Abstract:
Attenuated recombinant viruses containing DNA coding for a cytokine and/or a tumor associated antigen, as well as methods and compositions employing the viruses, are disclosed and claimed. The recombinant viruses can be NYVAC or ALVAC recombinant viruses. The DNA can code for at least on of: human tumor necrosis factor; nuclear phosphoprotein p53, wildtype or mutant; human melanoma-associated antigen; IL-2; IFNg; IL-4; GNCSF; IL-12; B7; erb-B-2 and carcinoembryonic antigen. The recombinant viruses and gene products therefrom are useful for cancer therapy.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS  
       [0001]    This application is a continuation-in-part of application Ser. No. 08/007,115, filed Jan. 20, 1993, incorporated herein by reference. Application Ser. No. 08/007,115 is a continuation-in-part of application Ser. No. 07/847,951, filed Mar. 6, 1992, which in turn is a continuation-in-part of application Ser. No. 07/713,967, filed Jun. 11, 1991 which in turn is a continuation-in-part of application Ser. No. 07/666,056, filed Mar. 7, 1991; and, application Ser. No. 08/007,115 is also a continuation-in-part of application Ser. No. 07/805,567, filed Dec. 16, 1991, which in turn is a continuation-in-part of application Ser. No. 07/638,080, filed Jan. 7, 1991; and, application Ser. No. 08/007,115 is also a continuation-in-part of application Ser. No. 07/847,977 filed Mar. 3, 1992 as a division of application Ser. No. 07/478,179, filed Feb. 14, 1990 as a continuation-in-part of application Ser. No. 07/320,471, filed Mar. 8, 1989; all of which are hereby incorporated herein by reference. Reference is also made to copending U.S. applications Ser. Nos. 715,921, filed Jun. 14, 1991, 736,254, filed Jul. 26, 1991, 776,867, filed Oct. 22, 1991, and 820,077, filed Jan. 13, 1992, all of which are hereby incorporated herein by reference. 
     
    
     
       FIELD OF THE INVENTION  
         [0002]    The present invention relates to a modified poxvirus and to methods of making and using the same. More in particular, the invention relates to improved vectors for the insertion and expression of foreign genes for use as safe immunization vehicles to protect against a variety of pathogens, as well as for use in immunotherapy.  
           [0003]    Several publications are referenced in this application. Full citation to these references is found at the end of the specification immediately preceding the claims or where the publication is mentioned; and each of these publications is hereby incorporated herein by reference. These publications relate to the art to which this invention pertains.  
         BACKGROUND OF THE INVENTION  
         [0004]    Vaccinia virus and more recently other poxviruses have been used for the insertion and expression of foreign genes. The basic technique of inserting foreign genes into live infectious poxvirus involves recombination between pox DNA sequences flanking a foreign genetic element in a donor plasmid and homologous sequences present in the rescuing poxvirus (Piccini et al., 1987).  
           [0005]    Specifically, the recombinant poxviruses are constructed in two steps known in the art and analogous to the methods for creating synthetic recombinants of poxviruses such as the vaccinia virus and avipox virus described in U.S. Pat. Nos. 4,769,330, 4,772,848, 4,603,112, 5,100,587, and 5,179,993, the disclosures of which are incorporated herein by reference.  
           [0006]    First, the DNA gene sequence to be inserted into the virus, particularly an open reading frame from a non-pox source, is placed into an  E. coli  plasmid construct into which DNA homologous to a section of DNA of the poxvirus has been inserted. Separately, the DNA gene sequence to be inserted is ligated to a promoter. The promoter-gene linkage is positioned in the plasmid construct so that the promoter-gene linkage is flanked on both ends by DNA homologous to a DNA sequence flanking a region of pox DNA containing a nonessential locus. The resulting plasmid construct is then amplified by growth within  E. coli  bacteria (Clewell, 1972) and isolated (Clewell et al., 1969; Maniatis et al., 1982).  
           [0007]    Second, the isolated plasmid containing the DNA gene sequence to be inserted is transfected into a cell culture, e.g. chick embryo fibroblasts, along with the poxvirus. Recombination between homologous pox DNA in the plasmid and the viral genome respectively gives a poxvirus modified by the presence, in a nonessential region of its genome, of foreign DNA sequences. The term “foreign” DNA designates exogenous DNA, particularly DNA from a non-pox source, that codes for gene products not ordinarily produced by the genome into which the exogenous DNA is placed.  
           [0008]    Genetic recombination is in general the exchange of homologous sections of DNA between two strands of DNA. In certain viruses RNA may replace DNA. Homologous sections of nucleic acid are sections of nucleic acid (DNA or RNA) which have the same sequence of nucleotide bases.  
           [0009]    Genetic recombination may take place naturally during the replication or manufacture of new viral genomes within the infected host cell. Thus, genetic recombination between viral genes may occur during the viral replication cycle that takes place in a host cell which is co-infected with two or more different viruses or other genetic constructs. A section of DNA from a first genome is used interchangeably in constructing the section of the genome of a second co-infecting virus in which the DNA is homologous with that of the first viral genome.  
           [0010]    However, recombination can also take place between sections of DNA in different genomes that are not perfectly homologous. If one such section is from a first genome homologous with a section of another genome except for the presence within the first section of, for example, a genetic marker or a gene coding for an antigenic determinant inserted into a portion of the homologous DNA, recombination can still take place and the products of that recombination are then detectable by the presence of that genetic marker or gene in the recombinant viral genome. Additional strategies nave recently been reported for generating recombinant vaccinia virus (Scheiflinger et al., 1992; Merchlinsky and Moss, 1992).  
           [0011]    Successful expression of the inserted DNA genetic sequence by the modified infectious virus requires two conditions. First, the insertion must be into a nonessential region of the virus in order that the modified the inserted DNA. The promoter must be placed so that it is located upstream from the DNA sequence to be expressed.  
           [0012]    Vaccinia virus has been used successfully to immunize against smallpox, culminating in the worldwide eradication of smallpox in 1980. In the course of its history, many strains of vaccinia have arisen. These different strains demonstrate varying immunogenicity and are implicated to varying degrees with potential complications, the most serious of which are post-vaccinial encephalitis and generalized vaccinia (Behbehani, 1983).  
           [0013]    With the eradication of smallpox, a new role for vaccinia became important, that of a genetically engineered vector for the expression of foreign genes. Genes encoding a vast number of heterologous antigens have been expressed in vaccinia, often resulting in protective immunity against challenge by the corresponding pathogen (reviewed in Tartaglia et al., 1990a,b).  
           [0014]    The genetic background of the vaccinia vector has been shown to affect the protective efficacy of the expressed foreign immunogen. For example, expression of Epstein Barr Virus (EBV) gp340 in the Wyeth vaccine strain of vaccinia virus did not protect cottontop tamarins against EBV virus induced lymphoma, while expression of the same gene in the WR laboratory strain of vaccinia virus was protective (Morgan et al., 1988).  
           [0015]    A fine balance between the efficacy and the safety of a vaccinia virus-based recombinant vaccine candidate is extremely important. The recombinant virus must present the immunogen(s) in a manner that elicits a protective immune response in the vaccinated animal but lacks any significant pathogenic properties. Therefore attenuation of the vector strain would be a highly desirable advance over the current state of technology.  
           [0016]    A number of vaccinia genes have been identified which are non-essential for growth of the virus in tissue culture and whose deletion or inactivation reduces virulence in a variety of animal systems.  
           [0017]    The gene encoding the vaccinia virus thymidine kinase (TK) has been mapped (Hruby et al., 1982) and sequenced (Hruby et al., 1983; Weir et al., 1983). Inactivation or complete deletion of the thymidine kinase gene does not prevent growth of vaccinia virus in a wide variety of cells in tissue culture. TK −  vaccinia virus is also capable of replication in vivo at the site of inoculation in a variety of hosts by a variety of routes.  
           [0018]    It has been shown for herpes simplex virus type 2 that intravaginal inoculation of guinea pigs with TK −  virus resulted in significantly lower virus titers in the spinal cord than did inoculation with TK +  virus (Stanberry et al., 1985). It has been demonstrated that herpesvirus encoded TK activity in vitro was not important for virus growth in actively metabolizing cells, but was required for virus growth in quiescent cells (Jamieson et al., 1974).  
           [0019]    Attenuation of TK −  vaccinia has been shown in mice inoculated by the intracerebral and intraperitoneal routes (Buller et al., 1985). Attenuation was observed both for the WR neurovirulent laboratory strain and for the Wyeth vaccine strain. In mice inoculated by the intradermal route, TK −  recombinant vaccinia generated equivalent anti-vaccinia neutralizing antibodies as compared with the parental TK +  vaccinia virus, indicating that in this test system the loss of TK function does not significantly decrease immunogenicity of the vaccinia virus vector. Following intranasal inoculation of mice with TK −  and TK +  recombinant vaccinia virus (WR strain), significantly less dissemination of virus to other locations, including the brain, has been found (Taylor et al., 1991a).  
           [0020]    Another enzyme involved with nucleotide metabolism is ribonucleotide reductase. Loss of virally encoded ribonucleotide reductase activity in herpes simplex virus (HSV) by deletion of the gene encoding the large subunit was shown to have no effect on viral growth and DNA synthesis in dividing cells in vitro, but severely compromised the ability of the virus to grow on serum starved cells (Goldstein et al., 1988). Using a mouse model for acute HSV infection of the eye and reactivatable latent infection in the trigeminal ganglia, reduced virulence was demonstrated for HSV deleted of the large subunit of ribonucleotide reductase, compared to the virulence exhibited by wild type HSV (Jacobson et al., 1989).  
           [0021]    Both the small (Slabaugh et al., 1988) and large (Schmitt et al., 1988) subunits of ribonucleotide reductase have been identified in vaccinia virus. Insertional inactivation of the large subunit of ribonucleotide reductase in the WR strain of vaccinia virus leads to attenuation of the virus as measured by intracranial inoculation of mice (Child et al., 1990).  
           [0022]    The vaccinia virus hemagglutinin gene (HA) has been mapped and sequenced (Shida, 1986). The HA gene of vaccinia virus is nonessential for growth in tissue culture (Ichihashi et al., 1971). Inactivation of the HA gene of vaccinia virus results in reduced neurovirulence in rabbits inoculated by the intracranial route and smaller lesions in rabbits at the site of intradermal inoculation (Shida et al., 1988). The HA locus was used for the insertion of foreign genes in the WR strain (Shida et al., 1987), derivatives of the Lister strain (Shida et al., 1988) and the Copenhagen strain (Guo et al., 1989) of vaccinia virus. Recombinant HA −  vaccinia virus expressing foreign genes have been shown to be immunogenic (Guo et al., 1989; Itamura et al., 1990; Shida et al., 1988; Shida et al., 1987) and protective against challenge by the relevant pathogen (Guo et al., 1989; Shida et al., 1987).  
           [0023]    Cowpox virus (Brighton red strain) produces red (hemorrhagic) pocks on the chorioallantoic membrane of chicken eggs. Spontaneous deletions within the cowpox genome generate mutants which produce white pocks (Pickup et al., 1984). The hemorrhagic function (u) maps to a 38 kDa protein encoded by an early gene (Pickup et al., 1986). This gene, which has homology to serine protease inhibitors, has been shown to inhibit the host inflammatory response to cowpox virus (Palumbo et al., 1989) and is an inhibitor of blood coagulation.  
           [0024]    The u gene is present in WR strain of vaccinia virus (Kotwal et al., 1989b). Mice inoculated with a WR vaccinia virus recombinant in which the u region has been inactivated by insertion of a foreign gene produce higher antibody levels to the foreign gene product compared to mice inoculated with a similar recombinant vaccinia virus in which the u gene is intact (Zhou et al., 1990). The u region is present in a defective nonfunctional form in Copenhagen strain of vaccinia virus (open reading frames B13 and B14 by the terminology reported in Goebel et al., 1990a,b).  
           [0025]    Cowpox virus is localized in infected cells in cytoplasmic A type inclusion bodies (ATI) (Kato et al., 1959). The function of ATI is thought to be the protection of cowpox virus virions during dissemination from animal to animal (Bergoin et al., 1971). The ATI region of the cowpox genome encodes a 160 kDa protein which forms the matrix of the ATI bodies (Funahashi et al., 1988; Patel et al., 1987). Vaccinia virus, though containing a homologous region in its genome, generally does not produce ATI. In WR strain of vaccinia, the ATI region of the genome is translated as a 94 kDa protein (Patel et al., 1988). In Copenhagen strain of vaccinia virus, most of the DNA sequences corresponding to the ATI region are deleted, with the remaining 3′ end of the region fused with sequences upstream from the ATI region to form open reading frame (ORF) A26L (Goebel et al., 1990a,b).  
           [0026]    A variety of spontaneous (Altenburger et al., 1989; Drillien et al., 1981; Lai et al., 1989; Moss et al., 1981; Paez et al., 1985; Panicali et al., 1981) and engineered (Perkus et al., 1991; Perkus et al., 1989; Perkus et al., 1986) deletions have been reported near the left end of the vaccinia virus genome. A WR strain of vaccinia virus with a 10 kb spontaneous deletion (Moss et al., 1981; Panicali et al., 1981) was shown to be attenuated by intracranial inoculation in mice (Buller et al., 1985). This deletion was later shown to include 17 potential ORFs (Kotwal et al., 1988b). Specific genes within the deleted region include the virokine N1L and a 35 kDa protein (C3L, by the terminology reported in Goebel et al., 1990a,b). Insertional inactivation of N1L reduces virulence by intracranial inoculation for both normal and nude mice (Kotwal et al., 1989a). The 35 kDa protein is secreted like N1L into the medium of vaccinia virus infected cells. The protein contains homology to the family of complement control proteins, particularly the complement 4B binding protein (C4 bp) (Kotwal et al., 1988a). Like the cellular C4 bp, the vaccinia 35 kDa protein binds the fourth component of complement and inhibits the classical complement cascade (Kotwal et al., 1990). Thus the vaccinia 35 kDa protein appears to be involved in aiding the virus in evading host defense mechanisms.  
           [0027]    The left end of the vaccinia genome includes two genes which have been identified as host range genes, K1L (Gillard et al., 1986) and C7L (Perkus et al., 1990). Deletion of both of these genes reduces the ability of vaccinia virus to grow on a variety of human cell lines (Perkus et al., 1990).  
           [0028]    Two additional vaccine vector systems involve the use of naturally host-restricted poxviruses, avipoxviruses. Both fowlpoxvirus (FPV) and canarypoxvirus (CPV) have been engineered to express foreign gene products. Fowlpox virus (FPV) is the prototypic virus of the Avipox genus of the Poxvirus family. The virus causes an economically important disease of poultry which has been well controlled since the 1920&#39;s by the use of live attenuated vaccines. Replication of the avipox viruses is limited to avian species (Matthews, 1982b) and there are no reports in the literature of avipoxvirus causing a productive infection in any non-avian species including man. This host restriction provides an inherent safety barrier to transmission of the virus to other species and makes use of avipoxvirus based vaccine vectors in veterinary and human applications an attractive proposition.  
           [0029]    FPV has been used advantageously as a vector expressing antigens from poultry pathogens. The hemagglutinin protein of a virulent avian influenza virus was expressed in an FPV recombinant (Taylor et al., 1988a). After inoculation of the recombinant into chickens and turkeys, an immune response was induced which was protective against either a homologous or a heterologous virulent influenza virus challenge (Taylor et al., 1988a). FPV recombinants expressing the surface glycoproteins of Newcastle Disease Virus have also been developed (Taylor et al., 1990; Edbauer et al., 1990).  
           [0030]    Despite the host-restriction for replication of FPV and CPV to avian systems, recombinants derived from these viruses were found to express extrinsic proteins in cells of nonavian origin. Further, such recombinant viruses were shown to elicit immunological responses directed towards the foreign gene product and where appropriate were shown to afford protection from challenge against the corresponding pathogen (Tartaglia et al., 1993 a,b; Taylor et al., 1992; 1991b; 1988b).  
           [0031]    In the past, viruses have been shown to have utility in cancer immunotherapy, in that, they provide a means of enhancing tumor immunoresponsiveness. Examples exist showing that viruses such as Newcastle disease virus (Cassel et al., 1983), influenza virus (Lindenmann, 1974; Lindenmann, 1967), and vaccinia virus (Wallack et al., 1986; Shimizu et al., 1988; Shimizu et al. 1984; Fujiwara et al., 1984) may act as tumor-modifying antigens or adjuvants resulting in inducing tumor-specific and tumor-nonspecific immune effector mechanisms. Due to advances in the fields of immunology, tumor biology, and molecular biology, however, such approaches have yielded to more directed immunotherapeutic approaches for cancer. Genetic modification of tumor cells and immune effector cells (i.e. tumor-infiltrating lymphocytes; TILS) to express, for instance cytokines, have provided encouraging results in animal models and humans with respect to augmenting tumor-directed immune responses (Pardoll, 1992; Rosenberg, 1992). Further, the definition of tumor-associated antigens (TAAs) has provided the opportunity to investigate their role in the immunobiology of certain cancers which may eventually be applied to their use in cancer prevention or therapy (van der Bruggen, 1992).  
           [0032]    Advances in the use of eukaryotic vaccine vectors have provided a renewed interest in viruses in cancer prevention and therapy. Among the viruses engineered to express foreign gene products are adenoviruses, adeno-associated virus, baculovirus, herpesviruses, poxviruses, and retroviruses. Most notably, retrovirus-, adenovirus-, and poxvirus-based recombinant viruses have been developed with the intent of in vivo utilization in the areas of vector-based vaccines, gene therapy, and cancer therapy (Tartaglia, in press; Tartaglia, 1990).  
           [0033]    Immunotherapeutic approaches to combat cancers or neoplasia can take the form of classical vaccination schemes or cell-based therapies. Immunotherapeutic vaccination is the concept of inducing or enhancing immune responses of the cancer patient to antigenic determinants that are uniquely expressed or expressed at increased levels on tumor cells. Tumor-associated antigens (TAAs) are usually of such weak immunogenicity as to allow progression of the tumor unhindered by the patient&#39;s immune system. Under normal circumstances, the severity of the disease-state associated with the tumor progresses more rapidly than the elaboration of immune responses, if any, to the tumor cells. Consequently, the patient may succumb to the neoplasia before a sufficient immune response is mounted to control and prevent growth and spread of the tumor.  
           [0034]    Poxvirus vector technology has been utilized to elicit immunological responses to TAAs. Examples exist demonstrating the effectiveness of poxvirus-based recombinant viruses expressing TAAs in animal models in the immunoprophylaxis and immunotherapy of experimentally-induced tumors. The gene encoding carcinoembryonic antigen (CEA) was isolated from human colon tumor cells and inserted into the vaccinia virus genome (Kaufman et al., 1991). Inoculation of the vaccinia-based CEA recombinant elicited CEA-specific antibodies and an antitumor effect in a murine mouse model. This recombinant virus has been shown to elicit humoral and cell-mediated responses in rhesus macaques (Kantor et al., 1993). The human melanoma TAA, p97, has also been inserted into vaccinia virus and shown to protect mice from tumor transplants (Hu et al., 1988; Estin et al., 1988). A further example was described by Bernards et al. (1987). These investigators constructed a vaccinia recombinant that expressed the extracellular domain of the rat neu-encoded transmembrane glycoprotein, p185. Mice immunized with this recombinant virus developed a strong humoral response against the neu gene product and were protected against subsequent tumor challenge. Vaccinia virus recombinants expressing either a secreted or membrane-anchored form of a breast cancer-associated epithelial tumor antigen (ETA) have been generated for evaluation in the active immunotherapy of breast cancer (Hareuveni et al., 1991; 1990). These recombinant viruses have been shown to elicit anti-ETA antibodies in mice and to protect mice against a tumorigenic challenge with a ras-transformed Fischer rat fibroblast line expressing either form of ETA (Hareuveni et al., 1990). Further, vaccinia virus recombinants expressing the polyoma virus-derived T-Ag were shown efficacious for prevention and therapy in a mouse tumor model system (Lathe et al., 1987).  
           [0035]    Recombinant vaccinia viruses have also been used to express cytokine genes (Reviewed by Ruby et al., 1992). Expression of certain cytokines (IL-2, IFN-α, TNF-α) lead to self-limiting vaccinia virus infection in mice and, in essence, act to attenuate the virus. Expression of other cytokines (i.e. IL-5, IL-6) were found to modulate the immune response to co-expressed extrinsic immunogens (Reviewed by Ruby et al., 1992).  
           [0036]    Frequently, immune responses against tumor cells are mediated by T cells, particularly cytotoxic T lymphocytes (CTLs); white blood cells capable of killing tumor cells and virus-infected cells (Greenberg, 1991). The behavior of CTLs is regulated by soluble factors termed cytokines. Cytokines direct the growth, differentiation, and functional properties of CTLs, as well as, other immune effector cells.  
           [0037]    Cell-based immunotherapy has been shown to provide effective therapy for viruses and tumors in animal models (Greenberg, 1991; Pardoll, 1992; Riddel et al., 1992). Cytomegalovirus (CMV)-specific CTL clones from bone marrow donors have recently been isolated. These clones were propagated and expanded in vitro and ultimately returned to immunodeficient bone marrow patients. These transferred CMV-specific CTL clones provided no toxic-effects and provided persistent reconstitution of CD8 +  CMV-specific CTL responses preventing CMV infection in the transplant patient (Riddel et al., 1992).  
           [0038]    There exists two forms of cell-based immunotherapy. These are adoptive immunotherapy, which involves the expansion of tumor reactive lymphocytes in vitro and reinfusion into the host, and active immunotherapy, which involves immunization of tumor cells to potentially enhance existing or to elicit novel tumor-specific immune responses and provide systemic anti-tumor immunity. Immunotherapeutic vaccination is the concept of inducing or enhancing immune responses of the cancer patient to antigenic determinants that are uniquely expressed or expressed at increased levels on tumor cells.  
           [0039]    It can be appreciated that provision of novel strains, such as NYVAC, ALVAC, and TROVAC having enhanced safety would be a highly desirable advance over the current state of technology. For instance, so as to provide safer vaccines or safer products from the expression of a gene or genes by a virus.  
         OBJECTS OF THE INVENTION  
         [0040]    It is therefore an object of this invention to provide modified recombinant viruses, which viruses have enhanced safety, and to provide a method of making such recombinant viruses.  
           [0041]    It is an additional object of this invention to provide a recombinant poxvirus vaccine having an increased level of safety compared to known recombinant poxvirus vaccines.  
           [0042]    It is a further object of this invention to provide a modified vector for expressing a gene product in a host, wherein the vector is modified so that it has attenuated virulence in the host.  
           [0043]    It is another object of this invention to provide a method for expressing a gene product in a cell cultured in vitro using a modified recombinant virus or modified vector having an increased level of safety.  
           [0044]    These and other objects and advantages of the present invention will become more readily apparent after consideration of the following.  
         STATEMENT OF THE INVENTION  
         [0045]    In one aspect, the present invention relates to a modified recombinant virus having inactivated virus-encoded genetic functions so that the recombinant virus has attenuated virulence and enhanced safety. The functions can be non-essential, or associated with virulence. The virus is advantageously a poxvirus, particularly a vaccinia virus or an avipox virus, such as fowlpox virus and canarypox virus. The modified recombinant virus can include, within a non-essential region of the virus genome, a heterologous DNA sequence which encodes an antigenic protein, e.g., derived from a pathogen, a tumor associated antigen, a cytokine, or combination thereof.  
           [0046]    In another aspect, the present invention relates to a vaccine for inducing an antigenic response in a host animal inoculated with the vaccine, said vaccine including a carrier and a modified recombinant virus having inactivated nonessential virus-encoded genetic functions so that the recombinant virus has attenuated virulence and enhanced safety. The virus used in the vaccine according to the present invention is advantageously a poxvirus, particularly a vaccinia virus or an avipox virus, such as fowlpox virus and canarypox virus. The modified recombinant virus can include, within a non-essential region of the virus genome, a heterologous DNA sequence which encodes an antigenic protein, e.g., derived from a pathogen, a tumor associated antigen, a cytokine, or combination thereof.  
           [0047]    In yet another aspect, the present invention relates to an immunogenic composition containing a modified recombinant virus having inactivated nonessential virus-encoded genetic functions so that the recombinant virus has attenuated virulence and enhanced safety. The modified recombinant virus includes, within a non-essential region of the virus genome, a heterologous DNA sequence which encodes an antigenic protein (e.g., derived from a pathogen, a tumor associated antigen, a cytokine, or combination thereof) wherein the composition, when administered to a host, is capable of inducing an immunological response specific to the protein encoded by the pathogen.  
           [0048]    In a further aspect, the present invention relates to a method for expressing a gene product in a cell cultured in vitro by introducing into the cell a modified recombinant virus having attenuated virulence and enhanced safety. The modified recombinant virus can include, within a non-essential region of the virus genome, a heterologous DNA sequence which encodes an antigenic protein, e.g., derived from a pathogen, a tumor associated antigen, a cytokine, or combination thereof.  
           [0049]    In a still further aspect, the present invention relates to a modified recombinant virus having nonessential virus-encoded genetic functions inactivated therein so that the virus has attenuated virulence, and wherein the modified recombinant virus further contains DNA from a heterologous source in a nonessential region of the virus genome. The DNA can code for a tumor associated antigen, a cytokine, or a combination thereof. In particular, the genetic functions are inactivated by deleting an open reading frame encoding a virulence factor or by utilizing naturally host restricted viruses. The virus used according to the present invention is advantageously a poxvirus, particularly a vaccinia virus or an avipox virus, such as fowlpox virus and canarypox virus. Advantageously, the open reading frame is selected from the group consisting of J2R, B13R+B14R, A26L, A56R, C7L−K1L, and I4L (by the terminology reported in Goebel et al., 1990a,b); and, the combination thereof. In this respect, the open reading frame comprises a thymidine kinase gene, a hemorrhagic region, an A type inclusion body region, a hemagglutinin gene, a host range gene region or a large subunit, ribonucleotide reductase; or, the combination thereof. The modified Copenhagen strain of vaccinia virus is identified as NYVAC (Tartaglia et al., 1992). 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0050]    The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings, in which:  
         [0051]    [0051]FIG. 1 schematically shows a method for the construction of plasmid pSD460 for deletion of thymidine kinase gene and generation of recombinant vaccinia virus vP410;  
         [0052]    [0052]FIG. 2 schematically shows a method for the construction of plasmid pSD486 for deletion of hemorrhagic region and generation of recombinant vaccinia virus vP553;  
         [0053]    [0053]FIG. 3 schematically shows a method for the construction of plasmid pMP494Δ for deletion of ATI region and generation of recombinant vaccinia virus vP618;  
         [0054]    [0054]FIG. 4 schematically shows a method for the construction of plasmid pSD467 for deletion of hemagglutinin gene and generation of recombinant vaccinia virus vP723;  
         [0055]    [0055]FIG. 5 schematically shows a method for the construction of plasmid pMPCK1Δ for deletion of gene cluster [C7L−K1L] and generation of recombinant vaccinia virus vP804;  
         [0056]    [0056]FIG. 6 schematically shows a method for the construction of plasmid pSD548 for deletion of large subunit, ribonucleotide reductase and generation of recombinant vaccinia virus vP866 (NYVAC);  
         [0057]    [0057]FIG. 7 schematically shows a method for the construction of plasmid pRW842 for insertion of rabies glycoprotein G gene into the TK deletion locus and generation of recombinant vaccinia virus vP879;  
         [0058]    [0058]FIG. 8 shows the DNA sequence (SEQ ID NO:68) of a canarypox PvuII fragment containing the C5 ORF.  
         [0059]    [0059]FIGS. 9A and 9B schematically show a method for the construction of recombinant canarypox virus vCP65 (ALVAC-RG);  
         [0060]    [0060]FIG. 10 shows schematically the ORFs deleted to generate NYVAC;  
         [0061]    [0061]FIG. 11 shows the nucleotide sequence (SEQ ID NO:77) of a fragment of TROVAC DNA containing an F8 ORF;  
         [0062]    [0062]FIG. 12 shows the DNA sequence (SEQ ID NO:78) of a 2356 base pair fragment of TROVAC DNA containing the F7 ORF;  
         [0063]    [0063]FIGS. 13A to  13 D show graphs of rabies neutralizing antibody titers (RFFIT, IU/ml), booster effect of HDC and vCP65 (10 5.5  TCID50) in volunteers previously immunized with either the same or the alternate vaccine (vaccines given at days 0, 28 and 180, antibody titers measured at days 0, 7, 28, 35, 56, 173, 187 and 208); days 0, 28 and 180, antibody titers measured at days 0, 7, 28, 35, 56, 173, 187 and 208);  
         [0064]    [0064]FIGS. 14A to  14 C show the nucleotide sequence of a 7351 bp fragment containing the ALVAC C3 insertion site (SEQ ID NO:127);  
         [0065]    [0065]FIG. 15 shows the nucleotide sequences of H6/TNF-α expression cassette and flanking regions from vCP245 (SEQ ID NO:79);  
         [0066]    [0066]FIG. 16 shows the nucleotide sequence of the H6/TNF-α expression cassette and flanking regions from vP1200 (SEQ ID NO:89);  
         [0067]    [0067]FIG. 17 shows the nucleotide sequence of the H6/p53 (wildtype) expression cassette and flanking regions from vP1101 (SEQ ID NO:99);  
         [0068]    [0068]FIG. 18 shows the nucleotide sequence of the H6/p53 (wildtype) expression cassette and flanking regions from vCP207 (SEQ ID NO:99);  
         [0069]    [0069]FIG. 19 shows the nucleotide sequence of the H6/MAGE-1 expression cassette and flanking region from vCP235 (SEQ ID NO:109);  
         [0070]    [0070]FIG. 20 shows the nucleotide sequence of the H6/MAGE-1 expression cassette and flanking regions from pMAW037 (SEQ ID NO:110);  
         [0071]    [0071]FIGS. 21A and B show the nucleotide sequence of the p126.15 SERA cDNA insert along with the predicted amino acid sequence (SEQ ID NOS:119; 120);  
         [0072]    [0072]FIG. 22 shows the nucleotide sequence of the H6/CEA expression cassette and flanking regions from pH6.CEA.C3.2 (SEQ ID NO:144);  
         [0073]    [0073]FIG. 23 shows the nucleotide sequence of the H6/CEA expression cassette and flanking regions from pH6.CEA.HA (SEQ ID NO:145);  
         [0074]    [0074]FIG. 24 shows the nucleotide sequence of murine IL-2 from the translation initiation codon through the stop codon (SEQ ID NO:150);  
         [0075]    [0075]FIG. 25 shows the corrected nucleotide sequence of human IL-2 from the translation initiation codon through the stop codon (SEQ ID NO:159);  
         [0076]    [0076]FIG. 26 shows the nucleotide sequence of the I3L/murine IFNγ expression cassette (SEQ ID NO:163);  
         [0077]    [0077]FIG. 27 shows the nucleotide sequence of the I3L/human IFNγ expression cassette (SEQ ID NO:168);  
         [0078]    [0078]FIG. 28 shows the nucleotide sequence of the canarypox insert in pC6HIII3kb (SEQ ID NO:169);  
         [0079]    [0079]FIG. 29 shows the nucleotide sequence pC6L (SEQ ID NO:174);  
         [0080]    [0080]FIG. 30 shows the nucleotide sequence of the E3L/murine IL-4 expression cassette (SEQ ID NO:178);  
         [0081]    [0081]FIG. 31 shows the nucleotide sequence of the expression cassette comprising the E3L promoted IL-4 gene (SEQ ID NO:186);  
         [0082]    [0082]FIG. 32 shows the nucleotide sequence of the vaccinia E3L/hGMCSF expression cassette (SEQ ID NO:191);  
         [0083]    [0083]FIG. 33 shows the sequence of the EPV 42 kDa/human IL-12 P40 expression cassette (SEQ ID NO:194);  
         [0084]    [0084]FIG. 34 shows the nucleotide sequence of the vaccinia E3L/human IL-12 P35 expression cassette (SEQ ID NO:199);  
         [0085]    [0085]FIG. 35 shows the nucleotide sequence of the murine B7 gene (SEQ ID NO:202);  
         [0086]    [0086]FIG. 36 shows flow cytometric analysis of murine B7 expression in NYVAC and ALVAC infected murine tumor cell lines;  
         [0087]    [0087]FIG. 37 shows the nucleotide sequence for the human B7 gene (SEQ ID NO:207);  
         [0088]    [0088]FIG. 38 shows the murine p53 gene (SEQ ID NO:214); and  
         [0089]    [0089]FIG. 39 shows the coding sequence for the human p53 gene (SEQ ID NO:215). 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0090]    To develop a new vaccinia vaccine strain, NYVAC (vP866), the Copenhagen vaccine strain of vaccinia virus was modified by the deletion of six nonessential regions of the by the deletion of six nonessential regions of the genome encoding known or potential virulence factors. The sequential deletions are detailed below. All designations of vaccinia restriction fragments, open reading frames and nucleotide positions are based on the terminology reported in Goebel et al., 1990a,b.  
         [0091]    The deletion loci were also engineered as recipient loci for the insertion of foreign genes.  
         [0092]    The regions deleted in NYVAC are listed below. Also listed are the abbreviations and open reading frame designations for the deleted regions (Goebel et al., 1990a,b) and the designation of the vaccinia recombinant (vP) containing all deletions through the deletion specified:  
         [0093]    (1) thymidine kinase gene (TK; J2R) vP410;  
         [0094]    (2) hemorrhagic region (u; B13R+B14R) vP553;  
         [0095]    (3) A type inclusion body region (ATI; A26L) vP618;  
         [0096]    (4) hemagglutinin gene (HA; A56R) vP723;  
         [0097]    (5) host range gene region (C7L−K1L) vP804; and  
         [0098]    (6) large subunit, ribonucleotide reductase (I4L) vP866 (NYVAC).  
         [0099]    NYVAC is a genetically engineered vaccinia virus strain that was generated by the specific deletion of eighteen open reading frames encoding gene products associated with virulence and host range. NYVAC is highly attenuated by a number of criteria including i) decreased virulence after intracerebral inoculation in newborn mice, ii) inocuity in genetically (nu + /nu + ) or chemically (cyclophosphamide) immunocompromised mice, iii) failure to cause disseminated infection in immunocompromised mice, iv) lack of significant induration and ulceration on rabbit skin, v) rapid clearance from the site of inoculation, and vi) greatly reduced replication competency on a number of tissue culture cell lines including those of human origin. Nevertheless, NYVAC based vectors induce excellent responses to extrinsic immunogens and provided protective immunity.  
         [0100]    TROVAC refers to an attenuated fowlpox that was a plaque-cloned isolate derived from the FP-1 vaccine strain of fowlpoxvirus which is licensed for vaccination of 1 day old chicks. ALVAC is an attenuated canarypox virus-based vector that was a plaque-cloned derivative of the licensed canarypox vaccine, Kanapox (Tartaglia et al., 1992). ALVAC has some general properties which are the same as some general properties of Kanapox. ALVAC-based recombinant viruses expressing extrinsic immunogens have also been demonstrated efficacious as vaccine vectors (Tartaglia et al., 1993 a,b). This avipox vector is restricted to avian species for productive replication. On human cell cultures, canarypox virus replication is aborted early in the viral replication cycle prior to viral DNA synthesis. Nevertheless, when engineered to express extrinsic immunogens, authentic expression and processing is observed in vitro in mammalian cells and inoculation into numerous mammalian species induces antibody and cellular immune responses to the extrinsic immunogen and provides protection against challenge with the cognate pathogen (Taylor et al., 1992; Taylor et al., 1991). Recent Phase I clinical trials in both Europe and the United States of a canarypox/rabies glycoprotein recombinant (ALVAC-RG) demonstrated that the experimental vaccine was well tolerated and induced protective levels of rabiesvirus neutralizing antibody titers (Cadoz et al., 1992; Fries et al., 1992). Additionally, peripheral blood mononuclear cells (PBMCs) derived from the ALVAC-RG vaccinates demonstrated significant levels of lymphocyte proliferation when stimulated with purified rabies virus (Fries et al., 1992).  
         [0101]    NYVAC, ALVAC and TROVAC have also been recognized as unique among all poxviruses in that the National Institutes of Health (“NIH”)(U.S. Public Health Service), Recombinant DNA Advisory Committee, which issues guidelines for the physical containment of genetic material such as viruses and vectors, i.e., guidelines for safety procedures for the use of such viruses and vectors which are based upon the pathogenicity of the particular virus or vector, granted a reduction in physical containment level: from BL2 to BL1. No other poxvirus has a BL1 physical containment level. Even the Copenhagen strain of vaccinia virus—the common smallpox vaccine—has a higher physical containment level; namely, BL2. Accordingly, the art has recognized that NYVAC, ALVAC and TROVAC have a lower pathogenicity than any other poxvirus.  
         [0102]    Both NYVAC- and ALVAC-based recombinant viruses have been shown to stimulate in vitro specific CD8 +  CTLs from human PBMCs (Tartaglia et al., 1993a). Mice immunized with NYVAC or ALVAC recombinants expressing various forms of the HIV-1 envelope glycoprotein generated both primary and memory HIV specific CTL responses which could be recalled by a second inoculation (Tartaglia et al., 1993a). ALVAC-env and NYVAC-env recombinants (expressing the HIV-1 envelope glycoprotein) stimulated strong HIV-specific CTL responses from peripheral blood mononuclear cells (PBMC) of HIV-1 infected individuals (Tartaglia et al., 1993a). Acutely infected autologous PBMC were used as stimulator cells for the remaining PBMC. After 10 days incubation in the absence of exogenous IL-2, the cells were evaluated for CTL activities. NYVAC-env and ALVAC-env stimulated high levels of anti-HIV activities. Thus, these vectors lend themselves well to ex vivo stimulation of antigen reactive lymphocytes; for example, adoptive immunotherapy such as the ex vivo expression of tumor reactive lymphocytes and reinfusion into the host (patient).  
         [0103]    Immunization of the patient with NYVAC-, ALVAC-, or TROVAC-based recombinant viruses expressing TAAs produced by the patient&#39;s tumor cells can elicit anti-tumor immune responses more rapidly and to sufficient levels to impede or halt tumor spread and potentially eliminate the tumor burden.  
         [0104]    Clearly based on the attenuation profiles of the NYVAC, ALVAC, and TROVAC vectors and their demonstrated ability to elicit both humoral and cellular immunological responses to extrinsic immunogens (Tartaglia et al., 1993a,b; Taylor et al., 1992; Konishi et al., 1992) such recombinant viruses offer a distinct advantage over previously described vaccinia-based recombinant viruses.  
         [0105]    The immunization procedure for such recombinant viruses as immunotherapeutic vaccines or compositions may be via a parenteral route (intradermal, intramuscular or subcutaneous). Such an administration enables a systemic immune response against the specific TAA(s). Alternatively, the vaccine or composition may be administered directly into the tumor mass (intratumor). Such a route of administration can enhance the anti-tumor activities of lymphocytes specifically associated with tumors (Rosenberg, 1992). Immunization of the patient with NYVAC-, ALVAC-or TROVAC-based recombinant viruses expressing TAAs produced by the patient&#39;s tumor cells can elicit anti-tumor immune responses more rapidly and to sufficient levels to impede or halt tumor spread and potentially eliminate the tumor burden. The heightened tumor-specific immune response resulting from vaccinations with these poxvirus-based recombinant vaccines can result in remission of the tumor, including permanent remission of the tumor. Examples of known TAAs for which recombinant poxviruses can be generated and employed with immunotherapeutic value in accordance with this invention include, but are not limited to p53 (Hollstein et al., 1991), p21-ras (Almoguera et al., 1988), HER-2 (Fendly et al., 1990), and the melanoma-associated antigens (MAGE-1; MZE-2) (van der Bruggen et al., 1991), and p97 (Hu et al., 1988) and the carcinoembryonic antigen (CEA) associated with colorecteal cancer (Kantor et al., 1993; Fishbein et al., 1992; Kaufman et al., 1991).  
         [0106]    More generally, the inventive vaccines or compositions (vaccines or compositions containing the poxvirus art. Such vaccines or compositions can be administered to a patient in need of such administration in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular patient, and the route of administration. The vaccines or compositions can be co-administered or sequentially administered with other antineoplastic, anti-tumor or anti-cancer agents and/or with agents which reduce or alleviate ill effects of antineoplastic, anti-tumor or anti-cancer agents; again taking into consideration such factors as the age, sex, weight, and condition of the particular patient, and, the route of administration.  
         [0107]    Examples of vaccines or compositions of the invention include liquid preparations for orifice, e.g., oral, nasal, anal, vaginal, etc., administration such as suspensions, syrups or elixirs; and, preparations for parental, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration) such as sterile suspensions or emulsions. In such compositions the recombinant poxvirus may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like. The recombinant poxvirus of the invention can be provided in lyophilized form for reconstituting, for instance, in isotonic aqueous, saline buffer. Further, the invention also comprehends a kit wherein the recombinant poxvirus is provided. The kit can include a separate container containing a suitable carrier, diluent or excipient. The kit can also include an additional anti-cancer, anti-tumor or antineoplastic agent and/or an agent which reduces or alleviates ill effects of antineoplastic, anti-tumor or anti-cancer agents for co-or sequential-administration. Additionally, the kit can include instructions for mixing or combining ingredients and/or administration.  
         [0108]    The poxvirus vector technology provides an appealing approach towards manipulating lymphocytes and tumor cells for use in cell-based immunotherapeutic modalities for cancer. Characteristics of the NYVAC, ALVAC and TROVAC vectors providing the impetus for such applications include 1) their apparent independence for specific receptors for entry into cells, 2) their ability to express foreign genes in cell substrates despite their species-or tissue-specific origin, 3) their ability to express foreign genes independent of host cell regulation, 4) the demonstrated ability of using poxvirus recombinant viruses to amplify specific CTL reactivities from peripheral blood mononuclear cells (PBMCs), and 5) their highly attenuated properties compared to existing vaccinia virus vaccine strains (Reviewed by Tartaglia et al., 1993a; Tartaglia et al., 1990).  
         [0109]    The expression of specific cytokines or the co-expression of specific cytokines with TAAs by NYVAC-, ALVAC-, and TROVAC-based recombinant viruses can enhance the numbers and anti-tumor activities of CTLs associated with tumor cell depletion or elimination. Examples of cytokines which have a beneficial effect in this regard include tumor necrosis factor-α (TNF-α ). interferon-gamma (INF-gamma), interleukin-2 (IL-2), interleukin-4 (IL-4), and interleukin-7 (IL-7) (reviewed by Pardoll, 1992). Cytokine interleukin 2 (IL-2) plays a major role in promoting cell mediated immunity. Secreted by the T H 1 subset of lymphocytes, IL-2 is a T cell growth factor which stimulates division of both CD4 +  and CD8 +  T cells. In addition, IL-2 also has been shown to activate B cells, monocytes and natural killer cells. To a large degree the biological effects of IL-2 are due to its role in inducing production of IFNγ. Recombinant vaccinia virus expressing IL-2 is attenuated in mice compared to wild-type vaccinia virus. This is due to the ability of the vaccinia-expressed IL-2 to stimulate mouse NK cells to produce IFNγ, which limits the growth of the recombinant vaccinia virus (Karupiah et al., 1990). Similarly, it has been shown that inoculation of immunodeficient athymic nude mice with recombinant vaccinia virus expressing both IL-2 and the HA gene of influenza can protect these mice from subsequent challenge with influenza virus (Karupiah et al., 1992).  
         [0110]    Cytokine interferon γ (IFNγ) is secreted by the T H 1 subset of lymphocytes. IFNγ promotes the T H 1 cell mediated immune response, while inhibiting the T H 2 (antibody) response. IFNγ induces the expression of major histocompatibility, complex (MHC) molecules on antigen presenting cells, and induces the expression of the B7 costimulatory molecule on macrophages. In addition to enhancing the phaqocytic activity of macrophages, IFNγ enhances the cytotoxic activity of NK cells. When expressed in replicating recombinant vaccinia virus, IFNγ limits the growth of the recombinant virus. This allows T cell immunodeficient mice to resolve the infection (Kohonen-Corish et al., 1990).  
         [0111]    Cytokine interleukin 4 (IL-4) is secreted by the T H 2 subset of lymphocytes. IL-4 promotes the T H 2 (antibody) response, while inhibiting the T H 1 cell mediated immune response. Recombinant vaccinia virus expressing IL-4 shows increased pathogenicity in mice compared to wild-type vaccinia virus (Ramshaw et al., 1992).  
         [0112]    Cytokine granulocyte macrophage colony stimulating factor (GMCSF) is pleiotropic. In addition to stimulating the proliferation of cells of both the granulocyte and macrophage cell lineages, GMCSF, in cross-competition with interleukins 3 and 5 (IL-3 and IL-5), influences many other aspects of hematopoiesis and may play a role in facilitation of tumor cell growth (Lopez et al., 1992). GMCSF is used clinically for hematopoietic reconstitution following bone marrow transplantation.  
         [0113]    Cytokine interleukin 12 (IL-12), formerly known as natural killer (NK) cell stimulatory factor, is a heterodimer composed of 35 kDa and 40 kDa subunits. IL-12 is produced by monocytes, macrophages, B cells and other accessory cells. IL-12 has pleiotropic effects on both NK cells and T cells. Partly through its role in inducing IFNγ production, IL-12 plays a major role in promoting the T H 1 cell mediated immune response, while inhibiting the T H 2 response (reviewed in Trinchieri, 1993). Recently, recombinant murine IL-12 has been demonstrated to have potent antitumor and antimetastatic effects in mice (Brunda et al., 1993).  
         [0114]    B7(BB-1), a member of the immunoglobin superfamily, is present on the surface of antigen presenting cells. Interaction of the B7 molecule on antigen presenting cells with its receptors on T cells provides costimulatory signals, including IL-2, which are necessary for T cell activation (Schwartz, 1992). Recently it was shown that experimental co-expression of B7 along with a tumor antigen on murine melanoma cells can lead to regression of tumors in mice. This was accomplished by the B7-assisted activation of tumor-specific cytotoxic T cells (Chen et al, 1992).  
         [0115]    The c-erb-B-2 gene, which is conserved among vertebrates, encodes a possible receptor protein. The 185 kDa translation product contains a kinase domain which is highly homologous to the kinase domain of the epidermal growth factor (EGF) receptor. The c-erb-B-2 gene is conserved among vertebrates, and is the same as the rat neu gene, which has been detected in a number of rat neuro/glioblastomas. The human c-erb-B-2 gene, also known as HER2, is amplified in certain neoplasias, most notably breast cancer. In the gastric cancer cell line, MKN-7, both the normal 4.6 kb transcript encoding c-erb-B-2 and a 2.3 kb transcript which specifies only the extracellular domain of the putative receptor are synthesized at elevated levels (Yamamoto et al., 1986). The extracellular domain has been suggested as a potential immunogen for active specific immunotherapy of breast cancer (Fendly et al., 1990).  
         [0116]    Utility of NYVAC-, ALVAC-, and TROVAC-based recombinant viruses expressing TAAs plus or minus specific cytokines for adoptive immunotherapy can take several forms. For one, genetic modification of PBMCs can be accomplished by vector-mediated introduction of TAAs, cytokine genes, or other genes and then directly reintroduced into the patient. Such administration relies on the drainage or movement of modified PBMCs to lymphoid tissue (i.e. spleen; lymph nodes) via the reticuloendothelial system (RES) for elicitation of the tumor-specific immune response. PBMCs modified by infection with the pertinent NYVAC-, ALVAC-, and TROVAC-based recombinant can be employed, for instance, in vitro, to expand TAA-specific CTLs for reinfusion into the patient. Tumor-infiltrating lymphocytes (TILs) derived from the tumor mass can be isolated, expanded, and modified to express pertinent genes using NYVAC-, ALVAC-, or TROVAC-based recombinants viruses prior to reinfusion into the patient. TILs retain the capability of returning to tumors (homing) when re-introduced into the subject (Rosenberg, 1992). Thus, they provide a convenient vehicle for delivery of cytotoxic or cytostatic cytokines to tumor masses.  
         [0117]    Cell-based active immunotherapy can also take on several potential modalities using the NYVAC-, ALVAC-, and TROVAC vectors. Tumor cells can be modified to express TAAs, cytokines, or other novel antigens (i.e. class I or class II major histocompatibility genes). Such modified tumor cells can subsequently be utilized for active immunization. The therapeutic potential for such an administration is based on the ability of these modified tumor cells to secrete cytokines and to alter the presentation of TAAs to achieve systemic anti-tumor activity. The modified tumor cells can also be utilized to expand tumor-specific CTLs in vitro for reinfusion into the patient.  
         [0118]    A better understanding of the present invention and of its many advantages will be had from the following examples, given by way of illustration.  
       EXAMPLES  
       [0119]    DNA Cloning and Synthesis. Plasmids were constructed, screened and grown by standard procedures (Maniatis et al., 1982; Perkus et al., 1985; Piccini et al., 1987). Restriction endonucleases were obtained from Bethesda Research Laboratories, Gaithersburg, Md., New England Biolabs, Beverly, Mass.; and Boehringer Mannheim Biochemicals, Indianapolis, Ind. Kienow fragment of  E. coli  polymerase was obtained from Boehringer Mannheim Biochemicals. BAL-31 exonuclease and phage T4 DNA ligase were obtained from New England Biolabs. The reagents were used as specified by the various suppliers.  
         [0120]    Synthetic oligodeoxyribonucleotides were prepared on a Biosearch 8750 or Applied Biosystems 380B DNA synthesizer as previously described (Perkus et al., 1989). DNA sequencing was performed by the dideoxy-chain termination method (Sanger et al., 1977) using Sequenase (Tabor et al., 1987) as previously described (Guo et al., 1989). DNA amplification by polymerase chain reaction (PCR) for sequence verification (Engelke et al., 1988) was performed using custom synthesized oligonucleotide primers and GeneAmp DNA amplification Reagent Kit (Perkin Elmer Cetus, Norwalk, Conn.) in an automated Perkin Elmer Cetus DNA Thermal Cycler. Excess DNA sequences were deleted from plasmids by restriction endonuclease digestion followed by limited digestion by BAL-31 exonuclease and mutagenesis (Mandecki, 1986) using synthetic oligonucleotides.  
         [0121]    Cells, Virus, and Transfection. The origins and conditions of cultivation of the Copenhagen strain of vaccinia virus has been previously described (Guo et al., 1989). Generation of recombinant virus by recombination, in situ hybridization of nitrocellulose filters and screening for B-galactosidase activity are as previously described (Piccini et al., 1987).  
         [0122]    The origins and conditions of cultivation of the Copenhagen strain of vaccinia virus and NYVAC has been previously described (Guo et al., 1989; Tartaglia et al., 1992). Generation of recombinant virus by recombination, in situ hybridization of nitrocellulose filters and screening for B-galactosidase activity are as previously described (Panicali et al., 1982; Perkus et al., 1989).  
         [0123]    The parental canarypox virus (Rentschler strain) is a vaccinal strain for canaries. The vaccine strain was obtained from a wild type isolate and attenuated through more than 200 serial passages on chick embryo fibroblasts. A master viral seed was subjected to four successive plaque purifications under agar and one plaque clone was amplified through five additional passages after which the stock virus was used as the parental virus in in vitro recombination tests. The plaque purified canarypox isolate is designated ALVAC.  
         [0124]    The strain of fowlpox virus (FPV) designated FP-1 has been described previously (Taylor et al., 1988a). It is an attenuated vaccine strain useful in vaccination of day old chickens. The parental virus strain Duvette was obtained in France as a fowlpox scale from a chicken. The virus was attenuated by approximately 50 serial passages in chicken embryonated eggs followed by 25 passages on chicken embryo fibroblast cells. The virus was subjected to four successive plaque purifications. One plaque isolate was further amplified in primary CEF cells and a stock virus, designated as TROVAC, established.  
         [0125]    NYVAC, ALVAC and TROVAC viral vectors and their derivatives were propagated as described previously (Piccini et al., 1987; Taylor et al., 1988a,b). Vero cells and chick embryo fibroblasts (CEF) were propagated as described previously (Taylor et al., 1988a,b).  
       Example 1  
     Construction of Plasmid pSD460 for Deletion of Thymidine Kinase Gene (J2R)  
       [0126]    Referring now to FIG. 1, plasmid pSD406 contains vaccinia HindIII J (pos. 83359-88377) cloned into pUC8. pSD406 was cut with HindIII and PvuII, and the 1.7 kb fragment from the left side of HindIII J cloned into pUC8 cut with HindIII/SmaI, forming pSD447. pSD447 contains the entire gene for J2R (pos. 83855-84385). The initiation codon is contained within an NlaIII site and the termination codon is contained within an SspI site. Direction of transcription is indicated by an arrow in FIG. 1.  
         [0127]    To obtain a left flanking arm, a 0.8 kb HindIII/EcoRI fragment was isolated from pSD447, then digested with NlaIII and a 0.5 kb HindIII/NlaIII fragment isolated. Annealed synthetic oligonucleotides MPSYN43/MPSYN44 (SEQ ID NO:1/SEQ ID NO:2)  
                                                                 SmaI                   MPSYN43   5      TAATTAACTAGCTACCCGGG     3′                       MPSYN44   3′ GTACATTAATTGATCGATGGGCCCTTAA 5′                  Nla III                   Eco RI          
 
         [0128]    were ligated with the 0.5 kb HindIII/NlaIII fragment into pUC18 vector plasmid cut with HindIII/EcoRI, generating plasmid pSD449.  
         [0129]    To obtain a restriction fragment containing a vaccinia right flanking arm and pUC vector sequences, pSD447 was cut with SspI (partial) within vaccinia sequences and HindIII at the pUC/vaccinia junction, and a 2.9 kb vector fragment isolated. This vector fragment was ligated with annealed synthetic oligonucleotides MPSYN45/MPSYN46 (SEQ ID NO:3/SEQ ID NO:4)  
                                      HindIII   SmaI                       NotI                Ssp I           MPSYN45   5′  AGCTTCCCGGGTAAGTAATACGTCAAGGAGAAAACGAAACGATCTGTAGTTAGCGGCCGCCTAATTAACTAAT  3′ MPSYN45       MPSYN46   3′      AGGGCCCATTCATTATGCAGTTCCTCTTTTGCTTTGCTAGACATCAATCGCCGGCGGATTAATTGATTA  5′ MPSYN46          
 
         [0130]    generating pSD459.  
         [0131]    To combine the left and right flanking arms into one plasmid, a 0.5 kb HindIII/SmaI fragment was isolated from pSD449 and ligated with pSD459 vector plasmid cut with HindIII/SmaI, generating plasmid pSD460. pSD460 was used as donor plasmid for recombination with wild type parental vaccinia virus Copenhagen strain VC-2.  32 P labelled probe was synthesized by primer extension using MPSYN45 (SEQ ID NO:3) as template and the complementary 20mer oligonucleotide MPSYN47 (SEQ ID NO:5) (5′ TTAGTTAATTAGGCGGCCGC 3′) as primer. Recombinant virus vP410 was identified by plaque hybridization.  
       Example 2  
     Construction of Plasmid pSD486 for Deletion of Hemorrhagic Region (B13R+B14R)  
       [0132]    Referring now to FIG. 2, plasmid pSD419 contains vaccinia SalI G (pos. 160,744-173,351) cloned into pUC8. pSD422 contains the contiguous vaccinia SalI fragment to the right, SalI J (pos. 173,351-182,746) cloned into pUC8. To construct a plasmid deleted for the hemorrhagic region, u, B13R−B14R (pos. 172,549-173,552), pSD419 was used as the source for the left flanking arm and pSD422 was used as the source of the right flanking arm. The direction of transcription for the u region is indicated by an arrow in FIG. 2.  
         [0133]    To remove unwanted sequences from pSD419, sequences to the left of the NcoI site (pos. 172,253) were removed by digestion of pSD419 with NcoI/SmaI followed by blunt ending with Klenow fragment of  E. coli  polymerase and ligation generating plasmid pSD476. A vaccinia right flanking arm was obtained by digestion of pSD422 with HpaI at the termination codon of B14R and by digestion with NruI 0.3 kb to the right. This 0.3 kb fragment was isolated and ligated with a 3.4 kb HincII vector fragment isolated from pSD476, generating plasmid pSD477. The location of the partial deletion of the vaccinia u region in pSD477 is indicated by a triangle. The remaining B13R coding sequences in pSD477 were removed by digestion with ClaI/HpaI, and the resulting vector fragment was ligated with annealed synthetic oligonucleotides SD22mer/SD20mer (SEQ ID NO:6/SEQ ID NO:7)  
                                               Cla I          Bam HI  Hpa I           SD22 mer   5′ CGATTACT ATG AAGGATCCGTT 3′           SD20 mer   3′   TAATGATACTTCCTAGGCAA 5′          
 
         [0134]    generating pSD479. pSD479 contains an initiation codon (underlined) followed by a BamHI site. To place  E. coli  Beta-galactosidase in the B13-B14 (u) deletion locus under the control of the u promoter, a 3.2 kb BamHI fragment containing the Beta-galactosidase gene (Shapira et al., 1983) was inserted into the BamHI site of pSD479, generating pSD479BG. pSD479BG was used as donor plasmid for recombination with vaccinia virus vP410. Recombinant vaccinia virus vP533 was isolated as a blue plaque in the presence of chromogenic substrate X-gal. In vP533 the B13R−B14R region is deleted and is replaced by Beta-galactosidase.  
         [0135]    To remove Beta-galactosidase sequences from vP533, plasmid pSD486, a derivative of pSD477 containing a polylinker region but no initiation codon at the u deletion junction, was utilized. First the ClaI/HpaI vector fragment from pSD477 referred to above was ligated with annealed synthetic oligonucleotides SD42mer/SD40mer (SEQ ID NO:8/SEQ ID NO:9)  
                                       Cla I           Sac I         Xho I         Hpa I               SD42mer       5′ CGATTACTAGATCTGAGCTCCCCGGGCTCGAGGGATCCGTT   3′               SD40mer       3′   TAATGATCTAGACTCGAGGGGCCCGAGCTCCCTAGGCAA   5′                   Bgl II        Sma I          Bam HI          
 
         [0136]    generating plasmid pSD478. Next the EcoRI site at the pUC/vaccinia junction was destroyed by digestion of pSD478 with EcoRI followed by blunt ending with Klenow fragment of  E. coli  polymerase and ligation, generating plasmid pSD478E − . pSD478E −  was digested with BamHI and HpaI and ligated with annealed synthetic oligonucleotides HEM5/HEM6 (SEQ ID NO:10/SEQ ID NO:11)  
                                                  Bam HI  Eco RI    Hpa I               HEM5   5′  GATCCGAATTCTAGCT 3′           HEM6   3′      GCTTAAGATCGA 5′          
 
         [0137]    generating plasmid pSD486. pSD486 was used as donor plasmid for recombination with recombinant vaccinia virus vP533, generating vP553, which was isolated as a clear plaque in the presence of X-gal.  
       Example 3  
     Construction of Plasmid pMP494Δ for Deletion of ATI Region (A26L)  
       [0138]    Referring now to FIG. 3, pSD414 contains SalI B cloned into pUC8. To remove unwanted DNA sequences to the left of the A26L region, pSD414 was cut with XbaI within vaccinia sequences (pos. 137,079) and with HindIII at the pUC/vaccinia junction, then blunt ended with Klenow fragment of  E. coli  polymerase and ligated, resulting in plasmid pSD483. To remove unwanted vaccinia DNA sequences to the right of the A26L region, pSD483 was cut with EcoRI (pos. 140,665 and at the pUC/vaccinia junction) and ligated, forming plasmid pSD484. To remove the A26L coding region, pSD484 was cut with NdeI (partial) slightly upstream from the A26L ORF (pos. 139,004) and with HpaI (pos. 137,889) slightly downstream from the A26L ORF. The 5.2 kb vector fragment was isolated and ligated with annealed synthetic oligonucleotides ATI3/ATI4 (SEQ ID NO:12/SEQ ID NO:13)  
                                      Nde I                                                           Bgl II  Eco RI  Hpa I           ATI3   5′  TATGAGTAACTTAACTCTTTTGTTAATTAAAAGTATATTCAAAAAATAAGTTATATAAATAGATCTGAATTCGTT  3′  ATI3               ATI4   3′    ACTCATTGAATTGAGAAAACAATTAATTTTCATATAAGTTTTTTATTCAATATATTTATCTAGACTTAAGCAA  3′  ATI4          
 
         [0139]    reconstructing the region upstream from A26L and replacing the A26L ORF with a short polylinker region containing the restriction sites BglII, EcoRI and HpaI, as indicated above. The resulting plasmid was designated pSD485. Since the BglII and EcoRI sites in the polylinker region of pSD485 are not unique, unwanted BglII and EcoRI sites were removed from plasmid pSD483 (described above) by digestion with BglII (pos. 140,136) and with EcoRI at the pUC/vaccinia junction, followed by blunt ending with Klenow fragment of  E. coli  polymerase and ligation. The resulting plasmid was designated pSD489. The 1.8 kb ClaI (pos. 137,198)/EcoRV (pos. 139,048) fragment from pSD489 containing the A26L ORF was replaced with the corresponding 0.7 kb polylinker-containing ClaI/EcoRV fragment from pSD485, generating pSD492. The BalII and EcoRI sites in the polylinker region of pSD492 are unique.  
         [0140]    A 3.3 kb BglII cassette containing the  E. coli  Beta-galactosidase gene (Shapira et al., 1983) under the control of the vaccinia 11 kDa promoter (Bertholet et al., 1985; Perkus et al., 1990) was inserted into the BglII site of pSD492, forming pSD493KBG. Plasmid pSD493KBG was used in recombination with rescuing virus vP553. Recombinant vaccinia virus, vP581, containing Beta-galactosidase in the A26L deletion region, was isolated as a blue plaque in the presence of X-gal.  
         [0141]    To generate a plasmid for the removal of Beta-galactosidase sequences from vaccinia recombinant virus vP581, the polylinker region of plasmid pSD492 was deleted by mutagenesis (Mandecki, 1986) using synthetic oligonucleotide MPSYN177 (SEQ ID NO:14) (5′ AAAATGGGCGTGGATTGTTAACTTTATATAACTTATTTTTTGAATATAC 3′). In the resulting plasmid, pMP494Δ, vaccinia DNA encompassing positions [137,889-138,937], including the entire A26L ORF is deleted. Recombination between the pMP494Δ and the Beta-galactosidase containing vaccinia recombinant, vP581, resulted in vaccinia deletion mutant vP618, which was isolated as a clear plaque in the presence of X-gal.  
       Example 4  
     Construction of Plasmid pSD467 for Deletion of Hemagglutinin Gene (A56R)  
       [0142]    Referring now to FIG. 4, vaccinia SalI G restriction fragment (pos. 160,744-173,351) crosses the HindIII A/B junction (pos. 162,539). pSD419 contains vaccinia SalI G cloned into pUC8. The direction of transcription for the hemagglutinin (HA) gene is indicated by an arrow in FIG. 4. Vaccinia sequences derived from HindIII B were removed by digestion of pSD419 with HindIII within vaccinia sequences and at the pUC/vaccinia junction followed by ligation. The resulting plasmid, pSD456, contains the HA gene, A56R, flanked by 0.4 kb of vaccinia sequences to the left and 0.4 kb of vaccinia sequences to the right. A56R coding sequences were removed by cutting pSD456 with RsaI (partial; pos. 161,090) upstream from A56R coding sequences, and with EagI (pos. 162,054) near the end of the gene. The 3.6 kb RsaI/EagI vector fragment from pSD456 was isolated and ligated with annealed synthetic oligonucleotides MPSYN59 (SEQ ID NO:15), MPSYN62 (SEQ ID NO:16), MPSYN60 (SEQ ID NO:17), and MPSYN61 (SEQ ID NO:18)  
                                       MPSYN59                  Rsa I           5′ ACACGAATGATTTTCTAAAGTATTTGGAAAGTTTTATAGGT-                       MPSYN62           3′ TGTGCTTACTAAAAGATTTCATAAACCTTTCAAAATATCCA-                       MPSYN59           AGTTGATAGAACAAAATACATAATTT 3′                       MPSYN62           TCAACTATCT 5′                       MPSYN60           5′                 TGTAAAAATAAATCACTTTTTATA-                       MPSYN61           3′ TGTTTTATGTATTAAAACATTTTTATTTAGTGAAAAATAT-                       MPSYN60               Bgl II  Sma I   Pst I   Eaq I           CTAAGATCTCCCGGGCTGCAGC      3′                       MPSYN61           GATTCTAGAGGGCCCGACGTCGCCGG5 ′          
 
         [0143]    reconstructing the DNA sequences upstream from the A56R ORF and replacing the A56R ORF with a polylinker region as indicated above. The resulting plasmid is pSD466. The vaccinia deletion in pSD466 encompasses positions [161,185-162,053]. The site of the deletion in pSD466 is indicated by a triangle in FIG. 4.  
         [0144]    A 3.2 kb BglII/BamHI (partial) cassette containing the  E. coli  Beta-galactosidase gene (Shapira et al., 1983) under the control of the vaccinia 11 kDa promoter (Bertholet et al., 1985; Guo et al., 1989) was inserted into the BglII site of pSD466, forming pSD466KBG. Plasmid pSD466KBG was used in recombination with rescuing virus vP618. Recombinant vaccinia virus, vP708, containing Beta-galactosidase in the A56R deletion, was isolated as a blue plaque in the presence of X-gal.  
         [0145]    Beta-galactosidase sequences were deleted from vP708 using donor plasmid pSD467. pSD467 is identical to pSD466, except that EcoRI, SmaI and BamHI sites were removed from the pUC/vaccinia junction by digestion of pSD466 with EcoRI/BamHI followed by blunt ending with Klenow fragment of  E. coli  polymerase and ligation. Recombination between vP708 and pSD467 resulted in recombinant vaccinia deletion mutant, vP723, which was isolated as a clear plaque in the presence of X-gal.  
       Example 5  
     Construction of Plasmid pMPCSK1Δ for Deletion of Open Reading Frames [C7L−K1L] 
       [0146]    Referring now to FIG. 5, the following vaccinia clones were utilized in the construction of pMPCSK1Δ. pSD420 is SalI H cloned into pUC8. pSD435 is KpnI F cloned into pUC18. pSD435 was cut with SphI and religated, forming pSD451. In pSD451, DNA sequences to the left of the SphI site (pos. 27,416) in HindIII M are removed (Perkus et al., 1990). pSD409 is HindIII M cloned into pUC8.  
         [0147]    To provide a substrate for the deletion of the [C7L−K1L] gene cluster from vaccinia,  E. coli  Beta-galactosidase was first inserted into the vaccinia M2L deletion locus (Guo et al., 1990) as follows. To eliminate the BglII site in pSD409, the plasmid was cut with BglII in vaccinia sequences (pos. 28,212) and with BamHI at the pUC/vaccinia junction, then ligated to form plasmid pMP409B. pMP409B was cut at the unique SphI site (pos. 27,416). M2L coding sequences were removed by mutagenesis (Guo et al., 1990; Mandecki, 1986) using synthetic oligonucleotide  
                                                                  Bgl II               MPSYN82   5′ TTTCTGTATATTTGCACCAATTTAGATCTT-ACTCAAAATATGTAACAATA 3′   (SEQ ID NO:19)          
 
         [0148]    The resulting plasmid, pMP409D, contains a unique BglII site inserted into the M2L deletion locus as indicated above. A 3.2 kb BamHI (partial)/BglII cassette containing the  E. coli  Beta-galactosidase gene (Shapira et al., 1983) under the control of the 11 kDa promoter (Bertholet et al., 1985) was inserted into pMP409D cut with BglII. The resulting plasmid, pMP409DBG (Guo et al., 1990), was used as donor plasmid for recombination with rescuing vaccinia virus vP723. Recombinant vaccinia virus, vP784, containing Beta-galactosidase inserted into the M2L deletion locus, was isolated as a blue plaque in the presence of X-gal.  
         [0149]    A plasmid deleted for vaccinia genes [C7L−K1L] was assembled in pUC8 cut with SmaI, HindIII and blunt ended with Klenow fragment of  E. coli  polymerase. The left flanking arm consisting of vaccinia HindIII C sequences was obtained by digestion of pSD420 with XbaI (pos. 18,628) followed by blunt ending with Klenow fragment of  E. coli  polymerase and digestion with BglII (pos. 19,706). The right flanking arm consisting of vaccinia HindIII K sequences was obtained by digestion of pSD451 with BglII (pos. 29,062) and EcoRV (pos. 29,778). The resulting plasmid, pMP581CK is deleted for vaccinia sequences between the BglII site (pos. 19,706) in HindIII C and the BglII site (pos. 29,062) in HindIII K. The site of the deletion of vaccinia sequences in plasmid pMP581CK is indicated by a triangle in FIG. 5.  
         [0150]    To remove excess DNA at the vaccinia deletion junction, plasmid pMP581CK, was cut at the NcoI sites within vaccinia sequences (pos. 18,811; 19,655), treated with Bal-31 exonuclease and subjected to mutagenesis (Mandecki, 1986) using synthetic oligonucleotide MPSYN233 (SEQ ID NO:20) 5′-TGTCATTTAACACTATACTCATATTAATAAAAATAATATTTATT-3′. The resulting plasmid, pMPCSK1Δ, is deleted for vaccinia sequences positions 18,805-29,108, encompassing 12 vaccinia open reading frames [C7L−K1L]. Recombination between pMPCSK1Δ and the Beta-galactosidase containing vaccinia recombinant, vP784, resulted in vaccinia deletion mutant, vP804, which was isolated as a clear plaque in the presence of X-gal.  
       Example 6  
     Construction of Plasmid pSD548 for Deletion of Large Subunit, Ribonucleotide Reductanse (I4L)  
       [0151]    Referring now to FIG. 6, plasmid pSD405 contains vaccinia HindIII I (pos. 63,875-70,367) cloned in pUC8. pSD405 was digested with EcoRV within vaccinia sequences (pos. 67,933) and with SmaI at the pUC/vaccinia junction, and ligated, forming plasmid pSD518. pSD518 was used as the source of all the vaccinia restriction fragments used in the construction of pSD548.  
         [0152]    The vaccinia I4L gene extends from position 67,371-65,059. Direction of transcription for I4L is indicated by an arrow in FIG. 6. To obtain a vector plasmid fragment deleted for a portion of the I4L coding sequences, pSD518 was digested with BamHI (pos. 65,381) and HpaI (pos. 67,001) and blunt ended using Klenow fragment of  E. coli  polymerase. This 4.8 kb vector fragment was ligated with a 3.2 kb SmaI cassette containing the  E. coli  Beta-galactosidase gene (Shapira et al., 1983) under the control of the vaccinia 11 kDa promoter (Bertholet et al., 1985; Perkus et al., 1990), resulting in plasmid pSD524KBG. pSD524KBG was used as donor plasmid for recombination with vaccinia virus vP804. Recombinant vaccinia virus, vP855, containing Beta-galactosidase in a partial deletion of the I4L gene, was isolated as a blue plaque in the presence of X-gal.  
         [0153]    To delete Beta-galactosidase and the remainder of the I4L ORF from vP855, deletion plasmid pSD548 was constructed. The left and right vaccinia flanking arms were assembled separately in pUC8 as detailed below and presented schematically in FIG. 6.  
         [0154]    To construct a vector plasmid to accept the left vaccinia flanking arm, pUC8 was cut with BamHI/EcoRI and ligated with annealed synthetic oligonucleotides 518A1/518A2 (SEQ ID NO:21/SEQ ID NO:22)  
                                       Bam HI    Rsa I                                                   Bgl II     Eco RI           518A1   5′ GATCCTGAGTACTTTGTAATATAATGATATATATTTTCACTTTATCTCATTTGAGAATAAAAAGATCTTAGG     3′ 518A1               518A2   3′     ACTCATGAAACATTATATTACTATATATAAAAGTGAAATAGAGTAAACTCTTATTTTTCTAGAATCCTTAA  5′ 518A2          
 
         [0155]    forming plasmid pSD531. pSD531 was cut with RsaI (partial) and BamHI and a 2.7 kb vector fragment isolated. pSD518 was cut with BglII (pos. 64,459)/RsaI (pos. 64,994) and a 0.5 kb fragment isolated. The two fragments were ligated together, forming pSD537, which contains the complete vaccinia flanking arm left of the I4L coding sequences.  
         [0156]    To construct a vector plasmid to accept the right vaccinia flanking arm, pUC8 was cut with BamHI/EcoRI and ligated with annealed synthetic oligonucleotides 518B1/518B2 (SEQ ID NO:23/SEQ ID NO:24)  
                                       Bam HI  Bgl II  Sma I                                                   Rsa I    Eco RI           518B1   5′  GATCCAGATCTCCCGGGAAAAAAATTATTTAACTTTTCATTAATAG-GGATTTGACGTATGTAGCGTACTAGG     3′ 518B1               518B2   3′      GTCTAGAGGGCCCTTTTTTTAATAAATTGAAAAGTAATTATC-CCTAAACTGCATACTACGCATGATCCTTAA 5′ 518B2          
 
         [0157]    forming plasmid pSD532. pSD532 was cut with RsaI (partial)/EcoRI and a 2.7 kb vector fragment isolated. pSD518 was cut with RsaI within vaccinia sequences (pos. 67,436) and EcoRI at the vaccinia/pUC junction, and a 0.6 kb fragment isolated. The two fragments were ligated together, forming pSD538, which contains the complete vaccinia flanking arm to the right of I4L coding sequences.  
         [0158]    The right vaccinia flanking arm was isolated as a 0.6 kb EcoRI/BglII fragment from pSD538 and ligated into pSD537 vector plasmid cut with EcoRI/BglII. In the resulting plasmid, pSD539, the I4L ORF (pos. 65,047-67,386) is replaced by a polylinker region, which is flanked by 0.6 kb vaccinia DNA to the left and 0.6 kb vaccinia DNA to the right, all in a pUC background. The site of deletion within vaccinia sequences is indicated by a triangle in FIG. 6. To avoid possible recombination of Beta-galactosidase sequences in the pUC-derived portion of pSD539 with Beta-galactosidase sequences in recombinant vaccinia virus vP855, the vaccinia I4L deletion cassette was moved from pSD539 into pRC11, a pUC derivative from which all Beta-galactosidase sequences have been removed and replaced with a polylinker region (Colinas et al., 1990). pSD539 was cut with EcoRI/PstI and the 1.2 kb fragment isolated. This fragment was ligated into pRC11 cut with EcoRI/PstI (2.35 kb), forming pSD548. Recombination between pSD548 and the Beta-galactosidase containing vaccinia recombinant, vP855, resulted in vaccinia deletion mutant vP866, which was isolated as a clear plaque in the presence of X-gal.  
         [0159]    DNA from recombinant vaccinia virus vP866 was analyzed by restriction digests followed by electrophoresis on an agarose gel. The restriction patterns were as expected. Polymerase chain reactions (PCR) (Engelke et al., 1988) using vP866 as template and primers flanking the six deletion loci detailed above produced DNA fragments of the expected sizes. Sequence analysis of the PCR generated fragments around the areas of the deletion junctions confirmed that the junctions were as expected. Recombinant vaccinia virus vP866, containing the six engineered deletions as described above, was designated vaccinia vaccine strain “NYVAC.” 
       Example 7  
     Insertion of a Rabies Glycoprotein G Gene into NYVAC  
       [0160]    The gene encoding rabies glycoprotein G under the control of the vaccinia H6 promoter (Taylor et al., 1988a,b) was inserted into TK deletion plasmid pSD513. pSD513 is identical to plasmid pSD460 (FIG. 1) except for the presence of a polylinker region.  
         [0161]    Referring now to FIG. 7, the polylinker region was inserted by cutting pSD460 with SmaI and ligating the plasmid vector with annealed synthetic oligonucleotides VQ1A/VQ1B (SEQ ID NO:25/SEQ ID NO:26)  
                                       VQ1A                 SmaI BglII XhoI  PstI  NarI  BamHI           5′  GGGAGATCTCTCGAGCTGCAGGGCGCCGGATCCTTTTTCT  3′                       VQ1B           3′  CCCTCTAGAGAGCTCGACGTCCCGCGGCCTAGGAAAAAGA  5′          
 
         [0162]    to form vector plasmid pSD513. pSD513 was cut with SmaI and ligated with a SmaI ended 1.8 kb cassette containing the gene encoding the rabies glycoprotein G gene under the control of the vaccinia H6 promoter (Taylor et al., 1988a,b). The resulting plasmid was designated pRW842. pRW842 was used as donor plasmid for recombination with NYVAC rescuing virus (vP866). Recombinant vaccinia virus vP879 was identified by plaque hybridization using  32 P-labelled DNA probe to rabies glycoprotein G coding sequences.  
         [0163]    The modified recombinant viruses of the present invention provide advantages as recombinant vaccine vectors. The attenuated virulence of the vector advantageously reduces the opportunity for the possibility of a runaway infection due to vaccination in the vaccinated individual and also diminishes transmission from vaccinated to unvaccinated individuals or contamination of the environment.  
         [0164]    The modified recombinant viruses are also advantageously used in a method for expressing a gene product in a cell cultured in vitro by introducing into the cell the modified recombinant virus having foreign DNA which codes for and expresses gene products in the cell.  
       Example 8  
     Construction of TROVAC-NDV Expressing the Fusion and Hemagglutinin-Neuraminidase Glycoproteins of Newcastle Disease Virus  
       [0165]    This example describes the development of TROVAC, a fowlpox virus vector and, of a fowlpox Newcastle Disease Virus recombinant designated TROVAC-NDV and its safety and efficacy. A fowlpox virus (FPV) vector expressing both F and HN genes of the virulent NDV strain Texas was constructed. The recombinant produced was designated TROVAC-NDV. TROVAC-NDV expresses authentically processed NDV glycoproteins in avian cells infected with the recombinant virus and inoculation of day old chicks protects against subsequent virulent NDV challenge.  
         [0166]    Cells and Viruses. The Texas strain of NDV is a velogenic strain. Preparation of cDNA clones of the F and HN genes has been previously described (Taylor et al., 1990; Edbauer et al., 1990). The strain of FPV designated FP-1 has been described previously (Taylor et al., 1988a). It is a vaccine strain useful in vaccination of day old chickens. The parental virus strain Duvette was obtained in France as a fowlpox scab from a chicken. The virus was attenuated by approximately 50 serial passages in chicken embryonated eggs followed by 25 passages on chicken embryo fibroblast cells. The virus was subjected to four successive plaque purifications. One plaque isolate was further amplified in primary CEF cells and a stock virus, designated as TROVAC, established. The stock virus used in the in vitro recombination test to produce TROVAC-NDV had been subjected to twelve passages in primary CEF cells from the plaque isolate.  
         [0167]    Construction of a Cassette for NDV-F. A 1.8 kbp BamHI fragment containing all but 22 nucleotides from the 5′ end of the F protein coding sequence was excised from pNDV81 (Taylor et al., 1990) and inserted at the BamHI site of pUC18 to form pCE13. The vaccinia virus H6 promoter previously described (Taylor et al., 1988a,b; Guo et al., 1989; Perkus et al., 1989) was inserted into pCE13 by digesting pCE13 with SalI, filling in the sticky ends with Klenow fragment of  E. coli  DNA polymerase and digesting with HindIII. A HindIII-EcoRV fragment containing the H6 promoter sequence was then inserted into pCE13 to form pCE38. A perfect 5′ end was generated by digesting pCE38 with KpnI and NruI and inserting the annealed and kinased oligonucleotides CE75 (SEQ ID NO:27) and CE76 (SEQ ID NO:28) to generate pCE47.  
                           CE75:           CGATATCCGTTAAGTTTGTATCGTAATGGGCTCCAGATCTTCTACCAGGA       TCCCGGTAC               CE76:       CGGGATCCTGGTAGAAGATCTGGAGCCCATTACGATACAAACTTAACGGA       TATCG.          
 
         [0168]    In order to remove non-coding sequence from the 3′ end of the NDV-F a SmaI to PstI fragment from pCE13 was inserted into the SmaI and PstI sites of pUCI8 to form pCE23. The non-coding sequences were removed by sequential digestion of pCE23 with SacI, BamHI, Exonuclease III, SI nuclease and EcoRI. The annealed and kinased oligonucleotides CE42 (SEQ ID NO:29) and CE43 (SEQ ID NO:30) were then inserted to form pCE29.  
                                           CE42:   AATTCGAGCTCCCCGGG                           CE43:   CCCGGGGAGCTCG          
 
         [0169]    The 3′ end of the NDV-F sequence was then inserted into plasmid pCE20 already containing the 5′ end of NDV-F by cloning a PstI-SacI fragment from pCE29 into the PstI and SacI sites of pCE20 to form pCE32. Generation of pCE20 has previously been described in Taylor et al., 1990.  
         [0170]    In order to align the H6 promoter and NDV-F 5′ sequences contained in pCE47 with the 3′ NDV-F sequences contained in pCE32, a HindIII-PstI fragment of pCE47 was inserted into the HindIII and PstI sites of pCE32 to form pCE49. The H6 promoted NDV-F sequences were then transferred to the de-ORFed F8 locus (described below) by cloning a HindIII-NruI fragment from pCE49 into the HindIII and SmaI sites of pJCA002 (described below) to form pCE54. Transcription stop signals were inserted into pCE54 by digesting pCE54 with SacI, partially digesting with BamHI and inserting the annealed and kinased oligonucleotides CE166 (SEQ ID NO:31) and CE167 (SEQ ID NO:32) to generate pCE58.  
                                           CE166:   CTTTTTATAAAAAGTTAACTACGTAG                           CE167:   GATCCTACGTAGTTAACTTTTTATAAAAAGAGCT          
 
         [0171]    A perfect 3′ end for NDV-F was obtained by using the polymerase chain reaction (PCR) with pCE54 as template and oligonucleotides CE182 (SEQ ID NO:33) and CE183 (SEQ ID NO:34) as primers.  
                               CE182:   CTTAACTCAGCTGACTATCC                   CE183:   TACGTAGTTAACTTTTTATAAAAATCATATTTTTGTAGTGGCTC          
 
         [0172]    The PCR fragment was digested with PvuII and HpaI and cloned into pCE58 that had been digested with HaI and partially digested with PvuII. The resulting plasmid was designated pCE64. Translation stop signals were inserted by cloning a HindIII-HpaI fragment which contains the complete H6 promoter and F coding sequence from pCE64 into the HindIII and HpaI sites of pRW846 to generate pCE71, the final cassette for NDV-F. Plasmid pRW846 is essentially equivalent to plasmid pJCA002 (described below) but containing the H6 promoter and transcription and translation stop signals. Digestion of pRW846 with HindIII and HvaI eliminates the H6 promoter but leaves the stop signals intact.  
         [0173]    Construction of Cassette for NDV-HN. Construction of plasmid pRW802 was previously described in Edbauer et al., 1990. This plasmid contains the NDV-HN sequences linked to the 3′ end of the vaccinia virus H6 promoter in a pUC9 vector. A HindIII-EcoRV fragment encompassing the 5′ end of the vaccinia virus H6 promoter was inserted into the HindIII and EcoRV sites of pRW802 to form pRW830. A perfect 3′ end for NDV-HN was obtained by inserting the annealed and kinased oligonucleotides CE162 (SEQ ID NO:35) and CE163 (SEQ ID NO:36) into the EcoRI site of pRW830 to form pCE59, the final cassette for NDV-HN.  
                           CE162:           AATTCAGGATCGTTCCTTTACTAGTTGAGATTCTCAAGGATGATGGGATT       TAATTTTTATAAGCTTG               CE163:       AATTCAAGCTTATAAAAATTAAATCCCATCATCCTTGAGAATCTCAACTA       GTAAAGGAACGATCCTG          
 
         [0174]    Construction of FPV Insertion Vector. Plasmid pRW731-15 contains a 10 kb PvuII-PvuII fragment cloned from genomic DNA. The nucleotide sequence was determined on both strands for a 3660 bp PvuII-EcoRV fragment. The limits of an open reading frame designated here as F8 were determined. Plasmid pRW761 is a sub-clone of pRW731-15 containing a 2430 bp EcoRV-EcoRV fragment. The F8 ORF was entirely contained between an XbaI site and an SspI site in pRW761. In order to create an insertion plasmid which on recombination with TROVAC genomic DNA would eliminate the F8 ORF, the following steps were followed. Plasmid pRW761 was completely digested with XbaI and partially digested with SspI. A 3700 bp XbaI-SspI band was isolated from the gel and ligated with the annealed double-stranded oligonucleotides JCA017 (SEQ ID NO:37) and JCA018 (SEQ ID NO:38).  
                           JCA017: 5′           CTAGACACTTTATGTTTTTTAATATCCGGTCTTAAAAGCTTCCCGGGGAT       CCTTATACGGGGAATAAT               JCA018: 5′       ATTATTCCCCGTATAAGGATCCCCCGGGAAGCTTTTAAGACCGGATATTA       AAAAACATAAAGTGT          
 
         [0175]    The plasmid resulting from this ligation was designated pJCA002.  
         [0176]    Construction of Double Insertion Vector for NDV F and HN. The H6 promoted NDV-HN sequence was inserted into the H6 promoted NDV-F cassette by cloning a HindIII fragment from pCE59 that had been filled in with Klenow fragment of  E. coli  DNA polymerase into the HpaI site of pCE71 to form pCE80. Plasmid pCE80 was completely digested with NdeI and partially digested with BglII to generate an NdeI-BglII 4760 bp fragment containing the NDV F and HN genes both driven by the H6 promoter and linked to F8 flanking arms. Plasmid pJCA021 was obtained by inserting a 4900 bp PvuII-HindII fragment from pRW731-15 into the SmaI and HindII sites of pBSSK+. Plasmid pJCA021 was then digested with NdeI and BglII and ligated to the 4760 bp NdeI-BglII fragment of pCE80 to form pJCA024. Plasmid pJCA024 therefore contains the NDV-F and HN genes inserted in opposite orientation with 3′ ends adjacent between FPV flanking arms. Both genes are linked to the vaccinia virus H6 promoter. The right flanking arm adjacent to the NDV-F sequence consists of 2350 bp of FPV sequence. The left flanking arm adjacent to the NDV-HN sequence consists of 1700 bp of FPV sequence.  
         [0177]    Development of TROVAC-NDV. Plasmid pJCA024 was transfected into TROVAC infected primary CEF cells by using the calcium phosphate precipitation method previously described (Panicali et al., 1982; Piccini et al., 1987). Positive plaques were selected on the basis of hybridization to specific NDV-F and HN radiolabelled probes and subjected to five sequential rounds of plaque purification until a pure indicate that expression of either HN or F alone is sufficient to elicit protective immunity against NDV challenge. Work on other paramyxoviruses has indicated, however, that antibody to both proteins may be required for full protective immunity. It has been demonstrated that SV5 virus could spread in tissue culture in the presence of antibody to the HN glycoprotein but not to the F glycoprotein (Merz et al., 1980). In addition, it has been suggested that vaccine failures with killed measles virus vaccines were due to inactivation of the fusion component (Norrby et al., 1975). Since both NDV glycoproteins have been shown to be responsible for eliciting virus neutralizing antibody (Avery et al., 1979) and both glycoproteins, when expressed individually in a fowlpox vector are able to induce a protective immune response, it can be appreciated that the most efficacious NDV vaccine should express both glycoproteins.  
       Example 9  
     Construction of ALVAC Recombinants Expressing Rabies Virus Glycoprotein G  
       [0178]    This example describes the development of ALVAC, a canarypox virus vector and, of a canarypox-rabies recombinant designated as ALVAC-RG (vCP65) and its safety and efficacy.  
         [0179]    Cells and Viruses. The parental canarypox virus (Rentschler strain) is a vaccinal strain for canaries. The vaccine strain was obtained from a wild type isolate and attenuated through more than 200 serial passages on chick embryo fibroblasts. A master viral seed was subjected to four successive plaque purifications under agar and one plaque clone was amplified through five additional passages after which the stock virus was used as the parental virus in in vitro recombination tests. The plaque purified canarypox isolate is designated ALVAC.  
         [0180]    Construction of a Canarypox Insertion Vector. An 880 bp canarypox PvuII fragment was cloned between the PvuII sites of pUC9 to form pRW764.5. The sequence of this fragment is shown in FIG. 8 between positions 1372 and 2251. The limits of an population was achieved. One representative plaque was then amplified and the resulting TROVAC recombinant was designated TROVAC-NDV (vFP96).  
         [0181]    Immunofluorescence. Indirect immunofluorescence was performed as described (Taylor et al., 1990) using a polyclonal anti-NDV serum and, as mono-specific reagents, sera produced in rabbits against vaccinia virus recombinants expressing NDV-F or NDV-HN.  
         [0182]    Immunoprecipitation. Immunoprecipitation reactions were performed as described (Taylor et al., 1990) using a polyclonal anti-NDV serum obtained from SPAFAS Inc., Storrs, Conn.  
         [0183]    The stock virus was screened by in situ plaque hybridization to confirm that the F8 ORF was deleted. The correct insertion of the NDV genes into the TROVAC genome and the deletion of the F8 ORF was also confirmed by Southern blot hybridization.  
         [0184]    In NDV-infected cells, the F glycoprotein is anchored in the membrane via a hydrophobic transmembrane region near the carboxyl terminus and requires post-translational cleavage of a precursor, F 0 , into two disulfide linked polypeptides F 1  and F 2 . Cleavage of F 0  is important in determining the pathogenicity of a given NDV strain (Homma and Ohuchi, 1973; Nagai et al., 1976; Nagai et al., 1980), and the sequence of amino acids at the cleavage site is therefore critical in determining viral virulence. It has been determined that amino acids at the cleavage site in the NDV-F sequence inserted into FPV to form recombinant vFP29 had the sequence Arg-Arg-Gln-Arg-Arg (SEQ ID NO:39) (Taylor et al., 1990) which conforms to the sequence found to be a requirement for virulent NDV strains (Chambers et al., 1986; Espion et al., 1987; Le et al., 1988; McGinnes and Morrison, 1986; Toyoda et al., 1987). The HN glycoprotein synthesized in cells infected with virulent strains of NDV is an uncleaved glycoprotein of 74 kDa. Extremely avirulent strains such as Ulster and Queensland encode an HN precursor (HNo) which requires cleavage for activation (Garten et al., 1980).  
         [0185]    The expression of F and HN genes in TROVAC-NDV was analyzed to confirm that the gene products were authentically processed and presented. Indirect-immunofluorescence using a polyclonal anti-NDV chicken serum confirmed that immunoreactive proteins were presented on the infected cell surface. To determine that both proteins were presented on the plasma membrane, mono-specific rabbit sera were produced against vaccinia recombinants expressing either the F or HN glycoproteins. Indirect immunofluorescence using these sera confirmed the surface presentation of both proteins.  
         [0186]    Immunoprecipitation experiments were performed by using ( 35 S) methionine labeled lysates of CEF cells infected with parental and recombinant viruses. The expected values of apparent molecular weights of the glycolysated forms of F 1  and F 2  are 54.7 and 10.3 kDa respectively (Chambers et al., 1986). In the immunoprecipitation experiments using a polyclonal anti-NDV serum, fusion specific products of the appropriate size were detected from the NDV-F single recombinant vFP29 (Taylor et al., 1990) and the TROVAC-NDV double recombinant vFP96. The HN glycoprotein of appropriate size was also detected from the NDV-HN single recombinant VFP-47 (Edbauer et al., 1990) and TROVAC-NDV. No NDV specific products were detected from uninfected and parental TROVAC infected CEF cells.  
         [0187]    In CEF cells, the F and HN glycoproteins are appropriately presented on the infected cell surface where they are recognized by NDV immune serum. Immunoprecipitation analysis indicated that the F 0  protein is authentically cleaved to the F 1  and F 2  components required in virulent strains. Similarly, the HN glycoprotein was authentically processed in CEF cells infected with recombinant TROVAC-NDV.  
         [0188]    Previous reports (Taylor et al., 1990; Edbauer et al., 1990; Boursnell et al., 1990a,b,c; Ogawa et al., 1990) would open reading frame designated as C5 were defined. It was determined that the open reading frame was initiated at position 166 within the fragment and terminated at position 487. The C5 deletion was made without interruption of open reading frames. Bases from position 167 through position 455 were replaced with the sequence (SEQ ID NO:39) GCTTCCCGGGAATTCTAGCTAGCTAGTTT. This replacement sequence contains HindIII, SmaI and EcoRI insertion sites followed by translation stops and a transcription termination signal recognized by vaccinia virus RNA polymerase (Yuen et al., 1987). Deletion of the C5 ORF was performed as described below. Plasmid pRW764.5 was partially cut with RsaI and the linear product was isolated. The RsaI linear fragment was recut with BglII and the pRW764.5 fragment now with a RsaI to BglII deletion from position 156 to position 462 was isolated and used as a vector for the following synthetic oligonucleotides:  
                                   RW145:   ACTCTCAAAAGCTTCCCGGGAATTCTAGCTAGCTAGTTTTTATAAA   (SEQ ID NO:40)                   RW146:   GATCTTTATAAAAACTAGCTAGCTAGAATTCCCGGGAAGCTTTTGAGAGT   (SEQ ID NO:41)          
 
         [0189]    Oligonucleotides RW145 and RW146 were annealed and inserted into the pRW 764.5 RsaI and BglII vector described above. The resulting plasmid is designated pRWS31.  
         [0190]    Construction of Insertion Vector Containing the Rabies G Gene. Construction of pRW838 is illustrated below. oligonucleotides A through E, which overlap the translation initiation codon of the H6 promoter with the ATG of rabies G, were cloned into pUC9 as pRW737. Oligonucleotides A through E contain the H6 promoter, starting at NruI, through the HindIII site of rabies G followed by BglII. Sequences of oligonucleotides A through E ((SEQ ID NO:42)-(SEQ ID NO:46)) are:  
                                             A:   CTGAAATTATTTCATTATCGCGATATCCGTTAAGTTTGTATCGTAATGGTTCCTCAGGCTCTCCTGTTTGT   (SEQ ID NO:42)                   B:   CATTACGATACAAACTTAACGGATATCGCGATAATGAAATAATTTCAG   (SEQ ID NO:43)               C:   ACCCCTTCTGGTTTTTCCGTTGTGTTTTGGGAAATTCCCTATTTACACGATCCCAGACAAGCTTAGATCTCAG   (SEQ ID NO:44)               D:   CTGAGATCTAAGCTTGTCTGGGATCGTGTAAATAGGGAATTTCCCAAAACA   (SEQ ID NO:45)               E:   CAACGGAAAAACCAGAAGGGGTACAAACAGGAGAGCCTGAGGAAC   (SEQ ID NO:46)            The diagram of annealed oligonucleotides A through E is as           follows:                  A                            C       -------------------------¦---------------------------       -----------------¦-------------------¦---------------             B                    E                 D          
 
         [0191]    Oligonucleotides A through E were kinased, annealed (95° C. for 5 minutes, then cooled to room temperature), and inserted between the PvuII sites of pUC9. The resulting plasmid, pRW737, was cut with HindIII and BglII and used as a vector for the 1.6 kbp HindIII-BglII fragment of ptg155PRO (Kieny et al., 1984) generating pRW739. The ptg155PRO HindIII site is 86 bp downstream of the rabies G translation initiation codon. BglII is downstream of the rabies G translation stop codon in ptg155PRO. pRW739 was partially cut with NruI, completely cut with BglII, and a 1.7 kbp NruI-BglII fragment, containing the 3′ end of the H6 promoter previously described (Taylor et al., 1988a,b; Guo et al., 1989; Perkus et al., 1989) through the entire rabies G gene, was inserted between the NruI and BamHI sites of pRW824. The resulting plasmid is designated pRW832. Insertion into pRW824 added the H6 promoter 5′ of NruI. The pRW824 sequence of BamHI followed by SmaI is (SEQ ID NO:47): GGATCCCCGGG. pRW824 is a plasmid that contains a nonpertinent gene linked precisely to the vaccinia virus H6 promoter. Digestion with NruI and BamHI completely excised this nonpertinent gene. The 1.8 kbp pRW832 SmaI fragment, containing H6 promoted rabies G, was inserted into the SmaI of pRW831, to form plasmid pRW838.  
         [0192]    Development of ALVAC-RG. Plasmid pRW838 was transfected into ALVAC infected primary CEF cells by using the calcium phosphate precipitation method previously described (Panicali et al., 1982; Piccini et al., 1987). Positive plaques were selected on the basis of hybridization to a specific rabies G probe and subjected to 6 sequential rounds of plaque purification until a pure population was achieved. One representative plaque was then amplified and the resulting ALVAC recombinant was designated ALVAC-RG (vCP65) (see also FIG. 9). The correct insertion of the rabies G gene into the ALVAC genome without subsequent mutation was confirmed by sequence analysis.  
         [0193]    Immunofluorescence. During the final stages of assembly of mature rabies virus particles, the glycoprotein component is transported from the golgi apparatus to the plasma membrane where it accumulates with the carboxy terminus extending into the cytoplasm and the bulk of the protein on the external surface of the cell membrane. In order to confirm that the rabies glycoprotein expressed in ALVAC-RG was correctly presented, immunofluorescence was performed on primary CEF cells infected with ALVAC or ALVAC-RG. Immunofluorescence was performed as previously described (Taylor et al., 1990) using a rabies G monoclonal antibody. Strong surface fluorescence was detected on CEF cells infected with ALVAC-RG but not with the parental ALVAC.  
         [0194]    Immunoprecipitation. Preformed monolayers of primary CEF, Vero (a line of African Green monkey kidney cells ATCC #CCL81) and MRC-5 cells (a fibroblast-like cell line derived from normal human fetal lung tissue ATCC #CCL171) were inoculated at 10 pfu per cell with parental virus ALVAC and recombinant virus ALVAC-RG in the presence of radiolabelled  35 S-methionine and treated as previously described (Taylor et al., 1990). Immunoprecipitation reactions were performed using a rabies G specific monoclonal antibody. Efficient expression of a rabies specific glycoprotein with a molecular weight of approximately 67 kDa was detected with the recombinant ALVAC-RG. No rabies specific products were detected in uninfected cells or cells infected with the parental ALVAC virus.  
         [0195]    Sequential Passaging Experiment. In studies with ALVAC virus in a range of non-avian species no proliferative infection or overt disease was observed (Taylor et al., 1991b). However, in order to establish that neither the parental nor recombinant virus could be adapted to grow in non-avian cells, a sequential passaging experiment was performed.  
         [0196]    The two viruses, ALVAC and ALVAC-RG, were inoculated in 10 sequential blind passages in three cell lines:  
         [0197]    (1) Primary chick embryo fibroblast (CEF) cells produced from 11 day old white leghorn embryos;  
         [0198]    (2) Vero cells—a continuous line of African Green monkey kidney cells (ATCC #CCL81); and  
         [0199]    (3) MRC-5 cells—a diploid cell line derived from human fetal lung tissue (ATCC #CCL171).  
         [0200]    The initial inoculation was performed at an m.o.i. of 0.1 pfu per cell using three 60 mm dishes of each cell line containing 2×10 6  cells per dish. One dish was inoculated in the presence of 40 μg/ml of Cytosine arabinoside (Ara C), an inhibitor of DNA replication. After an absorption period of 1 hour at 37° C., the inoculum was removed and the monolayer washed to remove unabsorbed virus. At this time the medium was replaced with 5 ml of EMEM+2% NBCS on two dishes (samples t0 and t7) and 5 ml of EMEM+2% NBCS containing 40 μg/ml Ara C on the third (sample t7A). Sample t0 was frozen at −70° C. to provide an indication of the residual input virus. Samples t7 and t7A were incubated at 37° C. for 7 days, after which time the contents were harvested and the cells disrupted by indirect sonication.  
         [0201]    One ml of sample t7 of each cell line was inoculated undiluted onto three dishes of the same cell line (to provide samples t0, t7 and t7A) and onto one dish of primary CEF cells. Samples t0, t7 and t7A were treated as for passage one. The additional inoculation on CEF cells was included to provide an amplification step for more sensitive detection of virus which might be present in the non-avian cells,  
         [0202]    This procedure was repeated for 10 (CEF and MRC-5) or 8 (Vero) sequential blind passages. Samples were then frozen and thawed three times and assayed by titration on primary CEF monolayers.  
         [0203]    Virus yield in each sample was then determined by plaque titration on CEF monolayers under agarose. Summarized results of the experiment are shown in Tables 1 and 2.  
         [0204]    The results indicate that both the parental ALVAC and the recombinant ALVAC-RG are capable of sustained replication on CEF monolayers with no loss of titer. In Vero cells, levels of virus fell below the level of detection after 2 passages for ALVAC and 1 passage for ALVAC-RG. In MRC-5 cells, a similar result was evident, and no virus was detected after 1 passage. Although the results for only four passages are shown in Tables 1 and 2 the series was continued for 8 (Vero) and 10 (MRC-5) passages with no detectable adaptation of either virus to growth in the non-avian cells.  
         [0205]    In passage 1 relatively high levels of virus were present in the t7 sample in MRC-5 and Vero cells. However this level of virus was equivalent to that seen in the t0 sample and the t7A sample incubated in the presence of Cytosine arabinoside in which no viral replication can occur. This demonstrated that the levels of virus seen at 7 days in non-avian cells represented residual virus and not newly replicated virus.  
         [0206]    In order to make the assay more sensitive, a portion of the 7 day harvest from each cell line was inoculated onto a permissive CEF monolayer and harvested at cytopathic effect (CPE) or at 7 days if no CPE was evident. The results of this experiment are shown in Table 3. Even after amplification through a permissive cell line, virus was only detected in MRC-5 and Vero cells for two additional passages. These results indicated that under the conditions used, there was no adaptation of either virus to growth in Vero or MRC-5 cells.  
         [0207]    Inoculation of Macapues. Four HIV seropositive macaques were initially inoculated with ALVAC-RG as described in Table 4. After 100 days these animals were re-inoculated to determine a booster effect, and an additional seven animals were inoculated with a range of doses. Blood was drawn at appropriate intervals and sera analyzed, after heat inactivation at 56° C. for 30 minutes, for the presence of anti-rabies antibody using the Rapid Fluorescent Focus Inhibition Assay (Smith et al., 1973).  
         [0208]    Inoculation of Chimpanzees. Two adult male chimpanzees (50 to 65 kg weight range) were inoculated intramuscularly or subcutaneously with 1×10 7  pfu of vCP65. Animals were monitored for reactions and bled at regular intervals for analysis for the presence of anti-rabies antibody with the RFFI test (Smith et al., 1973). Animals were re-inoculated with an equivalent dose 13 weeks after the initial inoculation.  
         [0209]    Inoculation of Mice. Groups of mice were inoculated with 50 to 100 μl of a range of dilutions of different batches of vCP65. Mice were inoculated in the footpad. On day 14, mice were challenged by intracranial inoculation of from 15 to 43 mouse LD 50  of the virulent CVS strain of rabies virus. Survival of mice was monitored and a protective dose 50% (PD 50 ) calculated at 28 days post-inoculation.  
         [0210]    Inoculation of Dogs and Cats. Ten beagle dogs, 5 months old, and 10 cats, 4 months old, were inoculated subcutaneously with either 6.7 or 7.7 log 10  TCID 50  of ALVAC-RG. Four dogs and four cats were not inoculated. Animals were bled at 14 and 28 days post-inoculation and anti-rabies antibody assessed in an RFFI test. The animals receiving 6.7 log 10  TCID 50  of ALVAC-RG were challenged at 29 days post-vaccination with 3.7 log 10  mouse LD 50  (dogs) or 4.3 log 10  mouse LD 50  (cats) of the NYGS rabies virus challenge strain.  
         [0211]    Inoculation of Squirrel Monkeys. Three groups of four squirrel monkeys ( Saimiri sciureus ) were inoculated with one of three viruses (a) ALVAC, the parental canarypox virus, (b) ALVAC-RG, the recombinant expressing the rabies G glycoprotein or (c) vCP37, a canarypox recombinant expressing the envelope glycoprotein of feline leukemia virus. Inoculations were performed under ketamine anaesthesia. Each animal received at the same time: (1) 20 μl instilled on the surface of the right eye without scarification; (2) 100 μl as several droplets in the mouth; (3) 100 μl in each of two intradermal injection sites in the shaven skin of the external face of the right arm; and (4) 100 μl in the anterior muscle of the right thigh.  
         [0212]    Four monkeys were inoculated with each virus, two with a total of 5.0 log 10  pfu and two with a total of 7.0 log 10  pfu. Animals were bled at regular intervals and sera analyzed for the presence of antirabies antibody using an RFFI test (Smith et al., 1973). Animals were monitored daily for reactions to vaccination. Six months after the initial inoculation the four monkeys receiving ALVAC-RG, two monkeys initially receiving vCP37, and two monkeys initially receiving ALVAC, as well as one naive monkey were inoculated with 6.5 log 10  pfu of ALVAC-RG subcutaneously. Sera were monitored for the presence of rabies neutralizing antibody in an RFFI test (Smith et al., 1973).  
         [0213]    Inoculation of Human Cell Lines with ALVAC-RG. In order to determine whether efficient expression of a foreign gene could be obtained in non-avian cells in which the virus does not productively replicate, five cell types, one avian and four non-avian, were analyzed for virus yield, expression of the foreign rabies G gene and viral specific DNA accumulation. The cells inoculated were:  
         [0214]    (a) Vero, African Green monkey kidney cells, ATCC #CCL81;  
         [0215]    (b) MRC-5, human embryonic lung, ATCC #CCL 171;  
         [0216]    (c) WISH human amnion, ATCC #CCL 25;  
         [0217]    (d) Detroit-532, human foreskin, Downs&#39;s syndrome, ATCC #CCL 54; and  
         [0218]    (e) Primary CEF cells.  
         [0219]    Chicken embryo fibroblast cells produced from 11 day old white leghorn embryos were included as a positive control. All inoculations were performed on preformed monolayers of 2×10 6  cells as discussed below.  
         [0220]    A. Methods for DNA analysis.  
         [0221]    Three dishes of each cell line were inoculated at 5 pfu/cell of the virus under test, allowing one extra dish of each cell line un-inoculated. One dish was incubated in the presence of 40 μg/ml of cytosine arabinoside (Ara C). After an adsorption period of 60 minutes at 37° C., the inoculum was removed and the monolayer washed twice to remove unadsorbed virus. Medium (with or without Ara C) was then replaced. Cells from one dish (without Ara C) were harvested as a time zero sample. The remaining dishes were incubated at 37° C. for 72 hours, at which time the cells were harvested and used to analyze DNA accumulation. Each sample of 2×10 6  cells was resuspended in 0.5 ml phosphate buffered saline (PBS) containing 40 mM EDTA and incubated for 5 minutes at 37° C. An equal volume of 1.5% agarose prewarmed at 42° C. and containing 120 mM EDTA was added to the cell suspension and gently mixed. The suspension was transferred to an agarose plug mold and allowed to harden for at least 15 min. The agarose plugs were then removed and incubated for 12-16 hours at 50° C. in a volume of lysis buffer (1% sarkosyl, 100 μg/ml proteinase K, 10 mM Tris HCl pH 7.5, 200 mM EDTA) that completely covers the plug. The lysis buffer was then replaced with 5.0 ml sterile 0.5×TBE (44.5 mM Tris-borate, 44.5 mM boric acid, 0.5 mM EDTA) and equilibrated at 4° C. for 6 hours with 3 changes of TBE buffer. The viral DNA within the plug was fractionated from cellular RNA and DNA using a pulse field electrophoresis system. Electrophoresis was performed for 20 hours at 180 V with a ramp of 50-90 sec at 15° C. in 0.5×TBE. The DNA was run with lambda DNA molecular weight standards. After electrophoresis the viral DNA band was visualized by staining with ethidium bromide. The DNA was then transferred to a nitrocellulose membrane and probed with a radiolabelled probe prepared from purified ALVAC genomic DNA.  
         [0222]    B. Estimation of virus yield.  
         [0223]    Dishes were inoculated exactly as described above, with the exception that input multiplicity was 0.1 pfu/cell. At 72 hours post infection, cells were lysed by three successive cycles of freezing and thawing. Virus yield was assessed by plaque titration on CEF monolayers.  
         [0224]    C. Analysis of expression of Rabies G gene.  
         [0225]    Dishes were inoculated with recombinant or parental virus at a multiplicity of 10 pfu/cell, allowing an additional dish as an uninfected virus control. After a one hour absorption period, the medium was removed and replaced with methionine free medium. After a 30 minute period, this medium was replaced with methionine-free medium containing 25 uCi/ml of  35 S-Methionine. Infected cells were labelled overnight (approximately 16 hours), then lysed by the addition of buffer A lysis buffer. Immunoprecipitation was performed as previously described (Taylor et al., 1990) using a rabies G specific monoclonal antibody.  
         [0226]    Results: Estimation of Viral Yield. The results of titration for yield at 72 hours after inoculation at 0.1 pfu per cell are shown in Table 5. The results indicate that while a productive infection can be attained in the avian cells, no increase in virus yield can be detected by this method in the four non-avian cell systems.  
         [0227]    Analysis of Viral DNA Accumulation. In order to determine whether the block to productive viral replication in the non-avian cells occurred before or after DNA replication, DNA from the cell lysates was fractionated by electrophoresis, transferred to nitrocellulose and probed for the presence of viral specific DNA. DNA from uninfected CEF cells, ALVAC-RG infected CEF cells at time zero, ALVAC-RG infected CEF cells at 72 hours post-infection and ALVAC-RG infected CEF cells at 72 hours post-infection in the presence of 40 μg/ml of cytosine arabinoside all showed some background activity, probably due to contaminating CEF cellular DNA in the radiolabelled ALVAC DNA probe preparation. However, ALVAC-RG infected CEF cells at 72 hours post-infection exhibited a strong band in the region of approximately 350 kbp representing ALVAC-specific viral DNA accumulation. No such band is detectable when the culture is incubated in the presence of the DNA synthesis inhibitor, cytosine arabinoside. Equivalent samples produced in Vero cells showed a very faint band at approximately 350 kbp in the ALVAC-RG infected Vero cells at time zero. This level represented residual virus. The intensity of the band was amplified at 72 hours post-infection indicating that some level of viral specific DNA replication had occurred in Vero cells which had not resulted in an increase in viral progeny. Equivalent samples produced in MRC-5 cells indicated that no viral specific DNA accumulation was detected under these conditions in this cell line. This experiment was then extended to include additional human cell lines, specifically WISH and Detroit-532 cells. ALVAC infected CEF cells served as a positive control. No viral specific DNA accumulation was detected in either WISH or Detroit cells inoculated with ALVAC-RG. It should be noted that the limits of detection of this method have not been fully ascertained and viral DNA accumulation may be occurring, but at a level below the sensitivity of the method. Other experiments in which viral DNA replication was measured by  3 H-thymidine incorporation support the results obtained with Vero and MRC-5 cells.  
         [0228]    Analysis of Rabies Gene Expression. To determine if any viral gene expression, particularly that of the inserted foreign gene, was occurring in the human cell lines even in the absence of viral DNA replication, immunoprecipitation experiments were performed on  35 S-methionine labelled lysates of avian and non-avian cells infected with ALVAC and ALVAC-RG. The results of immunoprecipitation using a rabies G specific monoclonal antibody illustrated specific immunoprecipitation of a 67 kDa glycoprotein in CEF, Vero and MRC-5, WISH and Detroit cells infected with ALVAC-RG. No such specific rabies gene products were detected in any of the uninfected and parentally infected cell lysates.  
         [0229]    The results of this experiment indicated that in the human cell lines analyzed, although the ALVAC-RG recombinant was able to initiate an infection and express a foreign gene product under the transcriptional control of the H6 early/late vaccinia virus promoter, the replication did not proceed through DNA replication, nor was there any detectable viral progeny produced. In the Vero cells, although some level of ALVAC-RG specific DNA accumulation was observed, no viral progeny was detected by these methods. These results would indicate that in the human cell lines analyzed the block to viral replication occurs prior to the onset of DNA replication, while in Vero cells, the block occurs following the onset of viral DNA replication.  
         [0230]    In order to determine whether the rabies glycoprotein expressed in ALVAC-RG was immunogenic, a number of animal species were tested by inoculation of the recombinant. The efficacy of current rabies vaccines is evaluated in a mouse model system. A similar test was therefore performed using ALVAC-RG. Nine different preparations of virus (including one vaccine batch (J) produced after 10 serial tissue culture passages of the seed virus) with infectious titers ranging from 6.7 to 8.4 log 10  TCID 50  per ml were serially diluted and 50 to 100 μl of dilutions inoculated into the footpad of four to six week old mice. Mice were challenged 14 days later by the intracranial route with 300 μl of the CVS strain of rabies virus containing from 15 to 43 mouse LD 50  as determined by lethality titration in a control group of mice. Potency, expressed as the PD 50  (Protective dose 50%), was calculated at 14 days post-challenge. The results of the experiment are shown in Table 6. The results indicated that ALVAC-RG was consistently able to protect mice against rabies virus challenge with a PD 50  value ranging from 3.33 to 4.56 with a mean value of 3.73 (STD 0.48). As an extension of this study, male mice were inoculated intracranially with 50 μl of virus containing 6.0 log 10  TCID 50  of ALVAC-RG or with an equivalent volume of an uninfected cell suspension. Mice were sacrificed on days 1, 3 and 6 post-inoculation and their brains removed, fixed and sectioned. Histopathological examination showed no evidence for neurovirulence of ALVAC-RG in mice.  
         [0231]    In order to evaluate the safety and efficacy of ALVAC-RG for dogs and cats, a group of 14, 5 month old beagles and 14, 4 month old cats were analyzed. Four animals in each species were not vaccinated. Five animals received 6.7 log 10  TCID 50  subcutaneously and five animals received 7.7 log 10  TCID 50  by the same route. Animals were bled for analysis for anti-rabies antibody. Animals receiving no inoculation or 6.7 log 10  TCID 50  of ALVAC-RG were challenged at 29 days post-vaccination with 3.7 log 10  mouse LD 50  (dogs, in the temporal muscle) or 4.3 log 10  mouse LD 50  (cats, in the neck) of the NYGS rabies virus challenge strain. The results of the experiment are shown in Table 7.  
         [0232]    No adverse reactions to inoculation were seen in either cats or dogs with either dose of inoculum virus. Four of 5 dogs immunized with 6.7 log 10  TCID 50  had antibody titers on day 14 post-vaccination and all dogs had titers at 29 days. All dogs were protected from a challenge which killed three out of four controls. In cats, three of five cats receiving 6.7 log 10  TCID 50  had specific antibody titers on day 14 and all cats were positive on day 29 although the mean antibody titer was low at 2.9 IU. Three of five cats survived a challenge which killed all controls. All cats immunized with 7.7 log 10  TCID 50  had antibody titers on day 14 and at day 29 the Geometric Mean Titer was calculated as 8.1 International Units.  
         [0233]    The immune response of squirrel monkeys ( Saimiri sciureus ) to inoculation with ALVAC, ALVAC-RG and an unrelated canarypox virus recombinant was examined. Groups of monkeys were inoculated as described above and sera analyzed for the presence of rabies specific antibody. Apart from minor typical skin reactions to inoculation by the intradermal route, no adverse reactivity was seen in any of the monkeys. Small amounts of residual virus were isolated from skin lesions after intradermal inoculation on days two and four post-inoculation only. All specimens were negative on day seven and later. There was no local reaction to intra-muscular injection. All four monkeys inoculated with ALVAC-RG developed anti-rabies serum neutralizing antibodies as measured in an RFFI test. Approximately six months after the initial inoculation all monkeys and one additional naive monkey were re-inoculated by the subcutaneous route on the external face of the left thigh with 6.5 log 10  TCID 50  of ALVAC-RG. Sera were analyzed for the presence of anti-rabies antibody. The results are shown in Table 8.  
         [0234]    Four of the five monkeys naive to rabies developed a serological response by seven days post-inoculation with ALVAC-RG. All five monkeys had detectable antibody by 11 days post-inoculation. Of the four monkeys with previous exposure to the rabies glycoprotein, all showed a significant increase in serum neutralization titer between days 3 and 7 post-vaccination. The results indicate that vaccination of squirrel monkeys with ALVAC-RG does not produce adverse side-effects and a primary neutralizing antibody response can be induced. An amnanestic response is also induced on re-vaccination. Prior exposure to ALVAC or to a canarypox recombinant expressing an unrelated foreign gene does not interfere with induction of an anti-rabies immune response upon re-vaccination.  
         [0235]    The immunological response of HIV-2 seropositive macaques to inoculation with ALVAC-RG was assessed. Animals were inoculated as described above and the presence of anti-rabies serum neutralizing antibody assessed in an RFFI test. The results, shown in Table 9, indicated that HIV-2 positive animals inoculated by the subcutaneous route developed anti-rabies antibody by 11 days after one inoculation. An anamnestic response was detected after a booster inoculation given approximately three months after the first inoculation. No response was detected in animals receiving the recombinant by the oral route. In addition, a series of six animals were inoculated with decreasing doses of ALVAC-RG given by either the intra-muscular or subcutaneous routes. Five of the six animals inoculated responded by 14 days post-vaccination with no significant difference in antibody titer.  
         [0236]    Two chimpanzees with prior exposure to HIV were inoculated with 7.0 log 10  pfu of ALVAC-RG by the subcutaneous or intra-muscular route. At 3 months post-inoculations both animals were re-vaccinated in an identical fashion. The results are shown in Table 10.  
         [0237]    No adverse reactivity to inoculation was noted by either intramuscular or subcutaneous routes. Both chimpanzees responded to primary inoculation by 14 days and a strongly rising response was detected following re-vaccination.  
                                                       TABLE 1                           Sequential Passage of ALVAC in Avian and non-Avian       Cells.                CEF   Vero   MRC-5                            Pass 1                       Sample           t0 a     2.4   3.0   2.6           t7 b     7.0   1.4   0.4           t7A c     1.2   1.2   0.4           Pass 2           Sample           t0   5.0   0.4   N.D. d             t7   7.3   0.4   N.D.           t7A   3.9   N.D.   N.D.           Pass 3           Sample           t0   5.4   0.4   N.D.           t7   7.4   N.D.   N.D.           t7A   3.8   N.D.   N.D.           Pass 4           Sample           t0   5.2   N.D.   N.D.           t7   7.1   N.D.   N.D.           t7A   3.9   N.D.   N.D.                                                                      
 
         [0238]    [0238]                                                       TABLE 2                           Sequential Passage of ALVAC-RG in Avian and non-       Avian Cells                CEF   Vero   MRC-5                            Pass 1                       sample           t0 a     3.0   2.9   2.9           t7 b     7.1   1.0   1.4           t7A c     1.8   1.4   1.2           Pass 2           Sample           t0   5.1   0.4   0.4           t7   7.1       N.D. d     N.D.           t7A   3.8   N.D.   N.D.           Pass 3           Sample           t0   5.1   0.4   N.D.           t7   7.2   N.D.   N.D.           t7A   3.6   N.D.   N.D.           Pass 4           Sample           t0   5.1   N.D.   N.D.           t7   7.0   N.D.   N.D.           t7A   4.0   N.D.    N.D                                                                        
         [0239]    [0239]                                                       TABLE 3                           Amplification of residual virus by passage in CEF       cells                CEF   Vero   MRC-5                            a) ALVAC                       Pass           2 a     7.0 b     6.0   5.2           3   7.5   4.1   4.9           4   7.5        N.D. c     N.D.           5   7.1   N.D.   N.D.           b) ALVAC-RG           Pass           2 a     7.2   5.5   5.5           3   7.2   5.0   5.1           4   7.2   N.D.   N.D.           5   7.2   N.D.   N.D.                                                            
         [0240]    [0240]                                       TABLE 4                           Schedule of inoculation of rhesus macaques with       ALVAC-RG (vCP65)            Animal   Inoculation                    176L   Primary:   1 × 10 8  pfu of vCP65 orally in TANG           secondary:   1 × 10 7  pfu of vCP65 plus 1 × 10 7                 pfu of vCP82 a  by SC route       185 L   Primary:   1 × 10 8  pfu of vCP65 orally in Tang           secondary:   1 × 10 7  pfu of vCP65 plus 1 × 10 7                 pfu of vCP82 by SC route       177 L   Primary:   5 × 10 7  pfu SC of vCP65 by SC route           Secondary:   1 × 10 7  pfu of vCP65 plus 1 × 10 7                 pfu of vCP82 by SC route       186L   Primary:   5 × 10 7  pfu of vCP65 by SC route           Secondary:   1 × 10 7  pfu of vCP65 plus 1 × 10 7                 pfu of vCP82 by SC route       178L   Primary:   1 × 10 7  pfu of vCP65 by SC route       182L   Primary:   1 × 10 7  pfu of vCP65 by IM route       179L   Primary:   1 × 10 6  pfu of vCP65 by SC route       183L   Primary:   1 × 10 6  pfu of vCP65 by IM route       180L   Primary:   1 × 10 6  pfu of vCP65 by SC route       184L   Primary:   1 × 10 5  pfu of vCP65 by IM route       187L   Primary   1 × 10 7  pfu of vCP65 orally                            
         [0241]    [0241]                                                           TABLE 5                           Analysis of yield in avian and non-avian cells       inoculated with ALVAC-RG                Sample Time                       Cell Type   t0   t72   t72A b                              Expt 1                       CEF   3.3 a     7.4   1.7           Vero   3.0   1.4   1.7           MRC-5   3.4   2.0   1.7           Expt 2           CEF   2.9   7.5   &lt;1.7           WISH   3.3   2.2   2.0           Detroit-532   2.8   1.7   &lt;1.7                                                
         [0242]    [0242]                                 TABLE 6                           Potency of ALVAC-RG as tested in mice                Test   Challenge Dose a     PD 50   b                         Initial seed   43   4.56           Primary seed   23   3.34           Vaccine Batch H   23   4.52           Vaccine Batch I   23   3.33           Vaccine Batch K   15   3.64           Vaccine Batch L   15   4.03           Vaccine Batch M   15   3.32           Vaccine Batch N   15   3.39           Vaccine Batch J   23   3.42                                                
         [0243]    [0243]                                     TABLE 7                           Efficacy of ALVAC-RG in dogs and cats                Dogs       Cats           Dose   Antibody a     Survival b     Antibody   Survival               6.7   11.9   5/5   2.9   3/5       7.7   10.1   N.T.   8.1   N.T.                                    
         [0244]    [0244]                                                                                                         TABLE 8                           Anti-rabies serological response of Squirrel monkeys       inoculated with canarypox recombinants            Monkey   Previous   Rabies serum-neutralizing antibody a              #   Exposure   −196 b     0   3   7   11   21   28                    22   ALVAC c     NT 9     &lt;1.2   &lt;1.2   &lt;1.2   2.1   2.3   2.2       51   ALVAC c     NT   &lt;1.2   &lt;1.2   1.7   2.2   2.2   2.2       39   vCP37 d     NT   &lt;1.2   &lt;1.2   1.7   2.1   2.2   N.T. g         55   vCP37 d     NT   &lt;1.2   &lt;1.2   1.7   2.2   2.1   N.T.       37   ALVAC-RG e     2.2   &lt;1.2   &lt;3.2   3.2   3.5   3.5   3.2       53   ALVAC-RG e     2.2   &lt;1.2   &lt;1.2   3.6   3.6   3.6   3.4       38   ALVAC-RG f     2.7   &lt;1.7   &lt;1.7   3.2   3.8   3.6   N.T.       54   ALVAC-RG f     3.2   &lt;1.7   &lt;1.5   3.6   4.2   4.0   3.6       57   None   NT   &lt;1.2   &lt;1.2   1.7   2.7   2.7   2.3                                                                            
         [0245]    [0245]                                                                                                                                                                 TABLE 9                           Inoculation of rhesus macaques with ALVAC-RG a         Route of Primary Inoculation            Days post-   or/Tang   SC   SC   SC   IM   SC   IM   SC   IM   OR            Inoculation   176L b     185L   177L   186L   178L   182L   179L   183L   180L   184L   187L b                      −84   —   —       —                                   −9   —   —   —   —   —       —       3   —   —   —   —       6   —   —   ±   ±       11   —   —   16 d     128       19   —   —   32   128   —       —       35   —   —   32   512       59   —   —   64   256       75   —   —   64   128   —       —       99 c     —   —   64   256   —       —   —   —   —   —       2   —   —   32   256   —   —   —   —   —   —   —       6   —   —   512   512   —   —   —   —   —   —   —       15   16   16   512   512   64   32   64   128   32   —   —       29   16   32   256   256   64   64   32   128   32   —   —       55       32               32       32   16   —       57   16       128   128   16       16               —                                                    
         [0246]    [0246]                                                   TABLE 10                           Inoculation of chimpanzees with ALVAC-RG                Weeks post-   Animal 431   Animal 457           Inoculation   I.M.   S.C.                            0   &lt;8 a     &lt;8           1   &lt;8   &lt;8           2   8   32           4   16   32           8   16   32           12 b /0   16   8           13/1   128   128           15/3   256   512           20/8   64   128           26/12   32   128                                                
       Example 10  
     Immunization of Humans Using Canarypox Expressing Rabies Glycoprotein (ALVAC-RG: vCP65)  
       [0247]    ALVAC-RG (vCP65) was generated as described in Example 9 and FIGS. 9A and 9B. For scaling-up and vaccine manufacturing ALVAC-RG (vCP65) was grown in primary CEF derived from specified pathogen free eggs. Cells were infected at a multiplicity of 0.01 and incubated at 37° C. for three days.  
         [0248]    The vaccine virus suspension was obtained by ultrasonic disruption in serum free medium of the infected cells; cell debris were then removed by centrifugation and filtration. The resulting clarified suspension was supplemented with lyophilization stabilizer (mixture of amino-acids), dispensed in single dose vials and freeze dried. Three batches of decreasing titer were prepared by ten-fold serial dilutions of the virus suspension in a mixture of serum free medium and lyophilization stabilizer, prior to lyophilization.  
         [0249]    Quality control tests were applied to the cell substrates, media and virus seeds and final product with emphasis on the search for adventitious agents and innocuity in laboratory rodents. No undesirable trait was found.  
         [0250]    Preclinical data. Studies in vitro indicated that VERO or MRC-5 cells do not support the growth of ALVAC-RG (vCP65); a series of eight (VERO) and 10 (MRC) blind serial passages caused no detectable adaptation of the virus to grow in these non avian lines. Analyses of human cell lines (MRC-5, WISH, Detroit 532, HEL, HNK or EBV-transformed lymphoblastoid cells) infected or inoculated with ALVAC-RG (vCP65) showed no accumulation of virus specific DNA suggesting that in these cells the block in replication occurs prior to DNA synthesis. Significantly, however, the expression of the rabies virus glycoprotein gene in all cell lines tested indicating that the abortive step in the canarypox replication cycle occurs prior to viral DNA replication.  
         [0251]    The safety and efficacy of ALVAC-RG (vCP65) were documented in a series of experiments in animals. A number of species including canaries, chickens, ducks, geese, laboratory rodents (suckling and adult mice), hamsters, guinea-pigs, rabbits, cats and dogs, squirrel monkeys, rhesus macaques and chimpanzees, were inoculated with doses ranging from 10 5  to 10 8  pfu. A variety of routes were used, most commonly subcutaneous, intramuscular and intradermal but also oral (monkeys and mice) and intracerebral (mice).  
         [0252]    In canaries, ALVAC-RG (vCP65) caused a “take” lesion at the site of scarification with no indication of disease or death. Intradermal inoculation of rabbits resulted in a typical poxvirus inoculation reaction which did not spread and healed in seven to ten days. There was no adverse side effects due to canarypox in any of the animal tests. Immunogenicity was documented by the development of anti-rabies antibodies following inoculation of ALVAC-RG (vCP65) in rodents, dogs, cats, and primates, as measured by Rapid Fluorescent Focus Inhibition Test (RFFIT). Protection was also demonstrated by rabies virus challenge experiments in mice, dogs, and cats immunized with ALVAC-RG (vCP65).  
         [0253]    Volunteers. Twenty-five healthy adults aged 20-45 with no previous history of rabies immunization were enrolled. Their health status was assessed by complete medical histories, physical examinations, hematological and blood chemistry analyses. Exclusion criteria included pregnancy, allergies, immune depression of any kind, chronic debilitating disease, cancer, injection of immune globins in the past three months, and seropositivity to human immunodeficiency virus (HIV) or to hepatitis B virus surface antigen.  
         [0254]    Study design. Participants were randomly allocated to receive either standard Human Diploid Cell Rabies Vaccine (HDC) batch no E0751 (Pasteur Merieux Serums &amp; Vaccine, Lyon, France) or the study vaccine ALVAC-RG (vCP65).  
         [0255]    The trial was designated as a dose escalation study. Three batches of experimental ALVAC-RG (vCP65) vaccine were used sequentially in three groups of volunteers (Groups A, B and C) with two week intervals between each step. The concentration of the three batches was 10 3.5 , 10 4.5 , 10 5.5  Tissue Culture Infectious Dose (TCID 50 ) per dose, respectively.  
         [0256]    Each volunteer received two doses of the same vaccine subcutaneously in the deltoid region at an interval of four weeks. The nature of the injected vaccine was not known by the participants at the time of the first injection but was known by the investigator.  
         [0257]    In order to minimize the risk of immediate hypersensitivity at the time of the second injection, the volunteers of Group B allocated to the medium dose of experimental vaccine were injected 1 h previously with the lower dose and those allocated to the higher dose (Group C) received successively the lower and the medium dose at hourly intervals.  
         [0258]    Six months later, the recipients of the highest dosage of ALVAC-RG (vCP65) (Group C) and HDC vaccine were offered a third dose of vaccine; they were then randomized to receive either the same vaccine as previously or the alternate vaccine. As a result, four groups were formed corresponding to the following immunization scheme: 1. HDC, HDC-HDC; 2. HDC, HDC-ALVAC-RG (vCP65); 3. ALVAC-RG (vCP65), ALVAC-RG (vCP65)-HDC; 4. ALVAC-RG (vCP65), ALVAC-RG (vCP65), ALVAC-RG (vCP65).  
         [0259]    Monitoring of Side Effects. All subjects were monitored for 1 h after injection and re-examined every day for the next five days. They were asked to record local and systemic reactions for the next three weeks and were questioned by telephone two times a week.  
         [0260]    Laboratory Investigators. Blood specimens were obtained before enrollment and two, four and six days after each injection. Analysis included complete blood cell count, liver enzymes and creatine kinase assays.  
         [0261]    Antibody assays. Antibody assays were performed seven days prior to the first injection and at days 7, 28, 35, 56, 173, 187 and 208 of the study.  
         [0262]    The levels of neutralizing antibodies to rabies were determined using the Rapid Fluorescent Focus Inhibition test (RFFIT) (Smith &amp; Yaeger, In Laboratory Techniques on Rabies). Canarypox antibodies were measured by direct ELISA. The antigen, a suspension of purified canarypox virus disrupted with 0.1% Triton X100, was coated in microplates. Fixed dilutions of the sera were reacted for two hours at room temperature and reacting antibodies were revealed with a peroxidase labelled anti-human IgG goat serum. The results are expressed as the optical density read at 490 nm.  
         [0263]    Analysis. Twenty-five subjects were enrolled and completed the study. There were 10 males and 15 females and the mean age was 31.9 (21 to 48). All but three subjects had evidence of previous smallpox vaccination; the three remaining subjects had no typical scar and vaccination history. Three subjects received each of the lower doses of experimental vaccine (10 3.5  and 10 4.5  TCID 50 ), nine subjects received 10 5.5  TCID 50  and ten received the HDC vaccine.  
         [0264]    Safety (Table 11). During the primary series of immunization, fever greater than 37.7° C. was noted within 24 hours after injection in one HDC recipient (37.8° C.) and in one vCP65 10 5.5  TCID 50  recipient (38° C.). No other systemic reaction attributable to vaccination was observed in any participant.  
         [0265]    Local reactions were noted in 9/10 recipients of HDC vaccine injected subcutaneously and in 0/3, 1/3 and 9/9 recipients of vCP65 10 3.5 , 10 4.5 , 10 5.5  TCID 50 , respectively.  
         [0266]    Tenderness was the most common symptoms and was always mild. Other local symptoms included redness and induration which were also mild and transient. All symptoms usually subsided within 24 hours and never lasted more than 72 hours.  
         [0267]    There was no significant change in blood cell counts, liver enzymes or creatine kinase values.  
         [0268]    Immune Responses: Neutralizing Antibodies to Rabies (Table 12). Twenty eight days after the first injection all the HDC recipients had protective titers (≧0.5 IU/ml). By contrast none in groups A and B (10 3.5  and 10 4.5  TCID 50 ) and only 2/9 in group C (10 5.5  TCID 50 ) ALVAC-RG (vCP65) recipients reached this protective titer.  
         [0269]    At day 56 (i.e. 28 days after the second injection) protective titers were achieved in 0/3 of Group A, 2/3 of Group B and 9/9 of Group C recipients of ALVAC-RG (vCP65) vaccine and persisted in all 10 HDC recipients.  
         [0270]    At day 56 the geometric mean titers were 0.05, 0.47, 4.4 and 11.5 IU/ml in groups A, B. C and HDC respectively.  
         [0271]    At day 180, the rabies antibody titers had substantially decreased in all subjects but remained above the minimum protective titer of 0.5 IU/ml in 5/10 HCD recipients and in 5/9 ALVAC-RG (vCP65) recipients; the geometric mean titers were 0.51 and 0.45 IU/ml in groups HCD and C, respectively.  
         [0272]    Antibodies to the CanaryDox virus (Table 13). The pre-immune titers observed varied widely with titers varying from 0.22 to 1.23 O.D. units despite the absence of any previous contact with canary birds in those subjects with the highest titers. When defined as a greater than two-fold increase between preimmunization and post second injection titers, a seroconversion was obtained in 1/3 subjects in group B and in 9/9 subjects in group C whereas no subject seroconverted in groups A or HDC.  
         [0273]    Booster Injection. The vaccine was similarly well tolerated six months later, at the time of the booster injection: fever was noted in 2/9 HDC booster recipients and in 1/10 ALVAC-RG (vCP65) booster recipients. Local reactions were present in 5/9 recipients of HDC booster and in 6/10 recipients of the ALVAC-RG (vCP65) booster.  
         [0274]    Observations. FIG. 13 shows graphs of rabies neutralizing antibody titers (Rapid Fluorescent Focus Inhibition Test or RFFIT, IU/ml): Booster effect of HDC and vCP65 (10 5.5  TCID 50 ) in volunteers previously immunized with either the same or the alternate vaccine. Vaccines were given at days 0, 28 and 180. Antibody titers were measured at days 0, 7, 28, 35, 56, 173, and 187 and 208.  
         [0275]    As shown in FIGS. 13A to  13 D, the booster dose given resulted in a further increase in rabies antibody titers in every subject whatever the immunization scheme. However, the ALVAC-RG (vCP65) booster globally elicited lower immune responses than the HDC booster and the ALVAC-RG (vCP65), ALVAC-RG (vCP65)-ALVAC-RG (vCP65) group had significantly lower titers than the three other groups. Similarly, the ALVAC-RG (vCP65) booster injection resulted in an increase in canarypox antibody titers in 3/5 subjects who had previously received the HDC vaccine and in all five subjects previously immunized with ALVAC-RG (vCP65).  
         [0276]    In general, none of the local side effects from administration of vCP65 was indicative of a local replication of the virus. In particular, lesions of the skin such as those observed after injection of vaccine were absent. In spite of the apparent absence of replication of the virus, the injection resulted in the volunteers generating significant amounts of antibodies to both the canarypox vector and to the expressed rabies glycoprotein.  
         [0277]    Rabies neutralizing antibodies were assayed with the Rapid Fluorescent Focus Inhibition Test (RFFIT) which is known to correlate well with the sero neutralization test in mice. Of 9 recipients of 10 5.5  TCID 50 , five had low level responses after the first dose. Protective titers of rabies antibodies were obtained after the second injection in all recipients of the highest dose tested and even in 2 of the 3 recipients of the medium dose. In this study, both vaccines were given subcutaneously as usually recommended for live vaccines, but not for the inactivated HDC vaccine. This route of injection was selected as it best allowed a careful examination of the injection site, but this could explain the late appearance of antibodies in HDC recipients: indeed, none of the HDC recipients had an antibody increase at day 7, whereas, in most studies where HDC vaccine is give intramuscularly a significant proportion of subjects do (Klietmann et al., Int&#39;l Green Cross—Geneva, 1981; Kuwert et al., Int&#39;l Green Cross—Geneva, 1981). However, this invention is not necessarily limited to the subcutaneous route of administration.  
         [0278]    The GMT (geometric mean titers) of rabies neutralizing antibodies was lower with the investigational vaccine than with the HDC control vaccine, but still well above the minimum titer required for protection. The clear dose effect response obtained with the three dosages used in this study suggest that a higher dosage might induce a stronger response. Certainly from this disclosure the skilled artisan can select an appropriate dosage for a given patient.  
         [0279]    The ability to boost the antibody response is another important result of this Example; indeed, an increase in rabies antibody titers was obtained in every subject after the 6 month dose whatever the immunization scheme, showing that preexisting immunity elicited by either the canarypox vector or the rabies glycoprotein had no blocking effect on the booster with the recombinant vaccine candidate or the conventional HDC rabies vaccine. This contrasts findings of others with vaccinia recombinants in humans that immune response may be blocked by pre-existing immunity (Cooney et al., Lancet 1991, 337:567-72; Etlinger et al., Vaccine 9:470-72, 1991).  
         [0280]    Thus, this Example clearly demonstrates that a non-replicating poxvirus can serve as an immunizing vector in humans, with all of the advantages that replicating agents confer on the immune response, but without the safety problem created by a fully permissive virus.  
                                                                                                                 TABLE 11                           Reactions in the 5 days following vaccination            vCP65                       dosage               H D C       (TCID50)   10 3.5     10 4.5     10 5.5     Control            Injection   1st   2nd   1st   2nd   1st   2nd   1st   2nd                    No.   3   3   3   3   9   9   10   10       vaccinees       temp &gt;37.7° C.   0   0   0   0   0   1   1   0       soreness   0   0   1   1   6   8   8   6       redness   0   0   0   0   0   4   5   4       induration   0   0   0   0   0   4   5   4                  
 
         [0281]    [0281]                                                                                                                 TABLE 11                           Reactions in the 5 days following vaccination            vCP65 dosage               H D C       (TCID50)   10 3.5     10 4.5     10 5.5     control            Injection   1st   2nd   1st   2nd   1st   2nd   1st   2nd                    No. vaccinees   3   3   3   3   9   9   10   10       temp &gt;37.7° C.   0   0   0   0   0   1   1   0       soreness   0   0   1   1   6   8   8   6       redness   0   0   0   0   0   4   5   4       induration   0   0   0   0   0   4   5   4                    
         [0282]    [0282]                                                                                                     TABLE 12                           Rabies neutralizing antibodies (REFIT; IU/ml)       Individual titers and geometric mean titers (GMT)                    TCID50/   Days                No.   dose   0   7   28   35   56                            1   10 3.5     &lt;0.1   &lt;0.1   &lt;0.1   &lt;0.1   0.2           3   10 3.5     &lt;0.1   &lt;0.1   &lt;0.1   &lt;0.1   &lt;0.1           4   10 3.5     &lt;0.1   &lt;0.1   &lt;0.1   &lt;0.1   &lt;0.1               G.M.T.   &lt;0.1   &lt;0.1   &lt;0.1   &lt;0.1   &lt;0.1           6   10 4.5     &lt;0.1   &lt;0.1   &lt;0.1   &lt;0.1   &lt;0.1           7   10 4.5     &lt;0.1   &lt;0.1   &lt;0.1   2.4   1.9           10   10 4.5     &lt;0.1   &lt;0.1   &lt;0.1   1.6   1.1               G.M.T.   &lt;0.1   &lt;0.1   0.1   0.58   0.47           11   10 5.5     &lt;0.1   &lt;0.1   1.0   3.2   4.3           13   10 5.5     &lt;0.1   &lt;0.1   0.3   6.0   8.8           14   10 5.5     &lt;0.1   &lt;0.1   0.2   2.1   9.4           17   10 5.5     &lt;0.1   &lt;0.1   &lt;0.1   1.2   2.5           18   10 5.5     &lt;0.1   &lt;0.1   0.7   8.3   12.5           20   10 5.5     &lt;0.1   &lt;0.1   &lt;0.1   0.3   3.7           21   10 5.5     &lt;0.1   &lt;0.1   0.2   2.6   3.9           23   10 5.5     &lt;0.1   &lt;0.1   &lt;0.1   1.7   4.2           25   10 5.5     &lt;0.1   &lt;0.1   &lt;0.1   0.6   0.9               G.M.T.   &lt;0.1   &lt;0.1   0.16   1.9   4.4*           2   HDC   &lt;0.1   &lt;0.1   0.8   7.1   7.2           5   HDC   &lt;0.1   &lt;0.1   9.9   12.8   18.7           8   HDC   &lt;0.1   &lt;0.1   12.7   21.1   16.5           9   HDC   &lt;0.1   &lt;0.1   6.0   9.9   14.3           12   HDC   &lt;0.1   &lt;0.1   5.0   9.2   25.3           15   HDC   &lt;0.1   &lt;0.1   2.2   5.2   8.6           16   HDC   &lt;0.1   &lt;0.1   2.7   7.7   20.7           19   HDC   &lt;0.1   &lt;0.1   2.6   9.9   9.1           22   HDC   &lt;0.1   &lt;0.1   1.4   8.6   6.6           24   HDC   &lt;0.1   &lt;0.1   0.8   5.8   4.7               G.M.T.   &lt;0.1   &lt;0.1   2.96   9.0   11.5*                                    
         [0283]    [0283]                                                   TABLE 13                           Canarypox antibodies: ELISA Geometric Mean Titers*            vCP65           dosage   Days            TCID50/dose   0   7   28   35   56               10 3.5     0.69   ND   0.76   ND   0.68       10 4.5     0.49   0.45   0.56   0.63   0.87       10 5.5     0.38   0.38   0.77   1.42   1.63       HDC control   0.45   0.39   0.40   0.35   0.39                            
       Example 11  
     Comparison of the LD 50  of ALVAC and NYVAC with Various Vaccinia Virus Strains  
       [0284]    Mice. Male outbred Swiss Webster mice were purchased from Taconic Farms (Germantown, N.Y.) and maintained on mouse chow and water ad libitum until use at 3 weeks of age (“normal” mice). Newborn outbred Swiss Webster mice were of both sexes and were obtained following timed pregnancies performed by Taconic Farms. All newborn mice used were delivered within a two day period.  
         [0285]    Viruses. ALVAC was derived by plaque purification of a canarypox virus population and was prepared in primary chick embryo fibroblast cells (CEF). Following purification by centrifugation over sucrose density gradients, ALVAC was enumerated for plaque forming units in CEF cells. The WR(L) variant of vaccinia virus was derived by selection of large plaque phenotypes of WR (Panicali et al., 1981). The Wyeth New York State Board of Health vaccine strain of vaccinia virus was obtained from Pharmaceuticals Calf Lymph Type vaccine Dryvax, control number 302001B. Copenhagen strain vaccinia virus VC-2 was obtained from Institut Merieux, France. Vaccinia virus strain NYVAC was derived from Copenhagen VC-2. All vaccinia virus strains except the Wyeth strain were cultivated in Vero African green monkey kidney cells, purified by sucrose gradient density centrifugation and enumerated for plaque forming units on Vero cells. The Wyeth strain was grown in CEF cells and enumerated in CEF cells.  
         [0286]    Inoculations. Groups of 10 normal mice were inoculated intracranially (ic) with 0.05 ml of one of several dilutions of virus-prepared by 10-fold serially diluting the stock preparations in sterile phosphate-buffered saline. In some instances, undiluted stock virus preparation was used for inoculation.  
         [0287]    Groups of 10 newborn mice, 1 to 2 days old, were inoculated ic similarly to the normal mice except that an injection volume of 0.03 ml was used.  
         [0288]    All mice were observed daily for mortality for a period of 14 days (newborn mice) or 21 days (normal mice) after inoculation. Mice found dead the morning following inoculation were excluded due to potential death by trauma.  
         [0289]    The lethal dose required to produce mortality for 50% of the experimental population (LD 50 ) was determined by the proportional method of Reed and Muench.  
         [0290]    Comparison of the LD 50  of ALVAC and NYVAC with Various Vaccinia Virus Strains for Normal. Young Outbred Mice by the ic Route. In young, normal mice, the virulence of NYVAC and ALVAC were several orders of magnitude lower than the other vaccinia virus strains tested (Table 14). NYVAC and ALVAC were found to be over 3,000 times less virulent in normal mice than the Wyeth strain; over 12,500 times less virulent than the parental VC-2 strain; and over 63,000,000 times less virulent than the WR(L) variant. These results would suggest that NYVAC is highly attenuated compared to other vaccinia strains, and that ALVAC is generally nonvirulent for young mice when administered intracranially, although both may cause mortality in mice at extremely high doses (3.85×10 8  PFUs, ALVAC and 3×10 8  PFUs, NYVAC) by an undetermined mechanism by this route of inoculation.  
         [0291]    Comparison of the LD 50  of ALVAC and NYVAC with Various Vaccinia Virus Strains for Newborn Outbred Mice by the ic Route. The relative virulence of 5 poxvirus strains for normal, newborn mice was tested by titration in an intracranial (ic) challenge model system (Table 15). With mortality as the endpoint, LD 50  values indicated that ALVAC is over 100,000 times less virulent than the Wyeth vaccine strain of vaccinia virus; over 200,000 times less virulent than the Copenhagen VC-2 strain of vaccinia virus; and over 25,000,000 times less virulent than the WR-L variant of vaccinia virus. Nonetheless, at the highest dose tested, 6.3×10 7  PFUs, 100% mortality resulted. Mortality rates of 33.3% were observed at 6.3×10 6  PFUS. The cause of death, while not actually determined, was not likely of toxicological or traumatic nature since the mean survival time (MST) of mice of the highest dosage group (approximately 6.3 LD 50 ) was 6.7±1.5 days. When compared to WR(L) at a challenge dose of 5 LD 50 , wherein MST is 4.8±0.6 days, the MST of ALVAC challenged mice was significantly longer (P=0.001).  
         [0292]    Relative to NYVAC, Wyeth was found to be over 15,000 times more virulent; VC-2, greater than 35,000 times more virulent; and WR(L), over 3,000,000 times more virulent. Similar to ALVAC, the two highest doses of NYVAC, 6×10 8  and 6×10 7  PFUs, caused 100% mortality. However, the MST of mice challenged with the highest dose, corresponding to 380 LD 50 , was only 2 days (9 deaths on day 2 and 1 on day 4). In contrast, all mice challenged with the highest dose of WR-L, equivalent to 500 LD 50 , survived to day 4.  
                             TABLE 14                           Calculated 50% Lethal Dose for mice by various vaccinia       virus strains and for canarypox virus (ALVAC) by the ic       route.                POXVIRUS   CALCULATED           STRAIN   LD 50  (PFUs)                       WR(L)   2.5           VC-2   1.26 × 10 4             WYETH   5.00 × 10 4             NYVAC   1.58 × 10 8             ALVAC   1.58 × 10 8                        
 
         [0293]    [0293]                             TABLE 15                           Calculated 50% Lethal Dose for newborn mice by various vaccinia       virus strains and for canarypox virus (ALVAC) by the ic route.                POXVIRUS   CALCULATED           STRAIN   LD 50  (PFUs)                       WR(L)   0.4           VC-2   0.1           WYETH   1.6           NYVAC   1.58 × 10 6             ALVAC   1.00 × 10 7                          
       Example 12  
     Evaluation of NYVAC (vP866) and NYVAC-RG (vP879)  
       [0294]    Immunoprecipitations. Preformed monolayers of avian or non-avian cells were inoculated with 10 pfu per cell of parental NYVAC (vP866) or NYVAC-RG (vP879) virus. The inoculation was performed in EMEM free of methionine and supplemented with 2% dialyzed fetal bovine serum. After a one hour incubation, the inoculum was removed and the medium replaced with EMEM (methionine free) containing 20 μCi/ml of  35 S-methionine. After an overnight incubation of approximately 16 hours, cells were lysed by the addition of Buffer A (1% Nonidet P-40, 10 mM Tris pH7.4, 150 mM NaCl, 1 mM EDTA, 0.01% sodium azide, 500 units per ml of aprotinin, and 0.02% phenyl methyl sulfonyl fluoride). Immunoprecipitation was performed using a rabies glycoprotein specific monoclonal antibody designated 24-3F10 supplied by Dr. C. Trinarchi, Griffith Laboratories, New York State Department of Health, Albany, N.Y., and a rat anti-mouse conjugate obtained from Boehringer Mannheim Corporation (Cat. #605-500). Protein A Sepharose CL-48 obtained from Pharmacia LKB Biotechnology Inc., Piscataway, N.J., was used as a support matrix. Immunoprecipitates were fractionated on 10% polyacrylamide gels according to the method of Dreyfuss et. al. (1984). Gels were fixed, treated for fluorography with 1M Na-salicylate for one hour, and exposed to Kodak XAR-2 film to visualize the immunoprecipitated protein species.  
         [0295]    Sources of Animals. New Zealand White rabbits were obtained from Hare-Marland (Hewitt, N.J.). Three week old male Swiss Webster outbred mice, timed pregnant female Swiss Webster outbred mice, and four week old Swiss Webster nude (nu + nu + ) mice were obtained from Taconic Farms, Inc. (Germantown, N.Y.). All animals were maintained according to NIH guidelines. All animal protocols were approved by the institutional IACUC. When deemed necessary, mice which were obviously terminally ill were euthanized.  
         [0296]    Evaluation of Lesions in Rabbits. Each of two rabbits was inoculated intradermally at multiple sites with 0.1 ml of PBS containing 10 4 , 10 5 , 10 6 , 10 7 , or 10 8  pfu of each test virus   
     
       
       
         1 
         
           
             
217 
 
           
           
             
               20 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              1 

TAATTAACTA GCTACCCGGG                                                 20 

 
           
           
             
               28 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              2 

GTACATTAAT TGATCGATGG GCCCTTAA                                        28 

 
           
           
             
               73 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              3 

AGCTTCCCGG GTAAGTAATA CGTCAAGGAG AAAACGAAAC GATCTGTAGT TAGCGGCCGC     60 

CTAATTAACT AAT                                                        73 

 
           
           
             
               69 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              4 

AGGGCCCATT CATTATGCAG TTCCTCTTTT GCTTTGCTAG ACATCAATCG CCGGCGGATT     60 

AATTGATTA                                                             69 

 
           
           
             
               20 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              5 

TTAGTTAATT AGGCGGCCGC                                                 20 

 
           
           
             
               22 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              6 

CGATTACTAT GAAGGATCCG TT                                              22 

 
           
           
             
               20 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              7 

TAATGATACT TCCTAGGCAA                                                 20 

 
           
           
             
               41 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              8 

CGATTACTAG ATCTGAGCTC CCCGGGCTCG AGGGATCCGT T                         41 

 
           
           
             
               39 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              9 

TAATGATCTA GACTCGAGGG GCCCGAGCTC CCTAGGCAA                            39 

 
           
           
             
               16 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              10 

GATCCGAATT CTAGCT                                                     16 

 
           
           
             
               12 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              11 

GCTTAAGATC GA                                                         12 

 
           
           
             
               75 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              12 

TATGAGTAAC TTAACTCTTT TGTTAATTAA AAGTATATTC AAAAAATAAG TTATATAAAT     60 

AGATCTGAAT TCGTT                                                      75 

 
           
           
             
               73 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              13 

ACTCATTGAA TTGAGAAAAC AATTAATTTT CATATAAGTT TTTTATTCAA TATATTTATC     60 

TAGACTTAAG CAA                                                        73 

 
           
           
             
               49 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              14 

AAAATGGGCG TGGATTGTTA ACTTTATATA ACTTATTTTT TGAATATAC                 49 

 
           
           
             
               67 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              15 

ACACGAATGA TTTTCTAAAG TATTTGGAAA GTTTTATAGG TAGTTGATAG AACAAAATAC     60 

ATAATTT                                                               67 

 
           
           
             
               51 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              16 

TGTGCTTACT AAAAGATTTC ATAAACCTTT CAAAATATCC ATCAACTATC T              51 

 
           
           
             
               46 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              17 

TGTAAAAATA AATCACTTTT TATACTAAGA TCTCCCGGGC TGCAGC                    46 

 
           
           
             
               66 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              18 

TGTTTTATGT ATTAAAACAT TTTTATTTAG TGAAAAATAT GATTCTAGAG GGCCCGACGT     60 

CGCCGG                                                                66 

 
           
           
             
               50 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              19 

TTTCTGTATA TTTGCACCAA TTTAGATCTT ACTCAAAATA TGTAACAATA                50 

 
           
           
             
               44 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              20 

TGTCATTTAA CACTATACTC ATATTAATAA AAATAATATT TATT                      44 

 
           
           
             
               72 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              21 

GATCCTGAGT ACTTTGTAAT ATAATGATAT ATATTTTCAC TTTATCTCAT TTGAGAATAA     60 

AAAGATCTTA GG                                                         72 

 
           
           
             
               72 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              22 

GACTCATGAA ACATTATATT ACTATATATA AAAGTGAAAT AGAGTAAACT CTTATTTTTC     60 

TAGAATCCTT AA                                                         72 

 
           
           
             
               72 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              23 

GATCCAGATC TCCCGGGAAA AAAATTATTT AACTTTTCAT TAATAGGGAT TTGACGTATG     60 

TAGCGTACTA GG                                                         72 

 
           
           
             
               72 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              24 

GTCTAGAGGG CCCTTTTTTT AATAAATTGA AAAGTAATTA TCCCTAAACT GCATACTACG     60 

CATGATCCTT AA                                                         72 

 
           
           
             
               40 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              25 

GGGAGATCTC TCGAGCTGCA GGGCGCCGGA TCCTTTTTCT                           40 

 
           
           
             
               40 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              26 

CCCTCTAGAG AGCTCGACGT CCCGCGGCCT AGGAAAAAGA                           40 

 
           
           
             
               59 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              27 

CGATATCCGT TAAGTTTGTA TCGTAATGGG CTCCAGATCT TCTACCAGGA TCCCGGTAC      59 

 
           
           
             
               55 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              28 

CGGGATCCTG GTAGAAGATC TGGAGCCCAT TACGATACAA ACTTAACGGA TATCG          55 

 
           
           
             
               17 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              29 

AATTCGAGCT CCCCGGG                                                    17 

 
           
           
             
               13 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              30 

CCCGGGGAGC TCG                                                        13 

 
           
           
             
               26 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              31 

CTTTTTATAA AAAGTTAACT ACGTAG                                          26 

 
           
           
             
               34 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              32 

GATCCTACGT AGTTAACTTT TTATAAAAAG AGCT                                 34 

 
           
           
             
               20 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              33 

CTTAACTCAG CTGACTATCC                                                 20 

 
           
           
             
               44 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              34 

TACGTAGTTA ACTTTTTATA AAAATCATAT TTTTGTAGTG GCTC                      44 

 
           
           
             
               67 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              35 

AATTCAGGAT CGTTCCTTTA CTAGTTGAGA TTCTCAAGGA TGATGGGATT TAATTTTTAT     60 

AAGCTTG                                                               67 

 
           
           
             
               67 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              36 

AATTCAAGCT TATAAAAATT AAATCCCATC ATCCTTGAGA ATCTCAACTA GTAAAGGAAC     60 

GATCCTG                                                               67 

 
           
           
             
               68 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              37 

CTAGACACTT TATGTTTTTT AATATCCGGT CTTAAAAGCT TCCCGGGGAT CCTTATACGG     60 

GGAATAAT                                                              68 

 
           
           
             
               65 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              38 

ATTATTCCCC GTATAAGGAT CCCCCGGGAA GCTTTTAAGA CCGGATATTA AAAAACATAA     60 

AGTGT                                                                 65 

 
           
           
             
               29 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              39 

GCTTCCCGGG AATTCTAGCT AGCTAGTTT                                       29 

 
           
           
             
               46 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              40 

ACTCTCAAAA GCTTCCCGGG AATTCTAGCT AGCTAGTTTT TATAAA                    46 

 
           
           
             
               50 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              41 

GATCTTTATA AAAACTAGCT AGCTAGAATT CCCGGGAAGC TTTTGAGAGT                50 

 
           
           
             
               71 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              42 

CTGAAATTAT TTCATTATCG CGATATCCGT TAAGTTTGTA TCGTAATGGT TCCTCAGGCT     60 

CTCCTGTTTG T                                                          71 

 
           
           
             
               48 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              43 

CATTACGATA CAAACTTAAC GGATATCGCG ATAATGAAAT AATTTCAG                  48 

 
           
           
             
               73 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              44 

ACCCCTTCTG GTTTTTCCGT TGTGTTTTGG GAAATTCCCT ATTTACACGA TCCCAGACAA     60 

GCTTAGATCT CAG                                                        73 

 
           
           
             
               51 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              45 

CTGAGATCTA AGCTTGTCTG GGATCGTGTA AATAGGGAAT TTCCCAAAAC A              51 

 
           
           
             
               45 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              46 

CAACGGAAAA ACCAGAAGGG GTACAAACAG GAGAGCCTGA GGAAC                     45 

 
           
           
             
               11 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              47 

GGATCCCCGG G                                                          11 

 
           
           
             
               60 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              48 

TCATTATCGC GATATCCGTG TTAACTAGCT AGCTAATTTT TATTCCCGGG ATCCTTATCA     60 

 
           
           
             
               60 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              49 

GTATAAGGAT CCCGGGAATA AAAATTAGCT AGCTAGTTAA CACGGATATC GCGATAATGA     60 

 
           
           
             
               24 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              50 

GACAATCTAA GTCCTATATT AGAC                                            24 

 
           
           
             
               18 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              51 

GGATTTTTAG GTAGACAC                                                   18 

 
           
           
             
               18 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              52 

TCATCGTCTT CATCATCG                                                   18 

 
           
           
             
               29 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              53 

GTCTTAAACT TATTGTAAGG GTATACCTG                                       29 

 
           
           
             
               61 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              54 

AACGATTAGT TAGTTACTAA AAGCTTGCTG CAGCCCGGGT TTTTTATTAG TTTAGTTAGT     60 

C                                                                     61 

 
           
           
             
               60 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              55 

GACTAACTAA CTAATAAAAA ACCCGGGCTG CAGCAAGCTT TTTGTAACTA ACTAATCGTT     60 

 
           
           
             
               99 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              56 

GCACGGAACA AAGCTTATCG CGATATCCGT TAAGTTTGTA TCGTAATGCT ATCAATCACG     60 

ATTCTGTTCC TGCTCATAGC AGAGGGCTCA TCTCAGAAT                            99 

 
           
           
             
               99 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              57 

ATTCTGAGAT GAGCCCTCTG CTATGAGCAG GAACAGAATC GTGATTGATA GCATTACGAT     60 

ACAAACTTAA CGGATATCGC GATAAGCTTT GTTCCGTGC                            99 

 
           
           
             
               66 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              58 

GAAAAATTTA AAGTCGACCT GTTTTGTTGA GTTGTTTGCG TGGTAACCAA TGCAAATCTG     60 

GTCACT                                                                66 

 
           
           
             
               66 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              59 

TCTAGCAAGA CTGACTATTG CAAAAAGAAG CACTATTTCC TCCATTACGA TACAAACTTA     60 

ACGGAT                                                                66 

 
           
           
             
               87 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              60 

ATCCGTTAAG TTTGTATCGT AATGGAGGAA ATAGTGCTTC TTTTTGCAAT AGTCAGTCTT     60 

GCTAGAAGTG ACCAGATTTG CATTGGT                                         87 

 
           
           
             
               49 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              61 

TACCACGCAA ACAACTCAAC AAAACAGGTC GACTTTAAAT TTTTCTGCA                 49 

 
           
           
             
               132 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              62 

GTACAGGTCG ACAAGCTTCC CGGGTATCGC GATATCCGTT AAGTTTGTAT CGTAATGAAT     60 

ACTCAAATTC TAATACTCAC TCTTGTGGCA GCCATTCACA CAAATGCAGA CAAAATCTGC    120 

CTTGGACATC AT                                                        132 

 
           
           
             
               132 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              63 

ATGATGTCCA AGGCAGATTT TGTCTGCATT TGTGTGAATG GCTGCCACAA GAGTGAGTAT     60 

TAGAATTTGA GTATTCATTA CGATACAAAC TTAACGGATA TCGCGATACC CGGGAAGCTT    120 

GTCGACCTGT AC                                                        132 

 
           
           
             
               51 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              64 

ATAACATGCG GTGCACCATT TGTATATAAG TTAACGAATT CCAAGTCAAG C              51 

 
           
           
             
               51 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              65 

GCTTGACTTG GAATTCGTTA ACTTATATAC AAATGGTGCA CCGCATGTTA T              51 

 
           
           
             
               55 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              66 

ATCATCTCGC GATATCCGTT AAGTTTGTAT CGTAATGAGC ACTGAAAGCA TGATC          55 

 
           
           
             
               37 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              67 

ATCATCTCTA GAATAAAAAT CACAGGGCAA TGATCCC                              37 

 
           
           
             
               3209 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              68 

TGAATGTTAA ATGTTATACT TTGGATGAAG CTATAAATAT GCATTGGAAA AATAATCCAT     60 

TTAAAGAAAG GATTCAAATA CTACAAAACC TAAGCGATAA TATGTTAACT AAGCTTATTC    120 

TTAACGACGC TTTAAATATA CACAAATAAA CATAATTTTT GTATAACCTA ACAAATAACT    180 

AAAACATAAA AATAATAAAA GGAAATGTAA TATCGTAATT ATTTTACTCA GGAATGGGGT    240 

TAAATATTTA TATCACGTGT ATATCTATAC TGTTATCGTA TACTCTTTAC AATTACTATT    300 

ACGAATATGC AAGAGATAAT AAGATTACGT ATTTAAGAGA ATCTTGTCAT GATAATTGGG    360 

TACGACATAG TGATAAATGC TATTTCGCAT CGTTACATAA AGTCAGTTGG AAAGATGGAT    420 

TTGACAGATG TAACTTAATA GGTGCAAAAA TGTTAAATAA CAGCATTCTA TCGGAAGATA    480 

GGATACCAGT TATATTATAC AAAAATCACT GGTTGGATAA AACAGATTCT GCAATATTCG    540 

TAAAAGATGA AGATTACTGC GAATTTGTAA ACTATGACAA TAAAAAGCCA TTTATCTCAA    600 

CGACATCGTG TAATTCTTCC ATGTTTTATG TATGTGTTTC AGATATTATG AGATTACTAT    660 

AAACTTTTTG TATACTTATA TTCCGTAAAC TATATTAATC ATGAAGAAAA TGAAAAAGTA    720 

TAGAAGCTGT TCACGAGCGG TTGTTGAAAA CAACAAAATT ATACATTCAA GATGGCTTAC    780 

ATATACGTCT GTGAGGCTAT CATGGATAAT GACAATGCAT CTCTAAATAG GTTTTTGGAC    840 

AATGGATTCG ACCCTAACAC GGAATATGGT ACTCTACAAT CTCCTCTTGA AATGGCTGTA    900 

ATGTTCAAGA ATACCGAGGC TATAAAAATC TTGATGAGGT ATGGAGCTAA ACCTGTAGTT    960 

ACTGAATGCA CAACTTCTTG TCTGCATGAT GCGGTGTTGA GAGACGACTA CAAAATAGTG   1020 

AAAGATCTGT TGAAGAATAA CTATGTAAAC AATGTTCTTT ACAGCGGAGG CTTTACTCCT   1080 

TTGTGTTTGG CAGCTTACCT TAACAAAGTT AATTTGGTTA AACTTCTATT GGCTCATTCG   1140 

GCGGATGTAG ATATTTCAAA CACGGATCGG TTAACTCCTC TACATATAGC CGTATCAAAT   1200 

AAAAATTTAA CAATGGTTAA ACTTCTATTG AACAAAGGTG CTGATACTGA CTTGCTGGAT   1260 

AACATGGGAC GTACTCCTTT AATGATCGCT GTACAATCTG GAAATATTGA AATATGTAGC   1320 

ACACTACTTA AAAAAAATAA AATGTCCAGA ACTGGGAAAA ATTGATCTTG CCAGCTGTAA   1380 

TTCATGGTAG AAAAGAAGTG CTCAGGCTAC TTTTCAACAA AGGAGCAGAT GTAAACTACA   1440 

TCTTTGAAAG AAATGGAAAA TCATATACTG TTTTGGAATT GATTAAAGAA AGTTACTCTG   1500 

AGACACAAAA GAGGTAGCTG AAGTGGTACT CTCAAAATGC AGAACGATGA CTGCGAAGCA   1560 

AGAAGTAGAG AAATAACACT TTATGACTTT CTTAGTTGTA GAAAAGATAG AGATATAATG   1620 

ATGGTCATAA ATAACTCTGA TATTGCAAGT AAATGCAATA ATAAGTTAGA TTTATTTAAA   1680 

AGGATAGTTA AAAATAGAAA AAAAGAGTTA ATTTGTAGGG TTAAAATAAT ACATAAGATC   1740 

TTAAAATTTA TAAATACGCA TAATAATAAA AATAGATTAT ACTTATTACC TTCAGAGATA   1800 

AAATTTAAGA TATTTACTTA TTTAACTTAT AAAGATCTAA AATGCATAAT TTCTAAATAA   1860 

TGAAAAAAAA GTACATCATG AGCAACGCGT TAGTATATTT TACAATGGAG ATTAACGCTC   1920 

TATACCGTTC TATGTTTATT GATTCAGATG ATGTTTTAGA AAAGAAAGTT ATTGAATATG   1980 

AAAACTTTAA TGAAGATGAA GATGACGACG ATGATTATTG TTGTAAATCT GTTTTAGATG   2040 

AAGAAGATGA CGCGCTAAAG TATACTATGG TTACAAAGTA TAAGTCTATA CTACTAATGG   2100 

CGACTTGTGC AAGAAGGTAT AGTATAGTGA AAATGTTGTT AGATTATGAT TATGAAAAAC   2160 

CAAATAAATC AGATCCATAT CTAAAGGTAT CTCCTTTGCA CATAATTTCA TCTATTCCTA   2220 

GTTTAGAATA CTTTTCATTA TATTTGTTTA CAGCTGAAGA CGAAAAAAAT ATATCGATAA   2280 

TAGAAGATTA TGTTAACTCT GCTAATAAGA TGAAATTGAA TGAGTCTGTG ATAATAGCTA   2340 

TAATCAGAGA AGTTCTAAAA GGAAATAAAA ATCTAACTGA TCAGGATATA AAAACATTGG   2400 

CTGATGAAAT CAACAAGGAG GAACTGAATA TAGCTAAACT ATTGTTAGAT AGAGGGGCCA   2460 

AAGTAAATTA CAAGGATGTT TACGGTTCTT CAGCTCTCCA TAGAGCTGCT ATTGGTAGGA   2520 

AACAGGATAT GATAAAGCTG TTAATCGATC ATGGAGCTGA TGTAAACTCT TTAACTATTG   2580 

CTAAAGATAA TCTTATTAAA AAAAAATAAT ATCACGTTTA GTAATATTAA AATATATTAA   2640 

TAACTCTATT ACTAATAACT CCAGTGGATA TGAACATAAT ACGAAGTTTA TACATTCTCA   2700 

TCAAAATCTT ATTGACATCA AGTTAGATTG TGAAAATGAG ATTATGAAAT TAAGGAATAC   2760 

AAAAATAGGA TGTAAGAACT TACTAGAATG TTTTATCAAT AATGATATGA ATACAGTATC   2820 

TAGGGCTATA AACAATGAAA CGATTAAAAA TTATAAAAAT CATTTCCCTA TATATAATAC   2880 

GCTCATAGAA AAATTCATTT CTGAAAGTAT ACTAAGACAC GAATTATTGG ATGGAGTTAT   2940 

AAATTCTTTT CAAGGATTCA ATAATAAATT GCCTTACGAG ATTCAGTACA TTATACTGGA   3000 

GAATCTTAAT AACCATGAAC TAAAAAAAAT TTTAGATAAT ATACATTAAA AAGGTAAATA   3060 

GATCATCTGT TATTATAAGC AAAGATGCTT GTTGCCAATA ATATACAACA GGTATTTGTT   3120 

TTTATTTTTA ACTACATATT TGATGTTCAT TCTCTTTATA TAGTATACAC AGAAAATTCA   3180 

TAATCCACTT AGAATTTCTA GTTATCTAG                                     3209 

 
           
           
             
               34 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              69 

ATCATCGAAT TCTGAATGTT AAATGTTATA CTTG                                 34 

 
           
           
             
               28 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              70 

GGGGGTACCT TTGAGAGTAC CACTTCAG                                        28 

 
           
           
             
               44 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              71 

GGGTCTAGAG CGGCCGCTTA TAAAGATCTA AAATGCATAA TTTC                      44 

 
           
           
             
               35 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              72 

ATCATCCTGC AGGTATTCTA AACTAGGAAT AGATG                                35 

 
           
           
             
               82 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              73 

GTACGTGACT AATTAGCTAT AAAAAGGATC CGGTACCCTC GAGTCTAGAA TCGATCCCGG     60 

GTTTTTATGA CTAGTTAATC AC                                              82 

 
           
           
             
               82 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              74 

GGCCGTGATT AACTAGTCAT AAAAACCCGG GATCGATTCT AGACTCGAGG GTACCGGATC     60 

CTTTTTATAG CTAATTAGTC AC                                              82 

 
           
           
             
               39 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              75 

TCGGGATCCG GGTTAATTAA TTAGTCATCA GGCAGGGCG                            39 

 
           
           
             
               40 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              76 

TAGCTCGAGG GTACCTACGA TACAAACTTA ACGGATATCG                           40 

 
           
           
             
               3659 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              77 

GATATCTGTG GTCTATATAT ACTACACCCT ACCGATATTA ACCAACGAGT TTCTCACAAG     60 

AAAACTTGTT TAGTAGATAG AGATTCTTTG ATTGTGTTTA AAAGAAGTAC CAGTAAAAAG    120 

TGTGGCATAT GCATAGAAGA AATAAACAAA AAACATATTT CCGAACAGTA TTTTGGAATT    180 

CTCCCAAGTT GTAAACATAT TTTTTGCCTA TCATGTATAA GACGTTGGGC AGATACTACC    240 

AGAAATACAG ATACTGAAAA TACGTGTCCT GAATGTAGAA TAGTTTTTCC TTTCATAATA    300 

CCCAGTAGGT ATTGGATAGA TAATAAATAT GATAAAAAAA TATTATATAA TAGATATAAG    360 

AAAATGATTT TTACAAAAAT ACCTATAAGA ACAATAAAAA TATAATTACA TTTACGGAAA    420 

ATAGCTGGTT TTAGTTTACC AACTTAGAGT AATTATCATA TTGAATCTAT ATTGTTTTTT    480 

AGTTATATAA AAACATGATT AGCCCCCAAT CGGATGAAAA TATAAAAGAT GTTGAGAATT    540 

TCGAATACAA CAAAAAGAGG AATCGTACGT TGTCCATATC CAAACATATA AATAAAAATT    600 

CAAAAGTAGT ATTATACTGG ATGTTTAGAG ATCAACGTGT ACAAGATAAT TGGGCTTTAA    660 

TTTACGCACA ACGATTAGCG TTAAAACTCA AAATACCTCT AAGAATATGC TTTTGTGTCG    720 

TGCCAAAATT TCACACTACT ACTTCTAGAC ACTTTATGTT TTTAATATCC GGTCTTAAAG    780 

AAGTCGCGGA AGAATGTAAA AGACTATGTA TAGGGTTTTC ATTGATATAT GGCGTACCAA    840 

AAGTAATAAT TCCGTGTATA GTAAAAAAAT ACAGAGTCGG AGTAATCATA ACGGATTTCT    900 

TTCCATTACG TGTTCCCGAA AGATTAATGA AACAGACTGT AATATCTCTT CCAGATAACA    960 

TACCTTTTAT ACAAGTAGAC GCTCATAATA TAGTACCTTG TTGGGAAGCT TCTGATAAAG   1020 

AAGAATACGG TGCACGAACT TTAAGAAAAA AGATATTTGA TAAATTATAT GAATATATGA   1080 

CAGAATTTCC TGTTGTTCGT AAACATCCAT ACGGTCCATT TTCTATATCT ATTGCAAAAC   1140 

CCAAAAATAT ATCATTAGAC AAGACGGTAT TACCCGTAAA ATGGGCAACG CCTGGAACAA   1200 

AAGCTGGAAT AATTGTTTTA AAAGAATTTA TAAAAAACAG ATTACCGTCA TACGACGCGG   1260 

ATCATAACAA TCCTACGTGT GACGCTTTGA GTAACTTATC TCCGTGGCTA CATTTTGGTC   1320 

ATGTATCCGC ACAACGTGTT GCCTTAGAAG TATTAAAATG TATACGAGAA AGCAAAAAAA   1380 

ACGTTGAAAC GTTTATAGAT GAAATAATTG TAAGAAGAGA ACTATCGGAT AATTTTTGTT   1440 

ACTATAACAA ACATTATGAT AGTATCCAGT CTACTCATTC ATGGGTTAGA AAAACATTAG   1500 

AAGATCACAT TAATGATCCT AGAAAGTATA TATATTCCAT TAAACAACTC GAAAAAGCGG   1560 

AAACTCATGA TCCTCTATGG AACGCGTCAC AAATGCAGAT GGTGAGAGAA GGAAAAATGC   1620 

ATAGTTTTTT ACGAATGTAT TGGGCTAAGA AGATACTTGA ATGGACTAGA ACACCTGAAG   1680 

ACGCTTTGAG TTATAGTATC TATTTGAACA ACAAGTACGA ACTAGACGGC ACGGATCCTA   1740 

ACGGATACGT AGGTTGTATG TGGTCTATTT GCGGATTACA CGATAGAGCG TGGAAAGCAA   1800 

GACCGATATT TGGAAAGATA AGATATATGA ATTATGAGAG TTCTAAGAAG AAATTTGATG   1860 

TTGCTGTATT TATACAGAAA TACAATTAAG ATAAATAATA TACAGCATTG TAACCATCGT   1920 

CATCCGTTAT ACGGGGAATA ATATTACCAT ACAGTATTAT TAAATTTTCT TACGAAGAAT   1980 

ATAGATCGGT ATTTATCGTT AGTTTATTTT ACATTTATTA ATTAAACATG TCTACTATTA   2040 

CCTGTTATGG AAATGACAAA TTTAGTTATA TAATTTATGA TAAAATTAAG ATAATAATAA   2100 

TGAAATCAAA TAATTATGTA AATGCTACTA GATTATGTGA ATTACGAGGA AGAAAGTTTA   2160 

CGAACTGGAA AAAATTAAGT GAATCTAAAA TATTAGTCGA TAATGTAAAA AAAATAAATG   2220 

ATAAAACTAA CCAGTTAAAA ACGGATATGA TTATATACGT TAAGGATATT GATCATAAAG   2280 

GAAGAGATAC TTGCGGTTAC TATGTACACC AAGATCTGGT ATCTTCTATA TCAAATTGGA   2340 

TATCTCCGTT ATTCGCCGTT AAGGTAAATA AAATTATTAA CTATTATATA TGTAATGAAT   2400 

ATGATATACG ACTTAGCGAA ATGGAATCTG ATATGACAGA AGTAATAGAT GTAGTTGATA   2460 

AATTAGTAGG AGGATACAAT GATGAAATAG CAGAAATAAT ATATTTGTTT AATAAATTTA   2520 

TAGAAAAATA TATTGCTAAC ATATCGTTAT CAACTGAATT ATCTAGTATA TTAAATAATT   2580 

TTATAAATTT TATAAATTTT AATAAAAAAT ACAATAACGA CATAAAGATA TTTAATCTTT   2640 

AATTCTTGAT CTGAAAAACA CATCTATAAA ACTAGATAAA AAGTTATTCG ATAAAGATAA   2700 

TAATGAATCG AACGATGAAA AATTGGAAAC AGAAGTTGAT AAGCTAATTT TTTTCATCTA   2760 

AATAGTATTA TTTTATTGAA GTACGAAGTT TTACGTTAGA TAAATAATAA AGGTCGATTT   2820 

TTACTTTGTT AAATATCAAA TATGTCATTA TCTGATAAAG ATACAAAAAC ACACGGTGAT   2880 

TATCAACCAT CTAACGAACA GATATTACAA AAAATACGTC GGACTATGGA AAACGAAGCT   2940 

GATAGCCTCA ATAGAAGAAG CATTAAAGAA ATTGTTGTAG ATGTTATGAA GAATTGGGAT   3000 

CATCCTCAAC GAAGAAATAG ATAAAGTTCT AAACTGGAAA AATGATACAT TAAACGATTT   3060 

AGATCATCTA AATACAGATG ATAATATTAA GGAAATCATA CAATGTCTGA TTAGAGAATT   3120 

TGCGTTTAAA AAGATCAATT CTATTATGTA TAGTTATGCT ATGGTAAAAC TCAATTCAGA   3180 

TAACGAACAT TGAAAGATAA AATTAAGGAT TATTTTATAG AAACTATTCT TAAAGACAAA   3240 

CGTGGTTATA AACAAAAGCC ATTACCCGGA TTGGAAACTA AAATACTAGA TAGTATTATA   3300 

AGATTTTAAA AACATAAAAT TAATAGGTTT TTATAGATTG ACTTATTATA TACAATATGG   3360 

ATAAAAGATA TATATCAACT AGAAAGTTGA ATGACGGATT CTTAATTTTA TATTATGATT   3420 

CAATAGAAAT TATTGTCATG TCGTGTAATC ATTTTATAAA TATATCAGCG TTACTAGCTA   3480 

AGAAAAACAA GGACTTTAAT GAATGGCTAA AGATAGAATC ATTTAGAGAA ATAATAGATA   3540 

CTTTAGATAA AATTAATTAC GATCTAGGAC AACGATATTG TGAAGAACTT ACGGCGCATC   3600 

ACATTCCAGT GTAATTATTG AGGTCAAAGC TAGTAACTTA ATAGATGACA GGACAGCTG    3659 

 
           
           
             
               2356 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              78 

TGTCTGGACT AACTGATTTC ATGGAACAAT TTTCATCAAA AATATCAGTT ATACCTAGTT     60 

CTACAAAGAC AGAACTTTGA TGTTATGTTT GTGTTTGTAT AGAAAATTTT GGGATACTAA    120 

CTGATATTTC TGAATATTTC TGAATATTTC ATGTTACTTA CTTACTCCTA TCTTAGACGA    180 

TAATAAAATT CGAGGCGTAA TATGTTTTTC CAAATATTTG AAATTCTTAT ACGTATCGGC    240 

GAAGAAAAGT AACATACTAT AAGTGTTATG CAAGTAAGGT ATGTTAATGA TATTGGATTT    300 

AATTTCATTG ACAATACATA TGTCCAAACA TTCCACTCGT AATTATGTAC GGAACGACTT    360 

TAGTTAAATA CTTAGTCACA AAAAACTTAT GACTGTCATT ATCTGAAAAC GGTGATTCCC    420 

ATAAATCAGA ATACTTAATA TTAAATAGAA TGCTCGCTTC TGGAGGTTTC CGGATACTAG    480 

ATAACATATC TTCTGTATTA TAGTTTAATT CACTCATTTT ATTACATAAT ACAGTAACAT    540 

CTCCCGAAAC CAATGATGTT ATATTAGATT TACTTACATA CTTCTTGTAA CTATCATGAA    600 

TACGTTTGTT ATGATCTATA AAGAAGATGG ATGTATATTC TGTTCTAGAT AGCAAGTTCT    660 

TTAAGTTATT CTTTGTCTGT ATTACTATCA TCGTCTTCAT CATCGTCTAA AGGTAGCATT    720 

ATATAATAAA TCTAATAGTT GATTTCTCGA TCTATCAGTA CTCGCTTTCA ATAACATTTT    780 

TACTATAAGC ATAATAGAAG GCGGTGATAT CACTATATTT TTATCGGGTA TTCTTTTAGT    840 

AATTAGTTAG TTCGTAGAAT TTCGTAGAGA TAAAAGCCAA TTTGTTGTTG ATACTGCTTA    900 

CGTTACTCAT GTTTCTTGTT TCTGTTAATT AACAGGTATA CCCTTACAAT AAGTTTAATT    960 

AACTTTTAGG TTTTTGTGAA GAACTTTTAG CTTCTAGTTC CCTTATCCAT AATTGGGTCT   1020 

TAGATCTAGA TTCTTCCCAT GTATAAAGGG GGACATACCC AAAATCTTTA AATGCTTTGT   1080 

CCGTTTCTAT AGTAAATGTC GTACATTCCT TAATCAAAGT ATAAGGATTT AGTAAAGGCG   1140 

TGTAAGAACA AATAGGTGAT AGTAATACTC TTAAACCTTT ATTAATATTA GCGATAAACC   1200 

TTAAACACCA TAAAGGAAGA CATGTATTCC GTAGATCCAT CCCTAATTGA TTAAAGAAAT   1260 

GCATGTTAAA ATCATGATAA TGTTCAGTAG GAGAGGTATC GTAACAGTAA TACACGTTAT   1320 

TGCAGAGAGG ACTATGTTGA CCATTTTCTA TCATATTTCT TGCTGCTAAA ATATGCATCC   1380 

AAGCTACGTT TCCTGCATAG ACTCTGCTAT GAAATACTTT ATCATCCGCA TATTTATACA   1440 

TTTTCCTGCT TTTATACGAT CTTCTGTATA AAGTTTCTAG TACTGGACAG TATTCTCCGA   1500 

AAACACCTAA TGGGCGTAGC GACAAGTGCA TAATCTAAGT CCTATATTAG ACATAGTACC   1560 

GTTAGCTTCT AGTATATATT TCTCAGATAA CTTGTTTACT AAGAGGATAA GCCTCTTTAT   1620 

GGTTAGATTG ATAATACGTA TTCTCGTTTC CTCTTATCAT CGCATCTCCG GAGAAAGTTA   1680 

GGACCTACCG CAGAATAACT ACTCGTATAT ACTAAGACTC TTACGCCGTT ATACAGACAA   1740 

GAATCTACTA CGTTCTTCGT TCCGTTGATA TTAACGTCCA TTATAGAGTC GTTAGTAAAC   1800 

TTACCCGCTA CATCATTTAT CGAAGCAATA TGAATGACCA CATCTGCTGA TCTAAGCGCT   1860 

TCGTCCAAAG TACTTTTATT TCTAACATCT CCAATCACGG GAACTATCTT TATTATATTA   1920 

CATTTTTCTA CAAGATCTAG TAACCATTGG TCGATTCTAA TATCGTAAAC ACGAACTTCT   1980 

TTTTAAAGAG GATTCGAACA AGATAAGATT ATTTATAATG TGTCTACCTA AAAATCCACA   2040 

CCCTCCGGTT ACCACGTATA CTAGTGTACG CATTTTGAGT ATTAACTATA TAAGACCAAA   2100 

ATTATATTTT CATTTTCTGT TATATTATAC TATATAATAA AAACAAATAA ATATACGAAT   2160 

ATTATAAGAA ATTTAGAACA CGTTATTAAA GTATTGCCTT TTTTATTAAC GGCGTGTTCT   2220 

TGTAATTGCC GTTTAGAATA GTCTTTATTT ACTTTAGATA ACTCTTCTAT CATAACCGTC   2280 

TCCTTATTCC AATCTTCTTC AGAAGTACAT GAGTACTTAC CGAAGTTTAT CATCATAGAG   2340 

ATTATATATG AAGAAA                                                   2356 

 
           
           
             
               965 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              79 

AAAAAGGATC CGGGTTAATT AATTAGTCAT CAGGCAGGGC GAGAACGAGA CTATCTGCTC     60 

GTTAATTAAT TAGAGCTTCT TTATTCTATA CTTAAAAAGT GAAAATAAAT ACAAAGGTTC    120 

TTGAGGGTTG TGTTAAATTG AAAGCGAGAA ATAATCATAA ATTATTTCAT TATCGCGATA    180 

TCCGTTAAGT TTGTATCGTA ATGAGCACTG AAAGCATGAT CCGGGACGTG GAGCTGGCCG    240 

AGGAGGCGCT CCCCAAGAAG ACAGGGGGGC CCCAGGGCTC CAGGCGGTGC TTGTTCCTCA    300 

GCCTCTTCTC CTTCCTGATC GTGGCAGGCG CCACCACGCT CTTCTGCCTG CTGCACTTTG    360 

GAGTGATCGG CCCCCAGAGG GAAGAGTCCC CCAGGGACCT CTCTCTAATC AGCCCTCTGG    420 

CCCAGGCAGT CAGATCATCT TCTCGAACCC CGAGTGACAA GCCTGTAGCC CATGTTGTAG    480 

CAAACCCTCA AGCTGAGGGG CAGCTCCAGT GGCTGAACCG CCGGGCCAAT GCCCTCCTGG    540 

CCAATGGCGT GGAGCTGAGA GATAACCAGC TGGTGGTGCC ATCAGAGGGC CTGTACCTCA    600 

TCTACTCCCA GGTCCTCTTC AAGGGCCAAG GCTGCCCCTC CACCCATGTG CTCCTCACCC    660 

ACACCATCAG CCGCATCGCC GTCTCCTACC AGACCAAGGT CAACCTCCTC TCTGCCATCA    720 

AGAGCCCCTG CCAGAGGGAG ACCCCAGAGG GGGCTGAGGC CAAGCCCTGG TATGAGCCCA    780 

TCTATCTGGG AGGGGTCTTC CAGCTGGAGA AGGGTGACCG ACTCAGCGCT GAGATCAATC    840 

GGCCCGACTA TCTCGACTTT GCCGAGTCTG GGCAGGTCTA CTTTGGGATC ATTGCCCTGT    900 

GATTTTTATT CTAGAATCGA TCCCGGGTTT TTATGACTAG TTAATCACGG CCGCTTATAA    960 

AGATC                                                                965 

 
           
           
             
               29 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              80 

ATCATCAAGC TTGATTCTTT ATTCTATAC                                       29 

 
           
           
             
               37 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              81 

CATGCTTTCA GTGCTCATTA CGATACAAAC TTAACGG                              37 

 
           
           
             
               21 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              82 

TTAACGGATA TCGCGATAAT G                                               21 

 
           
           
             
               35 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              83 

ACTACTAAGC TTCTTTATTC TATACTTAAA AAGTG                                35 

 
           
           
             
               18 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              84 

ATGAGCACTG AAAGCATG                                                   18 

 
           
           
             
               44 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              85 

GGGCTCAAGC TTGCGGCCGC TCATTAGACA AGCGAATGAG GGAC                      44 

 
           
           
             
               62 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              86 

AGATCTCCCG GGCTCGAGTA ATTAATTAAT TTTTATTACA CCAGAAAAGA CGGCTTGAGA     60 

TC                                                                    62 

 
           
           
             
               64 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              87 

TAATTACTCG AGCCCGGGAG ATCTAATTTA ATTTAATTTA TATAACTCAT TTTTTGAATA     60 

TACT                                                                  64 

 
           
           
             
               45 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              88 

TATCTCGAAT TCCCGCGGCT TTAAATGGAC GGAACTCTTT TCCCC                     45 

 
           
           
             
               947 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              89 

GATCTCAAGC CGTCTTTTCT GGTGTAATAA AAATTAATTA ATTACTCGAG CCCAGCTTGA     60 

TTCTTTATTC TATACTTAAA AAGTGAAAAT AAATACAAAG GTTCTTGAGG GTTGTGTTAA    120 

ATTGAAAGCG AGAAATAATC ATAAATTATT TCATTATCGC GATATCCGTT AAGTTTGTAT    180 

CGTAATGAGC ACTGAAAGCA TGATCCGGGA CGTGGAGCTG GCCGAGGAGG CGCTCCCCAA    240 

GAAGACAGGG GGGCCCCAGG GCTCCAGGCG GTGCTTGTTC CTCAGCCTCT TCTCCTTCCT    300 

GATCGTGGCA GGCGCCACCA CGCTCTTCTG CCTGCTGCAC TTTGGAGTGA TCGGCCCCCA    360 

GAGGGAAGAG TCCCCCAGGG ACCTCTCTCT AATCAGCCCT CTGGCCCAGG CAGTCAGATC    420 

ATCTTCTCGA ACCCCGAGTG ACAAGCCTGT AGCCCATGTT GTAGCAAACC CTCAAGCTGA    480 

GGGGCAGCTC CAGTGGCTGA ACCGCCGGGC CAATGCCCTC CTGGCCAATG GCGTGGAGCT    540 

GAGAGATAAC CAGCTGGTGG TGCCATCAGA GGGCCTGTAC CTCATCTACT CCCAGGTCCT    600 

CTTCAAGGGC CAAGGCTGCC CCTCCACCCA TGTGCTCCTC ACCCACACCA TCAGCCGCAT    660 

CGCCGTCTCC TACCAGACCA AGGTCAACCT CCTCTCTGCC ATCAAGAGCC CCTGCCAGAG    720 

GGAGACCCCA GAGGGGGCTG AGGCCAAGCC CTGGTATGAG CCCATCTATC TGGGAGGGGT    780 

CTTCCAGCTG GAGAAGGGTG ACCGACTCAG CGCTGAGATC AATCGGCCCG ACTATCTCGA    840 

CTTTGCCGAG TCTGGGCAGG TCTACTTTGG GATCATTGCC CTGTGATTTT TATTGGGAGA    900 

TCTAATTTAA TTTAATTTAT ATAACTTATT TTTTGAATAT ACTTTTA                  947 

 
           
           
             
               41 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              90 

GATCTGACTG CGGCTCCTCC ATTACGATAC AAACTTAACG G                         41 

 
           
           
             
               45 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              91 

GTGGGTAAGG GAATTCGGAT CCCCGGGTTA ATTAATTAGT GATAC                     45 

 
           
           
             
               34 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              92 

GTTTGTATCG TAATGGAGGA GCCGCAGTCA GATC                                 34 

 
           
           
             
               57 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              93 

CATTACGATA CAAACTTAAC GGATATCGCG ACGCGTTCAC ACAGGGCAGG TCTTGGC        57 

 
           
           
             
               49 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              94 

TACTACCTCG AGCCCGGGAT AAAAAACGCG TTCAGTCTGA GTCAGGCCC                 49 

 
           
           
             
               66 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              95 

GTGTGAACGC GTCGCGATAT CCGTTAAGTT TGTATCGTAA TGCAGCTGCG TGGGCGTGAG     60 

CGCTTC                                                                66 

 
           
           
             
               33 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              96 

ATCATCGGAT CCCCCGGGTT CTTTATTCTA TAC                                  33 

 
           
           
             
               66 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              97 

AGAAAAATCA GTTAGCTAAG ATCTCCCGGG CTCGAGGGTA CCGGATCCTG ATTAGTTAAT     60 

TTTTGT                                                                66 

 
           
           
             
               70 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              98 

GATCACAAAA ATTAACTAAT CAGGATCCGG TACCCTCGAG CCCGGGAGAT CTTAGCTAAC     60 

TGATTTTTCT                                                            70 

 
           
           
             
               1512 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              99 

GATTAAAGAA AGTTACTCTG AGACACAAAA GAGGTAGCTG AAGTGGTACT CTCAAAGGTA     60 

CCCCCGGGTT AATTAATTAG TCATCAGGCA GGGCGAGAAC GAGACTATCT GCTCGTTAAT    120 

TAATTAGGTC GACGGATCCC CGGGTTCTTT ATTCTATACT TAAAAAGTGA AAATAAATAC    180 

AAAGGTTCTT GAGGGTTGTG TTAAATTGAA AGCGAGAAAT AATCATAAAT TATTTCATTA    240 

TCGCGATATC CGTTAAGTTT GTATCGTAAT GGAGGAGCCG CAGTCAGATC CTAGCGTCGA    300 

GCCCCCTCTG AGTCAGGAAA CATTTTCAGA CCTATGGAAA CTACTTCCTG AAAACAACGT    360 

TCTGTCCCCC TTGCCGTCCC AAGCAATGGA TGATTTGATG CTGTCCCCGG ACGATATTGA    420 

ACAATGGTTC ACTGAAGACC CAGGTCCAGA TGAAGCTCCC AGAATGCCAG AGGCTGCTCC    480 

CCGCGTGGCC CCTGCACCAG CAGCTCCTAC ACCGGCGGCC CCTGCACCAG CCCCCTCCTG    540 

GCCCCTGTCA TCTTCTGTCC CTTCCCAGAA AACCTACCAG GGCAGCTACG GTTTCCGTCT    600 

GGGCTTCTTG CATTCTGGGA CAGCCAAGTC TGTGACTTGC ACGTACTCCC CTGCCCTCAA    660 

CAAGATGTTT TGCCAACTGG CCAAGACCTG CCCTGTGCAG CTGTGGGTTG ATTCCACACC    720 

CCCGCCCGGC ACCCGCGTCC GCGCCATGGC CATCTACAAG CAGTCACAGC ACATGACGGA    780 

GGTTGTGAGG CGCTGCCCCC ACCATGAGCG CTGCTCAGAT AGCGATGGTC TGGCCCCTCC    840 

TCAGCATCTT ATCCGAGTGG AAGGAAATTT GCGTGTGGAG TATTTGGATG ACAGAAACAC    900 

TTTTCGACAT AGTGTGGTGG TGCCCTATGA GCCGCCTGAG GTTGGCTCTG ACTGTACCAC    960 

CATCCACTAC AACTACATGT GTAACAGTTC CTGCATGGGC GGCATGAACC GGAGGCCCAT   1020 

CCTCACCATC ATCACACTGG AAGACTCCAG TGGTAATCTA CTGGGACGGA ACAGCTTTGA   1080 

GGTGCGTGTT TGTGCCTGTC CTGGGAGAGA CCGGCGCACA GAGGAAGAGA ATCTCCGCAA   1140 

GAAAGGGGAG CCTCACCACG AGCTGCCCCC AGGGAGCACT AAGCGAGCAC TGCCCAACAA   1200 

CACCAGCTCC TCTCCCCAGC CAAAGAAGAA ACCACTGGAT GGAGAATATT TCACCCTTCA   1260 

GATCCGTGGG CGTGAGCGCT TCGAGATGTT CCGAGAGCTG AATGAGGCCT TGGAACTCAA   1320 

GGATGCCCAG GCTGGGAAGG AGCCAGGGGG GAGCAGGGCT CACTCCAGCC ACCTGAAGTC   1380 

CAAAAAGGGT CAGTCTACCT CCCGCCATAA AAAACTCATG TTCAAGACAG AAGGGCCTGA   1440 

CTCAGACTGA ACGCGTTTTT ATCCCGGGCT CGAGTCTAGA ATCGATCCCG GGTTTTTATG   1500 

ACTAGTTAAT CA                                                       1512 

 
           
           
             
               71 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              100 

CATCTTAATT AATTAGTCAT CAGGCAGGGC GAGAACGAAG ACTATCTGCT CGTTAATTAA     60 

TTAGGTCGAC G                                                          71 

 
           
           
             
               69 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              101 

CATCCGTCGA CCTAATTAAT TAACGACGAC ATAGTCTCGT TCTCGCCTGC CTGATGACTA     60 

ATTAATTAA                                                             69 

 
           
           
             
               12 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              102 

AATTGCGGCC GC                                                         12 

 
           
           
             
               1484 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              103 

GGTACGTGAC TAATTAGCTA TAAAAAGGAT CTTAATTAAT TAGTCATCAG GCAGGGCGAG     60 

AACGAGACTA TCTGCTCGTT AATTAATTAG GTCGACGGAT CCCCCGGGTT CTTTATTCTA    120 

TACTTAAAAA GTGAAAATAA ATACAAAGGT TCTTGAGGGT TGTGTTAAAT TGAAAGCGAG    180 

AAATAATCAT AAATTATTTC ATTATCGCGA TATCCGTTAA GTTTGTATCG TAATGGAGGA    240 

GCCGCAGTCA GATCCTAGCG TCGAGCCCCC TCTGAGTCAG GAAACATTTT CAGACCTATG    300 

GAAACTACTT CCTGAAAACA ACGTTCTGTC CCCCTTGCCG TCCCAAGCAA TGGATGATTT    360 

GATGCTGTCC CCGGACGATA TTGAACAATG GTTCACTGAA GACCCAGGTC CAGATGAAGC    420 

TCCCAGAATG CCAGAGGCTG CTCCCCGCGT GGCCCCTGCA CCAGCAGCTC CTACACCGGC    480 

GGCCCCTGCA CCAGCCCCCT CCTGGCCCCT GTCATCTTCT GTCCCTTCCC AGAAAACCTA    540 

CCAGGGCAGC TACGGTTTCC GTCTGGGCTT CTTGCATTCT GGGACAGCCA AGTCTGTGAC    600 

TTGCACGTAC TCCCCTGCCC TCAACAAGAT GTTTTGCCAA CTGGCCAAGA CCTGCCCTGT    660 

GCAGCTGTGG GTTGATTCCA CACCCCCGCC CGGCACCCGC GTCCGCGCCA TGGCCATCTA    720 

CAAGCAGTCA CAGCACATGA CGGAGGTTGT GAGGCGCTGC CCCCACCATG AGCGCTGCTC    780 

AGATAGCGAT GGTCTGGCCC CTCCTCAGCA TCTTATCCGA GTGGAAGGAA ATTTGCGTGT    840 

GGAGTATTTG GATGACAGAA ACACTTTTCG ACATAGTGTG GTGGTGCCCT ATGAGCCGCC    900 

TGAGGTTGGC TCTGACTGTA CCACCATCCA CTACAACTAC ATGTGTAACA GTTCCTGCAT    960 

GGGCGGCATG AACCGGAGGC CCATCCTCAC CATCATCACA CTGGAAGACT CCAGTGGTAA   1020 

TCTACTGGGA CGGAACAGCT TTGAGGTGCG TGTTTGTGCC TGTCCTGGGA GAGACCGGCG   1080 

CACAGAGGAA GAGAATCTCC GCAAGAAAGG GGAGCCTCAC CACGAGCTGC CCCCAGGGAG   1140 

CACTAAGCGA GCACTGCCCA ACAACACCAG CTCCTCTCCC CAGCCAAAGA AGAAACCACT   1200 

GGATGGAGAA TATTTCACCC TTCAGATCCG TGGGCGTGAG CGCTTCGAGA TGTTCCGAGA   1260 

GCTGAATGAG GCCTTGGAAC TCAAGGATGC CCAGGCTGGG AAGGAGCCAG GGGGGAGCAG   1320 

GGCTCACTCC AGCCACCTGA AGTCCAAAAA GGGTCAGTCT ACCTCCCGCC ATAAAAAACT   1380 

CATGTTCAAG ACAGAAGGGC CTGACTCAGA CTGAACGCGT TTTTTATCCC GGGCTCGAGT   1440 

CTAGAATCGA TCCCGGGTTT TTATGACTAG TTAATCACGG CCGC                    1484 

 
           
           
             
               42 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              104 

CAGACTCCTC TGCTCAAGAG ACATTACGAT ACAAACTTAA CG                        42 

 
           
           
             
               24 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              105 

ATGTCTCTTG AGCAGAGGAG TCTG                                            24 

 
           
           
             
               21 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              106 

CAGGCCATCA TAGGAGAGAC C                                               21 

 
           
           
             
               24 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              107 

GTGGCTGATT TGGTTGGTTT TCTG                                            24 

 
           
           
             
               37 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              108 

ATCATCTCTA GAAAAAAAAT CACATAGCTG GTTTCAG                              37 

 
           
           
             
               1094 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              109 

AAAAAGGATC CGGGTTAATT AATTAGTCAT CAGGCAGGGC GAGAACGAGA CTATCTGCTC     60 

GTTAATTAAT TAGAGCTTCT TTATTCTATA CTTAAAAAGT GAAAATAAAT ACAAAGGTTC    120 

TTGAGGGTTG TGTTAAATTG AAAGCGAGAA ATAATCATAA ATTATTTCAT TATCGCGATA    180 

TCCGTTAAGT TTGTATCGTA ATGTCTCTTG AGCAGAGGAG TCTGCACTGC AAGCCTGAGG    240 

AAGCCCTTGA GGCCCAACAA GAGGCCCTGG GCCTGGTGTG TGTGCAGGCT GCCACCTCCT    300 

CCTCCTCTCC TCTGGTCCTG GGCACCCTGG AGGAGGTGCC CACTGCTGGG TCAACAGATC    360 

CTCCCCAGAG TCCTCAGGGA GCCTCCGCCT TTCCCACTAC CATCAACTTC ACTCGACAGA    420 

GGCAACCCAG TGAGGGTTCC AGCAGCCGTG AAGAGGAGGG GCCAAGCACC TCTTGTATCC    480 

TGGAGTCCTT GTTCCGAGCA GTAATCACTA AGAAGGTGGC TGATTTGGTT GGTTTTCTGC    540 

TCCTCAAATA TCGAGCCAGG GAGCCAGTCA CAAAGGCAGA AATGCTGGAG AGTGTCATCA    600 

AAAATTACAA GCACTGTTTT CCTGAGATCT TCGGCAAAGC CTCTGAGTCC TTGCAGCTGG    660 

TCTTTGGCAT TGACGTGAAG GAAGCAGACC CCACCGGCCA CTCCTATGTC CTTGTCACCT    720 

GCCTAGGTCT CTCCTATGAT GGCCTGCTGG GTGATAATCA GATCATGCCC AAGACAGGCT    780 

TCCTGATAAT TGTCCTGGTC ATGATTGCAA TGGAGGGCGG CCATGCTCCT GAGGAGGAAA    840 

TCTGGGAGGA GCTGAGTGTG ATGGAGGTGT ATGATGGGAG GGAGCACAGT GCCTATGGGG    900 

AGCCCAGGAA GCTGCTCACC CAAGATTTGG TGCAGGAAAA GTACCTGGAG TACGGCAGGT    960 

GCCGGACAGT GATCCCGCAC GCTATGAGTT CCTGTGGGGT CCAAGGGCCC TCGCTGAAAC   1020 

CAGCTATGTG ATTTTTATTC TAGAATCGAT CCCGGGTTTT TATGACTAGT TAATCACGGC   1080 

CGCTTATAAA GATC                                                     1094 

 
           
           
             
               1084 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              110 

ATAAATCACT TTTTATACTA ATATTTAATT AATTAAGCTT GGTACCCTCG AAGCTTCTTT     60 

ATTCTATACT TAAAAAGTGA AAATAAATAC AAAGGTTCTT GAGGGTTGTG TTAAATTGAA    120 

AGCGAGAAAT AATCATAAAT TATTTCATTA TCGCGATATC CGTTAAGTTT GTATCGTAAT    180 

GTCTCTTGAG CAGAGGAGTC TGCACTGCAA GCCTGAGGAA GCCCTTGAGG CCCAACAAGA    240 

GGCCCTGGGC CTGGTGTGTG TGCAGGCTGC CACCTCCTCC TCCTCTCCTC TGGTCCTGGG    300 

CACCCTGGAG GAGGTGCCCA CTGCTGGGTC AACAGATCCT CCCCAGAGTC CTCAGGGAGC    360 

CTCCGCCTTT CCCACTACCA TCAACTTCAC TCGACAGAGG CAACCCAGTG AGGGTTCCAG    420 

CAGCCGTGAA GAGGAGGGGC CAAGCACCTC TTGTATCCTG GAGTCCTTGT TCCGAGCAGT    480 

AATCACTAAG AAGGTGGCTG ATTTGGTTGG TTTTCTGCTC CTCAAATATC GAGCCAGGGA    540 

GCCAGTCACA AAGGCAGAAA TGCTGGAGAG TGTCATCAAA AATTACAAGC ACTGTTTTCC    600 

TGAGATCTTC GGCAAAGCCT CTGAGTCCTT GCAGCTGGTC TTTGGCATTG ACGTGAAGGA    660 

AGCAGACCCC ACCGGCCACT CCTATGTCCT TGTCACCTGC CTAGGTCTCT CCTATGATGG    720 

CCTGCTGGGT GATAATCAGA TCATGCCCAA GACAGGCTTC CTGATAATTG TCCTGGTCAT    780 

GATTGCAATG GAGGGCGGCC ATGCTCCTGA GGAGGAAATC TGGGAGGAGC TGAGTGTGAT    840 

GGAGGTGTAT GATGGGAGGG AGCACAGTGC CTATGGGGAG CCCAGGAAGC TGCTCACCCA    900 

AGATTTGGTG CAGGAAAAGT ACCTGGAGTA CGGCAGGTGC CGGACAGTGA TCCCGCACGC    960 

TATGAGTTCC TGTGGGGTCC AAGGGCCCTC GCTGAAACCA GCTATGTGAT TTTTATTCTA   1020 

GAACTAGTGG ATCCCCCGGG TAGCTAGCTA ATTTTTCTTT TACGTATTAT ATATGTAATA   1080 

AACG                                                                1084 

 
           
           
             
               45 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              111 

TATCGCGATA TCCGTTAAGT TTGTATCGTA ATGGAGTCTC CCTCG                     45 

 
           
           
             
               31 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              112 

TGCTAGATCT TTATCTCTCG ACCACTGTAT G                                    31 

 
           
           
             
               23 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              113 

CTATGAGTGT GGAATCCAGA ACG                                             23 

 
           
           
             
               51 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              114 

TCAGAAGCTT CCCGGGTCTA GACTCGAGAT AAAAACTATA TCAGAGCAAC C              51 

 
           
           
             
               20 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              115 

GTCTCAGAAC GTGTTCATGT                                                 20 

 
           
           
             
               29 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              116 

CACGGATCCA TGAAGTCATA TATTTCCTT                                       29 

 
           
           
             
               30 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              117 

GTGAAGCTTA ATCCATAATC TTCAATAATT                                      30 

 
           
           
             
               30 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              118 

GTGAAGCTTT TATACATAAC AGAAATAACA                                      30 

 
           
           
             
               2981 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              119 

ATGAAGTCAT ATATTTCCTT GTTTTTCATA TTGTGTGTTA TATTTAACAA AAATGTTATA     60 

AAATGTACAG GAGAAAGTCA AACAGGTAAT ACAGGAGGAG GTCAAGCAGG TAATACAGGA    120 

GGAGGTCAAG CAGGTAATAC AGTAGGAGAT CAAGCAGGTA GTACAGGAGG AAGTCCACAA    180 

GGTAGTACGG GAGCAAGTCA ACCCGGAAGT TCCGAACCAA GCAATCCTGT AAGTTCCGGA    240 

CATTCTGTAA GTACTGTATC AGTATCACAA ACTTCAACTT CTTCAGAAAA ACAGGATACA    300 

ATTCAAGTAA AATCAGCTTT ATTAAAAGAT TATATGGGTT TAAAAGTTAC TGGTCCATGT    360 

AACGAAAATT TCATAATGTT CTTAGTTCCT CATATATATA TTGATGTTGA TACAGAAGAT    420 

ACTAATATCG AATTAAGAAC AACATTGAAA GAAACAAATA ATGCAATATC ATTTGAATCA    480 

AACAGTGGTT CATTAGAAAA AAAAAAATAT GTAAAACTAC CATCAAATGG TACAACTGGT    540 

GAACAAAGTT CTAGTTCAAG TTCAAGTTCT AGTTCAAATT CTAGTTCAAG TTCAAGTTCA    600 

AGTTCAAGTT CTAGTTCAAG TTCAAGTTCA AGTTCTAGTT CAAGTTCTAG TTCAAGTTCA    660 

GAAAGTCTTC CTGCTAATGG ACCTGATTCC CCTACTGTTA AACCGCCAAG AAATTTACAA    720 

AATATATGTG AAACTGGAAA AAACTTCAAG TTGGTAGTAT ATATTAAGGA GAATACATTA    780 

ATAATTAAAT GGAAAGTATA CGGAGAAACA AAAGATACTA CTGAAAATAA CAAAGTTGAT    840 

GTAAGAAAGT ATTTGATAAA TGAAAAGGAA ACCCCATTTA CTAGTATACT AATACATGCG    900 

TATAAAGAAC ATAATGGAAC AAACTTAATA GAAAGTAAAA ACTACGCATT AGGATCAGAC    960 

ATTCCAGAAA AATGTGATAC CTTAGCTTCC AATTGCTTTT TAAGTGGTAA TTTTAACATT   1020 

GAAAAATGCT TTCAATGTGC TCTTTTAGTA GAAAAAGAAA ATAAAAATGA CGTATGTTAC   1080 

AAATACCTAT CTGAAGATAT TGTAAGTAAA TTCAAAGAAA TAAAAGCTGA GACAGAAGAT   1140 

GATGATGAAG ATGATTATAC TGAATATAAA TTAACAGAAT CTATTGATAA TATATTAGTA   1200 

AAAATGTTTA AAACAAATGA AAATAATGAT AAATCAGAAT TAATAAAATT AGAAGAAGTA   1260 

GATGATAGTT TGAAATTAGA ATTAATGAAT TACTGTAGTT TACTTAAAGA CGTAGATACA   1320 

ACAGGTACCT TAGATAATTA TGGGATGGGA AATGAAATGG ATATATTTAA TAACTTAAAG   1380 

AGATTATTAA TTTATCATTC AGAAGAAAAT ATTAATACTT TAAAAAATAA ATTCCGTAAT   1440 

GCAGCTGTAT GTCTTAAAAA TGTTGATGAT TGGATTGTAA ATAAGAGAGG TTTAGTATTA   1500 

CCTGAATTAA ATTATGATTT AGAATATTTC AATGAACATT TATATAATGA TAAAAATTCT   1560 

CCAGAAGATA AAGATAATAA AGGAAAAGGT GTCGTACATG TTGATACAAC TTTAGAAAAA   1620 

GAAGATACTT TATCATATGA TAACTCAGAT AATATGTTTT GTAATAAAGA ATATTGTAAC   1680 

AGATTAAAAG ATGAAAATAA TTGTATATCT AATCTTCAAG TTGAAGATCA AGGTAATTGT   1740 

GATACTTCAT GGATTTTTGC TTCAAAATAT CATTTAGAAA CTATTAGATG TATGAAAGGA   1800 

TATGAACCTA CCAAAATTTC TGCTCTTTAT GTAGCTAATT GTTATAAAGG TGAACATAAA   1860 

GATAGATGTG ATGAAGGTTC TAGTCCAATG GAATTCTTAC AAATTATTGA AGATTATGGA   1920 

TTCTTACCAG CAGAATCAAA TTATCCATAT AACTATGTGA AAGTTGGAGA ACAATGTCCA   1980 

AAGGTAGAAG ATCACTGGAT GAATCTATGG GATAATGGAA AAATCTTACA TAACAAAAAT   2040 

GAACCTAATA GTTTAGATGG TAAGGGATAT ACTGCATATG AAAGTGAAAG ATTTCATGAT   2100 

AATATGGATG CATTTGTTAA AATTATTAAA ACTGAAGTAA TGAATAAAGG TTCAGTTATT   2160 

GCATATATTA AAGCTGAAAA TGTTATGGGA TATGAATTTA GTGGAAAGAA AGTACAGAAC   2220 

TTATGTGGTG ATGATACAGC TGATCATGCA GTTAATATTG TTGGTTATGG TAATTATGTG   2280 

AATAGCGAAG GAGAAAAAAA ATCCTATTGG ATTGTAAGAA ACAGTTGGGG TCCATATTGG   2340 

GGAGATGAAG GTTATTTTAA AGTAGATATG TATGGACCAA CTCATTGTCA TTTTAACTTT   2400 

ATTCACAGTG TTGTTATATT CAATGTTGAT TTACCTATGA ATAATAAAAC AACTAAAAAA   2460 

GAATCAAAAA TATATGATTA TTATTTAAAG GCCTCTCCAG AATTTTATCA TAACCTTTAC   2520 

TTTAAGAATT TTAATGTTGG TAAGAAAAAT TTATTCTCTG AAAAGGAAGA TAATGAAAAC   2580 

AACAAAAAAT TAGGTAACAA CTATATTATA TTCGGTCAAG ATACGGCAGG ATCAGGACAA   2640 

AGTGGAAAGG AAAGCAATAC TGCATTAGAA TCTGCAGGAA CTTCAAATGA AGTCTCAGAA   2700 

CGTGTTCATG TTTATCACAT ATTAAAACAT ATAAAGGATG GCAAAATAAG AATGGGTATG   2760 

CGTAAATATA TAGATACACA AGATGTAAAT AAGAAACATT CTTGTACAAG ATCCTATGCA   2820 

TTTAATCCAG AGAATTATGA AAAATGTGTA AATTTATGTA ATGTGAACTG GAAAACATGC   2880 

GAGGAAAAAA CATCACCAGG ACTTTGTTTA TCCAAATTGG ATACAAATAA CGAATGTTAT   2940 

TTCTGTTATG TATAAAATAA TATAACAAAA AAAAAAAAAA A                       2981 

 
           
           
             
               984 amino acids  
               amino acid  
               single  
               linear  
             
             
               peptide  
             
             internal  
              120 

Met Lys Ser Tyr Ile Ser Leu Phe Phe Ile Leu Cys Val Ile Phe Asn 
1               5                   10                  15 

Lys Asn Val Ile Lys Cys Thr Gly Glu Ser Gln Thr Gly Asn Thr Gly 
            20                  25                  30 

Gly Gly Gln Ala Gly Asn Thr Gly Gly Gly Gln Ala Gly Asn Thr Val 
        35                  40                  45 

Gly Asp Gln Ala Gly Ser Thr Gly Gly Ser Pro Gln Gly Ser Thr Gly 
    50                  55                  60 

Ala Ser Gln Pro Gly Ser Ser Glu Pro Ser Asn Pro Val Ser Ser Gly 
65                  70                  75                  80 

His Ser Val Ser Thr Val Ser Val Ser Ser Thr Ser Thr Ser Ser Glu 
                85                  90                  95 

Lys Gln Asp Thr Ile Gln Val Lys Ser Ala Leu Leu Lys Asp Tyr Met 
            100                 105                 110 

Gly Leu Lys Val Thr Gly Pro Cys Asn Glu Asn Phe Ile Met Phe Leu 
        115                 120                 125 

Val Pro His Ile Tyr Ile Asp Val Asp Thr Glu Asp Thr Asn Ile Glu 
    130                 135                 140 

Leu Arg Thr Thr Leu Lys Glu Thr Asn Asn Ala Ile Ser Phe Glu Ser 
145                 150                 155                 160 

Asn Ser Gly Ser Leu Glu Lys Lys Lys Tyr Val Lys Leu Pro Ser Asn 
                165                 170                 175 

Gly Thr Thr Gly Glu Gln Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 
            180                 185                 190 

Asn Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 
        195                 200                 205 

Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Glu Ser Leu Pro 
    210                 215                 220 

Ala Asn Gly Pro Asp Ser Pro Thr Val Lys Pro Pro Arg Asn Leu Gln 
225                 230                 235                 240 

Asn Ile Cys Glu Thr Gly Lys Asn Phe Lys Leu Val Val Tyr Ile Lys 
                245                 250                 255 

Glu Asn Thr Leu Ile Ile Lys Trp Lys Val Tyr Gly Glu Thr Lys Asp 
            260                 265                 270 

Thr Thr Glu Asn Asn Lys Val Asp Val Arg Lys Tyr Leu Ile Asn Glu 
        275                 280                 285 

Lys Glu Thr Pro Phe Thr Ser Ile Leu Ile His Ala Tyr Lys Glu His 
    290                 295                 300 

Asn Gly Thr Asn Leu Ile Glu Ser Lys Asn Tyr Ala Leu Gly Ser Asp 
305                 310                 315                 320 

Ile Pro Glu Lys Cys Asp Thr Leu Ala Ser Asn Cys Phe Leu Ser Gly 
                325                 330                 335 

Asn Phe Asn Ile Glu Lys Cys Phe Gln Cys Ala Leu Leu Val Glu Lys 
            340                 345                 350 

Glu Asn Lys Asn Asp Val Cys Tyr Lys Tyr Leu Ser Glu Asp Ile Val 
        355                 360                 365 

Ser Lys Phe Lys Glu Ile Lys Ala Glu Thr Glu Asp Asp Asp Glu Asp 
    370                 375                 380 

Asp Tyr Thr Glu Tyr Lys Leu Thr Glu Ser Ile Asp Asn Ile Leu Val 
385                 390                 395                 400 

Lys Met Phe Lys Thr Asn Glu Asn Asn Asp Lys Ser Glu Leu Ile Lys 
                405                 410                 415 

Leu Glu Glu Val Asp Asp Ser Leu Lys Leu Glu Leu Met Asn Tyr Cys 
            420                 425                 430 

Ser Leu Leu Lys Asp Val Asp Thr Thr Gly Thr Leu Asp Asn Tyr Gly 
        435                 440                 445 

Met Gly Asn Glu Met Asp Ile Phe Asn Asn Leu Lys Arg Leu Leu Ile 
    450                 455                 460 

Tyr His Ser Glu Glu Asn Ile Asn Thr Leu Lys Asn Lys Phe Arg Asn 
465                 470                 475                 480 

Ala Ala Val Cys Leu Lys Asn Val Asp Asp Trp Ile Val Asn Lys Arg 
                485                 490                 495 

Gly Leu Val Leu Pro Glu Leu Asn Tyr Asp Leu Glu Tyr Phe Asn Glu 
            500                 505                 510 

His Leu Tyr Asn Asp Lys Asn Ser Pro Glu Asp Lys Asp Asn Lys Gly 
        515                 520                 525 

Lys Gly Val Val His Val Asp Thr Thr Leu Glu Lys Glu Asp Thr Leu 
    530                 535                 540 

Ser Tyr Asp Asn Ser Asp Asn Met Phe Cys Asn Lys Glu Tyr Cys Asn 
545                 550                 555                 560 

Arg Leu Lys Asp Glu Asn Asn Cys Ile Ser Asn Leu Gln Val Glu Asp 
                565                 570                 575 

Gln Gly Asn Cys Asp Thr Ser Trp Ile Phe Ala Ser Lys Tyr His Leu 
            580                 585                 590 

Glu Thr Ile Arg Cys Met Lys Gly Tyr Glu Pro Thr Lys Ile Ser Ala 
        595                 600                 605 

Leu Tyr Val Ala Asn Cys Tyr Lys Gly Glu His Lys Asp Arg Cys Asp 
    610                 615                 620 

Glu Gly Ser Ser Pro Met Glu Phe Leu Gln Ile Ile Glu Asp Tyr Gly 
625                 630                 635                 640 

Phe Leu Pro Ala Glu Ser Asn Tyr Pro Tyr Asn Tyr Val Lys Val Gly 
                645                 650                 655 

Glu Gln Cys Pro Lys Val Glu Asp His Trp Met Asn Leu Trp Asp Asn 
            660                 665                 670 

Gly Lys Ile Leu His Asn Lys Asn Glu Pro Asn Ser Leu Asp Gly Lys 
        675                 680                 685 

Gly Tyr Thr Ala Tyr Glu Ser Glu Arg Phe His Asp Asn Met Asp Ala 
    690                 695                 700 

Phe Val Lys Ile Ile Lys Thr Glu Val Met Asn Lys Gly Ser Val Ile 
705                 710                 715                 720 

Ala Tyr Ile Lys Ala Glu Asn Val Met Gly Tyr Glu Phe Ser Gly Lys 
                725                 730                 735 

Lys Val Gln Asn Leu Cys Gly Asp Asp Thr Ala Asp His Ala Val Asn 
            740                 745                 750 

Ile Val Gly Tyr Gly Asn Tyr Val Asn Ser Glu Gly Glu Lys Lys Ser 
        755                 760                 765 

Tyr Trp Ile Val Arg Asn Ser Trp Gly Pro Tyr Trp Gly Asp Glu Gly 
    770                 775                 780 

Tyr Phe Lys Val Asp Met Tyr Gly Pro Thr His Cys His Phe Asn Phe 
785                 790                 795                 800 

Ile His Ser Val Val Ile Phe Asn Val Asp Leu Pro Met Asn Asn Lys 
                805                 810                 815 

Thr Thr Lys Lys Glu Ser Lys Ile Tyr Asp Tyr Tyr Leu Lys Ala Ser 
            820                 825                 830 

Pro Glu Phe Tyr His Asn Leu Tyr Phe Lys Asn Phe Asn Val Gly Lys 
        835                 840                 845 

Lys Asn Leu Phe Ser Glu Lys Glu Asp Asn Glu Asn Asn Lys Lys Leu 
    850                 855                 860 

Gly Asn Asn Tyr Ile Ile Phe Gly Gln Asp Thr Ala Gly Ser Gly Gln 
865                 870                 875                 880 

Ser Gly Lys Glu Ser Asn Thr Ala Leu Glu Ser Ala Gly Thr Ser Asn 
                885                 890                 895 

Glu Val Ser Glu Arg Val His Val Tyr His Ile Leu Lys His Ile Lys 
            900                 905                 910 

Asp Gly Lys Ile Arg Met Gly Met Arg Lys Tyr Ile Asp Thr Gln Asp 
        915                 920                 925 

Val Asn Lys Lys His Ser Cys Thr Arg Ser Tyr Ala Phe Asn Pro Glu 
    930                 935                 940 

Asn Tyr Glu Lys Cys Val Asn Leu Cys Asn Val Asn Trp Lys Thr Cys 
945                 950                 955                 960 

Glu Glu Lys Thr Ser Pro Gly Leu Cys Leu Ser Lys Leu Asp Thr Asn 
                965                 970                 975 

Asn Glu Cys Tyr Phe Cys Tyr Val 
            980 

 
           
           
             
               21 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              121 

TAGAATCTGC AGGAACTTCA A                                               21 

 
           
           
             
               54 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              122 

CTACACGAGC TCCCGGGCTC GAGATAAAAA TTATACATAA CAGAAATAAC ATTC           54 

 
           
           
             
               76 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              123 

CTAGAGAAGC TTCCCGGGAT CCTCAAAATT GAAAATATAT AATTACAATA TAAAATGAAG     60 

TCATATATTT CCTTGT                                                     76 

 
           
           
             
               19 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              124 

ACTTCCGGGT TGACTTGCT                                                  19 

 
           
           
             
               62 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              125 

GATCTTTTGT TAACAAAAAC TAATCAGCTA TCGCGAATCG ATTCCCGGGG GATCCGGTAC     60 

CC                                                                    62 

 
           
           
             
               62 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              126 

TCGAGGGTAC CGGATCCCCC GGGAATCGAT TCGCGATAGC TGATTAGTTT TTGTTAACAA     60 

AA                                                                    62 

 
           
           
             
               7351 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              127 

AGATATTTGT TAGCTTCTGC CGGAGATACC GTGAAAATCT ATTTTCTGGA AGGAAAGGGA     60 

GGTCTTATCT ATTCTGTCAG CAGAGTAGGT TCCTCTAATG ACGAAGACAA TAGTGAATAC    120 

TTGCATGAAG GTCACTGTGT AGAGTTCAAA ACTGATCATC AGTGTTTGAT AACTCTAGCG    180 

TGTACGAGTC CTTCTAACAC TGTGGTTTAT TGGCTGGAAT AAAAGGATAA AGACACCTAT    240 

ACTGATTCAT TTTCATCTGT CAACGTTTCT CTAAGAGATT CATAGGTATT ATTATTACAT    300 

CGATCTAGAA GTCTAATAAC TGCTAAGTAT ATTATTGGAT TTAACGCGCT ATAAACGCAT    360 

CCAAAACCTA CAAATATAGG AGAAGCTTCT CTTATGAAAC TTCTTAAAGC TTTACTCTTA    420 

CTATTACTAC TCAAAAGAGA TATTACATTA ATTATGTGAT GAGGCATCCA ACATATAAAG    480 

AAGACTAAAG CTGTAGAAGC TGTTATGAAG AATATCTTAT CAGATATATT AGATGCATTG    540 

TTAGTTCTGT AGATCAGTAA CGTATAGCAT ACGAGTATAA TTATCGTAGG TAGTAGGTAT    600 

CCTAAAATAA ATCTGATACA GATAATAACT TTGTAAATCA ATTCAGCAAT TTCTCTATTA    660 

TCATGATAAT GATTAATACA CAGCGTGTCG TTATTTTTTG TTACGATAGT ATTTCTAAAG    720 

TAAAGAGCAG GAATCCCTAG TATAATAGAA ATAATCCATA TGAAAAATAT AGTAATGTAC    780 

ATATTTCTAA TGTTAACATA TTTATAGGTA AATCCAGGAA GGGTAATTTT TACATATCTA    840 

TATACGCTTA TTACAGTTAT TAAAAATATA CTTGCAAACA TGTTAGAAGT AAAAAAGAAA    900 

GAACTAATTT TACAAAGTGC TTTACCAAAA TGCCAATGGA AATTACTTAG TATGTATATA    960 

ATGTATAAAG GTATGAATAT CACAAACAGC AAATCGGCTA TTCCCAAGTT GAGAAACGGT   1020 

ATAATAGATA TATTTCTAGA TACCATTAAT AACCTTATAA GCTTGACGTT TCCTATAATG   1080 

CCTACTAAGA AAACTAGAAG ATACATACAT ACTAACGCCA TACGAGAGTA ACTACTCATC   1140 

GTATAACTAC TGTTGCTAAC AGTGACACTG ATGTTATAAC TCATCTTTGA TGTGGTATAA   1200 

ATGTATAATA ACTATATTAC ACTGGTATTT TATTTCAGTT ATATACTATA TAGTATTAAA   1260 

AATTATATTT GTATAATTAT ATTATTATAT TCAGTGTAGA AAGTAAAATA CTATAAATAT   1320 

GTATCTCTTA TTTATAACTT ATTAGTAAAG TATGTACTAT TCAGTTATAT TGTTTTATAA   1380 

AAGCTAAATG CTACTAGATT GATATAAATG AATATGTAAT AAATTAGTAA TGTAGTATAC   1440 

TAATATTAAC TCACATTATG AATACTACTA ATCACGAAGA ATGCAGTAAA ACATATGATA   1500 

CAAACATGTT AACAGTTTTA AAAGCCATTA GTAATAAACA GTACAATATA ATTAAGTCTT   1560 

TACTTAAAAA AGATATTAAT GTTAATAGAT TATTAACTAG TTATTCTAAC GAAATATATA   1620 

AACATTTAGA CATTACATTA TGTAATATAC TTATAGAACG TGCAGCAGAC ATAAACATTA   1680 

TAGATAAGAA CAATCGTACA CCGTTGTTTT ATGCGGTAAA GAATAATGAT TATGATATGG   1740 

TTAAACTCCT ATTAAAAAAT GGCGCGAATG TAAATTTACA AGATAGTATA GGATATTCAT   1800 

GTCTTCACAT CGCAGGTATA CATAATAGTA ACATAGAAAT AGTAGATGCA TTGATATCAT   1860 

ACAAACCAGA TTTAAACTCC CGCGATTGGG TAGGTAGAAC ACCGCTACAT ATCTTCGTGA   1920 

TAGAATCTAA CTTTGAAGCT GTGAAATTAT TATTAAAGTC AGGTGCATAT GTAGGTTTGA   1980 

AAGACAAATG TAAGCATTTT CCTATACACC ATTCTGTAAT GAAATTAGAT CACTTAATAT   2040 

CAGGATTGTT ATTAAAATAT GGAGCAAATC CAAATACAAT TAACGGCAAT GGAAAAACAT   2100 

TATTAAGCAT TGCTGTAACA TCTAATAATA CACTACTGGT AGAACAGCTG CTGTTATATG   2160 

GAGCAGAAGT TAATAATGGT GGTTATGATG TTCCAGCTCC TATTATATCC GCTGTCAGTG   2220 

TTAACAATTA TGATATTGTT AAGATACTGA TACATAATGG TGCGAATATA AATGTATCCA   2280 

CGGAAGATGG TAGAACGTCT TTACATACAG CTATGTTTTG GAATAACGCT AAAATAATAG   2340 

ATGAGTTGCT TAACTATGGA AGTGACATAA ACAGCGTAGA TACTTATGGT AGAACTCCGT   2400 

TATCTTGTTA TCGTAGCTTA AGTTATGATA TCGCTACTAA ACTAATATCA CGTATCATTA   2460 

TAACAGATGT CTATCGTGAA GCACCAGTAA ATATCAGCGG ATTTATAATT AATTTAAAAA   2520 

CTATAGAAAA TAATGATATA TTCAAATTAA TTAAAGATGA TTGTATTAAA GAGATAAACA   2580 

TACTTAAAAG TATAACCCTT AATAAATTTC ATTCATCTGA CATATTTATA CGATATAATA   2640 

CTGATATATG TTTATTAACG AGATTTATTC AACATCCAAA GATAATAGAA CTAGACAAAA   2700 

AACTCTACGC TTATAAATCT ATAGTCAACG AGAGAAAAAT CAAAGCTACT TACAGGTATT   2760 

ATCAAATAAA AAAAGTATTA ACTGTACTAC CTTTTTCAGG ATATTTCTCT ATATTGCCGT   2820 

TTGATGTGTT AGTATATATA CTTGAATTCA TCTATGATAA TAATATGTTG GTACTTATGA   2880 

GAGCGTTATC ATTAAAATGA AATAAAAAGC ATACAAGCTA TTGCTTCGCT ATCGTTACAA   2940 

AATGGCAGGA ATTTTGTGTA AACTAAGCCA CATACTTGCC AATGAAAAAA ATAGTAGAAA   3000 

GGATACTATT TTAATGGGAT TAGATGTTAA GGTTCCTTGG GATTATAGTA ACTGGGCATC   3060 

TGTTAACTTT TACGACGTTA GGTTAGATAC TGATGTTACA GATTATAATA ATGTTACAAT   3120 

AAAATACATG ACAGGATGTG ATATTTTTCC TCATATAACT CTTGGAATAG CAAATATGGA   3180 

TCAATGTGAT AGATTTGAAA ATTTCAAAAA GCAAATAACT GATCAAGATT TACAGACTAT   3240 

TTCTATAGTC TGTAAAGAAG AGATGTGTTT TCCTCAGAGT AACGCCTCTA AACAGTTGGG   3300 

AGCGAAAGGA TGCGCTGTAG TTATGAAACT GGAGGTATCT GATGAACTTA GAGCCCTAAG   3360 

AAATGTTCTG CTGAATGCGG TACCCTGTTC GAAGGACGTG TTTGGTGATA TCACAGTAGA   3420 

TAATCCGTGG AATCCTCACA TAACAGTAGG ATATGTTAAG GAGGACGATG TCGAAAACAA   3480 

GAAACGCCTA ATGGAGTGCA TGTCCAAGTT TAGGGGGCAA GAAATACAAG TTCTAGGATG   3540 

GTATTAATAA GTATCTAAGT ATTTGGTATA ATTTATTAAA TAGTATAATT ATAACAAATA   3600 

ATAAATAACA TGATAACGGT TTTTATTAGA ATAAAATAGA GATAATATCA TAATGATATA   3660 

TAATACTTCA TTACCAGAAA TGAGTAATGG AAGACTTATA AATGAACTGC ATAAAGCTAT   3720 

AAGGTATAGA GATATAAATT TAGTAAGGTA TATACTTAAA AAATGCAAAT ACAATAACGT   3780 

AAATATACTA TCAACGTCTT TGTATTTAGC CGTAAGTATT TCTGATATAG AAATGGTAAA   3840 

ATTATTACTA GAACACGGTG CCGATATTTT AAAATGTAAA AATCCTCCTC TTCATAAAGC   3900 

TGCTAGTTTA GATAATACAG AAATTGCTAA ACTACTAATA GATTCTGGCG CTGACATAGA   3960 

ACAGATACAT TCTGGAAATA GTCCGTTATA TATTTCTGTA TATAGAAACA ATAAGTCATT   4020 

AACTAGATAT TTATTAAAAA AAGGTGTTAA TTGTAATAGA TTCTTTCTAA ATTATTACGA   4080 

TGTACTGTAT GATAAGATAT CTGATGATAT GTATAAAATA TTTATAGATT TTAATATTGA   4140 

TCTTAATATA CAAACTAGAA ATTTTGAAAC TCCGTTACAT TACGCTATAA AGTATAAGAA   4200 

TATAGATTTA ATTAGGATAT TGTTAGATAA TAGTATTAAA ATAGATAAAA GTTTATTTTT   4260 

GCATAAACAG TATCTCATAA AGGCACTTAA AAATAATTGT AGTTACGATA TAATAGCGTT   4320 

ACTTATAAAT CACGGAGTGC CTATAAACGA ACAAGATGAT TTAGGTAAAA CCCCATTACA   4380 

TCATTCGGTA ATTAATAGAA GAAAAGATGT AACAGCACTT CTGTTAAATC TAGGAGCTGA   4440 

TATAAACGTA ATAGATGACT GTATGGGCAG TCCCTTACAT TACGCTGTTT CACGTAACGA   4500 

TATCGAAACA ACAAAGACAC TTTTAGAAAG AGGATCTAAT GTTAATGTGG TTAATAATCA   4560 

TATAGATACC GTTCTAAATA TAGCTGTTGC ATCTAAAAAC AAAACTATAG TAAACTTATT   4620 

ACTGAAGTAC GGTACTGATA CAAAGTTGGT AGGATTAGAT AAACATGTTA TTCACATAGC   4680 

TATAGAAATG AAAGATATTA ATATACTGAA TGCGATCTTA TTATATGGTT GCTATGTAAA   4740 

CGTCTATAAT CATAAAGGTT TCACTCCTCT ATACATGGCA GTTAGTTCTA TGAAAACAGA   4800 

ATTTGTTAAA CTCTTACTTG ACCACGGTGC TTACGTAAAT GCTAAAGCTA AGTTATCTGG   4860 

AAATACTCCT TTACATAAAG CTATGTTATC TAATAGTTTT AATAATATAA AATTACTTTT   4920 

ATCTTATAAC GCCGACTATA ATTCTCTAAA TAATCACGGT AATACGCCTC TAACTTGTGT   4980 

TAGCTTTTTA GATGACAAGA TAGCTATTAT GATAATATCT AAAATGATGT TAGAAATATC   5040 

TAAAAATCCT GAAATAGCTA ATTCAGAAGG TTTTATAGTA AACATGGAAC ATATAAACAG   5100 

TAATAAAAGA CTACTATCTA TAAAAGAATC ATGCGAAAAA GAACTAGATG TTATAACACA   5160 

TATAAAGTTA AATTCTATAT ATTCTTTTAA TATCTTTCTT GACAATAACA TAGATCTTAT   5220 

GGTAAAGTTC GTAACTAATC CTAGAGTTAA TAAGATACCT GCATGTATAC GTATATATAG   5280 

GGAATTAATA CGGAAAAATA AATCATTAGC TTTTCATAGA CATCAGCTAA TAGTTAAAGC   5340 

TGTAAAAGAG AGTAAGAATC TAGGAATAAT AGGTAGGTTA CCTATAGATA TCAAACATAT   5400 

AATAATGGAA CTATTAAGTA ATAATGATTT ACATTCTGTT ATCACCAGCT GTTGTAACCC   5460 

AGTAGTATAA AGTGATTTTA TTCAATTACG AAGATAAACA TTAAATTTGT TAACAGATAT   5520 

GAGTTATGAG TATTTAACTA AAGTTACTTT AGGTACAAAT AAAATATTAT GTAATATAAT   5580 

AGAAAATTAT CTTGAGTCTT CATTTCCATC ACCGTCTAAA TTTATTATTA AAACCTTATT   5640 

ATATAAGGCT GTTGAGTTTA GAAATGTAAA TGCTGTAAAA AAAATATTAC AGAATGATAT   5700 

TGAATATGTT AAAGTAGATA GTCATGGTGT CTCGCCTTTA CATATTATAG CTATGCCTTC   5760 

AAATTTTTCT CTCATAGACG CTGACATGTA TTCAGAATTT AATGAAATTA GTAATAGACT   5820 

TCAAAAATCT AAAGATAGTA ACGAATTTCA ACGAGTTAGT CTACTAAGGA CAATTATAGA   5880 

ATATGGTAAT GATAGTGATA TTAATAAGTG TCTAACATTA GTAAAAACGG ATATACAGAG   5940 

TAACGAAGAG ATAGATATTA TAGATCTTTT GATAAATAAA GGAATAGATA TAAATATTAA   6000 

AGACGATTTA GGAAACACAG CTTTGCATTA CTCGTGTGAT TATGCTAAGG GATCAAAGAT   6060 

AGCTAAAAAG TTACTAGATT GTGGAGCAGA TCCTAACATA GTTAATGATT TAGGTGTTAC   6120 

ACCACTAGCG TGTGCCGTTA ATACTTGCAA CGAGATACTA GTAGATATTC TGTTAAATAA   6180 

TGATGCGAAT CCTGATTCAT CTTCCTCATA TTTTTTAGGT ACTAATGTGT TACATACAGC   6240 

CGTAGGTACC GGTAATATAG ATATTGTAAG ATCTTTACTT ACGGCTGGTG CCAATCCTAA   6300 

TGTAGGAGAT AAATCTGGAG TTACTCCTTT GCACGTTGCT GCAGCTGATA AAGACAGTTA   6360 

TCTGTTAATG GAGATGCTAC TAGATAGCGG GGCAGATCCA AATATAAAAT GCGCAAACGG   6420 

TTTTACTCCT TTGTTTAATG CAGTATATGA TCATAACCGT ATAAAGTTAT TATTTCTTTA   6480 

CGGGGCTGAT ATCAATATTA CTGACTCTTA CGGAAATACT CCTCTTACTT ATATGACTAA   6540 

TTTTGATAAT AAATATGTAA ATTCAATAAT TATCTTACAA ATATATCTAC TTAAAAAAGA   6600 

ATATAACGAT GAAAGATTGT TTCCACCTGG TATGATAAAA AATTTAAACT TTATAGAATC   6660 

AAACGATAGT CTTAAAGTTA TAGCTAAAAA GTGTAATTCG TTAATACGCT ATAAGAAAAA   6720 

TAAAGACATA GATGCAGATA ACGTATTATT GGAGCTTTTA GAGGAAGAGG AAGAAGATGA   6780 

AATAGACAGA TGGCATACTA CATGTAAAAT ATCTTAAATA GTAATTAAAT CATTGAAATA   6840 

TTAACTTACA AGATGATCGA GGTCACTTAT TATACTCTTT AATAATGGGT ACAAAGAGTA   6900 

TTCATACGTT AGTTAAATCT AACGATGTAA TACGTGTTCG TGAATTAATA AAGGATGATA   6960 

GATGTTTGAT AAATAAAAGA AATAGAAGAA ATCAGTCACC TGTATATATA GCTATATACA   7020 

AAGGACTTTA TGAAATGACT GAAATGTTAT TGCTAAATAA TGCAAGTCTA GATACTAAAA   7080 

TACCTTCTTT AATTATAGCA GCTAAAAATA ATGACTTACC TATGATAAAA TTATTGATAC   7140 

AATACGGGGC AAAATTAAAT GATATTTATT TAAGGGACAC AGCATTAATG ATAGCTCTCA   7200 

GAAATGGTTA CCTAGATATA GCTGAATATT TACTTTCATT AGGAGCAGAA TTTGTTAAAT   7260 

ACAGACATAA GGTAATATAT AAATATCTAT CAAAAGATGC GTATGAATTA CTTTTTAGAT   7320 

TTAATTATGC AGTTAATATA ATAGATTGAG A                                  7351 

 
           
           
             
               29 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              128 

CAGTTGGTAC CACTGGTATT TTATTTCAG                                       29 

 
           
           
             
               61 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              129 

TATCTGAATT CCTGCAGCCC GGGTTTTTAT AGCTAATTAG TCAAATGTGA GTTAATATTA     60 

G                                                                     61 

 
           
           
             
               66 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              130 

TCGCTGAATT CGATATCAAG CTTATCGATT TTTATGACTA GTTAATCAAA TAAAAAGCAT     60 

ACAAGC                                                                66 

 
           
           
             
               30 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              131 

TTATCGAGCT CTGTAACATC AGTATCTAAC                                      30 

 
           
           
             
               37 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              132 

TCCGGTACCG CGGCCGCAGA TATTTGTTAG CTTCTGC                              37 

 
           
           
             
               33 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              133 

TCGCTCGAGT AGGATACCTA CCTACTACCT ACG                                  33 

 
           
           
             
               29 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              134 

TCGCTCGAGC TTTCTTGACA ATAACATAG                                       29 

 
           
           
             
               30 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              135 

TAGGAGCTCT TTATACTACT GGGTTACAAC                                      30 

 
           
           
             
               17 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              136 

AATTCCTCGA GGGATCC                                                    17 

 
           
           
             
               15 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              137 

CGGGATCCCT CGAGG                                                      15 

 
           
           
             
               69 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              138 

CCGGTTAATT AATTAGTTAT TAGACAAGGT GAAAACGAAA CTATTTGTAG CTTAATTAAT     60 

TAGGTCACC                                                             69 

 
           
           
             
               70 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              139 

CCGGGGTCGA CCTAATTAAT TAAGCTACAA ATAGTTTCGT TTTCACCTTG TCTAATAACT     60 

AATTAATTAA                                                            70 

 
           
           
             
               39 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              140 

CCCCCCGAAT TCGTCGACGA TTGTTCATGA TGGCAAGAT                            39 

 
           
           
             
               68 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              141 

CCCGGGGGAT CCCTCGAGGG TACCAAGCTT AATTAATTAA ATATTAGTAT AAAAAGTGAT     60 

TTATTTTT                                                              68 

 
           
           
             
               77 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              142 

AAGCTTGGTA CCCTCGAGGG ATCCCCCGGG TAGCTAGCTA ATTTTTCTTT TACGTATTAT     60 

ATATGTAATA AACGTTC                                                    77 

 
           
           
             
               39 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              143 

TTTTTTCTGC AGGTAAGTAT TTTTAAAACT TCTAACACC                            39 

 
           
           
             
               2434 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              144 

GAAGCAATAG CTTGTATGCT TTTTATTTGA TTAACTAGTC ATAAAAATCG GGATCCTTCT     60 

TTATTCTATA CTTAAAAAGT GAAAATAAAT ACAAAGGTTC TTGAGGGTTG TGTTAAATTG    120 

AAAGCGAGAA ATAATCATAA ATTATTTCAT TATCGCGATA TCCGTTAAGT TTGTATCGTA    180 

ATGGAGTCTC CCTCGGCCCC TCCCCACAGA TGGTGCATCC CCTGGCAGAG GCTCCTGCTC    240 

ACAGCCTCAC TTCTAACCTT CTGGAACCCG CCCACCACTG CCAAGCTCAC TATTGAATCC    300 

ACGCCGTTCA ATGTCGCAGA GGGGAAGGAG GTGCTTCTAC TTGTCCACAA TCTGCCCCAG    360 

CATCTTTTTG GCTACAGCTG GTACAAAGGT GAAAGAGTGG ATGGCAACCG TCAAATTATA    420 

GGATATGTAA TAGGAACTCA ACAAGCTACC CCAGGGCCCG CATACAGTGG TCGAGAGATA    480 

ATATACCCCA ATGCATCCCT GCTGATCCAG AACATCATCC AGAATGACAC AGGATTCTAC    540 

ACCCTACACG TCATAAAGTC AGATCTTGTG AATGAAGAAG CAACTGGCCA GTTCCGGGTA    600 

TACCCGGAGC TGCCCAAGCC CTCCATCTCC AGCAACAACT CCAAACCCGT GGAGGACAAG    660 

GATGCTGTGG CCTTCACCTG TGAACCTGAG ACTCAGGACG CAACCTACCT GTGGTGGGTA    720 

AACAATCAGA GCCTCCCGGT CAGTCCCAGG CTGCAGCTGT CCAATGGCAA CAGGACCCTC    780 

ACTCTATTCA ATGTCACAAG AAATGACACA GCAAGCTACA AATGTGAAAC CCAGAACCCA    840 

GTGAGTGCCA GGCGCAGTGA TTCAGTCATC CTGAATGTCC TCTATGGCCC GGATGCCCCC    900 

ACCATTTCCC CTCTAAACAC ATCTTACAGA TCAGGGGAAA ATCTGAACCT CTCCTGCCAC    960 

GCAGCCTCTA ACCCACCTGC ACAGTACTCT TGGTTTGTCA ATGGGACTTT CCAGCAATCC   1020 

ACCCAAGAGC TCTTTATCCC CAACATCACT GTGAATAATA GTGGATCCTA TACGTGCCAA   1080 

GCCCATAACT CAGACACTGG CCTCAATAGG ACCACAGTCA CGACGATCAC AGTCTATGCA   1140 

GAGCCACCCA AACCCTTCAT CACCAGCAAC AACTCCAACC CCGTGGAGGA TGAGGATGCT   1200 

GTAGCCTTAA CCTGTGAACC TGAGATTCAG AACACAACCT ACCTGTGGTG GGTAAATAAT   1260 

CAGAGCCTCC CGGTCAGTCC CAGGCTGCAG CTGTCCAATG ACAACAGGAC CCTCACTCTA   1320 

CTCAGTGTCA CAAGGAATGA TGTAGGACCC TATGAGTGTG GAATCCAGAA CGAATTAAGT   1380 

GTTGACCACA GCGACCCAGT CATCCTGAAT GTCCTCTATG GCCCAGACGA CCCCACCATT   1440 

TCCCCCTCAT ACACCTATTA CCGTCCAGGG GTGAACCTCA GCCTCTCCTG CCATGCAGCC   1500 

TCTAACCCAC CTGCACAGTA TTCTTGGCTG ATTGATGGGA ACATCCAGCA ACACACACAA   1560 

GAGCTCTTTA TCTCCAACAT CACTGAGAAG AACAGCGGAC TCTATACCTG CCAGGCCAAT   1620 

AACTCAGCCA GTGGCCACAG CAGGACTACA GTCAAGACAA TCACAGTCTC TGCGGAGCTG   1680 

CCCAAGCCCT CCATCTCCAG CAACAACTCC AAACCCGTGG AGGACAAGGA TGCTGTGGCC   1740 

TTCACCTGTG AACCTGAGGC TCAGAACACA ACCTACCTGT GGTGGGTAAA TGGTCAGAGC   1800 

CTCCCAGTCA GTCCCAGGCT GCAGCTGTCC AATGGCAACA GGACCCTCAC TCTATTCAAT   1860 

GTCACAAGAA ATGACGCAAG AGCCTATGTA TGTGGAATCC AGAACTCAGT GAGTGCAAAC   1920 

CGCAGTGACC CAGTCACCCT GGATGTCCTC TATGGGCCGG ACACCCCCAT CATTTCCCCC   1980 

CCAGACTCGT CTTACCTTTC GGGAGCGAAC CTCAACCTCT CCTGCCACTC GGCCTCTAAC   2040 

CCATCCCCGC AGTATTCTTG GCGTATCAAT GGGATACCGC AGCAACACAC ACAAGTTCTC   2100 

TTTATCGCCA AAATCACGCC AAATAATAAC GGGACCTATG CCTGTTTTGT CTCTAACTTG   2160 

GCTACTGGCC GCAATAATTC CATAGTCAAG AGCATCACAG TCTCTGCATC TGGAACTTCT   2220 

CCTGGTCTCT CAGCTGGGGC CACTGTCGGC ATCATGATTG GAGTGCTGGT TGGGGTTGCT   2280 

CTGATATAGT TTTTATCTCG AGGAATTCCT GCAGCCCGGG GTGACCTAAT TAATTAAGCT   2340 

ACAAATAGTT TCGTTTTCAC CTTGTCTAAT AACTAATTAA TTAACCGGGT TTTTATAGCT   2400 

AATTAGTCAA ATGTGAGTTA ATATTAGTAT ACTA                               2434 

 
           
           
             
               2349 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              145 

TAAAAATAAA TCACTTTTTA TACTAATATT TAATTAATTA AGCTTGGTAC CCTCGAAGCT     60 

TCTTTATTCT ATACTTAAAA AGTGAAAATA AATACAAAGG TTCTTGAGGG TTGTGTTAAA    120 

TTGAAAGCGA GAAATAATCA TAAATTATTT CATTATCGCG ATATCCGTTA AGTTTGTATC    180 

GTAATGGAGT CTCCCTCGGC CCCTCCCCAC AGATGGTGCA TCCCCTGGCA GAGGCTCCTG    240 

CTCACAGCCT CACTTCTAAC CTTCTGGAAC CCGCCCACCA CTGCCAAGCT CACTATTGAA    300 

TCCACGCCGT TCAATGTCGC AGAGGGGAAG GAGGTGCTTC TACTTGTCCA CAATCTGCCC    360 

CAGCATCTTT TTGGCTACAG CTGGTACAAA GGTGAAAGAG TGGATGGCAA CCGTCAAATT    420 

ATAGGATATG TAATAGGAAC TCAACAAGCT ACCCCAGGGC CCGCATACAG TGGTCGAGAG    480 

ATAATATACC CCAATGCATC CCTGCTGATC CAGAACATCA TCCAGAATGA CACAGGATTC    540 

TACACCCTAC ACGTCATAAA GTCAGATCTT GTGAATGAAG AAGCAACTGG CCAGTTCCGG    600 

GTATACCCGG AGCTGCCCAA GCCCTCCATC TCCAGCAACA ACTCCAAACC CGTGGAGGAC    660 

AAGGATGCTG TGGCCTTCAC CTGTGAACCT GAGACTCAGG ACGCAACCTA CCTGTGGTGG    720 

GTAAACAATC AGAGCCTCCC GGTCAGTCCC AGGCTGCAGC TGTCCAATGG CAACAGGACC    780 

CTCACTCTAT TCAATGTCAC AAGAAATGAC ACAGCAAGCT ACAAATGTGA AACCCAGAAC    840 

CCAGTGAGTG CCAGGCGCAG TGATTCAGTC ATCCTGAATG TCCTCTATGG CCCGGATGCC    900 

CCCACCATTT CCCCTCTAAA CACATCTTAC AGATCAGGGG AAAATCTGAA CCTCTCCTGC    960 

CACGCAGCCT CTAACCCACC TGCACAGTAC TCTTGGTTTG TCAATGGGAC TTTCCAGCAA   1020 

TCCACCCAAG AGCTCTTTAT CCCCAACATC ACTGTGAATA ATAGTGGATC CTATACGTGC   1080 

CAAGCCCATA ACTCAGACAC TGGCCTCAAT AGGACCACAG TCACGACGAT CACAGTCTAT   1140 

GCAGAGCCAC CCAAACCCTT CATCACCAGC AACAACTCCA ACCCCGTGGA GGATGAGGAT   1200 

GCTGTAGCCT TAACCTGTGA ACCTGAGATT CAGAACACAA CCTACCTGTG GTGGGTAAAT   1260 

AATCAGAGCC TCCCGGTCAG TCCCAGGCTG CAGCTGTCCA ATGACAACAG GACCCTCACT   1320 

CTACTCAGTG TCACAAGGAA TGATGTAGGA CCCTATGAGT GTGGAATCCA GAACGAATTA   1380 

AGTGTTGACC ACAGCGACCC AGTCATCCTG AATGTCCTCT ATGGCCCAGA CGACCCCACC   1440 

ATTTCCCCCT CATACACCTA TTACCGTCCA GGGGTGAACC TCAGCCTCTC CTGCCATGCA   1500 

GCCTCTAACC CACCTGCACA GTATTCTTGG CTGATTGATG GGAACATCCA GCAACACACA   1560 

CAAGAGCTCT TTATCTCCAA CATCACTGAG AAGAACAGCG GACTCTATAC CTGCCAGGCC   1620 

AATAACTCAG CCAGTGGCCA CAGCAGGACT ACAGTCAAGA CAATCACAGT CTCTGCGGAG   1680 

CTGCCCAAGC CCTCCATCTC CAGCAACAAC TCCAAACCCG TGGAGGACAA GGATGCTGTG   1740 

GCCTTCACCT GTGAACCTGA GGCTCAGAAC ACAACCTACC TGTGGTGGGT AAATGGTCAG   1800 

AGCCTCCCAG TCAGTCCCAG GCTGCAGCTG TCCAATGGCA ACAGGACCCT CACTCTATTC   1860 

AATGTCACAA GAAATGACGC AAGAGCCTAT GTATGTGGAA TCCAGAACTC AGTGAGTGCA   1920 

AACCGCAGTG ACCCAGTCAC CCTGGATGTC CTCTATGGGC CGGACACCCC CATCATTTCC   1980 

CCCCCAGACT CGTCTTACCT TTCGGGAGCG AACCTCAACC TCTCCTGCCA CTCGGCCTCT   2040 

AACCCATCCC CGCAGTATTC TTGGCGTATC AATGGGATAC CGCAGCAACA CACACAAGTT   2100 

CTCTTTATCG CCAAAATCAC GCCAAATAAT AACGGGACCT ATGCCTGTTT TGTCTCTAAC   2160 

TTGGCTACTG GCCGCAATAA TTCCATAGTC AAGAGCATCA CAGTCTCTGC ATCTGGAACT   2220 

TCTCCTGGTC TCTCAGCTGG GGCCACTGTC GGCATCATGA TTGGAGTGCT GGTTGGGGTT   2280 

GCTCTGATAT AGTTTTTATC TCGAGGGATC CCCCGGGTAG CTAGCTAATT TTTCTTTTAC   2340 

GTATTATAT                                                           2349 

 
           
           
             
               42 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              146 

ATCATCGGAT CCCTGCAGCC CGGGTTAATT AATTAGTGAT AC                        42 

 
           
           
             
               38 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              147 

GAGCTGCATG CTGTACATTA CGATACAAAC TTAACGGA                             38 

 
           
           
             
               36 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              148 

CGTTAAGTTT GTATCGTAAT GTACAGCATG CAGCTG                               36 

 
           
           
             
               38 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              149 

GAGGAGGAAT TCCCCGGGTT ATTGAGGGCT TGTTGAGA                             38 

 
           
           
             
               510 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              150 

ATGTACAGCA TGCAGCTCGC ATCCTGTGTC ACATTGACAC TTGTGCTCCT TGTCAACAGC     60 

GCACCCACTT CAAGCTCCAC TTCAAGCTCT ACAGCGGAAG CACAGCAGCA GCAGCAGCAG    120 

CAGCAGCAGC AGCAGCAGCA CCTGGAGCAG CTGTTGATGG ACCTACAGGA GCTCCTGAGC    180 

AGGATGGAGA ATTACAGGAA CCTGAAACTC CCCAGGATGC TCACCTTCAA ATTTTACTTG    240 

CCCAAGCAGG CCACAGAATT GAAAGATCTT CAGTGCCTAG AAGATGAACT TGGACCTCTG    300 

CGGCATGTTC TGGATTTGAC TCAAAGCAAA AGCTTTCAAT TGGAAGATGC TGAGAATTTC    360 

ATCAGCAATA TCAGAGTAAC TGTTGTAAAA CTAAAGGGCT CTGACAACAC ATTTGAGTGC    420 

CAATTCGATG ATGAGTCAGC AACTGTGGTG GACTTTCTGA GGAGATGGAT AGCCTTCTGT    480 

CAAAGCATCA TCTCAACAAG CCCTCAATAA                                     510 

 
           
           
             
               17 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              151 

GGCCGCGTCG ACATGCA                                                    17 

 
           
           
             
               9 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              152 

TGTCGACGC                                                              9 

 
           
           
             
               14 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              153 

GGTCGACGGA TCCT                                                       14 

 
           
           
             
               22 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              154 

GATCAGGATC CGTCGACCTG CA                                              22 

 
           
           
             
               38 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              155 

GAGTTGCATC CTGTACATTA CGATACAAAC TTAACGGA                             38 

 
           
           
             
               36 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              156 

CGTTAAGTTT GTATCGTAAT GTACAGGATG CAACTC                               36 

 
           
           
             
               79 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              157 

TTGTAGCTGT GTTTTCTTTG TAGAACTTGA AGTAGGTGCA CTGTTTGTGA CAAGTGCAAG     60 

ACTTAGTGCA ATGCAAGAC                                                  79 

 
           
           
             
               79 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              158 

TTCTACAAAG AAAACACAGC TACAACTGGA GCATTTACTT CTGGATTTAC AGATGATTTT     60 

GAATGGAATT AATAATTAC                                                  79 

 
           
           
             
               462 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              159 

ATGTACAGGA TGCAACTCCT GTCTTGCATT GCACTAAGTC TTGCACTTGT CACAAACAGT     60 

GCACCTACTT CAAGTTCTAC AAAGAAAACA CAGCTACAAC TGGAGCATTT ACTTCTGGAT    120 

TTACAGATGA TTTTGAATGG AATTAATAAT TACAAGAATC CCAAACTCAC CAGGATGCTC    180 

ACATTTAAGT TTTACATGCC CAAGAAGGCC ACAGAACTGA AACATCTTCA GTGTCTAGAA    240 

GAAGAACTCA AACCTCTGGA GGAAGTGCTA AATTTAGCTC AAAGCAAAAA CTTTCACTTA    300 

AGACCCAGGG ACTTAATCAG CAATATCAAC GTAATAGTTC TGGAACTAAA GGGATCTGAA    360 

ACAACATTCA TGTGTGAATA TGCTGATGAG ACAGCAACCA TTGTAGAATT TCTGAACAGA    420 

TGGATTACCT TTTGTCAAAG CATCATCTCA ACACTGACTT GA                       462 

 
           
           
             
               38 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              160 

GAGGAGGAAT TCCCCGGGTC AAGTCAGTGT TGAGATGA                             38 

 
           
           
             
               23 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              161 

TAATCATGAA CGCTACACAC TGC                                             23 

 
           
           
             
               33 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              162 

CCCGGATCCC TGCAGTTATT GGGACAATCT CTT                                  33 

 
           
           
             
               598 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              163 

ACATCATGCA GTGGTTAAAC AAAAACATTT TTATTCTCAA ATGAGATAAA GTGAAAATAT     60 

ATATCATTAT ATTACAAAGT ACAATTATTT AGGTTTAATC ATGAACGCTA CACACTGCAT    120 

CTTGGCTTTG CAGCTCTTCC TCATGGCTGT TTCTGGCTGT TACTGCCACG GCACAGTCAT    180 

TGAAAGCCTA GAAAGTCTGA ATAACTATTT TAACTCAAGT GGCATAGATG TGGAAGAAAA    240 

GAGTCTCTTC TTGGATATCT GGAGGAACTG GCAAAAGGAT GGTGACATGA AAATCCTGCA    300 

GAGCCAGATT ATCTCTTTCT ACCTCAGACT CTTTGAAGTC TTGAAAGACA ATCAGGCCAT    360 

CAGCAACAAC ATAAGCGTCA TTGAATCACA CCTGATTACT ACCTTCTTCA GCAACAGCAA    420 

GGCGAAAAAG GATGCATTCA TGAGTATTGC CAAGTTTGAG GTCAACAACC CACAGGTCCA    480 

GCGCCAAGCA TTCAATGAGC TCATCCGAGT GGTCCACCAG CTGTTGCCGG AATCCAGCCT    540 

CAGGAAGCGG AAAAGGAGTC GCTGCTGATT CGGGGTGGGG AAGAGATTGT CCCAATAA      598 

 
           
           
             
               97 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              164 

TAATCATGAA ATATACAAGT TATATCTTGG CTTTTCAGCT CTGCATCGTT TTGGGTTCTC     60 

TTGGCTGTTA CTGCCAGGAC CCATATGTAA AAGAAGC                              97 

 
           
           
             
               106 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              165 

TTCTTCAAAA TGCCTAAGAA AAGAGTTCCA TTATCCGCTA CATCTGAATG ACCTGCATTA     60 

AAATATTTCT TAAGGTTTTC TGCTTCTTTT ACATATGGGT CCTGGC                   106 

 
           
           
             
               45 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              166 

TCTTTTCTTA GGCATTTTGA AGAATTGGAA AGAGGAGAGT GACAG                     45 

 
           
           
             
               34 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              167 

CCCGGATCCC TGCAGTTACT GGGATGCTCT TCGA                                 34 

 
           
           
             
               601 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              168 

ACATCATGCA GTGGTTAAAC AAAAACATTT TTATTCTCAA ATGAGATAAA GTGAAAATAT     60 

ATATCATTAT ATTACAAAGT ACAATTATTT AGGTTTAATC ATGAAATATA CAAGTTATAT    120 

CTTGGCTTTT CAGCTCTGCA TCGTTTTGGG TTCTCTTGGC TGTTACTGCC AGGACCCATA    180 

TGTAAAAGAA GCAGAAAACC TTAAGAAATA TTTTAATGCA GGTCATTCAG ATGTAGCGGA    240 

TAATGGAACT CTTTTCTTAG GCATTTTGAA GAATTGGAAA GAGGAGAGTG ACAGAAAAAT    300 

AATGCAGAGC CAAATTGTCT CCTTTTACTT CAAACTTTTT AAAAACTTTA AAGATGACCA    360 

GAGCATCCAA AAGAGTGTGG AGACCATCAA GGAAGACATG AATGTCAAGT TTTTCAATAG    420 

CAACAAAAAG AAACGAGATG ACTTCGAAAA GCTGACTAAT TATTCGGTAA CTGACTTGAA    480 

TGTCCAACGC AAAGCAATAC ATGAACTCAT CCAAGTGATG GCTGAACTGT CGCCAGCAGC    540 

TAAAACAGGG AAGCGAAAAA GGAGTCAGAT GCTGTTTCAA GGTCGAAGAG CATCCCAGTA    600 

A                                                                    601 

 
           
           
             
               3063 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              169 

AAGCTTCTAT CAAAAGTCTT AATGAGTTAG GTGTAGATAG TATAGATATT ACTACAAAGG     60 

TATTCATATT TCCTATCAAT TCTAAAGTAG ATGATATTAA TAACTCAAAG ATGATGATAG    120 

TAGATAATAG ATACGCTCAT ATAATGACTG CAAATTTGGA CGGTTCACAT TTTAATCATC    180 

ACGCGTTCAT AAGTTTCAAC TGCATAGATC AAAATCTCAC TAAAAAGATA GCCGATGTAT    240 

TTGAGAGAGA TTGGACATCT AACTACGCTA AAGAAATTAC AGTTATAAAT AATACATAAT    300 

GGATTTTGTT ATCATCAGTT ATATTTAACA TAAGTACAAT AAAAAGTATT AAATAAAAAT    360 

ACTTACTTAC GAAAAAATGT CATTATTACA AAAACTATAT TTTACAGAAC AATCTATAGT    420 

AGAGTCCTTT AAGAGTTATA ATTTAAAAGA TAACCATAAT GTAATATTTA CCACATCAGA    480 

TGATGATACT GTTGTAGTAA TAAATGAAGA TAATGTACTG TTATCTACAA GATTATTATC    540 

ATTTGATAAA ATTCTGTTTT TTAACTCCTT TAATAACGGT TTATCAAAAT ACGAAACTAT    600 

TAGTGATACA ATATTAGATA TAGATACTCA TAATTATTAT ATACCTAGTT CTTCTTCTTT    660 

GTTAGATATT CTAAAAAAAA GAGCGTGTGA TTTAGAATTA GAAGATCTAA ATTATGCGTT    720 

AATAGGAGAC AATAGTAACT TATATTATAA AGATATGACT TACATGAATA ATTGGTTATT    780 

TACTAAAGGA TTATTAGATT ACAAGTTTGT ATTATTGCGC GATGTAGATA AATGTTACAA    840 

ACAGTATAAT AAAAAGAATA CTATAATAGA TATAATACAT CGCGATAACA GACAGTATAA    900 

CATATGGGTT AAAAATGTTA TAGAATACTG TTCTCCTGGC TATATATTAT GGTTACATGA    960 

TCTAAAAGCC GCTGCTGAAG ATGATTGGTT AAGATACGAT AACCGTATAA ACGAATTATC   1020 

TGCGGATAAA TTATACACTT TCGAGTTCAT AGTTATATTA GAAAATAATA TAAAACATTT   1080 

ACGAGTAGGT ACAATAATTG TACATCCAAA CAAGATAATA GCTAATGGTA CATCTAATAA   1140 

TATACTTACT GATTTTCTAT CTTACGTAGA AGAACTAATA TATCATCATA ATTCATCTAT   1200 

AATATTGGCC GGATATTTTT TAGAATTCTT TGAGACCACT ATTTTATCAG AATTTATTTC   1260 

TTCATCTTCT GAATGGGTAA TGAATAGTAA CTGTTTAGTA CACCTGAAAA CAGGGTATGA   1320 

AGCTATACTC TTTGATGCTA GTTTATTTTT CCAACTCTCT ACTAAAAGCA ATTATGTAAA   1380 

ATATTGGACA AAGAAAACTT TGCAGTATAA GAACTTTTTT AAAGACGGTA AACAGTTAGC   1440 

AAAATATATA ATTAAGAAAG ATAGTCAGGT GATAGATAGA GTATGTTATT TACACGCAGC   1500 

TGTATATAAT CACGTAACTT ACTTAATGGA TACGTTTAAA ATTCCTGGTT TTGATTTTAA   1560 

ATTCTCCGGA ATGATAGATA TACTACTGTT TGGAATATTG CATAAGGATA ATGAGAATAT   1620 

ATTTTATCCG AAACGTGTTT CTGTAACTAA TATAATATCA GAATCTATCT ATGCAGATTT   1680 

TTACTTTATA TCAGATGTTA ATAAATTCAG TAAAAAGATA GAATATAAAA CTATGTTTCC   1740 

TATACTCGCA GAAAACTACT ATCCAAAAGG AAGGCCCTAT TTTACACATA CATCTAACGA   1800 

AGATCTTCTG TCTATCTGTT TATGCGAAGT AACAGTTTGT AAAGATATAA AAAATCCATT   1860 

ATTATATTCT AAAAAGGATA TATCAGCAAA ACGATTCATA GGTTTATTTA CATCTGTCGA   1920 

TATAAATACG GCTGTTGAGT TAAGAGGATA TAAAATAAGA GTAATAGGAT GTTTAGAATG   1980 

GCCTGAAAAG ATAAAAATAT TTAATTCTAA TCCTACATAC ATTAGATTAT TACTAACAGA   2040 

AAGACGTTTA GATATTCTAC ATTCCTATCT GCTTAAATTT AATATAACAG AGGATATAGC   2100 

TACCAGAGAT GGAGTCAGAA ATAATTTACC TATAATTTCT TTTATCGTCA GTTATTGTAG   2160 

ATCGTATACT TATAAATTAC TAAATTGCCA TATGTACAAT TCGTGTAAGA TAACAAAGTG   2220 

TAAATATAAT CAGGTAATAT ATAATCCTAT ATAGGAGTAT ATATAATTGA AAAAGTAAAA   2280 

TATAAATCAT ATAATAATGA AACGAAATAT CAGTAATAGA CAGGAACTGG CAGATTCTTC   2340 

TTCTAATGAA GTAAGTACTG CTAAATCTCC AAAATTAGAT AAAAATGATA CAGCAAATAC   2400 

AGCTTCATTC AACGAATTAC CTTTTAATTT TTTCAGACAC ACCTTATTAC AAACTAACTA   2460 

AGTCAGATGA TGAGAAAGTA AATATAAATT TAACTTATGG GTATAATATA ATAAAGATTC   2520 

ATGATATTAA TAATTTACTT AACGATGTTA ATAGACTTAT TCCATCAACC CCTTCAAACC   2580 

TTTCTGGATA TTATAAAATA CCAGTTAATG ATATTAAAAT AGATTGTTTA AGAGATGTAA   2640 

ATAATTATTT GGAGGTAAAG GATATAAAAT TAGTCTATCT TTCACATGGA AATGAATTAC   2700 

CTAATATTAA TAATTATGAT AGGAATTTTT TAGGATTTAC AGCTGTTATA TGTATCAACA   2760 

ATACAGGCAG ATCTATGGTT ATGGTAAAAC ACTGTAACGG GAAGCAGCAT TCTATGGTAA   2820 

CTGGCCTATG TTTAATAGCC AGATCATTTT ACTCTATAAA CATTTTACCA CAAATAATAG   2880 

GATCCTCTAG ATATTTAATA TTATATCTAA CAACAACAAA AAAATTTAAC GATGTATGGC   2940 

CAGAAGTATT TTCTACTAAT AAAGATAAAG ATAGTCTATC TTATCTACAA GATATGAAAG   3000 

AAGATAATCA TTTAGTAGTA GCTACTAATA TGGAAAGAAA TGTATACAAA AACGTGGAAG   3060 

CTT                                                                 3063 

 
           
           
             
               42 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              170 

ATCATCGAGC TCGCGGCCGC CTATCAAAAG TCTTAATGAG TT                        42 

 
           
           
             
               73 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              171 

GAATTCCTCG AGCTGCAGCC CGGGTTTTTA TAGCTAATTA GTCATTTTTT CGTAAGTAAG     60 

TATTTTTATT TAA                                                        73 

 
           
           
             
               72 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              172 

CCCGGGCTGC AGCTCGAGGA ATTCTTTTTA TTGATTAACT AGTCAAATGA GTATATATAA     60 

TTGAAAAAGT AA                                                         72 

 
           
           
             
               45 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              173 

GATGATGGTA CCTTCATAAA TACAAGTTTG ATTAAACTTA AGTTG                     45 

 
           
           
             
               1615 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              174 

GAGCTCGCGG CCGCCTATCA AAAGTCTTAA TGAGTTAGGT GTAGATAGTA TAGATATTAC     60 

TACAAAGGTA TTCATATTTC CTATCAATTC TAAAGTAGAT GATATTAATA ACTCAAAGAT    120 

GATGATAGTA GATAATAGAT ACGCTCATAT AATGACTGCA AATTTGGACG GTTCACATTT    180 

TAATCATCAC GCGTTCATAA GTTTCAACTG CATAGATCAA AATCTCACTA AAAAGATAGC    240 

CGATGTATTT GAGAGAGATT GGACATCTAA CTACGCTAAA GAAATTACAG TTATAAATAA    300 

TACATAATGG ATTTTGTTAT CATCAGTTAT ATTTAACATA AGTACAATAA AAAGTATTAA    360 

ATAAAAATAC TTACTTACGA AAAATGACTA ATTAGCTATA AAAACCCGGG CTGCAGCTCG    420 

AGGAATTCTT TTTATTGATT AACTAGTCAA ATGAGTATAT ATAATTGAAA AAGTAAAATA    480 

TAAATCATAT AATAATGAAA CGAAATATCA GTAATAGACA GGAACTGGCA GATTCTTCTT    540 

CTAATGAAGT AAGTACTGCT AAATCTCCAA AATTAGATAA AAATGATACA GCAAATACAG    600 

CTTCATTCAA CGAATTACCT TTTAATTTTT TCAGACACAC CTTATTACAA ACTAACTAAG    660 

TCAGATGATG AGAAAGTAAA TATAAATTTA ACTTATGGGT ATAATATAAT AAAGATTCAT    720 

GATATTAATA ATTTACTTAA CGATGTTAAT AGACTTATTC CATCAACCCC TTCAAACCTT    780 

TCTGGATATT ATAAAATACC AGTTAATGAT ATTAAAATAG ATTGTTTAAG AGATGTAAAT    840 

AATTATTTGG AGGTAAAGGA TATAAAATTA GTCTATCTTT CACATGGAAA TGAATTACCT    900 

AATATTAATA ATTATGATAG GAATTTTTTA GGATTTACAG CTGTTATATG TATCAACAAT    960 

ACAGGCAGAT CTATGGTTAT GGTAAAACAC TGTAACGGGA AGCAGCATTC TATGGTAACT   1020 

TGGCCTATGT TTAATAGCCA GATCATTTTA CTCTATAAAC ATTTTACCAC AAATAATAGG   1080 

ATCCTCTAGA TATTTAATAT TATATCTAAC AACAACAAAA AAATTTAACG ATGTATGGCC   1140 

AGAAGTATTT TCTACTAATA AAGATAAAGA TAGTCTATCT TATCTACAAG ATATGAAAGA   1200 

AGATAATCAT TTAGTAGTAG CTACTAATAT GGAAAGAAAT GTATACAAAA ACGTGGAAGC   1260 

TTTTATATTA AATAGCATAT TACTAGAAGA TTTAAAATCT AGACTTAGTA TAACAAAACA   1320 

GTTAAATGCC AATATCGATT CTATATTTCA TCATAACAGT AGTACATTAA TCAGTGATAT   1380 

ACTGAAACGA TCTACAGACT CAACTATGCA AGGAATAAGC AATATGCCAA TTATGTCTAA   1440 

TATTTTAACT TTAGAACTAA AACGTTCTAC CAATACTAAA AATAGGATAC GTGATAGGCT   1500 

GTTAAAAGCT GCAATAAATA GTAAGGATGT AGAAGAAATA CTTTGTTCTA TACCTTCGGA   1560 

GGAAAGAACT TTAGAACAAC TTAAGTTTAA TCAAACTTGT ATTTATGAAG GTACC        1615 

 
           
           
             
               83 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              175 

TTAATCAGGA TCCTTAATTA ATTAGTTATT AGACAAGGTG AAACGAAACT ATTTGTAGCT     60 

TAATTAATTA GCTGCAGCCC GGG                                             83 

 
           
           
             
               84 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              176 

CCCGGGCTGC AGCTAATTAA TTAAGCTACA AATAGTTTCG TTTTCACCTT GTCTAATAAC     60 

TAATTAATTA AGGATCCTGA TTAA                                            84 

 
           
           
             
               31 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              177 

CAAAATTGAA AATATATAAT TACAATATAA A                                    31 

 
           
           
             
               490 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              178 

GAATTCGAAT AAAAAAATGA TAAAGTAGGT TCAGTTTTAT TGCTGGTTGT GTTAGTTCTC     60 

TCTAAAAATG GGTCTCAACC CCCAGCTAGT TGTCATCCTG CTCTTCTTTC TCGAATGTAC    120 

CAGGAGCCAT ATCCACGGAT GCGACAAAAA TCACTTGAGA GAGATCATCG GCATTTTGAA    180 

CGAGGTCACA GGAGAAGGGA CGCCATGCAC GGAGATGGAT GTGCCAAACG TCCTCACAGC    240 

AACGAAGAAC ACCACAGAGA GTGAGCTCGT CTGTAGGGCT TCCAAGGTGC TTCGTATATT    300 

TTATTTAAAA CATGGGAAAA CTCCATGCTT GAAGAAGAAC TCTAGTGTTC TCATGGAGCT    360 

GCAGAGACTC TTTCGGGCTT TTCGATGCCT GGATTCATCG ATAAGCTGCA CCATGAATGA    420 

GTCCAAGTCC ACATCACTGA AAGACTTCCT GGAAAGCCTA AAGAGCATCA TGCAAATGGA    480 

TTACTCGTAG                                                           490 

 
           
           
             
               99 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              179 

CTCACCCGGG TACCGAATTC GAATAAAAAA ATGATAAAGT AGGTTCAGTT TTATTGCTGG     60 

TTGTGTTAGT TCTCTCTAAA AATGGGTCTC AACCCCCAG                            99 

 
           
           
             
               55 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              180 

TTAGGGATCC AGATCTCGAG ATAAAAACTA CGAGTAATCC ATTTGCATGA TGCTC          55 

 
           
           
             
               47 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              181 

GCTGGTTGTG TTAGTTCTCT CTAAAAATGG GTCTCACCTC CCAACTG                   47 

 
           
           
             
               53 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              182 

ATCATCTCTA GAATAAAAAT CAGCTCGAAC ACTTTGAATA TTTCTCTCTC ATG            53 

 
           
           
             
               73 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              183 

ATCATCAAGC TTGAATAAAA AAATGATAAA GTAGGTTCAG TTTTATTGCT GGTTGTGTTA     60 

GTTCTCTCTA AAA                                                        73 

 
           
           
             
               61 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              184 

TTTTAGAGAG AACTAACACA ACCAGCAATA AAACTGAACC TACTTTATCA TTTTTTTATT     60 

C                                                                     61 

 
           
           
             
               40 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              185 

ATCATCAAGC TTGAATAAAA AAATGATAAA GTAGGTTCAG                           40 

 
           
           
             
               523 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              186 

GAATAAAAAA ATGATAAAGT AGGTTCAGTT TTATTGCTGG TTGTGTTAGT TCTCTCTAAA     60 

AATGGGTCTC ACCTCCCAAC TGCTTCCCCC TCTGTTCTTC CTGCTAGCAT GTGCCGGCAA    120 

CTTTGTCCAC GGACACAAGT GCGATATCAC CTTACAGGAG ATCATCAAAA CTTTGAACAG    180 

CCTCACAGAG CAGAAGACTC TGTGCACCGA GTTGACCGTA ACAGACATCT TTGCTGCCTC    240 

CAAGAACACA ACTGAGAAGG AAACCTTCTG CAGGGCTGCG ACTGTGCTCC GGCAGTTCTA    300 

CAGCCACCAT GAGAAGGACA CTCGCTGCCT GGGTGCGACT GCACAGCAGT TCCACAGGCA    360 

CAAGCAGCTG ATCCGATTCC TGAAACGGCT CGACAGGAAC CTCTGGGGCC TGGCGGGCTT    420 

GAATTCCTGT CCTGTGAAGG AAGCCAACCA GAGTACGTTG GAAAACTTCT TGGAAAGGCT    480 

AAAGACGATC ATGAGAGAGA AATATTCAAA GTGTTCGAGC TGA                      523 

 
           
           
             
               47 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              187 

GCTGGTTGTG TTAGTTCTCT CTAAAAATGT GGCTGCAGAG CCTGCTG                   47 

 
           
           
             
               53 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              188 

ATCATCCTCG AGATAAAAAT CACTCCTGGA CTGGCTCCCA GCAGTCAAAG GGG            53 

 
           
           
             
               73 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              189 

ATCATCCCCG GGGAATAAAA AAATGATAAA GTAGGTTCAG TTTTATTGCT GGTTGTGTTA     60 

GTTCTCTCTA AAA                                                        73 

 
           
           
             
               40 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              190 

ATCATCCCCG GGGAATAAAA AAATGATAAA GTAGGTTCAG                           40 

 
           
           
             
               496 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              191 

GAATAAAAAA ATGATAAAGT AGGTTCAGTT TTATTGCTGG TTGTGTTAGT TCTCTCTAAA     60 

AATGTGGCTG CAGAGCCTGC TGCTCTTGGG CACTGTGGCC TGCAGCATCT CTGCACCCGC    120 

CCGCTCGCCC AGCCCCAGCA CGCAGCCCTG GGAGCATGTG AATGCCATCC AGGAGGCCCG    180 

GCGTCTCCTG AACCTGAGTA GAGACACTGC TGCTGAGATG AATGAAACAG TAGAAGTCAT    240 

CTCAGAAATG TTTGACCTCC AGGAGCCGAC CTGCCTACAG ACCCGCCTGG AGCTGTACAA    300 

GCAGGGCCTG CGGGGCAGCC TCACCAAGCT CAAGGGCCCC TTGACCATGA TGGCCAGCCA    360 

CTACAAGCAG CACTGCCCTC CAACCCCGGA AACTTCCTGT GCAACCCAGA CTATCACCTT    420 

TGAAAGTTTC AAAGAGAACC TGAAGGACTT TCTGCTTGTC ATCCCCTTTG ACTGCTGGGA    480 

GCCAGTCCAG GAGTGA                                                    496 

 
           
           
             
               69 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              192 

CATCATATCG ATGGTACCTC AAAATTGAAA ATATATAATT ACAATATAAA ATGTGTCACC     60 

AGCAGTTGG                                                             69 

 
           
           
             
               38 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              193 

TACTACGAGC TCTCAGATAG AAATTATATC TTTTTGGG                             38 

 
           
           
             
               1018 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              194 

CAAAATTGAA AATATATAAT TACAATATAA AATGTGTCAC CAGCAGTTGG TCATCTCTTG     60 

GTTTTCCCTG GTTTTTCTGG CATCTCCCCT CGTGGCCATA TGGGAACTGA AGAAAGATGT    120 

TTATGTCGTA GAATTGGATT GGTATCCGGA TGCCCCTGGA GAAATGGTGG TCCTCACCTG    180 

TGACACCCCT GAAGAAGATG GTATCACCTG GACCTTGGAC CAGAGCAGTG AGGTCTTAGG    240 

CTCTGGCAAA ACCCTGACCA TCCAAGTCAA AGAGTTTGGA GATGCTGGCC AGTACACCTG    300 

TCACAAAGGA GGCGAGGTTC TAAGCCATTC GCTCCTGCTG CTTCACAAAA AGGAAGATGG    360 

AATTTGGTCC ACTGATATTT TAAAGGACCA GAAAGAACCC AAAAATAAGA CCTTTCTAAG    420 

ATGCGAGGCC AAGAATTATT CTGGACGTTT CACCTGCTGG TGGCTGACGA CAATCAGTAC    480 

TGATTTGACA TTCAGTGTCA AAAGCAGCAG AGGCTCTTCT GACCCCCAAG GGGTGACGTG    540 

CGGAGCTGCT ACACTCTCTG CAGAGAGAGT CAGAGGGGAC AACAAGGAGT ATGAGTACTC    600 

AGTGGAGTGC CAGGAGGACA GTGCCTGCCC AGCTGCTGAG GAGAGTCTGC CCATTGAGGT    660 

CATGGTGGAT GCCGTTCACA AGCTCAAGTA TGAAAACTAC ACCAGCAGCT TCTTCATCAG    720 

GGACATCATC AAACCTGACC CACCCAAGAA CTTGCAGCTG AAGCCATTAA AGAATTCTCG    780 

GCAGGTGGAG GTCAGCTGGG AGTACCCTGA CACCTGGAGT ACTCCACATT CCTACTTCTC    840 

CCTGACATTC TGCGTTCAGG TCCAGGGCAA GAGCAAGAGA GAAAAGAAAG ATAGAGTCTT    900 

CACGGACAAG ACCTCAGCCA CGGTCATCTG CCGCAAAAAT GCCAGCATTA GCGTGCGGGC    960 

CCAGGACCGC TACTATAGCT CATCTTGGAG CGAATGGGCA TCTGTGCCCT GCAGTTAG     1018 

 
           
           
             
               63 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              195 

CATCATGGTA CCTCAAAATT GAAAATATAT AATTACAATA TAAAATGTGT CCAGCGCGCA     60 

GCC                                                                   63 

 
           
           
             
               31 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              196 

TACTACATCG ATTTAGGAAG CATTCAGATA G                                    31 

 
           
           
             
               92 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              197 

CATCATGGTA CCGAATAAAA AAATGATAAA GTAGGTTCAG TTTTATTGCT GGTTGTGTTA     60 

GTTCTCTCTA AAAATGTGTC CAGCGCGCAG CC                                   92 

 
           
           
             
               39 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              198 

CATCATATCG ATTTAGGAAG CATTCAGATA GCTCGTCAC                            39 

 
           
           
             
               721 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              199 

GAATAAAAAA ATGATAAAGT AGGTTCAGTT TTATTGCTGG TTGTGTTAGT TCTCTCTAAA     60 

AATGTGTCCA GCGCGCAGCC TCCTCCTTGT GGCTACCCTG GTCCTCCTGG ACCACCTCAG    120 

TTTGGCCAGA AACCTCCCCG TGGCCACTCC AGACCCAGGA ATGTTCCCAT GCCTTCACCA    180 

CTCCCAAAAC CTGCTGAGGG CCGTCAGCAA CATGCTCCAG AAGGCCAGAC AAACTCTAGA    240 

ATTTTACCCT TGCACTTCTG AAGAGATTGA TCATGAAGAT ATCACAAAAG ATAAAACCAG    300 

CACAGTGGAG GCCTGTTTAC CATTGGAATT AACCAAGAAT GAGAGTTGCC TAAATTCCAG    360 

AGAGACCTCT TTCATAACTA ATGGGAGTTG CCTGGCCTCC AGAAAGACCT CTTTTATGAT    420 

GGCCCTGTGC CTTAGTAGTA TTTATGAAGA CTTGAAGATG TACCAGGTGG AGTTCAAGAC    480 

CATGAATGCA AAGCTTCTGA TGGATCCTAA GAGGCAGATC TTTCTAGATC AAAACATGCT    540 

GGCAGTTATT GATGAGCTGA TGCAGGCCCT GAATTTCAAC AGTGAGACTG TGCCACAAAA    600 

ATCCTCCCTT GAAGAACCGG ATTTTTATAA AACTAAAATC AAGCTCTGCA TACTTCTTCA    660 

TGCTTTCAGA ATTCGGGCAG TGACTATTGA CAGAGTGACG AGCTATCTGA ATGCTTCCTA    720 

A                                                                    721 

 
           
           
             
               60 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              200 

TATCTGGAAT TCTATCGCGA TATCCGTTAA GTTTGTATCG TAATGGCTTG CAATTGTCAG     60 

 
           
           
             
               30 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              201 

ATCGTAAGCT TACTAAAGGA AGACGGTCTG                                      30 

 
           
           
             
               921 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              202 

ATGGCTTGCA ATTGTCAGTT GATGCAGGAT ACACCACTCC TCAAGTTTCC ATGTCCAAGG     60 

CTCATTCTTC TCTTTGTGCT GCTGATTCGT CTTTCACAAG TGTCTTCAGA TGTTGATGAA    120 

CAACTGTCCA AGTCAGTGAA AGATAAGGTA TTGCTGCCTT GCCGTTACAA CTCTCCTCAT    180 

GAAGATGAGT CTGAAGACCG AATCTACTGG CAAAAACATG ACAAAGTGGT GCTGTCTGTC    240 

ATTGCTGGGA AACTAAAAGT GTGGCCCGAG TATAAGAACC GGACTTTATA TGACAACACT    300 

ACCTACTCTC TTATCATCCT GGGCCTGGTC CTTTCAGACC GGGGCACATA CAGCTGTGTC    360 

GTTCAAAAGA AGGAAAGAGG AACGTATGAA GTTAAACACT TGGCTTTAGT AAAGTTGTCC    420 

ATCAAAGCTG ACTTCTCTAC CCCCAACATA ACTGAGTCTG GAAACCCATC TGCAGACACT    480 

AAAAGGATTA CCTGCTTTGC TTCCGGGGGT TTCCCAAAGC CTCGCTTCTC TTGGTTGGAA    540 

AATGGAAGAG AATTACCTGG CATCAATACG ACAATTTCCC AGGATCCTGA ATCTGAATTG    600 

TACACCATTA GTAGCCAACT AGATTTCAAT ACGACTCGCA ACCACACCAT TAAGTGTCTC    660 

ATTAAATATG GAGATGCTCA CGTGTCAGAG GACTTCACCT GGGAAAAACC CCCAGAAGAC    720 

CCTCCTGATA GCAAGAACAC ACTTGTGCTC TTTGGGGCAG GATTCGGCGC AGTAATAACA    780 

GTCGTCGTCA TCGTTGTCAT CATCAAATGC TTCTGTAAGC ACAGAAGCTG TTTCAGAAGA    840 

AATGAGGCAA GCAGAGAAAC AAACAACAGC CTTACCTTCG GGCCTGAAGA AGCATTAGCT    900 

GAACAGACCG TCTTCCTTTA G                                              921 

 
           
           
             
               26 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              203 

CGACATTTGG ATTTCAAGCT TCTACG                                          26 

 
           
           
             
               30 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              204 

GATCCGTAGA AGCTTGAAAT CCAAATGTCG                                      30 

 
           
           
             
               33 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              205 

ATCGTAAGCT TATTATACAG GGCGTACACT TTC                                  33 

 
           
           
             
               60 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              206 

TATCTGGAAT TCTATCGCGA TATCCGTTAA GTTTGTATCG TAATGGGCCA CACACGGAGG     60 

 
           
           
             
               867 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              207 

ATGGGCCACA CACGGAGGCA GGGAACATCA CCATCCAAGT GTCCATACCT CAATTTCTTT     60 

CAGCTCTTGG TGCTGGCTGG TCTTTCTCAC TTCTGTTCAG GTGTTATCCA CGTGACCAAG    120 

GAAGTGAAAG AAGTGGCAAC GCTGTCCTGT GGTCACAATG TTTCTGTTGA AGAGCTGGCA    180 

CAAACTCGCA TCTACTGGCA AAAGGAGAAG AAAATGGTGC TGACTATGAT GTCTGGGGAC    240 

ATGAATATAT GGCCCGAGTA CAAGAACCGG ACCATCTTTG ATATCACTAA TAACCTCTCC    300 

ATTGTGATCC TGGCTCTGCG CCCATCTGAC GAGGGCACAT ACGAGTGTGT TGTTCTGAAG    360 

TATGAAAAAG ACGCTTTCAA GCGGGAACAC CTGGCTGAAG TGACGTTATC AGTCAAAGCT    420 

GACTTCCCTA CACCTAGTAT ATCTGACTTT GAAATTCCAA CTTCTAATAT TAGAAGGATA    480 

ATTTGCTCAA CCTCTGGAGG TTTTCCAGAG CCTCACCTCT CCTGGTTGGA AAATGGAGAA    540 

GAATTAAATG CCATCAACAC AACAGTTTCC CAAGATCCTG AAACTGAGCT CTATGCTGTT    600 

AGCAGCAAAC TGGATTTCAA TATGACAACC AACCACAGCT TCATGTGTCT CATCAAGTAT    660 

GGACATTTAA GAGTGAATCA GACCTTCAAC TGGAATACAA CCAAGCAAGA GCATTTTCCT    720 

GATAACCTGC TCCCATCCTG GGCCATTACC TTAATCTCAG TAAATGGAAT TTTTGTGATA    780 

TGCTGCCTGA CCTACTGCTT TGCCCCAAGA TGCAGAGAGA GAAGGAGGAA TGAGAGATTG    840 

AGAAGGGAAA GTGTACGCCC TGTATAA                                        867 

 
           
           
             
               35 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              208 

ATTATTATTG GATCCTTAAT TAATTAGTGA TACGC                                35 

 
           
           
             
               35 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              209 

CTCCTCCATG GCAGTCATTA CGATACAAAC TTAAC                                35 

 
           
           
             
               38 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              210 

CGTTAAGTTT GTATCGTAAT GACTGCCATG GAGGAGTC                             38 

 
           
           
             
               36 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              211 

TAGTAGTAGT AGTAGCTTCT GGAGGAAGTA GTTTCC                               36 

 
           
           
             
               39 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              212 

CAGAAGCTAC TACTACTACT ACCCACCTGC ACAAGCGCC                            39 

 
           
           
             
               43 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              213 

AACTACTGTC CCGGGATAAA AATCAGTCTG AGTCAGGCCC CAC                       43 

 
           
           
             
               1173 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              214 

ATGACTGCCA TGGAGGAGTC ACAGTCGGAT ATCAGCCTCG AGCTCCCTCT GAGCCAGGAG     60 

ACATTTTCAG GCTTATGGAA ACTACTTCCT CCAGAAGATA TCCTGCCATC ACCTCACTGC    120 

ATGGACGATC TGTTGCTGCC CCAGGATGTT GAGGAGTTTT TTGAAGGCCC AAGTGAAGCC    180 

CTCCGAGTGT CAGGAGCTCC TGCAGCACAG GACCCTGTCA CCGAGACCCC TGGGCCAGTG    240 

GCCCCTGCCC CAGCCACTCC ATGGCCCCTG TCATCTTTTG TCCCTTCTCA AAAAACTTAC    300 

CAGGGCAACT ATGGCTTCCA CCTGGGCTTC CTGCAGTCTG GGACAGCCAA GTCTGTTATG    360 

TGCACGTACT CTCCTCCCCT CAATAAGCTA TTCTGCCAGC TGGCGAAGAC GTGCCCTGTG    420 

CAGTTGTGGG TCAGCGCCAC ACCTCCAGCT GGGAGCCGTG TCCGCGCCAT GGCCATCTAC    480 

AAGAAGTCAC AGCACATGAC GGAGGTCGTG AGACGCTGCC CCCACCATGA GCGCTGCTCC    540 

GATGGTGATG GCCTGGCTCC TCCCCAGCAT CTTATCCGGG TGGAAGGAAA TTTGTATCCC    600 

GAGTATCTGG AAGACAGGCA GACTTTTCGC CACAGCGTGG TGGTACCTTA TGAGCCACCC    660 

GAGGCCGGCT CTGAGTATAC CACCATCCAC TACAAGTACA TGTGTAATAG CTCCTGCATG    720 

GGGGGCATGA ACCGCCGACC TATCCTTACC ATCATCACAC TGGAAGACTC CAGTGGGAAC    780 

CTTCTGGGAC GGGACAGCTT TGAGGTTCGT GTTTGTGCCT GCCCTGGGAG AGACCGCCGT    840 

ACAGAAGAAG AAAATTTCCG CAAAAAGGAA GTCCTTTGCC CTGAACTGCC CCCAGGGAGC    900 

GCAAAGAGAG CGCTGCCCAC CTGCACAAGC GCCTCTCCCC CGCAAAAGAA AAAACCACTT    960 

GATGGAGAGT ATTTCACCCT CAAGATCCGC GGGCGTAAAC GCTTCGAGAT GTTCCGGGAG   1020 

CTGAATGAGG CCTTAGAGTT AAAGGATGCC CATGCTACAG AGGAGTCTGG AGACAGCAGG   1080 

GCTCACTCCA GCTACCTGAA GACCAAGAAG GGCCAGTCTA CTTCCCGCCA TAAAAAAACA   1140 

ATGGTCAAGA AAGTGGGGCC TGACTCAGAC TGA                                1173 

 
           
           
             
               1182 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              215 

ATGGAGGAGC CGCAGTCAGA TCCTAGCGTC GAGCCCCCTC TGAGTCAGGA AACATTTTCA     60 

GACCTATGGA AACTACTTCC TGAAAACAAC GTTCTGTCCC CCTTGCCGTC CCAAGCAATG    120 

GATGATTTGA TGCTGTCCCC GGACGATATT GAACAATGGT TCACTGAAGA CCCAGGTCCA    180 

GATGAAGCTC CCAGAATGCC AGAGGCTGCT CCCCGCGTGG CCCCTGCACC AGCAGCTCCT    240 

ACACCGGCGG CCCCTGCACC AGCCCCCTCC TGGCCCCTGT CATCTTCTGT CCCTTCCCAG    300 

AAAACCTACC AGGGCAGCTA CGGTTTCCGT CTGGGCTTCT TGCATTCTGG GACAGCCAAG    360 

TCTGTGACTT GCACGTACTC CCCTGCCCTC AACAAGATGT TTTGCCAACT GGCCAAGACC    420 

TGCCCTGTGC AGCTGTGGGT TGATTCCACA CCCCCGCCCG GCACCCGCGT CCGCGCCATG    480 

GCCATCTACA AGCAGTCACA GCACATGACG GAGGTTGTGA GGCGCTGCCC CCACCATGAG    540 

CGCTGCTCAG ATAGCGATGG TCTGGCCCCT CCTCAGCATC TTATCCGAGT GGAAGGAAAT    600 

TTGCGTGTGG AGTATTTGGA TGACAGAAAC ACTTTTCGAC ATAGTGTGGT GGTGCCCTAT    660 

GAGCCGCCTG AGGTTGGCTC TGACTGTACC ACCATCCACT ACAACTACAT GTGTAACAGT    720 

TCCTGCATGG GCGGCATGAA CCGGAGGCCC ATCCTCACCA TCATCACACT GGAAGACTCC    780 

AGTGGTAATC TACTGGGACG GAACAGCTTT GAGGTGCGTG TTTGTGCCTG TCCTGGGAGA    840 

GACCGGCGCA CAGAGGAAGA GAATCTCCGC AAGAAAGGGG AGCCTCACCA CGAGCTGCCC    900 

CCAGGGAGCA CTAAGCGAGC ACTGCCCAAC AACACCAGCT CCTCTCCCCA GCCAAAGAAG    960 

AAACCACTGG ATGGAGAATA TTTCACCCTT CAGATCCGTG GGCGTGAGCG CTTCGAGATG   1020 

TTCCGAGAGC TGAATGAGGC CTTGGAACTC AAGGATGCCC AGGCTGGGAA GGAGCCAGGG   1080 

GGGAGCAGGG CTCACTCCAG CCACCTGAAG TCCAAAAAGG GTCAGTCTAC CTCCCGCCAT   1140 

AAAAAACTCA TGTTCAAGAC AGAAGGGCCT GACTCAGACT GA                      1182 

 
           
           
             
               47 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              216 

CGATATCCGT TAAGTTTGTA TCGTAATGGA GCTCCTGCAG CCCGGGG                   47 

 
           
           
             
               51 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              217 

GATCCCCCGG GCTGCAGGAG CTCCATTACG ATACAAACTT AACGGATATC G              51