA T'.
Ala Leu Giu Pf TTA CMA AAA ie Leu Gin Lys .270 GCC CCC GTA Ala Pro Val 275 GAC AGT CTC CTG Asp Ser Leu Leu MAA ATG CCA Lys Met Pro GCT GMA ACA Ala Giu Thr 285 GAA GOT GMA Giu Gly Glu
ATA
Ile 290 ATA GMA GAG TTT Ile Giu Glu Phe TTC GTA CCA TCA Phe Val. Pro Ser
ATT
Ile 929 971 GML AAA GTT TTC Glu Lys Val Phe
CCA
Pro 305 TCC CAC CMA CCT Ser His Gin Pro
TTC
Phe 310 TTG GMA GMA TCA Leu Giu Giu Ser CCA TTG GCC AGA ATG AAA TCC OGA TCC TTT Ser Gly Ser Phe Pro 315 Leu Ala Arg Met
AAC
Asn 325 AAA GTA CCT Lys Val Pro TTG TTC A Leu Phe Lys 340 TTA TTA GTT Leu Leu Val 330 TTC TTC ATG Phe Phe Met 345 GCA GAT TTT Ala Asp Phe GGA TTT MAC AGT GCA GMA GGA CTT Gly Phe Asn Ser Ala Giu Gly Leu AMA GM MAA CCA Lys Giu Lys Pro
GAG
Glu 350 ATG CTG MAC CMA Met Leu Asn Gin OCT GMA Ala Glu 355
GMA
Giu 360 AGA CTC GTA CCA Arg. Leu Val Pro
GCC
Al a 365 GAA TTT'GMA TTA Glu Phe Giu Leu
GTC
Val1 370 1013 1055 1097 1139 1181 1223 1265 25 CAT GGA TCA.GAG His Gly Ser Glu
GMA
Giu 375 TCG AMA AMA CTT Ser Lys Lys Leu
GCA
Al a 380 GMA AAM ATC AGO Giu Lys Ile Arg
MOG
Lys 30 385 TTT TAC TTT GAC Phe Tyr Phe Asp
GAT
Asp 390 AMA CCC GTT CCA Lys Pro Val Pro
GMA
Glu 395 MAT GMA CAG Asn Giu Gin ACT AGA GGT Thr Arg Gly 410 AAA TTr ATT GAC TTG ATA Lys Phe Ile Asp Leu Ile 400
GGA
Gly 405 GAT ATT TOO TTT Asp Ile Trp Phe GTT GAC MAG CAT GTC MAG TTG Val Asp Lys His Val Lys Leu GTG GAG MAA CMA GAC GMA Val Glu Lys G~r. Asp Glu 1307 1349 CCA GTT TAT Pro Val Tyr
TAT
Tyr 430 TAT GMA TAT TCC TTC TCG GAA AGT CAT Tyr Oiu Tyr Ser Phe Ser Giu Ser His
CCT
Pro 440 -184- GCA AAA GGA ACA TTT Ala Lys Gly Thr Phe 445 GGT GAT CAT AAT Gly Asp His Asn
CTG
Leu 450 ACT GGT GCA TGC Thr Gly Ala Cys 1391 1433
CAT
His 455 GGA GA-A GAA CTT Gly Glu Glu Leu AAT TTA TTC AAA Asn Leu Phe Lys GAG ATG ATO Glu Met Met AAG CTG Lys Leu 470 AGA GTA Arg Val GA)A AAA GAT AAA Glu Lys Asp Lys
CCT
Pro 475
ACT
Thr AAT GTT CTA Asn Val Leu
CTT
Leu 485 GCC ATG TGG Ala Met Trp TTA ACA AAA GAT Leu Thr Lys Asp AA480 AAAAT GGA AAT Lys Asn Gly Asn 495
AAC
As n 490 TTC ATC Phe Ile CCT ACT CCT Pro Thr Pro GTA ACA GAA TTA Val Thr Glu Leu CCA GTT AAA TGG Pro Val Lys Trp
GAA
Glu 510 1475 1517 1559 1601 1643 C C
C.
C
C
C
CCT GCC ACA AAA Pro Ala Thr Lys
GAC
Asp 515 AAG TTG AAT TAT Lys Leu Asn Tyr
TTG
Leu 520 AAC ATT GAT GCC Asn Ile Asp Ala
ACC
Thr 525 TTA ACT TTG GGA Leu Thr Leu Gly
ACA
Thr 530 AAT CCT GAG GCA Asn Pro Glu Ala
AAC
Asn 535 CGA GTC AAA Arg Val Lys TTT TGG Phe Trp 540 GAA GAC GCC ACA Glu.Asp Ala Thr
AAA
Lys 545 TCT TTG CAC GGT CAA TAA Ser Leu His Gly Gin 550 1682
TAATTTATGA
AAAATTAAAA
CAGTTCTAAT
TGGTTTTAAT
ATAGATATGT
30 ATCAGTAAAA
TTTAAAGAA.A
TAACTTAAAA
TGATATCATT
ATAAAATTAT
AAATTGTTTT
AATAACA-ATT
TTTATTTATT
TTTCAAAACA
TATATTAAGT
ATACATTAAC
ATACCAAAAA
ATAAAATTAA
AGTGA-AAATT
GT
AAATACTTTA
TTTATGTTTT
TATTCTTGTT
CAACGTCGTT
ACTCTGAAGT
TAAAAATATA
TAAAGTAAAA
CTCTTCAATA
ATATTTTGAT
GGTAATATAT
ATGTATTGGC
TTGCTTGTTT
TGTTTTTAGC
ATTTTTATAT
AGATATTTTC
TTCCAAACGG
ATTTTGATAA
AATACGTATT
TAGGTAAATA
TTATGTGTAT
TGAAATATCA
AAAATTTCCA
ATACACTAAA
AATAATTTTr
AATTTTTGTT
TTAGTATTTC
TATATTTAAA
1*732 1782 1832 1882 1932 1962 2032 2082 2132 2144 INFORMATION FOR SEQ ID NO:58: SEQUENCE CEAkRACTERISTICS: LEN%, 550 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) SEQUENCE DESCRIPTION: SEQ ID NO:58: Met Ser 1 Phe Asn Gin Gly Arg Phe Glu Val1 Ile Leu Ile Phe Leu Ser 5 Lys Cys Asp Ser 20 Vai Giy Lys Ala -185- Cys Ile Pro Thr Leu Thr Phe Le Val Th Asn Gi Lys Giu Tyr Phe Ser Tyr Thr Gly Val Pro Ty2 r Pro Pro Cys 85 15 Giu Ser Gly 20 Asp Tyr 15 S 25 Gly~ Leu Pro Ser Tyr I 225 Val1 Trp Lys Asp 100 Asp Phe T'yr Arg k1 a 170 Siu lai .ys Gly Gin Ser Cys Lys 115 Val1 Leu Leu Pro Trp 185 Asn His Arg Git 60 Gly Leu Leu Lys Thr 130 Vai Gly Gly Thr Ile 200 Tyr kl~a ILet Val As n Val1 Leu Gly Asx 145 Pro Asn Lys Thr Leu 215 Ile 1Arg Phe Phe Asn Phe Phe 90 Val Asn 105 Pro Val Ser Gly Phe Asp Leu Gly 160 Val Gly 175 Giu Asn Ile Gly Leu Leu Aia Gin 230 50 Lys Al a Leu Val Phe 120 Asn Val Phe Lieu Ile 190 ,1y 3er 3er Pro Thr Lys Tyr Phe Leu 135 Ile Leu Leu Giu Val 205 His Giy Prc Leu Lys Al a Trp Glu Phe 150 As n Asp Lys Ser Thr 220 Ser Gin Tyr Ile Pro Vai Phe Val Leu 165 Gin Phe Ala Thr Ala 235 Al Gl Glu Lys 110 His Gin Thr Glu VIal 180 Giy
G
1 y ['hr Phe *u Phe *r Leu Asn 0 a. Lys Ala Asn Gly2 Thr I) Gly G 125 Ser P 1 Phe A Leu G Ala A Gly A 195 Gly A 2 Gly L Asn P Ser Pro Gly Pro Giu Val1 ksp 'hr iy 'ro s n iu ia sp la eu ro Trp Ala 240 Phe Gin Arg His Pro 245 Val Lys Arg Ser Leu Gin Leu 250 -186- Asn Asn Ala Giu Ile Leu Gly His Pro Thr 255 Thr Gin Asp Ala 265 260 Leu Lys 6* Val1 295 Pro Ser Glu 15 Met Ala 365 Leu 20 Val Ile 25 Val Phe 435 Asn Phe Val Phe Glu Met Phe Phe 310 Phe Gly Leu Giu Ala 380 Pro Trp Glu Ser Leu 450 Lys Leu Ile Phe Pro Val Leu Asn 325 Leu As n Phe Giu 395 Phe .iu rhr ,fal 465 4 e u
L
4 ys Leu 270 Al a Pro Glu Lys Leu 340 Gin Giu Lys Asn Thr 410 Gln Ser Gly Giu Thr 480 Asn Gin Giu 285 Ser Glu Vali Phe Al a 355 Leu Ile Giu Arg Asp 42S His Al a Met Lys Gly 495 Lys Thr Ile 300 Ser Pro Lys Glu.
Val1 370 Arg Gin Gly Glu Pro 440 Cys Met As n Glu Glu Pro 315 Leu Phe Ala His Lys 385 Lys Val1 Pro Ala His 455 Lys Arg Pro Gly Lys Le u Leu 330 Phe Asp Gly Phe Phe 400 Asp Val Lys Gly Leu 4-70 Val Thr 275 Glu Val1 Al a Val1 Met 345 Phe Ser Tyr Ilie Lys 415 Tyr Giu Eeu 485 Pro Ile 290 Phe Arg Gly Lys Giu 360 Glu Phe Asp His Tyr 430 Thr Giu Lys Ala Giu1 500 Ile Pro 305 Met Phe Gi u Arg Glu 375 Asp Leu Val1 Tyr Phe 445 Leu Asp M1et Val Glu Ser Lys 320 Asn Lys Leu Ser Asp 390 Ile Lys Giu Gly Val 460 Lys Trp Thr Ala Pro Val Asp Ser Let Leu Glu His Ser Ser 335 Pro Val1 Lys Lys Gly 405 Leu Tyr Asp Asn Pro 475 Thr Giu Lys 280 Phe Gin Gly Ala Glu 350 Pro Lys Pro Asp Ser 420 Ser Leu ksn 490 -187- Leu Pro Val Lys Trp Giu 510 Pro Ala Thr Lys Asp Lys Leu Asn 515 Gly Thr Asn Pro 530 Tyr Leu Asn 520 Ile Asp Ala Thr 525 Leu Thr Leu Giu Ala Asn 535 Arg Val Lys Phe Trp, 540 Giu Asp Ala Thr Lys Ser 545 Leu His Gly Gin 550 INFORMATION FOR SEQ ID NO:59: SEQUENCE CHARACTERISTICS: LENGTH: 2144 nucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) SEQUENCE DESCRIPTION: SEQ ID NO:59:
ACATAATTTT
CTAATGATAT
ATTTTTAAGT
20 ATTTTCTTTA
ATTTTTACTG
TAACATATCT
AAATTAAAAC
AAATTAGAAC
25 TTTTTTAATT
TTTCATAAAT
ATTTGACTCG
ATGTTCAAAT
CAATAATTCT
30 TCCACATGGC
TTTTCCAGCT
ATGGCATGCA
GACTTTCCGA
TCCACAGACA
TCCTATCAAG
CAAAGTAAAA
CCATGGACTA
AGCTTGGTTC
GTCCTTCTGC.
CCGGATTTCA
TGGGAAAACT
CACCTTCTGT
TTTTGTAAGA.
TATCTCAGCA
ATGGATTAAA
GTGGTTGTAT
ATTTTAAATA
CAGAAATACT
TAAACAAAAA
AAAAAAATTA
ATTTTAGTGT
ATTGGAAATT
CATGATATTT
TGATACACAT
TTTATTTACC
TATTATTGAC
GTTTGCCTCA
AATTCAACTT
GTTACTTCAG
AAGTACTCTA
TCATCATCTC
CCAGTCAGAT
GAAGGAATAT
ACTTGACATG
TCAATAAATT
CTTCCTGATT
ATTCAAATTC
AGCATCTCTG
ACTGTTAAAT
TTCTGGCCAA
TTTTCAATTG
TTCAGCTGGC
ATTCTAAAGC
AGTTGAAGAC
AGCACTTCCA
GAGATAACAA
TAAATACGTA
AATTATCAAA
TTCCGTTTGG
TTGAAAATAT
ATATATAAAA
TTGCTAAAAA
CAAAACAAGC
AAGCCAATAC
TAATATATTA
CGTGCAAAGA
GGATTTGTTC
GTCTTTTGTG
GAGTAGGATT
TCTTTTGTTA.
GACTTTGAAT
TATGATCACC
TCATAATAAT
CTTGTCAACA
TCTGTTCATT
TTTTCTGCAA.
GGCTGGTACG
GTTTTTCTTT
CCAACTAATA
TGGTGATTCT
ATGGTACGAA
ATTTTCTTCA
ATCTTGAGTA
TACGCTTTAC
CTTTGAGCAA.
AAGATAATGA
TTATCAAAAT
ATTATTGAAG
AATTTTACTT
CTTATATTTT
ATACTTCAGA
CAAACGACGT
AAAACAAGAA
ATAAAACATA
CCTAAAGTAT
TTTTGTGGCG
CCAAAGTTAA
GCAGGTTCCC
TCCATTT=G
ATAGAACATT
AAATTCACAA
AAATGTTCCT
AAACTGGTTC
CCTCTAGTA.A
TTCTGGAACG
GTTTTTTCGA
AG TCTTTC .AA
CATGAAC.AAT
AAGGTACTTT
TCCAAGAAAG
GACAAACTCT
GGAGACTGTC
TTGTTTGTGG
TGGATGTCTT
TTGCCCTT
ACACTTGCTC
ATAATTTTCA
AGTTAATTTT
TATTTTTGGT
TAGTTAATGT
GTACTTAATA
TGTGTTTTGA
TAAATAAATA
AAAATTGTTA
TTAAAACAAT
TCTTCCCAAA
GGTGGCATCA
ATTTAACTGG
ATGAAGTTAG
AGGTTTATCT
GTTCTTCTCC
TTTGCAGGAT
GTCTTGTTTC
ACCAAATATC
GGTTTATCGT
TTCCTCTGA.T
AATCTGCTTC
TTGAACAAAA
GTTAAAGGAT
GTTGGTGGGA
TCTATTATTT
TACGGGGGCT
GATGACCCAA
TGGAAGGCCC
GTAAAGTCCA
CACCAGCAGA
100 150 200 250 -300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 -188- AACACCACCA
ATTGTAATAT
TTTCTTTGGT
CCATTTCAGA
CCTGGAGCAC
CCTCCAACTC
GTAATTGAAA
GTTACGAA
TTTGGAATTC TAAATTTCCG CAGAAAATA
CTGGAAGTTT
GTTTACTACC
AAGCAGTCTT
TTAAAGATTT
ACACACATTT
CATGGCTCAG
CTTTCTGTGG
TTTAGCATAA
GGTACACCTG
TCGTCAAAGC
TTTTCCAACC
GAATCACATT
TTATAAAATT
TTTCTGGATC
GCTGCCACCT
CAAATTCAGA
TAACATcAAA
GATCCAGTCA
TTTATCAGAA
CGTCTCCTTC
CCGTATAATG
AGGCTTA1AAT
TGTAGCTAAA
AATTCGCCTT
AAAACTAAAC
TCCACCAAAT TTCTCAATGT GATCCAATAA TCCTACATTT AATCCGAGAG GTCCCAATCG ATYTACTAAA TAATCTGGGC CAAAACCACC ACCATGAACC GTTGTTTTTG
GTGCGTACAC
AATTTTCTTC AAGAAGAAAT TGGCGTTGAA
AACACCTTGC
CTAAGTTCTC
CAACAGGAGG
ATACTCTTTT
CCAT'ITTCGT
GGGGCAAAGT
TACAGTCGGG
AAAAAAATAC AACTTAAAAA 1550 1600 1.650 1700 1750 1800 1850 1900 1950 2000 2050 2100 2144 k~AAAAA GACATCTTGG ATCTAGACTA TTGACTATGT GTAC INFORMATION FOR SEQ ID Wi SEQUENCE CHARACTERISTICS: LENGTH: 1650 nucl~eotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: (iii) FEATURE:
NAME/KEY:
LOCATION:
FEATURE:
NAME/KEY:
LOCATION:
cDNA
CDS
1. .1650 Asx Asn or Asp 433 S V
V
V
.5
S
V
SEQUENCE DESCRIPTION: SEQ ID
ATG
Met 30 1 TCT CGT GTT ATT TTT TTA AGT Ser Arg Val Ile Phe Leu Ser
S
TGT ATT Cys Ile AAT TTT ATA AAA Asn Phe Ile Lys OAT TCC CCG ACT Asp Ser Pro Thr TTT TTG TTT AGT Phe Leu Phe Ser GTA ACT TTG CCC Val Thr Leu Pro AAC OAA AAT GGA Asn Glu Asn Gly CAA GGC GAA Giln Gly Glu AAA GAG TAT Ljys Giu Tyr CCT GTT OGA Pro Val Gly TTG GTT GGA AAA OCT TTG ACG Leu Val Gly Lys Ala Leu Thr TTT AGC TAC ACA Phe Ser Tyr Thr GTA CCT TAT OCT Val Pro Tyr Ala AAA CCT Lys Pro CTT AGA TTT AAG Leu Arg Phe Lys CCA CAG AAA GCT Pro Gin Lys Ala -189- CCA TGG CAA GGT GTT TTC Pro Trp Gin Gly Val Phe AAC GCC ACA TTA Asn Ala Thr Leu TTC TTG MAG AAA Phe Leu Lys Lys
TGT
Cys AAA TCT TTA MAT Lys Ser Leu Asn
TTC
Phe 90 TAC GGA MAT GTG Tyr Gly Asn Val ATT GMA GGA GAC Ile Giu Gly Asp CCA AMA ACA ACT Pro Lys Thr Thr 110 252 294 336 GMA GAC Glu Asp 100 TGC TTG GTA GTA Cys Leu Val Val
AAC
Asn 105 GTG TAC GCA Val Tyr Ala TCT GAT AMA Ser Asp Lys 115 GGT TTT GTG Gly Phe Val AMA CTT CCA GTA Lys Leu Pro Val
TTT
Phe 120 TTC TGG GTT CAT Phe Trp Val His GGT GGT Gly Gly 125 GGA TCC GGA MAT Giy Ser Gly Asn GMA TTC CMA AGC Giu Phe Gin Ser
CCA
Pro 140 S. Se S. 0 *0 S 9 *5*S
C
Je S
S
0O S*SS 0
S.*
9 9.
9 55*i GAT TAT TTA GTA Asp Tyr Leu Vai TTT GAT GTT ATT Phe Asp Val le
TTC
Phe GTA ACT TTC MAT Val Thr Phe Asn 378 420 462 504 546
TAC
Tyr 155 CGA TTG GGA CCT Arg Leu Giy Pro
CTC
Leu 160 GGA TTT CTG AAT Gly Phe Leu Asn GAG TTG GAG Glu Leu Giu GTG GCA GCT Vai Ala Ala 180 GGT GCT Gly Ala 170 CCA GGA MAT GTA Pro Gly Asn Val
GGA
Gly 175 TTA TTG GAT CAG Leu Leu Asp Gin 25 CTG AMA TGG Leu Lys Trp 185 ACC AMA GA MAC Thr Lys Giu Asn
ATT
Ile 190 GAG AAA TTT GGT Glu Lys Phe Gly GGA GAT Gly Asp 195 CCA GMA MT Pro Giu Asn
ATT
Ile 200 ACA ATT GGT GGT Thr Ile Gly Gly TCT GCT GGT GGA Ser Ala Gly Gly AGT GTT CAT TAT Ser Val His Tyr TTG TTA TCT CAT Leu Leu Ser His
ACA
Thr 220 ACC ACT GGA CTT Thr Thr Gly Leu
TAC
Tyr 225 AMA AGG GCA ATT Lys Arg Al a Ile
GCT
Ala 230 CMA AGT GGA AGT Gin Ser Gly Ser
GCT
Aia 235 TTT MAT CCA Phe Asn Pro CTT CMA CTT Leu Gin Leu 250 TGG GCC Trp Ala 240 TTC CMA AGA CAT Phe Gin Arg His
CCA
Pro 245 GTA MAG CGT AGT Val Lys Arg Ser GCT GAG ATA Ala Giu Ile 255 TTG GGT CAT CCC Leu Giy His Pro
ACA
Thr 260 MAC MAT ACT CMA Asn Asn Thr Gin GAT GCT Asp Ala 265 -190- TTA GAA TTC TTA CAA AAA GCC CCC Lea Glu Phe Gin Lys Ala Pro
GTA
Val 275 GAC AGT CTC CTG AAG Asp Ser Leu Lea Lys.
280 AAA ATG CCA GCT Lys Met Pro Ala
GAA
Glu 285 ACA GAA GGT GAA Thr Glu Giy Glu
ATA
Ile 290 ATA GAA GAG TTT Ile Glu Giu Phe 840 882 924- 966
GTC
Val 295 TTC GTA CCA TCA Phe Val Pro Ser
ATT
Ile 300 GAA AAA GTT TTC Glu Lys Val Phe TCC CAC CAA Ser His Gin AAA TCC GGA Lys Ser Gly 320 CCT TTC TTG GAA GAA Pro Phe 310 Leu Giu Glu TCA CCA Ser Pro 315 TTG GCC AGA ATG Leu Aia Arg Met TCC TTT AAC Ser Phe Asn 325 GAA GGA CTT Glu Giy Leu AAA GTA CCT TTA Lys Val Pro Leu
TTA
Leu 330 GTT GGA TTT AAC Val Giy Phe Asn AGT GCA Ser Ala 335 *e S S.
S
TTG
Lea 340 TTC AAA TTC TTC Phe Lys Phe Phe AAA GAA AAA CCA Lys Glu Lys Pro
GAG
Glu 350 ATG CTG AAC CAA Met Lea Asn Gin
GCT
Ala 355 GAA GCA GAT TTT Glu Ala Asp Phe
GAA
Glu 360 AGA CTC GTA CCA Arg Lea Val Pro GAA TTT GAA TTA Glu Phe Giu Lea CAT GGA TCA GAG His Gly Ser Glu
GAA
Glu 375 TCG AAA AAA Ser Lys Lys GAT AAA CCC Asp Lys Pro 390 1008 1050 1092 1134 1176 1218 1260 1302 CTT GCA Lea Ala 380 GAA AAA ATC AGG Glu Lys Ile Arg
AAG
Lys 385 TTT TAC TTT GAC Phe Tyr Phe Asp OTT CCA GAA Val Pro Glu 395 ATT TGG TTT Ile Trp Phe AAT GAA CAG AAA Asn Glu Gin Lys
TTT
Phe 400 ATT GAC TTG ATA Ile Asp Leu Ile OGA GAT Gly Asp 405
ACT
Thr 410 AGA GOT GTT GAC Arg Oly Vai Asp CAT GTC AAG TTO His Vai Lys Lea GTG GAG AAA CAA Vai Glu Lys Gin
GAC
Asp 425 GAA CCA OTT TAT Glu Pro Val Tyr
TAT
Tyr 430 TAT OAA TAT TCC Tyr. Glu Tyr Ser
TTC
Phe 435 TCG OAA AGT CAT CCT GCA AAA GGA ACA Ser Glu Ser His Pro Ala Lys Giy Thr
TTT
Phe 445 GGT GAT CAT Gly Asp His 1344 -191- AAT CTG Asn Leu 450 ACT GGT GCA TGC Thr Gly Ala Cys
CAT
His 455 GGA GAA GAA CTT GTG MAT TTA Gay Glu Giu Leu Val Asn Leu 1386 1428 TTC AAA GTC GAG ATG ATG MAG Phe Lys Val Glu Met Met Lys 465
CTG
Leu 470 GM- AAA GAT AAA CCT MAT Glu Lys Asp Lys Pro Asn 475 GTT CTA TTA Val Leu Leu
ACA
Thr 480 MAA GAT AGA GTA Lys Asp Arg Val
CTT
Leu 485 GCC ATG TGG ACT MAC Ala Met Trp Thr Asn 490 1470 TTC ATC MAA MAT GGA MAT CCT ACT CCT Phe Ile Lys Asn Gly Asn Pro Thr Pro 495
GMA
Giu 500 GTA ACA GMA TTA Val Thr Giu Leu 1512
TTG
Leu 5059 CCA GTT MAA TGG Pro Val Lys Trp
GMA
Giu 510 CCT GCC ACA Pro Ala Thr- AAA GAC MAG TTG Lys Asp Lys Leu.
515
MAT
Asn 1554 TAT TTG Tyr Leu 520 MAC ATT GAT GCC Asn Ile Asp Ala TTA ACT TTG GGA ACA MAT CCT Leu Thr Leu Gly Thr Asn Pro 530 GAG GCA MAC CGA 20 Glu Ala Asn Arg 535 TTG CAC GGT CMA Leu His Gly Gin 550 GTC AAA TTT Val Lys Phe TGG GAA GAC GCC ACA Trp Giu Asp Ala Thr 540 1596 1638 1650 MAA TCT Lys Ser 545 25- INFORMATION FOR SEQ ID NO:61: SEQUENCE CHARACTERISTICS: LENGTH: 1650 niucleotides TYPE: niucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: cDNA SEQUEN CE DESCRIPTION: SEQ ID NO:61:
TTGACCGTGC
CCTCAGGATT
MACTTGTCTT
TTCAGGAGTA
CTCTATCTTT
ATCTCGACTT
CAGATTATGA
MTATTCATA
ACATGCTTGT
MAATTTCTGT
TGATTTTTTC
MTTCGGCTG
AMGATTTTG
TGTTCCCA
TTGTGGCAGG
GGATTTCCAT
TGTTMATAGA
TGMATAATT
TCACCAAATG
ATMATMACT
CMACACCTCT
TCATTTTCTG
TGCMAGTTTT
GTACGAGTCT
TGGCGTCTTC
GTTMAGGTGG
TTCCCATTT-
TTTTGATGAA
ACATTAGGTT
CACMAGTTCT
TTCCTTTTGC
GGTTCGTCTT
AGTMAACCMA
GMACGGGTTT
TTCGATTCCT
TTCAAAATCT
CCAAAATTTG
C .kTCMATGTT
,CTGGCMATA
GTTAGTCCAC
TATCTTTTTC
TCTCCATGGC
AGGATGACTT
GTTTCTCCAC
ATATCTCC-TA
ATCGTCAAAG
CTGATCCATG
GCTTCAGCTT
ACTCGGTTTG
CMAATMATTC
ATTCTGTTAC
ATGGCMAGTA
CAGC'rCATc
ATGCACCAGT
TCCGAGMAGG
AGACAACTTG
TCMAGTCMAT
TAAAACTTCC
GACTMATTCA
GGTTCAGCAT
100 150 200 250 300 350 400 450 500 550 600 -192- CTCTGGTTTT
TCTTTCATGP
TAAATCCAAC TAATAAAGGI1 GCCAATGGTG
ATTCTTCCA
AATTGATGGT ACGAAGACAA CTGGCATTTT CTTCAGGAGA AAAGCATCTT
GAGTATTGTT
AAGACTACGC TTTACTGGAT TTCCACTTTG
AGCAATTGCC
AACAAAAGAT AATGAACACT AATATTTTCT GGATCTCCAC TCAGAGCTGC
CACCTGATCC
AACTCCAAAT
TCAGAAATC
GAAAATAACA TCAAAATYTA TTCCGGATCC AGTCACAAA AGTTTTTTAT
CAGAAGTTGT
GTCTTCGTCT
CCTTCA.ATTT*
CATTTCCGTA
TAATGTGGCG
TGTGGAGGCT
TAAATCTAAG
ACCTGTGTAG
CTAAAATACT
20 CAACCAATTC
GCCTTGGGGC
AAATTAAAAC TAAAcAAAAA
AGAATTTGAA
*ACTTTGTTAA
*GAAAGGTTGG
*ACTCTTCTAT
CTGTCTACGG
TGTGGGATGA
GTCTTTGGAA
CTTTTGTAAA
TGCTCCACCA
CAAATTTCTC
A-ATAATCCTA
GAGAGGTCCC
CTAAATAATC
CCACCACCAT
TTTTGGTGCG
TCTTCAAGAA
TTGAAAACAC
TTCTCCAACA
CTTTTCCATT
AAAGTTACAG
AATACAACTT
CAAAAGTCCT
AGGATCCGGA
TGGGATGGGA
TATTTCACCT
GGGCTTTTTG
CCCAATATCT
GGCCCATGGA
GTCCAGTGGT
GCAGAAACAC
AATGTTTTCT
CATTTCCTGG
AATCGGTAAT
TGGGCTTTGG
GAACCCAGAA
rACACGTTTA
GAAATTTAAAC
CTTGCCATGG C 3;GAGGTTTAG C
ETCGTTCGTC
rCGGGGAATC A %.AAAATAA
C
TCTGCACTGT
TTTCATTCTG
AAACTTTTTC
TCTGTTTCAG
TAAGAATTCT
CAGCAAGTTG
TTAAAAGCAC
TGTATGAGAT
CACCAATTGT
TTGGTCCATT
AGCAcccTcc rGAAAGTTAC kA'ITCTAAAT kAATACTGGA
.TACCAAGCA
ATTTACACA
TCAGCTTTC
ATAAGGTAC
L.AAGCTT'rTC
~CATTTTATA
ACGAGACAT
650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 INFORMATION FOR SEQ ID NO:62: SEQUENCE CHARACTERISTICS: LENGTH: 29 bases TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: primer (iii) SEQUENCE DESCRIPTION: SEQ ID NO:62: 30 AAACTCGAGT CCCCCGACTG
TAACTTTGC
INFORMATION FOR SEQ ID NO:63: Wi SEQUENCE CHARACTERISTICS: LENGTH: 36 bases TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: -linear (ii) MOLECULE TYPE: primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: TCATCTGCAG TTATTGACTG TGCAAAGTTT
TTGTGG
INFORMATION FOR SEQ ID NO:64: Wi SEQUENCE CHARACTERISTICS: LENGTH: 32 bases TYPE: nucleic acid STRANDEDNESS: single -193- TOPOLOGY: linear (ii) MOLECULE TYPE: primer (iii) SEQUENCE DESCRIPTION: SEQ ID NO:64: TTCCGGATCC GGCTGATCTA CAAGTGACTT
TG
INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 34 bases TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: primer (iii) SEQUENCE DESCRIPTION: SEQ ID TGGTACTCGA GTCATAAAAA TTTATTCCAA
AATC
INFORMATION FOR SEQ ID NO:66: 15 SEQUENCE CHARACTERISTICS: LENGTH: 39 bases TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 20 (ii) MOLECULE TYPE: primer (iii) SEQUENCE DESCRIPTION: SEQ ID NO:66: AAAACTGCAG TATAAATATG TTACCTCACA
GTGCATTAG
INFORMATION FOR SEQ ID NO:67: i) SEQUENCE CHARACTERISTICS: 25 LENGTH: 1987 nucleotides TYPE: nucleic acid STR.ANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY:
CDS
LOCATION: 1. .1650 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: AATTCACAGT GTAAATAATT TTATTTGATA TAAATGTATT TAATTT1TrAT TTTAATCTAA TTTTAATTTA AATATATATA GTTTTATTTA
TAAAAAAATA
TTTTTTTTAT GATCGAAAAG AAATTTTTAT T TATGTTTAT
GAGTGTGTGT
-194- TTTGGCTATG ATTTACATTA TTTTTGAGCT AGTATAAAAT
TAAACCATAT
TATATTTTGG ATATATAATA ACATTTTATA ATG TGT GAT CCA TTA Met Cys Asp Pro Leu 21 CTA AAA ACA ACA ACA-TAT GGA ATT CTG AAA GGC AAG AAA GTT Leu Lys Thr Thr Thr Tyr Gly Ie Leu Lys Gly Lys Lys Vai
GTA
Val1 20 AAC GAA AAT GGT Asn Giu Asn Gly AAA ATT TAC Lys Ile Tyr 25 TAT AGT TAC ACA GGT ATA Tyr Ser Tyr Thr Gly Ile GAT CTC -AGA TTC AAG CCA Asp Leu Arg Phe Lys Pro 329 371.
CCC TAT Pro Tyr GCA AAA TCT CCT Ala Lys Ser Pro
AAT
As n 0000.
CCA CAA AAA Pro Gin Lys so CAG TAT GGA Gin Tyr Gly CTT GAT CCT TGG Leu Asp Pro Trp GGT GTT TTT GAC Gly Val Phe Asp GCC ACT Ala Thr
AAT
Asn AAT TGT GCT GCT Asn Cys Ala Ala AAA TGG TTT TTG Lys Trp Phe Leu TCA GCT GGG GGT Ser Ala Gly Gly
TGC
Cys s0 GAA GAT TGC CTT TAC TTA AAT ATC TAT Giu Asp Cys Leu Tyr Leu Asn Ile Tyr
GTC
Vali 90
CCA
Pro CAA AAC ACT TCA Gin Asn Thr Ser CAT GGA GGA GCA His Gly Gly Ala GAA AAT CCT TTG Glu Asn Pro Leu GTA ATG TTT Val Met Phe GGA AAT TCT Gly Asn Ser 1125 TGG ATT Trp Ile 105
TTT
Phe 1120 GTG GTC GGA TCA Val Val Gly Ser GAT ATA CAT GGT CCT GAT TAT TTA Asp Ile His Gly Pro Asp Tyr Leu 120 .125 ATA GAA TAT GAT Ile Giu Tyr Asp ATT ATC Ile Ile 1.30 TTA GTA ACT ATT AAT TAT CGT CTA GGA CCA CTT GGT TTT CTT Leu Val Thr Ile Asn Tyr Arg Leu Gly Pro Leu Gly Phe Leu 2.35 140 145 AAT TTG GAA ATC GAA GAT GCG CCT GGG AAT GTT GGA ATG Asn Leu Giu Ile Giu Asp Ala Pro Giy Asn Val Gly Leu Met
GAT
Asp 2.60 CAA GTT GCA GCC Gin Val Ala Ala AAA TGG GTA AAT GAA AAT ATT GCA Lys Trp Val Asn Giu Asn Ile Ala -195- ACC TTT Thr Phe 175 AGT GGA GAC CCA Ser Gly Asp Pro
AAA
Lys 180 AAT ATT ACA Asn Ile Thr ATT TGT GGA GCA Ile Cys Gly Ala 185 ACT GCT GGA Thr Ala Gly 190 CTT ACC AAA Leu Thr Lys GCT GCA AGT GTA Ala Ala Ser Val TAT CAC ATT TTG TCA CAA Tyr His Ile Leu Ser Gin 200 833 875
GGT
Gly 205 TTA TTC CAC AAG Leu Phe His Lys ATA GCA CAA AGT Ile Ala Gin Ser
GGA
Giy 215
AGT
Ser GCT TTT AAT CCC TGG GCT TTC CAA AAA AAT Ala Phe Asn Pro 220 Trp Ala Phe -Gln Lys 225 AAT GCA Asn Ala 230 Asn
CTT
Leu 240 CCT GTT AAG pro Val Lys ACC ACA AAC Thr Thr Asn 917 CTT CGA CTA Leu Arg Leu
TGC
Cys 235 AAA ACC TTA GGC Lys Thr Leu Gly AAC CTT CAA GAA GCC TTG Asn Leu Gin 245 GAA ACA TTG 20 Glu Thr Leu 260 CAA CTG.CTG Gin Leu Leu Giu Ala Leu
GAT
Asp 250 TTT TTG AAA AAC Phe Leu Lys Asn CTA CCA GTA Leu Pro Val 255 TTA AAT ACC AAA Leu Asn Thr Lys CCC CAA GAA ATT Pro Gln Glu Ile GAT GGT Asp Gly 270
GAT
Asp 275 GAC TTC GTG TTT Asp Phe Val Ph e
GTA
Val 280 CCT TCG ATT GAA AAA Pro Ser le. Giu Lys 285 1001 1043 10s5 1127 1169 1211 ACA TTT CCA GAA Thr Phe Pro Giu
CAA
Gin 290 GAT TCG TAC TTA Asp Ser Tyr Leu
ACT
Thr 295 GAO TTG CCA ATA Asp Leu Pro* Ile
CCA
Pro 30 300 ATA ATA AAT TCA Ile Ile Asn Ser
GGA
Gly 305 AAA TTC CAC AAA Lys Phe His Lys
GTT
Val 310 CCA TTG, TTG Pro Leu Leu TTC ATG TAC Phe Met Tyr 325 ACA GOT Thr Gly 315 TAC AAC ACT CC Tyr Asn Ser Ala CCC AAT CTA TTT Cly Asn Leu Phe TTA AAA ACA GAT CCA GAT TTA Leu Lys Thr Asp Pro Asp Ld'.
330 AAT AAA TTT GAA Asn Lys Phe Glu GCT GAT Ala Asp 1253 TTT GAA AGA Phe Ciu Arg
TTT
Phe 345 ATA CCA ACT GAC Ile Pro Thr Asp GAA T TA CCT TTG Giu Leu Pro Leu
CGA
Arg 355 1295 -196- TCA CAA AAA TCT Ser Gin Lys Ser ATT GCA CTG GGT GAA GCA ATC AGG GAA TTT 1337 Ile 360 Ala Leu Gly Glu Ala Ile 365 Arg Glu Phe CAG AAT TTT Gin Asn Phe
TAT
Tyr 370 TTC CAA AAC AAA Phe Gin Asn Lys
ACC
Thr 375 ATA TCA GAA AAT Ile Ser Giu Asn 1379 1421 GTA GAT Val Asp 385 GTT TTA AGT GAT Val Leu Ser Asp
A.AT
Asn 390 TGG TTT ACA Trp Phe Thr CGT GOA ATT GAT Arg Giy Ile Asp 395 GAG CAA GTA Glu Gin Val 400 AAG TTA ACT GTT Lys Leu Thr Vai
AAA
Lys 40S MAT CAG GMA GMA Asn Gin Giu Giu CCA GTT Pro Val 410 1463 1505 TTT TAT TAT Phe Tyr Tyr
GTT
Val 415 TAT MAT TTT GAT Tyr Asn Phe Asp MAT TCT CCA AGT Asn Ser Pro Ser MAA GTT TTT GGT GAT TTT GGA ATA MAA GGC GGT GGT CAT GCT Lys Val Phe Gly Asp Phe Giy Ilie Lys Gly Gly Gly His Ala 430 435 1547 1589
GAT
20 Asp 440 GMA TTG GGT MAT ATA TTT MAA GCC AMA Glu Leu Gly Asn Ile Phe Lys Ala Lys 445
AGT
Ser 450 GCA MAT TTT Ala Asn Phe AGA AGO ATG Arg Arg Met 465 GGG MAG Gly Lys 455 GMA ACA CCA MAT Glu Thr Pro Asn
OCT
Al a 460 GTG TTG GTT CAG Val Leu Vai Gin CTG GAG ATG Leu Glu Met 470 CCA GCT ATT Pro Ala Ile TOO ACT MAT TTT GCT MAA TTT GGA MAT Trp Thr Asn Phe Ala Lys Phe Gly Asn CCT ACT Pro Thr 480
ACG
Thr 485 GAT ACA CTT CCA Asp Thr Leu Pro AAA TGG GMA CCT Lys Trp Glu Pro
OCT
Al a 495 1631 1673 1715 1757 1799 TTT AMA GMA MT Phe Lys Glu Asn ACT TTT GTT CMA Thr Phe Val Gin
ATT
Ile 505 GAC ATT CAT TTA Asp Ile Asp Leu
AAT
An 510 TTC ACT ACT GAT Leu Ser Thr Asp CTA MAA ACT CGT Leu Lys Ser Arg GMA TTT GGG Giu Phe Cly MAT AMA Asn Lys 525 ATA AMA TTA TTA AAA Ile Lys Leu Leu Lys 530 TAAGTAACTA TACTTAGCTA 1840 MACCATMATA TACCAMATM TAGTATAGGA ATACTTCACA
ATTTTTTGTT
ACTTCCTTMA GTAMATTTMA TTTTTTATMA AACCMACTTT TACCMATAMA 1890 1940 -197- AAATGTAATT ATTTTGGAA AAAAAAAGAA AAAAAAAAA AAAAAAC 18 INFORMATION FOR SEQ ID NO: 68: SEQUENCE CHARACTERISTICS: LENGTH: 530 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: Met Cys Asp Pro Leu Leu Lys Thr Thr Thr Tyr Gly Ile Leu 1 5 Lys Gly Lys Lys Val Val Asn Giu Asn Gly Lys Ile Tyr Tyr 20 Ser Tyr. Thr Gly Ile Pro Tyr Ala Lys Ser Pro Val Asn Asp 35 Leu Arg Phe Lys Pro Pro Gin Lys Leu Asp Pro Trp Asn Gly s0 .Val Phe Asp Ala Thr Gin Tyr Gly Asn Asn Cys Ala Ala Gly 65 Lys Trp Phe Leu Lys Ser Ala Gly Gly Cys Glu Asp Cys Leu 75 Tyr Leu Asn Ile Tyr Val Pro Gin Asn Thr Ser Glu Asn Pro .5..85 90 Leu Pro Val Met Phe Trp Ile His Gly Giy Ala Phe Val Val 100 105 110 Gly Ser Gly Asn Ser Asp Ile His Giy Pro Asp Tyr Leu Ile 115 120 125 Giu Tyr Asp Ile Ile Leu Vai Thr Ile Asn Tyr Arg Leu Gly 130 135 140 -Pro Leu Gly Phe Leu Asn Leu Glu Ile Giu Asp Ala Pro Gly 145 150 Asn Val Gly Leu Met Asp Gin Val Ala Ala Leu Lys Trp Val 155 160 165 Asn Giu Asn Ile Ala Thr Phe Ser Gly Asp Pro Lys Asn Ile 170 175 180 Thr Ile Cys Gly Ala Thr Ala Gly Ala Ala Ser Val His Tyr 185 190 -195 -1i98- Gly Leu His Ile Leu Ser Gln Leu Thr Lys 200 Phe His Lys Ala 205 210 Ile Ala Gln Ser Gly Ser Ala Phe Asn Pro Trp Ala Phe Gl 215 220 Lys Asn Pro Val Lys
I
r o ur r r c -e ur r a 225 Gly Lys Gln Pro 15 Thr 295 Lys Leu Lys Glu Ala 365 Asn Thr Gln Asn Gly 435 Leu 240 Asn Glu Ser Asp Val 310 Phe Phe Leu Ile Met 380 Arg Glu Ser Gly Thr Leu 255 Ile Ile Leu Pro Phe 325 Glu Pro Arg Gln Gly 395 Glu Pro Gly Thr Pro Asp 270 Glu Pro Leu Met Ala 340 Leu Glu Asn Ile Pro 410 Ser His Asn Val Gly Lys 285 Ile Leu Tyr Asp Arg 355 Phe Phe Asp Val Arg 425 Ala Asn 230 Asn Glu Gln Thr Pro 300 Thr Leu Phe Ser Tyr 370 Val Glu Phe Lys Asp 440 Ala Leu 245 Thr Leu Phe Ile Gly 315 Lys.
Glu Gln Phe Asp 385 Gln Tyr Val Clu Leu Gln Leu 260 Leu Pro Ile Tyr Thr 330 Arg Lys Gln Val Val 400 Tyr Phe Leu Arg Glu Leu Asp 275 Glu Asn Asn Asp Phe 345 Ser Asn Leu Lys Val 415 Gly Gly Leu Ala Asn Asp Gln 290 Ser Ser Pro Ile Ile 360 Lys Ser Leu Tyr Asp 430 Asn SCys 235 Leu Thr Phe Asp Gly 305 Ala Asp Pro Ala Thr 375 Asp Thr A-n Phe Ile 445 Lys Asp 250 Lys Val Ser Lys Glu 320 Leu Thr Leu Ile ksn 390 Jal Phe Gly Phe Thr Phe Leu 265 Phe Tyr Phe Gly Leu 335 Asp Gly Ser Trp Lys 405 Asp Ile Lys Leu Leu Pro Val 280 Leu His Asn Asn Leu 350 Glu Glu Phe Asn Glu 420 Lys Ala 199- Lys Ser Ala Asn Phe Gly Lys Giu Thr Pro ASn Ala Vai Leu Val Gin Arg 465 Phe Gly Asn Lys Trp Giu Arg Met Leu Glu Trp Thr Asn Phe Ala Lys 475 Pro 480 Thr Pro Ala Ile Asp Thr Leu Pro Ile 490 Met Thr Phe Val Gln 500 Pro Ala Phe Lys Giu Asn 495 Ile 505 Asp Ile Asp Leu Asn 510 Leu Ser Thr Asp Leu Lys Ser Arg Met Giu Phe Gly Asn Lys Ile 520 525 Lys Leu Leu INFORMATION FOR SEQ ID NO:69: i) SEQUENCE CHARACTERISTICS: LENGTH: 1987 nucleotides TYPE: nucleic acid STRANDEDNESS; single TOPOLOGY: linear MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: 0 0
*GTTTTTTTTT
ATTCGTAAAA
AAAAAATTGT
CTAAGTATAG
25 ACGACTTTTT
CAAAAGTCAT
TCCGTAATAG
CTCCAGCATC
AATTTGCACT
CCGCCTTTTA
ATCAAAATTA
TTAACTTTAC
ACATCTACAA
AAATTCCCTG
GTAATTCTAA
TTTAATAAAT
GGCACTGTTG
TTATTATTGG
GTTTTTTCAA
AATTTCTTGG
TCAAAAAATC
TTGCATAGTC
A'FrAAAAGCA
TAAGTTGTGA
TTTTTTTTTC
GTTGGTTTTA
GAAGTATTCC
TTACTTATTT
AGTGGATCAG
ATTTTCTTTA
CTGGAGTAGG
CTTCTCTGAA
TTTGGCTTTA
TTCCAAAATC
TAAACATAAT
TTGCTCATCA
AATTCTGCAT
ATTGCTTCAC
GTCAGTTGr;T CTGGA!j..f 3T
TAACCTGTCA
TATTGGCAAG
TCGAAGGTAC
GGTAATTTGG
CAAGGCTTCjT
GAAGTGCATT
CTTCCACTTT
CAAAATGTGA
TTTTTTTTTT
TAAAAAATTA
TATACTATTA
TAATAATTT
TACTCAAATT
AAAGCAGGTT
ATTTCCAAAT
CCAACACAGC
AATATATTAC
ACCAAAAACT
AAAAAACTGG
ATTCCACGTG
ATTTTCTGAT
CCAGTGCAAT
ATAAATCTTT
TTTTAAGTAC
ACAATGGAAC
TCAGTTAAGT
AAACACGAAG
TATTTAACAA
TGAAGGTTGT
CTTAACAGGA
GTGCTATAGC
TAATGTACAC
C.CAAkAATAAT
AATTTACTTA
TTTGGTATAT
ATTTTATTCC
TAAATCAATG
CCCATTrTAT
TTAGCAAAAT
ATTTGGTGTT
CCAATTCATC
TTCCGACTTG
TTCTTCCTGA
TAAACCAATT
ATGGTTTTGT
AGATTTTGT
CAAAATCAGC
ATGAAAAATA
TTTGTGGAAT
ACGAATCTTG
TCATCCAGCA
TGTTTCTACT
TTGTGGTAJ\G
TTTTTTTGGA
CTTGTGGAAT
TTGCAGCTCC
TACATTTTTT
ACGAAGTAAC
TATGGTTTAG
CAAATTCCAT
TCAATTTGAA
TGGAAGTGTA
TAGTCCACAT
TCCTTCCCAA
AGCATGACCA
GAGAATTTTC
TTTTTAACAG
ATCACTTAAA
TTTGGAAATA
GATCGCAAAG
TTCAAATTTA
GATTGCCTTC
TTTCCTGAAT
TTCTGGAAAT
GTTGACCATC
GGTAGGTTTT
GCCTAAGGTT
AAGCCCAGGG
AAACCTTTGG
AGCAGTTGCT
100 150 200 250 300 350 400 450 S00 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 -200-
CCACAAATTG
ATTTACCCAT
GCGCATCTTC
TTAATAGTTA
TATATCAGAA
ACATTACTOG
AAGTAAAGGC
AGCAGCACAA
GATCAAGTTT
GCATAGGGTA
AACTTTCTTG
CACACATTAT
TTATACTAGC
AACATAAATA
ATAAAACTAT
ACATTTATAT
TAATATTTTT
TTTAGGGCTG
GATTTCCAAA
CTAAGATAAT
TTTCCTGATC
CAAAGGATTT
AATCTTCGCA
TTATTTCCAT
TTGTGGTGGC
TACCTGTGTA
CCTTTCAGAA
AAAATGTTAT
TCAAAAATAA
AAAATTTCTT
ATATATTTAA
CAAATAAAAT
TGGGTCTCCA
CAACTTGATC
TTAAGAAAAC
ATCATATTCT
CGACCACAAA
TCTGAAGTGT
ACCCCCAGCT
ACTGAGTGGC
TTGAATCTGA
ACTATAGTAA
TTCCATATGT'
TATATATCCA
TGTAAATCAT
TTCGATCATA
ATTAAAATTA
TATTTACACT
CTAAAGGTTG
CATCAATCCA
CAAGTGGTCC
ATTAA-ATAAT
TGCTCCTCCA
TTTGTGGGAC
GATL-rCAAAA
GTCAAAAACA
GATCATTTAc
ATTITACCAT
TGTTGTrTTTT
AAATATAATA
AGCCAAAACA
A-AAAAAATAT
GATTAAAATA
GTGAATT
CAATATTTTC
ACATTCCCAG
TAGACGATAA~
CAGGACCATG
TGAATCCA
ATAGATATTT
ACCATTTCCC
CCATTCCAAG
TTTCGTTTAC
AGTAATGGAT
TGGTTTAATT
CACACTCATA
TTTTTTATA-A
AAAATTAAAT
1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 1987 9, INFORMATION FOR SEQ ID i) SEQUENCE CHARACTERISTICS: LENGTH: 1590 nucleotides 20 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: cDNA (ix) FEATURE: 25 NAME/KEY:
CDS
LOCATION: 1.-1590 (xi) SEQUENCE DESCRIPTION: SEQ ID ATG TOT GAT CCA TTA CTA AAA ACA ACA ACA TAT GGA ATT CTG Met Cys Asp Pro Leu Leu Lys Thr TI-r Thr Tyr Gly Ile Leu 1 rI r
S
AAA GOC AAG AAA GTT Lys Oly Lys Lys Val AAC GAA PAT GGT Asn Olu Asn Gly AAA ATT TAC TAT Lys Ile Tyr Tyr AGT TAC Ser Tyr 30 ACA GGT ATA CCC Thr Gly Ile Pro GCA AAA TCT CCT GTA AAT OAT Ala Lys Ser Pro Val Asn Asp 126 CTC AGA TTC AAG CCA CCA CAA Leu Arg Phe Lys Pro Pro Gin
A-AA
Lys CTT GAT CCT TG Leu Asp Pro Trp PAT GOT Asn Gly OTT TTT GAC 0CC ACT CAG TAT OGA Val Phe Asp Ala Thr Gin Tyr Gly AAT TGT OCT OCT Asn Cys Ala Ala -201 AAA TGG TTT TTG Lys TrP Phe Leu AAA TCA GCT Lys Ser Ala GGG GGT TGC GAA GAT TGC CTT Gly Gly Cys Glu Asp Cys Leu s0 TAC TTA AAT ATC TAT GTC Tyr Leu Asn Ile Tyr CCA CAA AAC ACT TCA GAA AAT CCT Pro Gin Asz Thr Ser Giu Asn Pro TITG CCA Leu Pro 100 GTA ATG TTT TGG Val Met Phe Trp
ATT
Ile 105 CAT GGA GGA GCA His Gly Gly Ala TTT GTG GTC Phe Vai Val 110 GGA TCA GGA Gly Ser Gly 115 GAA TAT GAT Glu Tyr Asp AAT TCT GAT ATA Asn Ser Asp Ile GGT CCT GAT TAT Gly Pro Asp Tyr TTA ATA Leu Ile 125
ATT
Ile 130 ATC TTA GTA ACT ATT AAT TAT CGT CTA Ile Leu Val Thr Ile Asn Tyr Arg Leu 135 CCA CTT GGT TTT CTT AAT TTG GAA Pro Leu Gly Phe Leu Asn Leu Glu ATC GAA GAT Ile Giu Asp 150
AAT
Asn 155 GTT GGA TTG ATG Vai Gly Leu Met CAA GTT GCA GCC Gin Val Ala Ala GCG CCT GGG Ala Pro Gly AAA~ TGG GTA Lys Trp Vai AAA AAT ATT Lys Asn Ile 180 420 462 504 546 AAt GAA Asn Giu 170 AAT ATT GCA ACC Asn Ile Ala Thr AGT GGA GAC CCA Ser Gly Asp Pro 25 ACA ATT TGT Thr Ile Cys 185 GGA GCA ACT GCT Gly Ala Thr Ala
GGA
Gly 190 GCT GCA AGT GTA Ala Ala Ser Val CAT TAT His Tyr 195 CAC ATT TTG His Ile Leu
TCA
Ser 200 CAA CTT ACC AAA Gin Leu Thr Lys
GGT
Gly 205 TTA TTC CAC AAG Leu Phe His Lys
GCT
Ala -2 630 672 ATA GCA CAA AGT GGA AGT GCT TTT A-AT Ile Ala Gin Ser Gly Ser Ala Phe Asn 215
CCC
Pro 220 TGG GCT TTC CAA Trp Ala Phe Gin
AAA
Lys 225 AAT CCT GTT AAG Asn Pro Vai Lys GCA CTT CGA CTA Ala Leu Arg Leu AAA ACC TTA Lys Thr Leu GGC CTT Gly Leu 240 ACC ACA AAC AAC Thr Thr Asn Asn CAA GAA GCC TTG GAT TTT TTG Gin Giu Ala Leu Asp Phe Leu -202- AAA AAC CTA CCA GTA GAA Lys Asn Leu 255- Pro Val Giu ACA TTG TTA AAT ACC AAA TTA CCC Thr Leu Leu Asn Thr Lys Leu Pro 260 265 CAA GAA ATT Gin Giu Ile
GAT
Asp 270 GGT CAA CTG CTG GAT GAC TTC GTG TTT Gly Gin Leu Leu Asp Asp Phe Val Phe 275
GTA
Vai 280 CCT TCG ATT GAA Pro Ser Ile Giu
AAA
Lys 285 ACA TTT CCA GAA Thr Phe Pro Giu
CA
Gin 290 GAT TCG TAC TTA Asp Ser Tyr Leu 882 924
ACT
Thr 295 GAC TTG CCA ATA Asp Leu Pro Ile ATA ATA AAT Ile Ile Asn TCA GGA AAA TTC CAC Ser Gly 305 Lys Phe His AAA GTT CCA Lys Vai Pro 15 310 CTA TTT TTC Leu Phe Phe 325 AAA TTT GAA Lys Phe Giu TTG TTG ACA Leu Leu Thr
GGT
Giy 315 TAC AAC AGT GCC GAA GGC AAT Tyr Asn Ser Ala Giu Gly Asn ATG TAC TTA AAA Met Tyr Leu Lys
ACA
Thr 330 GAT CCA GAT TTA Asp Pro Asp Leu TTA AAT Leu Asn 335 966 1008 1050 1092 GAT TTT GAA AGA Asp Phe Glu Arg ATA CCA ACT GAC Ile Pro Thr Asp Leu 350 GAA TTA CCT TTG Giu Leu Pro Leu
CGA
Arg 355 TCA CAA AA.A TCT Ser Gin Lys Ser
ATT
Ile 360 GCA. CTG GGT GAA Aia Leu Gly Giu 25 GCA Al a 365 ATC AGG GAA TTT TAT TTC CAA PAC AAA Ile Arg Giu Phe Tyr Phe Gin Asn Lys 370 ATA TCA GPA Ile Ser Glu PAT TGG TTT Asn Trp Phe 390 1134 PAT ATG Asn Met 30 .380 CAG PAT TTT GTA Gin Asn Phe Val
GAT
Asp 385 GTT TTA AGT GAT Vai Leu Ser Asp 1176 ACA CGT GGA Thr Arg Giy 395 CAG GPA GAA -Gin Glu Giu ATT GAT GAG Ile Asp Giu CAA GTA Gin Val 400 PAG TTA ACT GTT Lys Leu Thr Val AAA PAT Lys Asn 405
CCA
Pro 410 GTT TTT TAT TAT Val Phe Tyr Tyr T'.T PAT TTT GAT yr Asa Phe Asp 1218 1260 1302 PAT TCT CCA AGT Asn Ser Pro Ser
CCC
Arg 425 AAA GTT TTT GGT Lys Val Phe Gly
GAT
Asp 430 TTT GGA ATA AAA Phe Gly Ile Lys -203-
GC
Gly 435 GGT GGT CAT GCT GAT GAA TTG GGT AAT Gly Gly His Ala Asp Giu Leu Gly Asn 440 ATA TTT AAA GCC Ile Phe Lys Ala 445 AAT GCT GTG TTG Asn Ala Val Leu 460 1344 AAA AGT Lys Ser 450 GCA AAT TTT GGG AAG GAA ACA CCA Ala Asn Phe Gly Lys Glu Thr Pro 455 1386 GTT CAG AGA Val. Gin Arg 465 TTT GGA AAT Phe Gly Asn AGG ATG CTG GAG Arg Met Leu Glu
ATG
Met 470 TGG ACT AAT TTT Trp Thr Asn Phe GCT AAA Ala Lys 475 1428 1470
CCT
Pro 480 ACT CCA GCT ATT ACG Thr Pro Ala Ile Thr 485 GAT ACA CTT CCA ATA Asp Thr Leu Pro Ile 490 AAA TGG GAA CCT Lys Trp Giu Pro TTT AAA GAA AAT ATG ACT TTT OTT CAA Phe Lys Giu Asn Met Thr Phe Val Gin 500 GAC ATT OAT TTA AAT TTG AGT ACT GAT Asp Ile Asp Leu Asn Leu Ser Thr Asp 510 CTA AAA AGT Leu Lys Ser
AAA
Lys 530 1512 1554 1590 CGT ATG Arg Met 520 GAA TTT GGG AAT AAA ATA AAA TTA TTA Oiu Phe Oly Asn Lys Ile Lys Leu Leu 525 INFORMATION FOR SEQ ID NO:71: i) SEQUENCE CHARACTERISTICS: LENGTH: 1590 nucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:
TTTTAATAAT
CAGTACTCAA
TTAAAAGCAG
AGGATTTCCA
GAACCAACAC
TTAAATATAT
ATCACCAAAA
AATAAAAAAC
TCAATTCCAC
CATATTTTCT
CACCCAGTGC
OGTATAAATC
TGTTTTTAAO
TTTATTTTAT
ATTTAAATCA
GTI'CCCATTT
AP FrTAGCAA
A-;CATTTGGT
TACCCAATTC
ACTTTCCGAC
TGGTTCTTCC
GTGTAAACCA
GATATGGTTT
AATAGATTTT
TTTCAAAATC
TACATGAAAA
TCCCAAATrc
ATGTCAATTT
TATTGGAAGT
AATTAGTCCA
GTTTCCTTCC
.ATCAGCATGA
TTGGAGAATT
TGATTTTTAA
ATTATCACTT
TGTTTTGGAA
TGTGATCGCA
AGCTTCAAAT
ATAGATTGCC
CATACGACTT
GAACAAAAGT
GTATCCGTAA
CATCTCCAGC
CAAAArTTTGC
CCACCGCCTT
TTCATCAAAA
CAGTTAACTT
AAJAACATCTA
ATAAAATTCC
AAGGTAATTC
TTATTTAATA
TTCGGCACTG
TTTAGTGGAT
CATATTrI'CT
TAGCTOGAGT
ATCCTTCTCT
ACTTTTGGCT
TTATTCCAAA
TTATAAACAT
TACTTGCTCA
CAAAATTCTG
CTGATTGCTT
TAAGTCAGTT
AATCTGGATC
TTGTAACCTG
so 100.
150 200 250 300 350 400 450 500 550 600 650 -204- TCAACAATGG
AACTTTGTGG
AAGTCAGTTA
AGTACGAATC
TACAAACACG AAGTCATCCA TGGTATTTAA
CAATGTTTCT
TCTTGAAGGT
TGTTTGTGGT
ATTCTTAACA
GGATTTTTTT
TTTGTGCTAT
AGCCTTGTGG
TGATAATGTA
CACTTGCAGC
TTTTGGGTCT
CCACTAAAGG
CTGCAACTTG
ATCCATCAAT
AAATTAAGAA
AACCAAGTGG
AATATCATAT
TCTATTAAAT
ATCCGACCAC
AAATGCTCCT
TTTTCTGAAG
TGTTTTGTGG
GCAACCCCCA
GCTGATTTCA
CATACTGAGT GGCGTCAAAA GGCTTGAATC
TGAGATCATT
GTAACTATAG
TAAATTTTAC
GAATTCCATA
TGTTGTTGTT
AATTTTCCTG
TTGTTCTGGA
GCAGTTGACC
ACTGGTAGGT
AAGGCCTAAG
GGAAAGCCCA
AATAAACCTT
TCCAGCAGTT
TTGCAATATT
CCAACATTCC
TCCTAGACGA
AATCAGGACC
CCATGAATCC
GACATAGATA
AAAACCATTT
ACACCATTCC
TACAGGAGAT
CATTTTCG3TT TrTTAGTAATG
AATTTATTAT
AATGTTTTTT
ATCAATTTCT
TTTTCAAAAA
GTTTTGCATA
GGGATTAAAA
TGGTAAGTTG
GCTCCACAAA
TTCATTTACC
CAGGCGCATC
TAATTAATAG
ATGTATATCA
AAAACATTAC
TTTAAGTAAA
CCCAGCAGCA
AAGGATCAAG
TTTGCATAGG
TACAACTTTC
GATCACACAT
TGGTATTGGC
CAATCGAAGG
TGGGGTAATT
ATCCAAGGCT
GTCGAAGTGC
GCACTTCCAC
TGACAAAATG
TTGTAATATT
CATTTTAGGG
TTCGATTTCC
TTACTAAGAT
GAATTTCCTG
TGGCAAAGGA
GGCAATCTTC
CAATTATTTC
TTTTTGTGGT
GTATACCTGT
TTGCCTTTCA
700 750 S00 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1590 20 INFORMATION FOR SEQ ID NO:72: SEQUENCE CHARACTERISTICS: LENGTH: 650 nucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: cDN'A (ix) FEATURE: NAME/KEY: CDS LOCATION: 650 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: OG ATC CAT GGA GGC OCA TrrC AAC CAA GGA TCA GGA TCT TAT Ile His Gly Gly Ala Phe Asn Gln Gly Ser Gly Ser Tyr 1 5 AAT' TTT TTT GGA CCT GAT TAT TTG ATC AGG GAA GGA ATT ATT Asn Phe Phe Gly Pro Asp Tyr Leu Ile Arg Glu Gly Ile Ile is 20 TTG GTC ACT ATC AAC TAT AGA TTA GGA GTT TTC GOT TTT CTA Leu Val.Thr Ile Asn Tyr Arg Leu Gly Val Phe Gly Phe Leu 35 TCA GCG CCG GAA TOG GAT ATC CAT GGA AAT ATG GGT CTA AAA Ser Ala Pro Glu Trp Asp Ile His Gly Asn Met Gly Leu Lys 50 -205- GAC CAG AGA TTG GCA CTA AAA Asp Gin Arg Leu Ala Leu Lys TGG GTT TAC Trp Val Tyr AAA ATT ACA Lys Ile Thr GAC AAC ATC GAA Asp Asn Ile Giu ATT GCT GGA GAA Ile Ala Gly Giu 209 251
AAG
Lys TTT GGT GGA GAC Phe Gly Gly Asp AGA GAA Arg Giu 75 TCT GCT Ser Ala GGA GCA GCA AGT Giy Ala Ala Ser GTC CAT Val His 90 TTT CTG ATG ATG GAC AAC Phe Leu Met Met Asp Asn TCG ACT AGA AAA TAC TAC CAA AGG GCC ATT TTG CAG AGT GGG Ser Thr Arg Lys Tyr Tyr Gin Arg Ala Ile Leu Gin Ser Gly 100 105 110.
.01.
0. .0 0 0 ACA TTA CTA Thr Leu Leu AGA TTT GAA Arg Phe Giu
AAT
Asn 115 CCG ACT GCT AAT Pro Thr Ala Asn CAA ATT Gin Ile 120 CTA AAC Leu Asn 135 CAA CTT CTG Gin LeU Leu
CAT
His AAA CTC Lys Leu 130 AAA CAA GTG Lys Gin Val ATC ACG CAA AAA Ile Thr Gin Lys 377 419 461
CAA
Gin 140 GAA CTC CTA AAC Glu Leu Leu Asn
CTG
Leu 145 GAT AAA AAC CTA ATT TTA CGA GCA Asp Lys Asn Leu Ile Leu Arg Ala 150 GCC TTA Ala Leu 155 AAC AGA GTT CCT Asn Arg Vai Pro
GAT
Asp 160 AGC AAC GAC CAT Ser Asn Asp His GAC CGA GAC Asp Arg Asp 165 ACA GTA CCA Thr Val Pro 170 GTA TTT AAT CCA Val Phe Asn Pro
GTC
Val1 175 TTA GAA TCA CCA Leu Glu Ser Pro GAA TCT Giu Ser 180 CCA GAT CCA Pro Asp Pro ACA T?1' CCA .TCT Thr Phe Pro Ser GCC TTG Ala Leu 190 GAA AGA ATG Giu Arg met
AGA
Arg 195 AAT GGT GAA TTT Asn Gly Glu Phe GAT GTC GAT GTC Asp Vai Asp Val ATC ATT GGT TTC AAT Ile Ile Gly Phe Asn 205
AGT
Ser 210 GCT GAA GGT TTA Ala Giu Gly Leu AGA TCT Arg Ser 215 INFORMATION FOR SEQ ID NO:73: i) SEQUENCE
CHARACTERISTICS:
LENGTH: 216 flucleotides TYPE: amino acid -206-
C
C
Ile 1 Phe Val1 *Ala Gin Phe 15 Ala Thr Leu Phe Clu 25 Leu Val1 Asp Gly Ala (ii) (xi) His Glyi Phe Gly Thr Ile Pro Glu Arg Leu Gly Gly Gly Ala Arg Lys 100 Leu Asn 115 Glu Lys Leu Leu Asn Arg Pro Val 170 Pro Ile 185 Glu Phe Glu Gly Pro Asp Tyr Leu Ile Asn Trp Al a 60 Asp Ala Tyr Pro Leu 130 Asn Val1 Phe Thr Pro 200 Leu Tyr Asp Leu Arg 75 Ser Tyr Thr Lys Leu 145 Pro Asn Phe Asp Arg 215 Arg Ile Lys Glu Val 90 Gln Ala Gln Asp Asp 160 Pro Pro Val Ser Leu 35 His Trp Lys His Arg 105 Asn Val Lys Ser Val 175 Ser Asp Gly Gly s0 Val1 Ile Phe Ala Gln 120 Leu Asn Asn Leu A-a 190 Val Arg Val As n Tyr 65 Thr Leu Ile Ile Asnr 135 Leu Asp Glu Leu Ile 20s Giu Phe Met Asp Ilie Met Leu Gin Ile Ile 150 His Ser G1u Ile Gly Gly Gly Asn Ala Met Gin Leu Thr Leu Asp 165 Pro Arg Gly Ile Phe Leu Ile Gly Asp Ser 110 Leu Glm Arg Arg Giu 180 Miet Phe Ile Leu Lys Giu Giu Asn Gly His 125 Lys Ala Asp Ser Arg 195 Asn *Leu Ser Asp Lys Ser Ser Thr Arg Gin 140 Al a Thr Pro Asn Ser 210 TOPOLOGY: linear MOLECULE TYPE: protein SEQUENCE DESCRIPTION: SEQ ID NO:73: Gly Ala Phe Asn Gin.Gly Ser Gly Ser Tyr Asn -207-
S
S
S
S
*SSS
INFORMATION FOR SEQ ID NO:74: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Xxn Tyr or Gly LOCATION: 3 NAME/KEY: Xxn Lys or Tyr'or Gly LOCATION: NAME/KRY: Xxn Val or Gin or Asn LOCATION: 6 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74: 15 Asp Leu Xxn Val Xxn Xxn Leu Gln Gly Thr Leu Lys Gly Lys 1 5 Glu INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 21 bases TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear.
25 (ii) MOLECULE TYPE: primer (xi) SEQUENCE DESCRIPTION: SEQ ID CGCGGATCCG CTGATCTACA AGTGACTTTG
C
INFORMATION FOR SEQ ID NO:76: SEQUENCE CHARACTERISTICS: LENGTH: 1488 nucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 3..1487 -208- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76: CC CAG GGC GAA TTG GTT GGA AAA GCT TTG ACG AAC GAA AAT GGA Gin Giy Glu Leu Val Gly Lys Ala Leu Thr Asn Giu Asn Gly
AAA
Lys is GAG TAT TTT AGC TAC ACA GGT GTG Giu Tyr Phe Ser Tyr Thr Gly Val CCT TAT Pro Tyr GCT AAA CCT Ala Lys Pro CCA GTT GGA GAA CTT AGA TTT AAG Pro Val Gly Giu Leu Arg Phe Lys 35 CCT CCA CAG AAA GCT GAG Pro Pro Gin Lys Ala Glu CCA TGG AAT Pro Trp Asn GGT GTT TTC AAC Gly Val Phe Asn GCC ACA Ala Thr so TCA CAT GGA AAT GTG Ser His Gly Asn Val 170 TGC AAA GCT Cys Lys Ala
TTG
Leu A.AT TTC TTC TTG Asn Phe Phe Leu
AAA
Lys 65 AAA ATT GAA GGA Lys Ile Giu Gly
GAC
Asp 212 254 GAA GAC TGC TTG Glu Asp Cys Leu GTG AAT GTG TAC Val Asn Val Tyr
GCA
Al a CCA AAA ACA ACT Pro Lys Thr Thr GAC AAA AAA CTT Asp Lys Lys Leu
S.
S S GTA TTT TTC TGG Val Phe Phe Trp, CAT GGT GGC His Gly Gly GGT TTT GTG Gly Phe Val 100 GAT TAT TTA Asp Tyr Leu 115 ACT GGA TCC Thr Gly Ser
GGA
Gly 105 AAT TTA GAA TTT CAA AGC CCA Asn Leu Glu Phe Gin Ser Pro GTA AAT TAT GAT Val Asn Tyr Asp ATT TTT GTA ACT Ile Phe Val Thr TTC AAT Phe Asn 125 TAC CGA TTG Tyr Arg Leu
GGA
Gly 130 CCA CTC GGA TTT TTG AAT TTG GAG TTG Pro Leu Gly Phe Leu Asn Leu Giu Leu 135
GAA
Glu 140 422 GGT GCT CCT GGA Gly Ala Pro Gly GTA GGA TTA TTG Vai Gly Leu Leu
GAT
Asp 150 CAG GTA GCA GCT Gin Val Ala Ala
TTG
Leu 155 AAA TGG ACC AAA Lys Trp, Thr Lys
GAA
Giu 160 AAT ATT GAG AAA Asn Ile Glu Lys GGT GGA GAT Gly Gly Asp GGT GGA GCA Gly Gly Ala 180 CCA GAA Pro Glu 170 AAT ATT ACA ATT GGT GGT GTT TCT GCT Asn Ile Thr Ile Gly Gly Val Ser Ala -209- AGT GTT CAT TAT CTT TTA TTG TCA Ser Val His Tyr Leu Leu Leu Ser 185 190 CAT ACA ACC ACT GGA CTT His Thr Thr Thr Gly Leu 195 TAC AAA AGG Tyr Lys Arg
GCA
Ala 200 ATT GCT CAA AGT Ile Ala Gin Ser
GGA
Gly 205 AGT GCT TTA AAT Ser Ala Leu Asn
CCA
Pro 210 TGG GCC TTC CAA Trp Ala Phe Gin
AGA
Arg 215 CAT CCA GTA AAG His Pro Val Lys
CGT
Arg 220 AGT CTT CPA CTT Ser Leu Gin Leu
GCT
Ala 225 GAG ATA TTA GGT Glu Ile Leu Gly
CAT
His 230 CCC ACA AAC AAC Pro Thr Asn Asn
ACT
Thr 235 CAA GAT GCT Gin Asp Ala CTC CTG AAA Leu Leu Lys 250 TTA GAA Leu Giu 240 TTC TTA CAA Phe Leu Gin AAA GCC Lys Ala 245 CCA GTA GAC AGT Pro Val Asp Ser 20 AAA ATG CCA Lys Met Pro 255 GTC TTC GTA Val Phe Val GCT GPA ACA GAA Ala Giu Thr Glu GAA ATA ATA GAA Glu Ile Ile Glu GAG TTC Glu Phe 265
CCA
Pro 270 TCA ATT GPA AAA Ser Ile Giu Lys
GTT
Val 275 TTC CCA TCC CAC Phe Pro Ser His
CAA
Gin 280 632 674 716 758 800 842 884 926 968 1010 1052 1094 CCT TTC TTG GAA Pro Phe Leu Glu
GAA
Glu 285 TCA CCA TTG GCC Ser Pro Leu Ala
AGA
Arg 290 ATG AAA TCT GGA Met Lys Ser Gly
TCC
Ser 295 TTT PAC AAA GTA Phe Asn Lys Val TTA TTA GTT GGA Leu Leu Val Gly
TTC
Phe 305 AAC AGC GCA Asn Ser Ala AAA CCA GAG Lys Pro Glu 320 GAA GGA Glu Gly 310 CTT TTG TAC AAA Leu Leu Tyr Lys TTT ATG AAA GAA Phe Met Lys Glu ATG CTG PAC Met Leu Asn 325 CAA GCT GPA GCA Gin Ala Giu Ala TTC GAA AGA CTC Phe Giu Arg Leu GTA CCA Val Pro 335 GCC GAA TTT Ala Giu Phe
GAA
Glu 340 TTA GCC CAT GGA Leu Ala His Gly
TCA
Ser 345 GAA GAA TCG AAA Glu Glu Scr Lys.
AAA
Lys 350 CTT GCA GAA AAA Leu Ala Glu Lys ATC AGG Ile Arg 355 AAG TTT TAC Lys Phe Tyr
TTT
Phe 360 GAC GAT AAA CCC Asp Asp Lys Pro -2 GTT CCT GAA AAT GAG CAG AAA. TTT ATT GAC TTG ATA GGA GAT 1136 Pro Giu Asn Glu Gin Lys .370 Phe Ile Asp ATT TGG Ile Trp 380 TTT ACT AGA GGC Phe Thr Arg Gly Ile Gly Asp AAG TTG TCT LYS Leu Ser 390
ATT
Ile 385 GAC AAG CAT GTC Asp Lys His Val GTA GAA AAA Val Giu Lys 395 CAA GAC GAG CCA Gin Asp Glu Pro
GTA
Val.
400 TAT TAT TAT GAA Tyr Tyr Tyr Glu TAT TCT Tyr Ser 405 1178 1220 1262 TTC TCT GAA Phe Ser Giu
AGT
Ser 410 CAT CCT GCA AAA His Pro Ala Lys
GGA
Gly 415 ACA 'ITT GGT GAC Thr Phe Gly Asp
CAT
His 420 AAC TTG ACT GGA Asn Leu Thr Gly
GCA
Aila 425 TGT CAT GGT GAA Cys His Gly Giu
GAA
Giu 430 CTT GTG AAT TTA Leu Vai Asn Leu
S
S
TTC
Phe 435 AAA GTC GAG ATG Lys Val Giu Met
ATG
Met 440 AAG CTG GAA AAA Lys Leu Giu Lys AAA CCG AAT Lys Pro Asn TGG ACG AAC Trp, Thr Asn 460 1304 1346 1388 GTT TTA Val Leu 450 TTA ACA AAA GAT Leu Thr Lys Asp
AGG
Arg 455 GTA CTT GCT ATG Val Leu Ala Met TTC ATC AAA 30 Phe Ile Lys 465 AAT GGA AAT CCT Asn Gly Asn Pro
ACT
Thr 470 CCT GAA GTA ACT Pro Giu Val Thr GAA TTA Glu Leu 475 TTG AAT Leu Asn 490 1430 TTG CCA GTT Leu-Pro Val
AAA
Lys 480 TGG GAA CCT GCC ACA AAA GAC AAG Trp Giu Pro Ala Thr Lys Asp Lys 485 1472 TAT TTG AAC ATT Tyr Leu Asn Ile GAT G Asp 495 1488 -211- While various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. Itis to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention, as set forth in the following claims.
oo* 0 0* 000 0 **0
S*

Claims (50)

1. An isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID 10 NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76 and a nucleic acid molecule encoding a protein comprising amino acid sequence SEQ ID NO:74.
2. An isolated nucleic acid molecule that hybridizes under stringent 15 hybridization conditions with a nucleic acid molecule having a nucleic acid sequence encoding a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 1, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:68, SEQ ID NO:73 and SEQ ID NO:74.
3. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEy ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID -213- NO:71, SEQ ID NO:72, SEQ ID NO:76 a nucleic acid molecule encoding a protein comprising amino acid sequence SEQ ID NO:74; and a nucleic acid molecule comprising an allelic variant of a nucleic acid molecule comprising any of said nucleic acid sequences.
4. An isolated protein encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions to a nucleic acid molecule selected from the group consisting of: a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:22, SEQ ID 10 NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:52, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:69, SEQ ID NO:71; and a nucleic acid molecule encoding a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 and SEQ ID NO:74.
5. An isolated flea protein selected from the group consisting of: a protein comprising an amino acid sequence selected from the group consisting of SEQ-ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:68, SEQ ID NO:73 and SEQ ID NO:74; and a protein encoded by an allelic variant of a nucleic acid molecule encoding a protein comprising any of said amino acid sequences.
6. A formulation of flea carboxylesterase proteins, wherein said proteins, when submitted to 14% Tris-glycine SDS-PAGE, comprise a fractionation profile as depicted in Fig. 3, wherein said proteins have caroxylesterase activity.
7. A formulation of flea carboxylesterase proteins, wherein said proteins, when submitted to IEF-PAGE, comprise a fractionation profile as depicted in Fig. 4 in a lane selected from the group consisting of lane 3, lane 4, lane 5, lane 6 and lane 7, wherein said proteins have carboxylesterase activity. -214-
8. An isolated flea protein that hydrolyzes a-napthyl acetate to produce a- napthol, when said protein is incubated in the presence of a-napthyl acetate contained in about 20 mM Tris at about pH 8.0 for about 15 minutes at about 37 0 C.
9. A formulation comprising flea proteins that hydrolyze a-napthyl acetate to produce a-napthol, when said proteins are incubated in the presence of a-napthyl acetate contained in about 20 mM Tris at about pH 8.0 for about 15 minutes at about 370C. An isolated flea protein that hydrolyzes the methyl ester group of juvenile hormone to produce a juvenile hormone acid.
11. A formulation comprising flea proteins that hydrgoyze the methyl ester group of juvenile hormone to produce a juvenile hormone acid.
12. A therapeutic composition that, when administered to an animal, reduces hematophagous ectoparasite infestation, said therapeutic composition comprising an excipient and a protective compound selected from the group consisting of: an isolated hematophagous ectoparasite carboxylesterase protein; a mimetope of an isolated •hematophagous ectoparasite carboxylesterase protein; an isolated hematophagous ectoparasite carboxylesterase nucleic acid molecule that hybridizes under stringent hybridization conditions with a Ctenocephalidesfelis carboxylesterase gene; an isolated antibody that selectively binds to a hematophagous ectoparasite carboxylesterase protein; and an inhibitor ofcarboxylesterase activity identified by its ability to inhibit the activity of a flea carboxylesterase.
13. A method to reduce hematophagous ectoparasite infestation comprising treating an animal with a therapeutic composition comprising a protective compound selected from the group consisting of: an isolated hematophagous ectoparasite carboxylesterase protein; a mimetope of a hematophagous ectoparasite carboxylesterase protein; an isolated hematophagous ectoparasite carboxylesterase nucleic acid molecule that hybridizes under stringent hybridization conditions with a Ctenocephalidesfelis carboxylesterase gene; an isolated antibody that selectively binds to a hematophagous ectoparasite carboxylesterase protein; and an inhibitor of carboxylesterase activity identified by its ability to inhibit the activity of a flea carboxylesterase. -215-
14. A method to produce a carboxylesterase protein, said method comprising culturing a cell capable of expressing said protein, said protein being encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a Ctenocephalidesfelis carboxylesterase gene.
15. A method to identify a compound capable of inhibiting flea carboxylesterase activity, said method comprising: contacting an isolated flea carboxylesterase with a putative inhibitory compound under conditions in which, in the absence of said compound, said protein has carboxylesterase activity; and determining if said putative inhibitory compound inhibits said Sactivity. .16. A test kit to identify a compound capable of inhibiting flea carboxylesterase activity, said test kit comprising an isolated flea carboxylesterase protein having esterase activity and a means for determining the extent of inhibition of said activity in the presence of a putative inhibitory compound.
17. The nucleic acid molecule of Claim 1, wherein said nucleic acid molecule is a flea nucleic acid molecule.
18. The nucleic acid molecule of Claim 1, wherein said nucleic acid molecule is selected from the group consisting of Ctenocephalides, Ceratophyllus, Diamanus, Echidnophaga, Nosopsyllus, Pulex, Tunga, Oropsylla, Orchopeus and Xenopsylla nucleic acid molecules.
19. The nucleic acid molecule of Claims 1 or 2, wherein said nucleic acid molecule is selected from the group consisting of Ctenocephalidesfelis, Ctenocephalides canis, Ceratophyllus pulicidae, Pulex irritans, Oropsylla (Thrassis) bacchi, Oropsylla (Diamanus) montana, Orchopeus howardi, Xenopsylla cheopis and Pulex simulans nucleic acid molecules. The nucleic acid molecule of Claims 1 or 2, wherein said nucleic acid molecule comprises a Ctenocephalidesfelis nucleic acid molecule.
21. The nucleic acid molecule of Claim 1, wherein said nucleic acid molecule hybridizes under stringent hybridization conditions with a nucleic acid molecule selected -216- from the group consisting of nifE 1 40 1 nfE2 3 64, nfE3 42 .nfE-4 524 nfE51 982 nf]E5 5 1 5 2 4 ,7 nfE5 1 650 nfE6 1488 nfE6 1792 nfE6 6 so, nfE7 2836 nfE71 788 nfE7 Z710' nfE7 650 nfEi8 2801 nfE8 1785 nfE81 71 0 nfE9 2 00 7 nfE9 15 g4, nfTE9 Z540, nfEl0 1987 and nfEl01 9 0.
22. The nucleic acid molecule of Claim 1, wherein said nucleic acid molecule comprises a nucleic acid molecule selected ftrm the group consisting of nfE 4 01 nfE2 3 ,1 nfE3 42 nfE-4 524 nfE-5 1982 nfE-5 515 nfE5 21 44, nfE5 165 0 nfE6 1 488 nfE-6 1 792 nfE6 1650 nfE7 2836 nfE-7 1788 nfE7 1 71 0 nfE7 650 nfE8 2801 nfE-8, 785 nfB8 171 0 nfE9 2 007, nfB-9 584 nfE9 540 nfB 10 9 7 and nfE 10 1590
23. The nucleic acid molecule of Claims 1 or 2, wherein said nucleic acid molecule is selected from the group consisting of:- a nucleic acid molecule comprising a nucleic acid sequence that encodes a protein having an amino acid sequence selected j from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:l 11, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO:25, SEQ ID NO:3 1, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ DD NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:68, SEQ ID NO:73 and SEQ ID NO:74; and a nucleic acid molecule comprising an allelic variant of a nucleic acid molecule encoding a protein :having any of said amino acid sequences. *24. The nucleic acid molecule of Claims I or 2, wherein said nucleic acid molecule is selected from the group consisting of:- a. nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:'J5, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:5 1, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76, a nucleic acid molecule encoding a protein comprising amino acid sequence SEQ ID NO:74 SEQ ID NO:74; and a nucleic acid -217- molecule comprising an allelic variant of a nucleic acid molecule comprising any of said nucleic acid sequences. The nucleic acid molecule of Claim 1, wherein said nucleic acid molecule comprises an oligonucleotide.
26. A recombinant molecule comprising a nucleic acid molecule as set forth in Claims 1 or 2 operatively linked to a transcription control sequence.
27. A recombinant virus comprising a nucleic acid molecule as set forth in Claims I or 2.
28. A recombinant cell comprising a nucleic acid molecule as set forth in Claims 1 or 2.
29. The nucleic acid molecule of Claim 2, wherein said nucleic acid molecule comprises a nucleic acid sequence that encodes a carboxylesterase protein. i
30. The nucleic acid molecule of Claim 2, wherein said nucleic acid molecule hybridizes under stringent hybridization conditions with the complement of a nucleic acid sequence encoding said protein.
31. The protein of Claim 4, wherein said protein, when administered to an animal, elicits an immune response against a carboxylesterase protein.
32. The protein of Claim 4, wherein said protein is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with nucleic acid molecule nfE6 1792
33. The protein of Claim 4, wherein said protein is selected from the group consisting of: a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:5, jEQ ID NO:55, SEQ ID NO:58 SEQ ID NO:68, SEQ ID NO:73 and SEQ ID NO:74; and a protein encoded by an allelic variant of a nucleic acid molecule encoding a protein selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ ID -218- SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:68, SEQ ID NO:73 and SEQ ID NO:74.
34. An isolated antibody that selectively binds to a protein as set forth in Claim 4. The formulation of Claim 6, wherein said proteins are selected from the group consisting of proteins having molecular weights ranging from about 60 kD to about 75 kD as determined by 14% Tris-glycine SDS-PAGE.
36. The formulation of Claim 6, wherein said proteins are isolated by the method comprising: applying soluble proteins of a flea extract to a p-aminobenzamidine S agarose bead column; collecting unbound protein from said p-aminobenzamidine agarose bead column and applying said unbound protein to a gel filtration column; eluting proteins bound to said gel filtration column and applying said eluted proteins to a cation exchange chromatography column; eluting proteins bound to said cation exchange column and applying said eluted proteins to an anion exchange chromatography column; and eluting proteins bound to said anion exchange column with about 170 mM NaCI to obtain said flea carboxylesterase proteins.
37. The formulation of Claim 7, wherein said proteins are selected from the group consisting of proteins having pi values ranging from about pi 4.7 to about pi 5.2 as determined by IEF-PAGE.
38. The invention of Claims 8 or 9, wherein said incubation results in the production of from about 0.3 to at least about 2.5 absorbance units in the presence of Fast Blue when measured at 590 nm.
39. The protein of Claim 8, wherein said protein does not hydrolyze the methyl ester group of juvenile hormone. The invention of Claims 8 or 9 or 10 or 11, wherein the mature form of said protein has a molecular weight ranging from about 60 kD to about 75 kD. -219-
41. The invention of Claims 8 or 9, wherein said protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:19, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:53.
42. The invention of Claims 10 or 11, wherein hydrolyzation of said methyl ester group results in the release of at least about 120 counts per minute of H-juvenile hormone acid when determined by the method comprising: incubating said flea protein in the presence of H-juvenile hormone to create a reaction mixture; combining said reaction mixture with isooctane to produce an aqueous *phase and an organic phase; recovering said aqueous phase; and determining the amount of 3 H-juvenile hormone acid present in said aqueous phase.
43. The protein of Claim 10, wherein said protein does not hydrolyze ca- napthyl acetate.
44. The invention of Claims 12 or 13, wherein said hematophagous ectoparasite carboxylesterase comprises a flea carboxylesterase. The invention of Claims 12 or 13, wherein said hematophagous ectoparasite is a flea of a genus selected from the group consisting of Ctenocephalides, Ceratophyllus, Diamanus, Echidnophaga, Nosopsyllus, Pulex, Tunga, Oropsylla, Orchopeus and Xenopsylla.
46. The invention of Claims 12 or 13, wherein said hematophagous ectoparasite is a flea of a species selected from the group consisting of Ctenocephalides felis, Ctenocephalides canis, Ceratophyllus pulicidae, Pulex irritans, Oropsylla (Thrassis) bacchi, Oropsylla (Diamanus) montana, Orchopeus howardi, Xenopsylla cheopis and Pulex simulans.
47. The invention of Claims 12 or 13, wherein said hematophagous ectoparasite carboxylesterase comprises a juvenile hormone esterase. -220-
48. The invention of Claims 12 or 13, wherein said composition further comprises a component selected from the group consisting of an adjuvant and a carrier.
49. The invention of Claims 12 or 13, wherein said composition further comprises a compound that reduces hematophagous ectoparasite burden by a method other than by reducing hematophagous ectoparasite carboxylesterase activity. The invention of Claims 12 or 13, wherein said protective compound is selected from the group consisting of a naked nucleic acid vaccine, a recombinant virus vaccine and a recombinant cell vaccine.
51. The invention of Claims 12 or 13, wherein said inhibitor comprises a substrate analog of a hematophagous ectoparasite carboxylesterase.
52. The method of Claim 13, wherein said animal is selected from the group S. consisting of adult hematophagous ectoparasites, hematophagous ectoparasite larvae and animals susceptible to hematophagous ectoparasite infestation.
53. The method of Claim 13, wherein said animal is selected from the group consisting of adult fleas, flea larvae and animals susceptible to flea infestation.
54. The method of Claim 13, wherein hematophagous ectoparasite infestation is reduced by hematophagous ectoparasite larvae ingesting adult hematophagous ectoparasite feces comprising said therapeutic composition.
55. The method of Claim 13, wherein said adult hematophagous ectoparasite feces comprises anti-hematophagous ectoparasite carboxylesterase antibodies.
56. The method of Claim 13, wherein anti-hematophagous ectoparasite carboxylesterase antibodies are elicited in a host animal in response to administration of a reagent selected from the group consisting of one or more of said isolated hematophagous ectoparasite carboxylesterase proteins and one or more of said hematophagous ectoparasite carboxylesterase nucleic acid molecules, said adult hematophagous ectoparasite having fed from said host animal aLer said administration.
57. The method of Claim 13, wherein said animal is selected from the group consisting of mammals and birds.
58. The method of Claim 13, wherein said animal is selected from the group consisting of cats and dogs. -221-
59. The method of Claim 14, wherein said cell expresses a nucleic acid molecule selected from the group consisting of pCro-nfE6 1 4 8 8 pTrc-nfE7 650 pTrc- nfE7 1 71 0 pTrc-nfE 81 71 0o. pTrc-nfE51650' pTrc-nfE9] 540 pFB-nfE6 16 79 7 pVL-nfE7 1802 pVL- fE8 1792 and pVL-nfE9 1600 DATED this 3rd Day of may, 2001 HESKA CORPORATION Attorney: DAVID ADAMTHWAITE Fellow Institute of Patent and Trade Mark Attorneys of Australia of BALDWIN SHELSTON WATERS
AU42063/01A 1996-11-12 2001-05-03 Novel carboxylesterase nucleic acid molecules, proteins and uses thereof Abandoned AU4206301A (en)

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