Abstract:
A transgenic non-human mammal is provided where the genome of the mammal comprises a DNA construct comprising in operable association a rabbit WAP promoter and a DNA sequence encoding a heterologous protein. Methods of using the mammal in the production of recoverable amounts of a heterologous protein in the mammal&#39;s milk are discussed and described such that the mammal is a bio-reactor for a heterologous protein of interest. Furthermore, cells isolated from the mammal are provided for the production of a heterologous protein of interest in vitro, as well as, DNA constructs of the rabbit WAP promoter for the production of the transgenic non-human mammal.

Description:
BACKGROUND 
     The present invention relates to a process for the preparation of a protein of interest in the milk of a transgenic animal. It also relates to the constructs which make it possible to obtain these animals, the animals obtained as well as the cells containing the constructs which permit the expression of a heterologous protein. 
     Several routes have been pursued in order to obtain proteins of biological, therapeutic or industrial interest, and which are naturally produced in small quantities or in a form difficult to purify. 
     It has thus been possible to produce proteins using genetic recombination techniques, by microorganisms such as bacteria or yeasts. Nevertheless, most of the proteins require, after their synthesis, a maturation stage consisting in chemical modifications of certain reacting groups, glycosylation, and the like. Prokaryotic cells do not have the adequate enzymatic content for carrying out this maturation, hence the production of inactive proteins and/or proteins with high antigenicity. 
     It is therefore preferable to synthesize these proteins in eukaryotic cells, which will perform the appropriate enzymatic conversions. Nevertheless, the large-scale culture of tissue cells poses a number of technical and economical difficulties. 
     Another approach therefore consists in causing these proteins to be produced by cells in vivo, using transgenic animals. It is desirable that the system used permits the production of proteins in large quantities and which are easily recoverable. It is therefore advantageous that the recombinant protein is produced in the mammary gland of transgenic animals, and excreted in the milk. It is indeed a biological fluid which can be easily collected, having a relatively limited complexity and a low proteolytic activity; in addition, the processes of maturation of the recombinant proteins will be probably ensured (glycosylation, phosphorylation, cleavage and the like). 
     SUMMARY OF THE INVENTION 
     Mouse or ewe mammary gland has thus been successfully made to synthesize milk proteins of another species or proteins normally absent from milk (Ref. 1 to 15). 
     However, the level of proteins thus produced is extremely variable. It is different from one transgenic animal to another, since it is a function of the process of integration of the transgene which is itself variable from one animal to another. The nature of the gene constructs is also essential, the elements regulating the expression of the milk protein genes being possibly many and situated at various points of the promoter region and the transcribed portion of the gene. Thus, the promoters of ovine beta-lactoglobulin, of rat WAP and of rat beta-casein are capable of causing these proteins to be synthesized in transgenic mice. The levels are, however, systematically high only in the case of beta-lactoglobulin. Likewise, the beta-lactoglobulin promoter directs the synthesis of human alpha 1  -antitrypsin which reaches the value of 7 mg/ml of milk in the milk of a transgenic mouse. The alpha S1  -casein promoter permits the synthesis of human urokinase, but the promoters of the rat beta-casein and rabbit beta-casein genes used up until now are of a limited activity. The promoter of the mouse WAP gene directs the synthesis of several foreign proteins (plasminogen activator, CD4) which are secreted in the milk of transgenic mice. The quantities of proteins obtained with this promoter are however relatively small. 
     Furthermore, it may happen that the specificity of the promoter is modified by its association with a foreign gene. In this way Gunzburg et al. (Molecular Endocrinology 1991) obtain the secretion of growth hormone by means of a recombinant DNA under the dependence of the mouse WAP promoter, in transgenic animals; but the growth hormone is, in this case, also produced in the cerebellum, in Bergman glial cells. Such phenomena can result in toxicity and in the premature death of the animal. 
     The present invention is based on the demonstration of the special interest of the promoter of the rabbit WAP gene. Indeed, the rabbit WAP (&#34;whey acidic protein&#34;) is a relatively abundant rabbit milk protein (15 mg/ml of milk), and rabbits are potentially transgenic animals which can be used on a large scale. 
     The present invention relates to a process for the preparation of a heterologous protein of interest in mature form or in fused form in the milk of a mammalian female, in which process: 
     said female is bred and, 
     the milk is recovered and said protein is recovered from it and is separated if necessary, 
     characterized in that said female is a transgenic animal in the genome of which has been integrated a sequence encoding said protein of interest under the control of at least one sequence present among the elements for expression of the rabbit WAP protein and situated on the fragment having a length of at least 3 Kb from the 3&#39; end of the complete WAP promoter. 
     Preferably, the present invention relates to a process in which the sequence controlling the expression of the protein of interest comprises, in addition, expression elements situated on the fragment between 3 Kb and 6.3 Kb from the 3&#39; end of the WAP promoter. 
     The production of transgenic animals is known and has been widely described with similar constructs in the documents mentioned above, but also in Gunzburg et al., Hennighausen et al., Burdon et al., Reddy et al. as well as in Patent WO 90 05188. 
     The detailed description of the methods of transgenesis will not be repeated in detail, the above documents are incorporated in the present description by direct reference. 
     &#34;Heterologous protein of interest&#34; is understood to essentially designate a protein which is not naturally under the control of the rabbit WAP promoter. 
     In the process according to the invention, the mammalian female used is preferably a rabbit, but these constructs are also effective in other mammalians, for example mice. 
     The milk obtained contains the protein of interest which can be isolated or otherwise, and then, according to whether it is in mature of fused form, it can be subjected to a chemical or enzymatic cleavage if necessary. 
     The DNA constructs used are preferably introduced by microinjection into fertilized egg at the one cell up to the 8 cell stage, and then the animals corresponding to the criteria described above, that is to say transgenic and expressing said protein in the milk, are selected. 
     The promoter region of this WAP gene, grafted on to the reporter gene for bacterial chloramphenicol acetyl transferase (CAT) contains the elements sensitive to the two most important lactogenic hormones, prolactin and glucocorticoids. These hormones intensely stimulate the expression of the CAT gene when the hybrid gene is transfected into rabbit mammary primary epithelial cells. The hormone response depends on the length of the promoter used. The promoter of the rabbit WAP gene is therefore much more effective than the promoters of the mouse and rat WAP genes used up until now. 
     In particular, in the process according to the present invention, the sequence encoding the protein of interest can be preceded toward its 5&#39; end, by a sequence corresponding to the promoter of the complete rabbit WAP gene, or by an equivalent sequence, which ensures the function of the promoter. It is even possible, in this case, to use the entire WAP gene and a WAP promoter of said gene or of an equivalent sequence. The FIG. 5 accompanying the present application represents said rabbit WAP gene. 
     &#34;Equivalent sequence&#34; is understood to preferably designate a sequence having at least a length of 3 Kb from the 3∝ end of the WAP promoter and in particular comprising the expression elements situated on the fragment with a length of at least 6.3 Kb from the 3&#39; end of the complete rabbit WAP promoter, especially situated between the HindIII and BamHI sites (FIG. 1). 
     The promoter may contain a 17-Kb sequence between the HindIII and EcoRI sites, or a sequence containing the expression elements situated on this fragment. The essential elements of the constructs according to the invention are situated on the fragment with a length of 17.6 Kb from the 3&#39; end of the promoter. 
     This long promoter is capable of providing elements which further promote the expression of the foreign gene which is attached to it, or of making its expression more regularly high by making them more independent of the site of insertion of the transgene into the genome. 
     The 6.3 Kb short promoter will give a response to the lactogenic hormones which is practically identical to that obtained with the 17 Kb long promoter, and can direct the synthesis of proteins, whose genes are associated with it in a vector, at very high levels. 
     The invention also relates to constructs as defined above but in which, in the sequence corresponding to the rabbit WAP protein gene, the initiator AUG codon is deleted. 
     This modification can be obtained especially by site-directed mutagenesis. 
     When the sequence encoding the protein of interest is in a form fused with the sequence of all or part of the WAP gene, it is possible to suppress the ATG sequence of this protein. 
     The rabbit WAP protein can thus, with this type of construct, be expressed for example in transgenic mice. 
     In this type of construct comprising the rabbit WAP protein gene, which may have lost the initiator AUG codon, the gene or cDNA for the protein of interest can be placed at different sites which may result in different levels and types of constructs, as will emerge from the examples. 
     According to another of its aspects, the present invention provides a process for recovering the milk produced by a mammalian female and the protein which it contains, characterized in that, in order to recover the protein of interest from the milk of said mammalian female, 
     a) the mammary glands are recovered, 
     b) the mammary glands are incubated at a temperature of about 0° C. for a period ranging from two hours to 18 hours, 
     c) the milk having spontaneously exuded from the glands is recovered, 
     d) the protein of interest is isolated from the milk and said purified protein is obtained. 
     This process was developed within the framework of the production of a heterologous protein by a transgenic animal having integrated into its genome fragments of the WAP protein gene, but it can be applied to the purification of any protein which it is desired to isolate from milk. 
     It represents a considerable advantage compared with conventional processes for milking non-ruminant mammalians, with respect to the quantities obtained, especially for small animals such as mice (24). The composition of the milk recovered is identical to that of the milk produced naturally. This process permits, in addition, an easier transfer of the products obtained from the site of production to the site of purification and treatment, by transporting the mammary glands in ice. 
     The process for the preparation of heterologous protein of the present invention, according to anyone of its variants, makes it possible to obtain a large number of proteins. Among these proteins, there should be mentioned: 
     growth factors, 
     interleukins, 
     stimulating factors, 
     kinases, 
     coagulation factors, 
     alpha-antitrypsin, 
     hirudin, 
     and in particular: 
     erythropoietin, 
     G-CSF, 
     alpha-antitrypsin, 
     urokinase, 
     factor VIII. 
     The following constructs can be mentioned: 
     WAP-human alpha 1  -antitrypsin construct (Arg 358 analog): the entire human alpha 1 ,-antitrypsin gene having Arg 358 in place of Meth 358 (Courtney, Bull. Inst. Pasteur (1988) 86, 85-94) was fused to the long promoter (17.6 Kb) of the rabbit WAP gene at the HindIII site of the untranslated 5&#39;P sequence, on the model of the WAP-GH constructs. Several mouse lines express the human gene at the concentration of 2 and 5 mg/ml of milk. 
     WAP-human erythropoietin construct: the entire human erythropoietin gene (Semenza et al., Proc. Natl. Acad. Sci. U.S.A. (1989) 86, 2301-2305) is fused to the promoter (6.3 Kb and 17.6 Kb) of the rabbit WAP gene. 
     WAP-human factor VIII-deltaII construct: the cDNA of the human factor VIII-deltaII (deleted from the B region [Meulien et al., Prot. Engin (1988) 2, 301-306]) preceded by the first intron of the factor VIII gene and lacking its polyadenylation sequence, was introduced inside the entire rabbit WAP gene at the HindIII site introduced by site-directed mutagenesis and which precedes the natural AUG. 
     The constructs according to the invention make it possible to obtain completely unexpected results, in particular, the 6.3 Kb promoter of WAP combined with the human and bovine growth hormone genes is capable of directing the synthesis of these proteins in transgenic mouse milk at very high levels (1-21 mg/ml). 
     The hGH contained in the milk of the transgenic mice is structurally intact. The hGH also conserved its biological activity (evaluated not by its growth hormone activity but by its prolactin activity). The biological test indicates that the concentration of the hormone is 10 mg/ml, a value which is in agreement with the values found by the radioimmunological test and by electrophoresis. 
     The promoter of the rabbit WAP gene is therefore much more effective than the promoters of the mouse and rat WAP genes used up until now. 
     Table 1 below gives a summary of the results of publications 2 to 15 and therefore projects the advantages of the results obtained with the constructs according to the invention relative to those published in the prior art. 
     
                                           TABLE 1__________________________________________________________________________Summary of the different recombinant proteins expressed in the milk oftransgenic animals.                            Concentration ofNumber of animals the protein inexpressing the pro- milk in  Promoter used Protein encoded Animal used tein in milk microgram/ml__________________________________________________________________________Bovine alpha-casein    Human urokinase             Mouse 1 out of 3                            1000  Rabbit beta-casein Human interleukin-2 Rabbit 4 out of 4 0.001 to 0.01                             Ovine beta-lacto- Human coagulation Ewe                            2 out of 2 0.01  globulin factor IX  Ovine beta-lacto- Human alpha.sub.1 -anti- Ewe 2 out of 2 5  globulin trypsin Mouse 7 out of 13 6 to 7000  Mouse WAP Human cD4 [sic] Mouse 5 out of 7 0.4  Mouse WAP Huxaan tPA Mouse 4 out of 6 0.08 to 50  Mouse WAP Protein PS2 Mouse 1 out of 1 1.5  Mouse WAP Human GH Mouse 8 out of 8 1000  Rabbit short WAP Human GH Mouse 6 out of 6 5 to 21,000  Rabbit short WAP Bovine GH Mouse 8 out of 8 1200 to 16,700  Rabbit long WAP Bovine GH Mouse 4 out of 4 87 to 6900__________________________________________________________________________ 
    
     One of the essential reasons may consist in the fact that the rabbit DNA fragment (6.3 Kb) was much longer than its mouse and rat homolog (2.6 Kb and 0.9 Kb). Essential regulatory elements can be missing in the mouse and rat DNA fragments used. 
     The present invention also relates to the constructs which make it possible to obtain transgenic animals according to the invention. 
     The present invention relates especially to the DNA sequences and the vectors which make it possible to implement the process; in particular the DNA sequences comprising at least one heterologous gene encoding a protein of interest, under the control of at least one sequence present among the elements for expression of the rabbit WAP protein and located on the fragment having a length of at least 3 Kb from the 3&#39; end of the complete WAP promoter. 
     The present invention also relates to transgenic animals which can be used in the process according to the present invention, as well as transformed cells containing the constructs according to the invention. 
     Although the animals in question can be very different, rabbits are animals which can be potentially used to obtain recombinant proteins in abundance. Up to 100 ml of milk can be collected each day. This milk is highly rich in proteins (much richer than the milk of ruminants). It is moreover easier and less costly to obtain rabbits than large transgenic animals. The promoter of the rabbit WAP gene has, furthermore, every chance of best directing the synthesis of recombinant proteins in rabbits. 
     Other characteristics and advantages of the present invention will emerge on reading the examples below in which reference is made to the following figures: 
    
    
     DESCRIPTION OF THE FIGURES 
     FIG. 1: Map of the rabbit WAP gene. 
     FIG. 2a: Schematic representation of the construction of the plasmid pW 3 . 
     FIG. 2b: Polylinker of the plasmid p-polyIII-I (SEQ ID NO:1) 
     FIG. 3: Schematic representation of the construction of the plasmid pJ 4 . 
     FIG. 4: Production of human and bovine growth hormone in transgenic mouse lines harboring the constructs pW 3  and pJ 4 . 
     FIG. 5 (A-E): Sequence of the rabbit WAP gene (SEQ ID NOS 2 and 3). 
     FIG. 6: Schematic representations of the different constructs used in vivo. 
     FIG. 7: Schematic representations of the constructs containing the CAT reporter gene and variable lengths of the promoter WAP. 
     FIG. 8: Efficiency of the constructs described in FIG. 7. 
    
    
     DESCRIPTION OF THE INVENTION 
     EXAMPLE 1 
     CONSTRUCTION OF THE PLASMID PW 3   
     The plasmid p26C is first prepared. 
     The plasmid p26C was obtained by introducing the BamH 1  -HindIII sequence of the WAP gene (6.3 Kb fragment of FIG. 1) into the polylinker of the p-polyIII-I vector (between the BamH 1  [sic] and HindIII sites). 
     During this cloning, the BamH 1  [sic] site was suppressed and replaced by the ClaI site which is present in the vector p26C (FIG. 2A). The vector p26C is therefore a plasmid capable of receiving a foreign gene placed under the dependency of the 6.3 Kb WAP promoter. The introduction of the foreign gene can be carried out for example in the SalI site of the polylinker (FIG. 2B). The inserts containing the entire promoter and foreign genes can be isolated from the plasmid after cutting at the two NotI sites which are at the ends of the polylinker of the plasmid p-polyIII-I. 
     The plasmid pW 3  obtained from the plasmid p26C (according to FIG. 2A) contains the promoter of the rabbit WAP gene (6.3 Kb) and the human growth hormone gene (hGH). The fragment used to obtain the transgenic mice is between the two NotI sites. 
     A HindIII site was introduced into the leader sequence of the gene by site-directed mutagenesis so as to serve as cloning site. 
     EXAMPLE 2 
     CONSTRUCTION OF THE PLASMID pJ 4   
     The plasmid pJ 4  obtained from the plasmid p26 (according to FIG. 3) contains the promoter of the rabbit WAP gene (6.3 Kb) and the bovine growth hormone gene (bGH). The fragment used to obtain transgenic mice is between the two NotI sites. 
     The E. coli strain containing the plasmid p26 was deposited on June 12, 1991, under the number I-1116 at the Collection Nationale de Culture de Microorganisms of Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS CEDEX 15. 
     EXAMPLE 3 
     PRODUCTION OF TRANSGENIC MICE 
     The pW 3  and pJ 4  fragments were used to obtain transgenic animals. Transgenic mice were obtained by the conventional technique of microinjection (Brinster et al., Proc. Natl. Acad. Sci. U.S.A. (1985) 82, 4438-4442). 1-2-pl containing 500 copies of the gene were injected into the male pronucleus of mouse embryos. The constructs were prepared in the vector p-polyIII-I (Lathe et al., Gene (1987) 57, 193-201). The NotI-NotI fragments of this vector containing the recombinant genes were microinjected. The embryos were then transferred into the oviduct of hormonally prepared adoptive females. About 10% of the engineered embryos gave birth to young mice and 2-5% of the engineered embryos to transgenic young mice. The presence of the transgenes was revealed by the technique of Southern blotting from the DNA extracted from the mouse tails. The concentrations of growth hormone in the blood and in the milk of the animals were evaluated by means of specific radioimmunological tests. 
     The biological activity of the hGH was evaluated by adding milk to the culture medium of cells or of mammary explants of rabbits. The hGH content in the milk induced the expression of the β-casein gene evaluated by the measurement of the mRNAs and the protein. 
     EXAMPLE 4 
     PRODUCTION OF HUMAN OR BOVINE GROWTH HORMONE IN THE MILK OF TRANSGENIC MICE HAVING INCORPORATED THE CONSTRUCTS pW3 AND PJ4 
     The hormone concentrations were determined by specific radioimmunological tests. 
     The identification of hGH in the milk of a transgenic mouse is carried out in the following manner. The mouse milk is centrifuged at 150,000 g for one hour in order to sediment the casein micelles. The supernatant (1 μl per well) was recovered and examined by polyacryl-amide gel electrophoresis in the presence of control human growth hormone and a control milk. The results are reported in FIG. 4. 
     The animals having integrated the construct pW 3  give hGH concentrations of the order of 10 mg/ml of milk and can reach 21 mg/ml. 
     The animals having integrated the construct pJ 4  produce of the order of 5 mg of bGH/ml of milk and up to 17 mg/ml. 
     The process according to the present invention makes it possible to harvest 1.5 ml of milk/mouse mammary gland (by placing the mammary gland in ice). 200 suckling mice expressing a foreign protein at the concentration of 3-5 mg/ml therefore provide 1 g of crude protein. 
     EXAMPLE 5 
     GENE CONSTRUCTS 
     The gene constructs used for expressing recombinant proteins in the milk of transgenic animals contain in all cases the regulatory region of the rabbit WAP gene: BamHI-HindIII fragment (6.3 Kb) or EcoRI-HindIII fragment (17.6 Kb). The plasmids WAP-hGH, WAP-bGH, WAP-α-AT, and WAP-EPO contain the entire genes (leader sequence, exons, introns, and transcription terminator) of the human growth hormone (hGH), of the bovine growth hormone (bGH), of the human α 1  -antitrypsin mutated at Arg 358 and of human erythropoietin, respectively. In these constructs, the genes were associated with the regulatory region of the WAP gene at the HindIII site. The construct WAP-FVIII-ΔII contains the cDNA of human factor VIII in its Δ II form preceded by an intron of the human factor VIII gene. This intron- cDNA assembly was introduced into the HindIII site of the entire rabbit WAP gene (FIG. 6). 
     EXAMPLE 6 
     IDENTIFICATION OF THE ACTIVITY OF THE REGULATORY REGION OF THE RABBIT WAP GENE IN VITRO 
     The variable lengths of the region situated upstream of the transcription initiation site of the WAP gene were combined with a reporter gene (the CAT gene: chloramphenicol acetyl transferase) (FIG. 7). These constructs were introduced into mammary epithelial cells cultured on a rat tail collagen I gel by transfection by means of lipofectin. The cells were then maintained for three days in the presence of hormones (insulin, cortisol, prolactin). The enzyme was then measured in the cellular extracts. The constructs containing only 1806 bp or less of the regulatory region do not express the CAT gene. The construct containing 3000 bp is weakly active while the constructs containing 6300 and 17,600 bp are clearly expressed in the presence of the hormones. Prolactin alone exerts a weak but significant inducing role on the CAT gene. Insulin and especially cortisol, which are inactive alone, amplify the action of prolactin. The sensitivity of the gene toward the hormones is exactly identical to that of the endogenous WAP gene of the cells. The -3000-1806 bp and -6300-3000 bp regions therefore contain the regulatory elements essential for the WAP gene to be intensely expressed (FIG. 8). The 17,600-6300 bp fragment does not provide additional stimulation in vitro, which does not rule out that it can have such an action in vitro in transgenic animals. These experiments reveal for the first time the activity of the regulatory regions of the WAP gene in vitro via transfections of the cells. 
     REFERENCES 
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     2. SIMONS J. P. et al. Nature (1987) 328, 530-532 
     3. SIMONS J. P. et al. Biotechnology (1988) 6, 179-183 
     4. CLARK A. J. Biotechnology (1989) 7, 487-492 
     5. ARCHIBALD A. L. et al. Proc. Natl. Acad. Sci. U.S.A. (1990) 87, 5178-5182 
     6. HARRIS et al. J. Reprod. Fert. (1990) 88, 707-715 
     7. GORDON K. et al. Biotechnology (1987) 5, 1183-1187 
     8. PITTIUS C. W. Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 5874-5878 
     9. PITTIUS C. W. et al. Mol. Endocr. (1988) 2, 1027-1032 
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     20. GUNZBURG W. H. et al. Molecular Endocrinology (1991). A mammary-specific Promoter Directs Expression of Growth Hormone not only to the Mammary Gland, but also to Bergman Glia cells in Transgenic Mice. 
     21. HENNIGHAUSEN L. Protein Expression and Purification (1990) 1, 3-8 
     22. BURDON T. et al. Expression of a whey acidic protein transgene during mammary development: Evidence for different mechanisms of regulation during pregnancy and lactation 
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         __________________________________________________________________________#             SEQUENCE LISTING   - -  - - (1) GENERAL INFORMATION:   - -    (iii) NUMBER OF SEQUENCES: 3   - -  - - (2) INFORMATION FOR SEQ ID NO:1:   - -      (i) SEQUENCE CHARACTERISTICS:       (A) LENGTH: 137 base - #pairs       (B) TYPE: nucleic acid       (C) STRANDEDNESS: double       (D) TOPOLOGY: linear   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:   - - GGATCTGCGG CCGCCGGCCT CGAGGGCCGG ATCCGAATTC CCGGGAGAGC TC -#GATATCGC     60   - - ATGCGGTACC TCTAGAAGAA GCTTGGCCAG CTGGTCGACC TGCAGATCCG GC -#CCTCGAGG    120   - - CCGGCGGCCG CAGATCT             - #                  - # - #  137  - -  - - (2) INFORMATION FOR SEQ ID NO:2:  - -      (i) SEQUENCE CHARACTERISTICS:      (A) LENGTH: 4157 base - #pairs      (B) TYPE: nucleic acid      (C) STRANDEDNESS: double      (D) TOPOLOGY: linear  - -     (ix) FEATURE:      (A) NAME/KEY: CDS      (B) LOCATION: join(1868..1 - #949, 2462..2587, 2888..3046,3416            ..3429)   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:   - - AGATCTTGTG CTCGCTCGCT CTCTCGCTCT CTCTCTCTCT CCTTCTGTCT CT -#CTGGAACT     60   - - TTGCCTTTCA AATAAATAAA TAATTTTTTT AAAAGACTAC TGTTTTGTTT TT -#TTTATTTA    120   - - CTTAAAGCAG AGTAACAGAG AAAGAAATAC ATTCCGTTTG CTGGTTCACT CC -#CCAAATGG    180   - - CCGCTAGATC CAGGGCTAGG CCAGGCTGAA GCCAGAACCC CTACCTGGGT CT -#CCCACGTG    240   - - AGTGACAGGG GCCCAAGCAC CTGGGCCAAC CACCTCTGCT TTCCCAGGGA CA -#TTGGCAGG    300   - - GAGATGGGTC AGGAGCAGAG CAGCCAGAAC TCAGGCTGCC CTCCAATCTG AG -#ACATCAGC    360   - - TTTGCAAGTG GTAGCTTAAC CCACGTGTCA CCCAGCCCCA AGATTCATGT TA -#ATGATAGG    420   - - AAATTTTAAT TTTATTTGCT CAGATTGAAA CATTATAAAG GCACCACAAT AA -#GCAGAGTC    480   - - CAGAGATGAG AGAAAAACAA AAAATAAAAT AAAAAAATCT TGTATTTCGG TT -#CCTTTGCA    540   - - GGCACTTTCT TCCCTTTGTG GAACAAGGAG CCCAAAAACC GCAGCAGGGG GC -#CCAGTGGA    600   - - GATGGGAGAT GCCTGGGAAG AACACCCTGG GAGGAGCTCC GGGAGGCGCA GG -#AGGAGGGG    660   - - GTTCCTGACG GGGTCAGCTC TGGCCTCGGG CCCAGCACCC CAGTGAGAAG GA -#TGGGAGCC    720   - - GCCCAGCCCA GCCTGGCTCG GGCAGGAAGG GGCAGGCCCA ACCACAGCCC CT -#CTGCTCCT    780   - - TCGCAGGGAG CGGAACAGCC CACGGAAGCA TCTTTCGGAC TTAGAGCCGT GA -#ACCTCGCC    840   - - ACGCCGTGTC CAGCCCACTG TCTGAGAGCC CTCACTGGCC AGTCCAGGCC CA -#GGCCCAGG    900   - - ACTCTGTGGG CAGCTGCAGG GCTGGAACAG AGTTACCCGA GCCTGGGGCT GC -#GAGGGGTG    960   - - CCTTTGTGGA ACCCACAAAG GACGCTTTGT GGAAGGACAT TTGGGGCTGG AG -#CCTCCCCA   1020   - - CGGCACAGCC TGAGGCCCAG GAAGCTGCGA GGAGCTCTGT GCCTGAGGCC GG -#AGCAGGGT   1080   - - CGCTGGCTGG ACAGGGCTGT GGCCCCCAGC CATCCTGCCC TGGGGTCTCC GC -#AGTCCCCA   1140   - - TGGCCCCTTC CCTGTCTGGG TCCTGGGGGG GCGGGTGCAG GAACTACACG GC -#CAGCAGCA   1200   - - CATCCGCCCC TGCCCTGTGG CACCTGCTCC CCTGGCACAG GGCACAGGAG GG -#CCTTCCGA   1260   - - GAAGAGACCT TCTGTCCCCT CGCCCCTCCA CAGTCGGCAA GCCTGCACTG GG -#GTCCCCAG   1320   - - GGCAGGGGCC CAGGCTCTGC AGTCCGCTTC TCCTGTCCCC TCGCCCCTCC AC -#AGGTGGCA   1380   - - AGCAGCACAT TCTTGCTTAC AGAGTCCAGA AAACCACACA CACACACACA CA -#CACACACA   1440   - - CACACACACA CACACAAAAA AAAACACTTG CCGACGAGAC AGCCCGCACT TG -#GTACCCGC   1500   - - CTCCCATGCT GCTTCTCCCG GCTCTGAGCC GTGGGTACAA CCCCTCGGGG GG -#GGGGGGGG   1560   - - GAGGATTTCT CTCCCCCACC CCCAGTCTTC CTAGCAGATG TGCATCCCGG CC -#AACATGGA   1620   - - GGGAAATGGA CAACCTTGCC GGGGACTTTT TTTTCTTTCA TTTGAAACCA TG -#ACCGCAGC   1680   - - CGTTCCTCCA ACCTGGCCTG ACCTCTCCAC GTGTCCAAGG AGGAAGCCCC CT -#GGCCCAGT   1740   - - TGAGGCCTCG CCAACCTGGC ACCCCTCCAG GCTCCTCCTC CTGCTCCAAC CT -#TTAAATGC   1800   - - ATCCCGGGGC CCCAGAACAC CATCCGACAC CTGCCTGCTG CCCACCACCA GC -#CTACCACC   1860   - - TGCCACC ATG CGC TGT CTC ATC AGC CTG GCC CTC - #GGC CTG CTC GCC CTG    1909    Met Arg Cys Leu Ile Ser - #Leu Ala Leu Gly Leu Leu Ala Leu      1        - #       5           - #       10  - - GAG GCG GCC CTC GCT CTG GCC CCC AAA TTC AT - #C GCT CCA G  - #  1949 Glu Ala Ala Leu Ala Leu Ala Pro Lys Phe Il - #e Ala Pro  15                 - # 20                 - # 25  - - GTAGGCCCAG CTGCCTTCCT CACTCCGGGA CGCACTCAGG AGGGGTCCCC TT -#GTCTCATA   2009   - - TCTGCTCCAG AGTCCACCCA AGACTCGTGG CCTTGGTGGC TCCGTGACAG GG -#ACACAGCC   2069   - - GGCCAGGAGA GGAGCAGAGG AGGCTCACCC TTGGGAGGGG GTCCTGGGTG GC -#AGGAAACC   2129   - - AGCGCCCTGT CCCCACGCAG GGGGCCACGA GCTGCCAGGC CAAGGACTGG TC -#ACCTCCGG   2189   - - CCAGGACCTG ACTGGCCTGC TCCTGCAGTG GACCTGTGTC TTGTGTCCCC AC -#TTCCACAG   2249   - - CTGACTTCAC TCGCTTTTGT CAGCCGTATC GCAGTTCTGG CCACGGGTTT TT -#GTTTTGTT   2309   - - TTGTTTTGTT TTGTTTTGTT TTGCCCTCCT TCCTGGGCTG CTGGGGGCCA GG -#CTCCCACG   2369   - - GTTCTGTCCT CGCCCTCCTC CAAGGAGCCC TGGGGGTGGG AGGGGCAGGG CT -#GCGGGCCC   2429   - - CCACACACTT GCTCGTCCTG CCCCGTGTGC AG  TG CAG GTC - #ATG TGC CCC GAG    2481              - #                  - #Val Gln Val Met Cys Pro Glu              - #                  - #         30  - - CCC AGC TCT TCC GAG GAG ACG CTC TGC CTC AG - #T GAC AAC GAC TGT CTC    2529 Pro Ser Ser Ser Glu Glu Thr Leu Cys Leu Se - #r Asp Asn Asp Cys Leu  35                 - # 40                 - # 45                 - # 50  - - GGC AGC ACC GTG TGC TGT CCC AGC GCC GCC GG - #C GGC TCC TGC AGA ACC    2577 Gly Ser Thr Val Cys Cys Pro Ser Ala Ala Gl - #y Gly Ser Cys Arg Thr             55 - #                 60 - #                 65  - - CCC ATC ATC G GTAACGTAGC CACACTGCAG GCCTCTCCGG AAGC - #CCACAC    2627 Pro Ile Ile ACCTGCCCCA TGGCGCAGTC TCTCTGGGCC CCATCCACCT GCCCCGAGGC CT - #CTGTGCCA2687   - - CCCCACAGGT CCCTGAGGGC TCCAGGATGC CCCAGTGCTG CGGGAGGTCC TG -#CGGTGAGA   2747   - - CCAGCAAGAG GGAGGCCACA GAGACCCAGC TGACCTCAGG GGTCCCCCGG CG -#CTCAACTT   2807   - - GTCTCAGTGG GGTCTTGCGG GTCAGGTCTG GGGGGGCCCA TGTTACAGGG TG -#TGACCAGA   2867   - - AAAGGCCTGT CTCTCCCCAG  TC CCT ACC CCC AAG GCT - #GGC CGC TGC CCC    2916              - #    Val Pro Thr Pro Lys Ala Gly Arg - #Cys Pro              - #     70             - #     75  - - TGG GTG CAG GCG CCA ATG CTG TCC CAG TTG TG - #T GAG GAG CTG AGC GAC    2964 Trp Val Gln Ala Pro Met Leu Ser Gln Leu Cy - #s Glu Glu Leu Ser Asp  80                 - # 85                 - # 90                 - # 95  - - TGT GCC AAC GAC ATC GAG TGC AGG GGC GAC AA - #G AAG TGC TGC TTC AGC    3012 Cys Ala Asn Asp Ile Glu Cys Arg Gly Asp Ly - #s Lys Cys Cys Phe Ser            100  - #               105  - #               110  - - CGC TGC GCC ATG CGC TAT CTG GAA CCC ATC CT - #A G GTATGTGTCC    305 - #6 Arg Cys Ala Met Arg Tyr Leu Glu Pro Ile Le - #u        115      - #           120  - - TGAGCCCTCC CCAGGCAGGG CTGTCCCTTC AGCAGGGCCC AGGGCTCAGG AG -#TGGATGTG   3116   - - GGTGAGTGAA GGGCACTCGG GGACGCAGGT GGCAGGCGGG ACTTGGCCCT GG -#GTGGCTCA   3176   - - CAGGCCAGCC TGTACCTTTG CCACTGATCT GAGAGGGAGT GCAGCACAGC TC -#CAGGTATC   3236   - - GGAGGAGTCG AAGGTTAGGA GCCTGGGGTG TTGTCCACCA GCTGTGGCCT GC -#ATATTCCT   3296   - - TCCTACAGAG GGGGGGGGGC AGAGGCGGGG AGGGGGCTCT GCTTGCGCAC TA -#GGGTCCCT   3356   - - GGCAGTGAAC CACAGCCGAC ACTGACCTCC CACCTTGTCC CCACCTGTGT CT -#CCTGCAG AG 3417               - #                  - #                  - # Glu  - - AGC ACT CCC CAG TGAGCCGCCT ACCCAGGAGT CCCTGGCTGC CA - #GGAGAGTT    3469 Ser Thr Pro Gln125  - - GGGCCTGAGT CCCCCTCTTG GACCCAGAGA GCTTGTGACG CGTCCTCCCT GC -#TGCTAATA   3529   - - AAACTACTCA GCTTCATGGC TCTGGTTGTC TGTCCATCTG CCCTGGGAGC TT -#GGGAAACC   3589   - - AGTGACCCCA AGTAGGCACA GCTCTGCCTG GCTCAGCAGC CCAGCACGAC GT -#CCGAGGGA   3649   - - ATGGACTAGA CCCCAAGATA ACGCTTACCT CCCTCCACCC CTGTTTGAGC TT -#GCCAGGAA   3709   - - GGGCAGCAGG CCATTCAGGG TGAGCCACGC CCTCAGGGAG CCCCCACGTA CC -#TGTGAGGT   3769   - - CACTTCCCTG GGCTTCAGTG CCCACGAACC CCTGTCCTTT TCCGTGGCAG TC -#AGTGAACA   3829   - - GAGTAAGAAG AGGAGAGTGA GCTCCAGCCT GTGAAGTTCA GCCCTTCCTG GG -#TGTGGCAC   3889   - - AGAGACAGGC CAGGCTGTCC CAGGCTGTCC CAGGCTGCTG GCCGGGGGGT GC -#ACAGAGGC   3949   - - CTCGCAGAAG AAAGAGCCAT CATGTGCAGA GTGAGAGGAA AGGCCCCCCC AG -#ACAGAGGC   4009   - - ATGTGCAGGA CGCCTCGGCC GGGACGTGGA TCGCCAGAGG CCCCTGCGCG CC -#ATGCTGGG   4069   - - GTGAGGGGAC GTTTAGGACA CAGGGCCTAA TGGAGAGCAG CTAGGTCATG GG -#GGTGCTGC   4129   - - CTCCTGAGAC TGGATTCGTC CCCTCGAG         - #                  - #  4157  - -  - - (2) INFORMATION FOR SEQ ID NO:3:  - -      (i) SEQUENCE CHARACTERISTICS:      (A) LENGTH: 127 amino - #acids      (B) TYPE: amino acid      (D) TOPOLOGY: linear  - -     (ii) MOLECULE TYPE: protein  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:  - - Met Arg Cys Leu Ile Ser Leu Ala Leu Gly Le - #u Leu Ala Leu Glu Ala   1               5 - #                 10 - #                 15  - - Ala Leu Ala Leu Ala Pro Lys Phe Ile Ala Pr - #o Val Gln Val Met Cys         20     - #             25     - #             30  - - Pro Glu Pro Ser Ser Ser Glu Glu Thr Leu Cy - #s Leu Ser Asp Asn Asp     35         - #         40         - #         45  - - Cys Leu Gly Ser Thr Val Cys Cys Pro Ser Al - #a Ala Gly Gly Ser Cys 50             - #     55             - #     60  - - Arg Thr Pro Ile Ile Val Pro Thr Pro Lys Al - #a Gly Arg Cys Pro Trp  65                 - # 70                 - # 75                 - # 80  - - Val Gln Ala Pro Met Leu Ser Gln Leu Cys Gl - #u Glu Leu Ser Asp Cys             85 - #                 90 - #                 95  - - Ala Asn Asp Ile Glu Cys Arg Gly Asp Lys Ly - #s Cys Cys Phe Ser Arg        100      - #           105      - #           110  - - Cys Ala Met Arg Tyr Leu Glu Pro Ile Leu Gl - #u Ser Thr Pro Gln    115          - #       120          - #       125__________________________________________________________________________