EP0573613A1 - Entomopoxvirus expression system comprising spheroidin or thymidine-kinase sequences - Google Patents

Abstract

The invention relates to novel entomopoxvirus (EPV) polynucleotide sequences which are not associated with other viral sequences with which they are naturally associated, to recombinant polynucleotide vectors containing these sequences, to recombinant viruses containing also these sequences, and to host cells infected with these recombinant viruses, as well as to methods of using these sequences for the expression of heterologous proteins in both insect and mammalian host cells .

Description

NOVEL ENTOMOPOXVIRUS EXPRESSION SYSTEM

Field of the Invention

This invention relates generally to the field of recombinantly-produced proteins and specifically to novel, recombinant Entomopoxvirus proteins, protein regulatory sequences and their uses in expressing heterologous genes in transformed hosts. Background of the Invention

Poxviruses are taxonomically classified into the family Chordopoxvirinae, whose members infect vertebrate hosts, e.g., the Orthopoxvirus vaccinia, or into the family Entomopoxvirinae. Very little is known about members of the Entomopoxvirinae family other than the insect host range of individual members. One species of Entomopoxvirus (EPV) is the Amsacta moorei Entomopoxvirus (AmEPV), which was first isolated from larvae of the red hairy caterpillar Amsacta moorei

[Roberts and Granados, J. Invertebr. Pathol., 12:141-143 (1968)]. AmEPV is the type species of genus B of EPVs and is one of three known EPVs which will replicate in cultured insect cells [R. R. Granados et al, "Replication of Amsacta moorei Entomopoxvirus and Autographa

californica Nuclear Polyhedrosis Virus in Hemocyte Cell Lines from Estigmene acrea", in Invertebrate Tissue

Culture Applications in Medicine, Biology, and

Agriculture. E. Kurstak and K. Maramorosch (ed.).

Academic Press, New York, pp. 379-389 (1976); T. Hukuhara et al, J. Invertebr. Pathol.. 56:222-232 (1990); and T. Sato, "Establishment of Eight Cell Lines from Neonate Larvae of Torticids (Lepidoptera), and Their Several Characteristics Including Susceptibility to Insect

Viruses", in Invertebrate Cell Systems Applications, J. Mitsuhashi (ed.). Vol. II, pp. 187-198, CRC Press, Inc., Boca Raton, Florida (1989)].

AmEPV is one of the few insect poxviruses which can replicate in insect cell culture; AmEPV is unable to replicate in vertebrate cell lines. The AmEPV doublestranded DNA genome is about 225 kb unusually A+T rich (18.5% G+C) [W. H. R. Langridge et al, Virology, 76:616620 (1977)]. Recently, a series of restriction maps for AmEPV were published [R. L. Hall et al. Arch. Virol., 110:77-90 (1990)]. No DNA homology to vaccinia has been detected [W. H. Langridge, J. Invertebr. Pathol., 42:77- 82 (1983); W. H. Langridge, J. Invertebr. Pathol., 43:41- 46 (1984)].

The viral replication cycle of AmEPV resembles that of other poxviruses except for the appearance of occluded virus late in infection. For AmEPV, once a cell is infected, both occluded and extracellular virus particles are generated. The mature occlusion body particle, which is responsible for environmentally protecting the virion during infection, consists of virus embedded within a crystalline matrix consisting primarily of a single protein, spheroidin. Spheroidin, the major structural protein of AmEPV, has been reported to be 110 kDa in molecular weight and to consist of a high

percentage of charged and sulfur-containing amino acids

[Langridge and Roberts, J. Invertebr. Pathol.. 39:346-353 (1982)]. The use of viruses and virus proteins in

eukaryotic host-vector systems has been the subject of a considerable amount of investigation and speculation.

Many existing viral vector systems suffer from

significant disadvantages and limitations which diminish their utility. For example, a number of eukaryotic viral vectors are either tumorigenic or oncogenic in mammalian systems, creating the potential for serious health and safety problems associated with resultant gene products and accidental infections. Further, in some eukaryotic host-viral vector systems, the gene product itself exhibits antiviral activity, thereby decreasing the yield of that protein.

In the case of simple viruses, the amount of exogenous DNA which can be packaged into a simple virus is limited. This limitation becomes a particularly acute problem when the genes used are eukaryotic. Because eukaryotic genes usually contain intervening sequences, they are too large to fit into simple viruses. Further, because they have many restriction sites, it is more difficult to insert exogenous DNA into complex viruses at specific locations.

Vaccinia virus has recently been developed as a eukaryotic cloning and expression vector [M. Mackett et al, DNA Cloning, Vol. II, ed. D. M. Glover, pp. 191-212, Oxford: IRL Press (1985); D. Panicali et al, Proc. Natl. Acad. Sci. USA. 8j3: 5364-5368 (1982)]. Numerous viral antigens have been expressed using vaccinia virus vectors [E. Paoletti et al, Proc. Natl. Acad. Sci. USA. 81:193-197 (1984); A. Piccine et al, BioEssays, 5:248-252

(1986)] including, among others, HBsAg, rabies G protein and the gp120/gp41 of human immunodeficiency virus (HIV). Regulatory sequences from the spruce budworm EPV have been used previously with vaccinia [L. Yuen et al,

Virology, 175:427-433 (1990)].

Additionally, studies with vaccinia virus have demonstrated that poxviruses have several advantageous features as vaccine vectors. These include the ability of poxvirus-based vaccines to stimulate both cell-mediated and humoral immunity, minimal cost to mass produce vaccine and the stability of the lyophilized vaccine without refrigeration, ease of administration under non-sterile condition, and the ability to insert at least 25,000 base pairs of foreign DNA into an infectious recombinant, thereby permitting the simultaneous expression of many antigens from one recombinant.

There exists a need in the art for additional viral compositions and methods for use in expressing heterologous genes in selected host cells, and in performing other research and production techniques associated therewith.

Summary of the Invention

As one aspect, the invention provides an

Entomopoxvirus polynucleotide sequence, free from other viral sequences with which it is associated in nature, which comprises a sequence encoding the Entomopoxvirus spheroidin gene and/or its regulatory sequences, an allelic variant, an analog or a fragment thereof. In a particular embodiment, the spheroidin DNA sequence is isolated from the Amsacta moorei Entomopoxvirus and is illustrated in Fig. 2 [SEQ ID NO:1].

Another aspect of the invention is the

polynucleotide sequence encoding the Entomopoxvirus spheroidin promoter or an allelic variant, analog or fragment thereof. The spheroidin promoter sequence is characterized by the ability to direct the expression of a heterologous gene to which the sequence or fragment is operably linked in a selected host cell.

As another aspect, the present invention provides a recombinant polynucleotide sequence comprising a sequence encoding the Entomopoxvirus spheroidin protein and/or its regulatory sequences, an allelic variant, analog or fragment thereof, linked to a second

polynucleotide sequence encoding a heterologous gene.

One embodiment of this polynucleotide sequence provides the spheroidin promoter sequence operably linked to the heterologous gene to direct the expression of the

heterologous gene in a selected host cell. Another embodiment provides the sequence encoding the spheroidin protein linked to the heterologous gene in a manner permitting expression of a fusion protein. Still another embodiment provides the heterologous gene inserted into a site in the spheroidin gene so that the heterologous gene is flanked on both termini by spheroidin sequences.

As yet a further aspect, the invention provides an Entomopoxvirus polynucleotide sequence free from other viral sequences with which it is associated in nature, comprising a sequence encoding the Entomopoxvirus

thymidine kinase (tk) gene and/or its regulatory

sequences, an allelic variant, an analog or a fragment thereof. In a particular embodiment, the sequence originates from the Amsacta moorei Entomopoxvirus and is illustrated in Fig. 3 [SEQ ID NO: 8].

In still another aspect the sequence encodes the Entomopoxvirus tk promoter, allelic variant or a fragment thereof. The tk promoter sequence is

characterized by the ability to direct the expression of a heterologous gene to which the sequence or fragment is operably linked in a selected host cell.

Yet a further aspect of the invention provides a recombinant polynucleotide sequence described above encoding the Entomopoxvirus tk gene and/or its regulatory sequences, an allelic variant, or a fragment thereof, linked to a heterologous gene. One embodiment of this polynucleotide sequence provides the tk promoter sequence operably linked to the heterologous gene to direct the expression of the heterologous gene in a selected host cell. Another embodiment provides the sequence encoding the tk protein linked to the heterologous gene in a manner permitting expression of a fusion protein. Still another embodiment provides the heterologous gene

inserted into a site in the tk gene so that the heterologous gene is flanked on both termini by tk sequences.

Another aspect of the invention is an

Entomopoxvirus spheroidin polypeptide, a fragment thereof, or an analog thereof, optionally fused to a heterologous protein or peptide. Also provided is an Entomopoxvirus tk polypeptide, a fragment thereof, or an analog thereof, optionally linked to a heterologous protein or peptide.

Yet another aspect of the invention is provided by recombinant polynucleotide molecules which comprise one or more of the polynucleotide sequences described above. This molecule may be an expression vector or shuttle vector. The molecule may also contain viral sequences originating from a virus other than the

Entomopoxvirus which contributed the spheroidin or tk polynucleotide sequence, e.g., vaccinia.

In another aspect, the present invention provides a recombinant virus comprising a polynucleotide sequence as described above. Also provided are host cells infected with one or more of the described

recombinant viruses.

The present invention also provides a method for producing a selected polypeptide comprising culturing a selected host cell infected with a recombinant virus, as described above, and recovering said polypeptide from the culture medium.

As a final aspect, the invention provides a method for screening recombinant host cells for insertion of heterologous genes comprising infecting the cells with a recombinant virus containing a polynucleotide molecule comprising the selected heterologous gene sequence linked to an incomplete spheroidin or tk polynucleotide sequence or inserted into and interrupting the coding sequences thereof so that the heterologous gene is flanked at each termini by an Entomopoxvirus spheroidin or tk

polynucleotide sequence. The absence of occlusion bodies formed by the expression of the spheroidin protein in the spheroidin containing cell indicates the integration of the heterologous gene. Alternatively, the absence of the thymidine kinase function, i.e., resistance to

methotrexate or a nucleotide analogue of methotrexate, formed by the integration of the inactive thymidine kinase sequence indicates the insertion of the

heterologous gene.

Other aspects and advantages of the present invention are described further in the following detailed description of embodiments of the present invention.

Brief Description of the Drawings

Fig. 1 is a physical map of AmEPV illustrating restriction fragments thereof and showing the spheroidin gene just to the right of site #29 in the Hindlll-G fragment.

Fig. 2 provides the AmEPV DNA sequence of the Amsacta moorei Entomopoxvirus spheroidin gene and

flanking sequences [SEQ ID NO:1], the deduced amino acid sequences of the spheroidin protein [SEQ ID NO: 6], and five additional open reading frames (ORFs) .

Fig. 3 provides the DNA sequence of the Amsacta moorei Entomopoxvirus thymidine kinase (tk) gene and flanking sequences [SEQ ID NO: 8], the deduced amino acid sequences of the tk protein [SEQ ID NO: 11], and two additional ORFs.

Fig. 4 provides the nucleotide sequences of the synthetic oligonucleotides designated RM58 [SEQ ID

NO:12], RM82 [SEQ ID NO: 13], RM83 [SEQ ID NO:14], RM92 [SEQ ID NO: 15], RM118 [SEQ ID NO: 16], RM165 [SEQ ID

NO:17], RM03 [SEQ ID NO:18], RM04 [SEQ ID NO:19], and RM129 [SEQ ID NO:20]. Fig. 5 is a schematic map of an AmEPV fragment illustrating the orientation of the spheroidin ORF on the physical map and indicating homologies.

Detailed Description of the Invention

The present invention provides novel

Entomopoxvirus (EPV) polynucleotide sequences free from association with other viral sequences with which they are naturally associated. Recombinant polynucleotide vectors containing the sequences, recombinant viruses containing the sequences, and host cells infected with the recombinant viruses are also disclosed herein. These compositions are useful in methods of the invention for the expression of heterologous genes and production of selected proteins in both insect and mammalian host cells.

Novel polynucleotide sequences of the invention encode the EPV spheroidin gene and/or its flanking sequences, including sequences which provide regulatory signals for the expression of the gene. The invention also provides novel polynucleotide sequences encoding the EPV thymidine kinase (tk) gene and/or its flanking

sequences. The polynucleotide sequences of this

invention may be either RNA or DNA sequences. More preferably, the polynucleotide sequences of this

invention are DNA sequences.

Specifically disclosed by the present invention are spheroidin and tk polynucleotide sequences obtained from the Amsacta moorei Entomopoxvirus (AmEPV). While this is the presently preferred species for practice of the methods and compositions of this invention, it is anticipated that, utilizing the techniques described herein, substantially homologous sequences may be

obtained by one of skill in the art from other available Entomopoxvirus species. The AmEPV spheroidin DNA sequence, including flanking and regulatory sequence, is reported in Fig. 2 as spanning nucleotides # 1 through 6768 [SEQ ID NO:1]. Within this sequence, the spheroidin gene coding sequence spans nucleotides #3080 to #6091 [SEQ ID NO:21]. A fragment which is likely to contain the promoter

sequences spans nucleotide #2781-3199 [SEQ ID NO:22]. Other regions of that sequence have also been identified as putative coding regions for other structural or regulatory genes associated with spheroidin. These other fragments of interest include the following sequences: nucleotide # 1472 through 2151 [SEQ ID NO: 23] encoding the G2R ORF [SEQ ID NO:3]; nucleotide #2502 through 2987 [SEQ ID NO: 24] encoding the G4R ORF [SEQ ID NO: 5]; and the following sequences transcribed left to right on Fig. 2: nucleotide #65 through 1459 [SEQ ID NO: 25] encoding the GIL ORF [SEQ ID NO: 2]; nucleotide #2239 through 2475 [SEQ ID NO: 26] encoding the G3L ORF [SEQ ID NO: 4];

and nucleotide #677 through 6768 [SEQ ID NO: 27] encoding the G6L ORF [SEQ ID NO:7]. These ORFs are identified in Fig. 2.

The AmEPV ORF G4R [SEQ ID NO: 5] which is immediately upstream of the spheroidin gene has

significant homology to the capripoxvirus HM3 ORF. A homolog of the HM3 ORF is found in vaccinia virus just upstream of a truncated version of the cowpox virus ATI gene. Therefore, the microenvironments in this region are similar in the two viruses. Two other ORFs relate to counterparts in vaccinia virus. These ORFs include the 17 ORF of the vaccinia virus HindIII-I fragment (17) [J. F. C. Schmitt et al, J. Virol.. 62:1889-1897 (1988)] which relates to the AmEPV GIL ORF [SEQ ID NO: 2] and the NTPase I (NPH I) ORF of the HindIII-D fragment which relates to the AmEPV G6L ORF [SEQ ID NO: 7] [S. S. Broyles et al, J. Virol., 61:1738-1742 (1987); and J. F. Rodriguez et al, Proc. Natl. Acad. Sci. USA. 83:9566-9570 (1986)]. The genomic location of the AmEPV ORFs compared with that of the vaccinia virus ORFs suggests that the arrangement of essential "core genes", which are

centrally located and colinear in many, if not all, of the vertebrate poxviruses on a more macroscopic scale, is quite different in the insect virus.

As set out in detail in the accompanying examples below, the spheroidin gene of AmEPV was

identified through direct microsequencing of the protein, and the results were used for the design of

oligonucleotide probes. Transcription of the spheroidin gene is inhibited by cycloheximide, suggesting it is a late gene. Consistent with this prediction are the observations that spheroidin transcripts were initiated within a TAATG motif (See Fig. 2, nucleotide #3077- 3082) and that there was a 5' poly(A) sequence, both characteristic of late transcripts.

The AmEPV tk DNA sequence, including flanking and regulatory sequence, is reported in Fig. 3, as

spanning nucleotides #1 through 1511 [SEQ ID NO: 8],

Within this sequence, the tk gene coding sequence spans nucleotides # 234 to 782 [SEQ ID NO: 28] (transcribed right to left on Fig. 3) . Another fragment of interest may include nucleotides #783 through #851 [SEQ ID NO: 29] of that sequence or fragments thereof. A fragment likely to contain the promoter regions spans nucleotide #750 - 890 [SEQ ID NO:30]. Other regions of that sequence have also been identified as putative coding regions for other structural or regulatory genes associated with tk. These other fragments of interest include the following

sequences (transcribed left to right on Fig. 3:

nucleotide # 18 through 218 [SEQ ID NO: 31] encoding ORF Q1 [SEQ ID NO: 10]); and nucleotide # 852 through 1511 [SEQ ID NO: 32] encoding ORF Q3 [SEQ ID NO: 10].

The location of the AmEPV tk gene maps in the EcoRI-Q fragment near the left end of the physical map of the AmEPV genome (Fig. 1) [see, also, R. L. Hall et al, Arch. Virol.. 110:77-90 (1990), incorporated by reference herein]. Because of the orientation of the gene within the AmEPV genome, transcription of the gene is likely to occur toward the terminus. There are believed to be similar tk genes, or variations thereof, in other

systems, including mammalian systems. As set out in detail in the examples below, the tk gene of AmEPV was identified through direct microsequencing of the protein, and the results were used for the design of

oligonucleotide probes.

The term "polynucleotide sequences" when used with reference to the invention can include the entire EPV spheroidin or tk genes with regulatory sequences flanking the coding sequences. The illustrated AmEPV sequences are also encompassed by that term. Also included in the definition are fragments of the coding sequences with flanking regulatory sequences. The definition also encompasses the regulatory sequences only, e.g., the promoter sequences, transcription sites, termination sequences, and other regulatory sequences.

Sequences of the invention may also include all or portions of the spheroidin or tk genes linked in frame to a heterologous gene sequence. Additionally,

polynucleotide sequences of the invention may include sequences of the spheroidin or tk genes into which have been inserted a foreign or heterologous gene sequence, so that the EPV sequences flank the heterologous gene sequence.

Polynucleotide sequences of this invention also include sequences which are capable of hybridizing to the sequences of Figs. 2 and 3, under stringent conditions, which sequences retain the same biological or regulatory activities as those of the figures. Also sequences capable of hybridizing to the sequences of Figs. 2 and 3 under non-stringent conditions may fall within this definition providing that the biological or regulatory characteristics of the sequences of Figs. 2 and 3, respectively, are retained. Examples of stringent and non-stringent conditions of hybridization are

conventional [See, e.g., Sambrook et al. Molecular

Cloning. A Laboratory Manual. 2d edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989)].

Similarly, polynucleotide sequences of this invention also include allelic variations (naturally- occurring base changes in the EPV species population which may or may not result in an amino acid change) of DNA sequences encoding the spheroidin or tk protein sequences or DNA sequences encoding the other ORFs or regulatory sequences illustrated in Figs. 2 and 3.

Similarly, DNA sequences which encode spheroidin or tk proteins of the invention but which differ in codon sequence due to the degeneracies of the genetic code or variations in the DNA sequences which are caused by point mutations or by induced modifications to enhance a

biological property or the usefulness of a desired

polynucleotide sequence encoded thereby are also

encompassed in the invention.

Utilizing the sequence data in Figs. 2 or 3, as well as the denoted characteristics of spheroidin or thymidine kinase, it is within the skill of the art to obtain other DNA sequences encoding these polypeptides. For example, the structural gene may be manipulated by varying individual nucleotides, while retaining the correct amino acid(s), or varying the nucleotides, so as to modify the amino acids, without loss of enzymatic activity. Nucleotides may be substituted, inserted, or deleted by known techniques, including, for example, in vitro mutagenesis and primer repair.

The structural gene may be truncated at its 3'- terminus and/or its 5'-terminus while retaining its biological activity. It may also be desirable to ligate a portion of the polypeptide sequence to a heterologous coding sequence, and thus to create a fusion peptide.

The polynucleotide sequences of the present invention may be prepared synthetically or can be derived from viral RNA or from available cDNA-containing plasmids by chemical and genetic engineering techniques or

combinations thereof which are standard in the art.

The AmEPV proteins, spheroidin, thymidine kinase and their respective regulatory sequences, as described herein, may be encoded by polynucleotide sequences that differ in sequence from the sequences of Figs. 2 and 3 due to natural allelic or species

variations. Thus, the terms spheroidin or tk

polypeptides also refer to any of the naturally occurring sequences and various analogs, e.g., processed or

truncated sequences or fragments, including the mature spheroidin or tk polypeptides and mutant or modified polypeptides or fragments that retain the same biological activity and preferably have a homology to Fig. 2 or 3, respectively, of at least 80%, more preferably 90%, and most preferably 95%.

Another aspect of the present invention is provided by the proteins encoded by the EPV spheroidin and tk polynucleotide sequences. Putative amino acid sequences of the two EPV proteins as well as additional putative proteins encoded by the ORFs of these sequences which are identified in Figs. 2 and 3, respectively. EPV spheroidin has no significant amino acid homology to any previously reported protein, including the polyhedrin protein of baculovirus. Both spheroidin and tk are nonessential proteins, which makes them desirable as sites for insertion of exogenous DNA.

Comparison of the AmEPV tk amino acid sequence with other tk genes reveals that the AmEPV tk gene is not highly related to any of the vertebrate poxvirus tk genes (43.4 to 45.7%). The relatedness of the vertebrate tk proteins to AmEPV is still lower (39.3 to 41.0%), while African Swine Fever (ASF) showed the least homology of all the tk proteins tested (31.4%). Although ASF has many similarities to poxviruses, and both ASF and AmEPV infect vertebrate hosts, the tk genes indicate little commonality and/or indication of common origin stemming from invertebrate hosts.

The spheroidin and thymidine kinase polypeptide sequences may include isolated naturally-occurring spheroidin or tk amino acid sequences identified herein or deliberately modified sequences which maintain the biological or regulatory functions of the AmEPV

polypeptides, respectively identified in Figs. 2 and 3. Therefore, provided that the biological activities of these polypeptides are retained in whole or part despite such modifications, this invention encompasses the use of all amino acid sequences disclosed herein as well as analogs thereof retaining spheroidin or tk biological activity. Typically, such analogs differ by only 1, 2, 3, or 4 codon changes. Similarly, proteins or functions encoded by the other spheroidin or tk ORFs may include sequences containing minor amino acid modifications but which retain their regulatory or other biological

functions. Examples of such modifications include

polypeptides with minor amino acid variations from the natural amino acid sequence of Entomopoxvirus spheroidin or thymidine kinase; in particular, conservative amino acid replacements. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into four families:

(1) acidic = aspartate, glutamate; (2) basic = lysine, arginine, histidine; (3) non-polar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar = glycine,

asparagine, glutamine, cystine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.

For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid will not have a major effect on biological activity, especially if the

replacement does not involve an amino acid at an active site of the polypeptides.

As used herein, the term "polypeptide" refers to a polymer of amino acids and does not refer to a specific length of the product; thus, peptides,

oligopeptides, and proteins are included within the definition of polypeptide. This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with

substituted linkages, as well as other modifications known in the art, both naturally occurring and

non-naturally occurring.

The proteins or polypeptides of the present invention may be expressed in host cells and purified from the cells or media by conventional means [Sambrook et al. Molecular Cloning. A Laboratory Manual, 2d edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989)].

This invention also relates to novel viral recombinant polynucleotide molecules or vectors, which permit the expression of heterologous genes in a selected host cell. Such a polynucleotide vector of the invention comprises the polynucleotide sequence encoding all or a portion of the spheroidin or tk gene, the RNA polymerase from a selected poxvirus, and the polynucleotide sequence encoding a desired heterologous gene. Preferably, the sequence includes the regulatory region, and most

preferably, the promoter region, of either the EMV spheroidin or tk gene. In addition, the source of the polymerase is not limited to EMV; rather, any poxvirus RNA polymerase may be utilized.

Therefore, the viral vectors may contain other viral elements contributed by another poxvirus, either vertebrate or invertebrate, with the only EPV sequences being provided by the presence of the EPV spheroidin or tk gene sequences, or fragments thereof. Numerous conventional expression viral vectors and expression systems are known in the art. Particularly desirable vectors systems are those of vertebrate or invertebrate poxviruses. The Entomopoxvirus spheroidin and tk gene regulatory sequences may be used in other virus vector systems which contain a poxvirus RNA polymerase to

enhance the performance of those systems, e.g., in

vaccinia vectors. Methods for the construction of

expression systems, in general, and the components thereof, including expression vectors and transformed host cells, are within the art. See, generally, methods described in standard texts, such as Sambrook et al, supra. The present invention is therefore not limited to any particular viral expression system or vector into which a polynucleotide sequence of this invention may be inserted, provided that the vector or system contains a poxvirus RNA polymerase.

The vectors of the invention provide a helper independent vector system, that is, the presence or absence of a functional spheroidin or tk gene in a poxvirus contributing elements to the vector, e.g. , contributing the RNA polymerase, does not affect the usefulness of the resulting recombinant viral vector. Because both spheroidin and tk are non-essential genes, the viral vectors of this invention do not require the presence of any other viral proteins, which in helperdependent systems are contributed by additional viruses to coinfect the selected host cell.

Selected host cells which, upon infection by the viral vectors will permit expression of the

heterologous gene, include insect and mammalian cells. Specifically, if the viral vector comprises the EPV spheroidin or tk gene sequences of the invention inserted into any member of the family Entomopoxvirinae, e.g., EPVs of any species, the host cell will be limited to cells of insects normally infected by EPVs. If the viral vector comprises the EPV spheroidin or tk gene sequences of the invention inserted into a vertebrate poxvirus, such as vaccinia or swinepox, the host cells may be selected from among the mammalian species normally infected by the wild-type vertebrate poxvirus. Most desirably, such mammalian cells may include human cells, rodent cells and primate cells, all known and available to one of skill in the art. According to one aspect of the subject

invention, therefore, vectors of the present invention may utilize a fragment of the polynucleotide sequence of EPV spheroidin, particularly the promoter and ancillary regulatory sequences which are responsible for the naturally high levels of expression of the gene.

Desirably, spheroidin sequences may be found within the sequence of Fig. 2 [SEQ ID NO:1], more particularly within the region of nucleotides # 2781 through 3199 [SEQ ID NO:22]. Smaller fragments within that region may also provide useful regulatory sequences. The desired

spheroidin promoter sequence can be utilized to produce large quantities of a desired protein by placing it in operative association with a selected heterologous gene in viral expression vectors capable of functioning in either a vertebrate or invertebrate host cell.

As used herein, the term "operative association" defines the relationship between a

regulatory sequence and a selected protein gene, such that the regulatory sequence is capable of directing the replication and expression of the protein in the

appropriate host cell. One of skill in the art is capable of operatively associating such sequences by resort to conventional techniques.

Where the spheroidin polynucleotide sequence in the vector contains all or a portion of the spheroidin coding sequence in association with, or linked to, the heterologous gene, the resulting protein expressed in the host cell may be a fusion protein consisting of all or a portion of the spheroidin protein and the heterologous protein. Where the spheroidin polynucleotide sequence in the vector does not contain sufficient coding sequence for the expression of a spheroidin protein or peptide fragment, the heterologous protein may be produced alone. In an analogous manner, the promoter and regulatory sequences of tk (Fig. 3 SEQ ID NO: 8) may be employed in the construction of an expression vector to drive expression of a heterologous protein, or a fusion protein, in a selected known expression system. These tk regulatory sequences are desirably obtained from the sequence of Fig. 3 [SEQ ID NO: 8], particularly in the fragment occurring between nucleotide #750 through 890 [SEQ ID NO: 30]. Smaller fragments within that region may also provide useful regulatory sequences.

An advantage of the use of the novel EPV spheroidin or tk promoter sequences of this invention is that these regulatory sequences are capable of operating in a vertebrate poxvirus (e.g., vaccinia)-mammalian cell expression vector system. For example, the strong spheroidin promoter can be incorporated into the vaccinia virus system through homologous recombination. Unlike the promoter for the baculovirus polyhedrin gene, the promoter for the EPV spheroidin gene can be utilized directly in the vaccinia or swinepox virus expression vector.

To construct a vector according to the present invention, the spheroidin or tk polynucleotide sequence may be isolated and purified from a selected

Entomopoxvirus, e.g., AmEPV, and digested with

appropriate restriction endonuclease enzymes to produce a fragment comprising all or part of the spheroidin or tk gene. Alternatively such a fragment may be chemically synthesized.

Still alternatively, the desired AmEPV sequences may be obtained from bacterial cultures

containing the plasmids pRH512, pMEGtk-1 or pRH7. The construction of the plasmid pRH512 is described in the examples below. This plasmid contains the 4.51 kb BglII fragment AmEPV DNA sequence inserted into a BamHI site in the conventional vector pUC9. The plasmid pRH7 was constructed by digesting AmEPV genomic DNA, obtained as described in Example 1, with Bsp1286I, and the resulting fragments with HaeII. T4 DNA polymerase is employed to blunt end the AmEPV DNA and the fragment containing the spheroidin gene is ligated to the large fragment of a Smal digested pUC9 fragment. This fragment contains the entire spheroidin open reading frame and some flanking sequence, included within the nucleotide sequence

spanning #2274-6182 [SEQ ID NO: 33] of Fig. 2. The construction of plasmid pMEGtk-1 comprising the

regulatory sequences of the tk gene as well as the structural gene is described below in the Example 8. It was constructed by inserting the EcoRI-Q fragment of AmEPV into the conventional vector pUC18.

Bacterial cultures containing plasmids pRH512, pMEG tk-1, and pRH7 have been deposited in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, USA. The deposited cultures are as follows: - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Culture Accession No. Deposit Date

E. coli SURE strain ATCC 68532 26 Feb 91

(Stratagene) pMEG-tkl

E. coli SURE strain ATCC 68533 26 Feb 91 (Stratagene) pRH512

E. coli SURE strain ATCC

(Stratagene) pRH7

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

The plasmids can be obtained from the deposited bacterial cultures by use of standard procedures, for example, using cleared lysate-isopycnic density gradient procedures, and the like.

These ATCC deposits were made under conditions that assure that access to the cultures will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademark to be entitled thereto under 37 CFR 1.14 and 35 USC 122. The deposits will be available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by government action.

Further, the subject culture deposit will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., it will be stored with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposit, and in any case, for a period of at least 30 (thirty) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the

culture. The depositor acknowledges the duty to replace the deposit should the depository be unable to furnish a sample when requested, due to the condition of the deposit. All restrictions on the availability to the public of the subject culture deposit will be irrevocably removed upon the granting of a patent disclosing it.

The molecular biology procedures referred to herein in describing construction of the vectors of this invention are standard, well-known procedures. The various methods employed in the preparation of the plasmid vectors and transformation or infection of host organisms are well-known in the art. These procedures are all described in, for example, Sambrook et al, cited above. Thus, it is within the skill of those in the genetic engineering art to extract DNA from microbial cells, perform restriction enzyme digestions,

electrophorese DNA fragments, tail and anneal plasmid and insert DNA, ligate DNA, transform cells, prepare plasmid DNA, electrophorese proteins, and sequence DNA.

Because the AmEPV genome has no known unique restriction sites into which selected genes may be effectively introduced in a site-specific manner so as to be under the control of the spheroidin or tk promoter sequences, such restriction sites must be introduced into desired sites in the selected EPV polynucleotide

sequence. For example, the unique BstB1 site located at nucleotide #3172 downstream from the start of the

spheroidin gene is the closest site to genetically engineer a usable insertion sequence for cloning.

Therefore, restriction sites closer to the initiating Met of the spheroidin gene must be deliberately inserted.

Methods for the insertion of restriction sites are known to those of skill in the art and include, the use of an intermediate shuttle vector, e.g., by cloning the EPV sequence into the site of an appropriate cloning vehicle. It will be recognized by those skilled in the art that any suitable cloning vehicle may be utilized provided that the spheroidin or tk gene and flanking viral DNA may be functionally incorporated.

A spheroidin shuttle vector may be constructed to include elements of the spheroidin structural gene, a cloning site located or introduced in the gene to enable the selected heterologous gene to be properly inserted into the viral genome adjacent to, and under the control of, the spheroidin promoter, and flanking viral DNA linked to either side of the spheroidin gene to

facilitate insertion of the spheroidin-foreign geneflanking sequence into another expression vector. The presence of flanking viral DNA also facilitates

recombination with the wild type Entomopoxvirus, allowing the transfer of a selected gene into a replicating viral genome. The shuttle vectors may thereafter be modified for insertion of a selected gene by deleting some or all of the sequences encoding for spheroidin or tk synthesis near the respective transcriptional start sites.

Examples of such sites in spheroidin are nucleotides #3077 and 3080 and in tk includes nucleotide #809.

Conventional procedures are available to delete

spheroidin or tk coding sequences.

As an alternative to or in addition to the restriction site, a variety of synthetic or natural oligonucleotide linker sequences may be inserted at the site of the deletion. A polynucleotide linker sequence, which may be either a natural or synthetic

oligonucleotide, may be inserted at the site of the deletion to allow the coupling of DNA segments at that site. One such linker sequence may provide an

appropriate space between the two linked sequences, e.g., between the promoter sequence and the gene to be

expressed. Alternatively, this linker sequence may encode, if desired, a polypeptide which is selectively cleavable or digestible by conventional chemical or enzymatic methods. For example, the selected cleavage site may be an enzymatic cleavage site, including sites for cleavage by a proteolytic enzyme, such as

enterokinase, factor Xa, trypsin, collegenase and

thrombin. Alternatively, the cleavage site in the linker may be a site capable of being cleaved upon exposure to a selected chemical, e.g. cyanogen bromide or

hydroxylamine. The cleavage site, if inserted into a linker useful in the sequences of this invention, does not limit this invention. Any desired cleavage site, of which many are known in the art, may be used for this purpose. In another alternative, the linker sequence may encode one or a series of restriction sites. It will be recognized by those skilled in the art who have the benefit of this disclosure that linker sequences bearing an appropriate restriction site need not be inserted in place of all or a portion of the spheroidin structural sequence, and that it would be possible to insert a linker in locations in the

Entomopoxvirus genome such that both the sequence coding for the selected polypeptide and the spheroidin

structural sequence would be expressed. For instance, the sequence coding for the selected polypeptide could be inserted into the tk gene in place of all or a portion of the tk structural sequence and under the transcriptional control of the tk promoter.

Polymerase chain reaction (PCR) techniques can also be used to introduce convenient restriction sites into the EPV DNA, as well as to amplify specific regions of the EPV DNA. These techniques are well known to those skilled in this art. See, for example, PCR Protocols: A Guide to Methods and Applications, M. A. Innis, D. H.

Gelfand, J. J. Sninsky, and T. J. White, (1990).

By use of these techniques, a variety of alternative modified shuttle vectors into which a

selected gene or portion thereof may be incorporated may be suitably utilized in the present invention.

As one embodiment of the invention, therefore, the polynucleotide sequence, described above, may be used as a shuttle vector to transfer a selected heterologous gene to a selected virus. In this embodiment, the polynucleotide sequence encoding the EMV spheroidin gene or EMV tk gene, or a fragment thereof, is linked to a heterologous gene. The polynucleotide sequence further contains a flanking region on either side of the

spheroidin-heterologous gene or tk-heterologous gene to enable ready transfer into a selected virus. This

resulting construct is termed a cassette. Such a flanking region may be derived from EPV, or

alternatively, may be complementary to the target virus. For example, if it is desirable to insert a selected heterologous gene into a vaccinia virus to create a recombinant virus, one would utilize flanking regions complementary to the targeted vaccinia virus. Similarly if the heterologous gene is inserted within the EPV spheroidin or tk gene, so that the selected EPV

regulatory sequence and heterologous gene are flanked by the EPV gene's own sequences, this cassette may be used for transfer into a wild type EPV having homologous sequences to the flanking sequences.

The insertion or linkage of the foreign gene into the tk or spheroidin sequences of the present invention or the linkage of flanking sequences foreign to the spheroidin or tk genes may be accomplished as

described above. The vectors of the subject invention may use cDNA clones of foreign genes, because poxvirus genes contain no introns, presumably as a consequence of a totally cytoplasmic site of infection.

In accordance with standard cloning techniques, any selected gene may be inserted into the vector at an available restriction site to produce a recombinant shuttle vector. Virtually any gene of interest could be inserted into the vectors described herein in order to obtain high expression of the desired protein.

Restriction sites in the fragment may thereafter be removed so as to produce a preferred spheroidin or tk shuttle vector, having one or more cleavage or cloning sites located in the 3' direction downstream from the spheroidin promoter sequence. Thus, the present

invention is not limited by the selection of the

heterologous gene.

Alternatively, a vector of this invention may comprise a heterologous gene which is inserted into all or a portion of the EMV spheroidin or tk protein encoding sequence to interrupt the protein's natural processing. However, when the vector is transferred to another virus which contains a wild-type spheroidin or tk gene,

expression of the inserted heterologous gene is obtained. Thus, the Entomopoxvirus spheroidin gene (Fig. 2 SEQ ID NO:1) and/or the tk gene (Fig. 3 SEQ ID NO: 8) can be used as the location for the insertion of exogenous or

heterologous DNA in any of the above-mentioned expression systems. A shuttle vector so constructed may be useful as a marker for research and production techniques for identifying the presence of successfully integrated heterologous genes into the selected expression system.

The tk gene is a particularly desirable site for insertion of a selected heterologous gene. Unlike spheroidin, tk is produced early in infection and in lesser quantities. Additionally, many poxviruses possess tk genes which may be sufficiently homologous to the EPV tk to provide easy recombination. For example, in

vaccinia virus expression systems for mammalian cells, the vaccinia tk gene is a common insertion site.

Therefore, the use of this gene is particularly desirable for construction of a shuttle vector to shuttle selected genes directly between vector systems. More

specifically, a foreign gene may be desirably inserted into the EPV tk gene sequence between nucleotide #460 and #560 (See Fig. 3).

Insertion of cassettes containing foreign genes into wild-type poxviruses can be accomplished by

homologous recombination. The homologous recombination techniques used to insert the genes of interest into the viruses of the invention are well known to those skilled in the art. The shuttle vectors, when co-infected into host cells with a wild-type virus, transfer the cassette containing the selected gene into the virus by homologous recombination, thereby creating recombinant virus vectors.

Expression of a selected gene is accomplished by infecting susceptible host insect cells with the recombinant viral vector of this invention in an

appropriate medium for growth. An EPV expression vector is propagated in insect cells or insects through

replication and assembly of infectious virus particles. These infectious vectors can be used to produce the selected gene in suitable insect cells, thus facilitating the efficient expression of the selected DNA sequence in the infected cell. If the EPV spheroidin gene (or tk gene) - heterologous gene fragment is inserted into a vertebrate poxvirus by the same methods as described above, the recombinant virus may be used to infect mammalian cells and produce the heterologous protein in the mammalian cells.

For example, a gene inserted into the tk site of a vaccinia virus system could be transferred directly to the tk locus of an Entomopoxvirus vector of the subject invention or vice versa. This shuttling could be accomplished, for example, using homologous

recombination. Similarly insertion of a selected gene into the spheroidin gene or tk gene in a viral vector permits the gene to be shuttled into other viruses having homologous spheroidin or tk sequences, respectively.

The following description illustrates an exemplary vector of this invention, employing the gene coding for human β-interferon (IFN-β) synthesis as the heterologous gene. A DNA fragment containing the IFN-β gene is prepared conventionally with restriction enzyme digested or blunt ended termini and cloned into a

suitable site in the AmEPV spheroidin gene, into which a restriction site has been engineered by the methods described above. The insertion of the IFN-β gene produces a hybrid or fused spheroidin-IFN-β gene capable of

producing a fused polypeptide product if only a portion of the spheroidin gene was deleted as described above. If the entire spheroidin structural sequence was deleted, only interferon will be produced. Further, the hybrid gene may comprise the spheroidin promoter, the IFN-β protein coding sequences, and sequences encoding a portion of the polypeptide sequence of the spheroidin protein, provided all such coding sequences are not deleted from the particular shuttle vector utilized.

The resulting shuttle vector contains the AmEPV spheroidin gene sequence coupled to the IFN-β gene. The hybrid spheroidin-IFN-β gene of the recombinant shuttle vector is thereafter transferred into the genome of an appropriate Entomopoxvirus, such as the preferred

Entomopoxvirus AmEPV, to produce a recombinant viral expression vector capable of expressing the gene encoding for β-interferon in a host insect cell. Transfer of the hybrid gene to a wild-type virus is accomplished by processes which are well known to those skilled in the art. For example, appropriate insect cells may be

infected with the wild type Entomopoxvirus. These

infected cells are then transfected with the shuttle vector of the subject invention. These procedures are described, for example, in DNA Cloning: A Practical

Approach, Vol. II, Edited by D. M. Glover, Chapter 7, 1985. A person skilled in the art could choose

appropriate insect cells to be used according to the subject invention. By way of example, salt marsh

caterpillars and cultured gypsy moth cells can be used.

During replication of the AmEPV DNA after transfection, the hybrid gene is transferred to the wild-type AmEPV by homologous recombination between the

recombinant shuttle vector and AmEPV DNA. Accordingly, a mixture is produced comprising wild-type, nonrecombinant EPVs and recombinant EPVs capable of expressing the IFN-β gene.

While transfection is the preferred process for transfer of the hybrid gene into the EPV genome, it will be understood by those skilled in the art that other procedures may be suitably utilized so as to effect the insertion of the gene into the EPV genome and that recombination may be accomplished between the recombinant shuttle vector and other strains of EPV (or other

poxviruses) so long as there is sufficient homology between the sequence of the hybrid gene and the

corresponding sequence of the other strain to allow recombination to occur.

The preferred recombinant AmEPV expression vector, comprising a hybrid spheroidin-IFN-β gene

incorporated into the AmEPV genome, can thereafter be selected from the mixture of nonrecombinant and

recombinant Entomopoxviruses. The preferred, but by no means only, method of selection is by screening for plaques formed by host insect cells infected with viruses that do not produce viral occlusions. Selection may be performed in this manner because recombinant EPV viruses which contain the spheroidin or tk protein coding

sequences interrupted by the heterologous gene are defective in the production of viral occlusions due to the insertional inactivation of the spheroidin gene.

Also, the selection procedure may involve the use of the β-galactosidase gene to facilitate color selection. This procedure involves the incorporation of the E. coli β-galactosidase gene into the shuttle vector and is well known to those skilled in the art. This technique may be of particular value if the exogenous DNA is inserted into the tk gene so that the spheroidin gene is still expressed. It will be recognized by those skilled in the art that alternative selection procedures may also be utilized in accordance with the present invention.

Accordingly, the DNA from a recombinant virus is thereafter purified and may be analyzed with

appropriate restriction enzymes, or PCR technology, to confirm that the recombinant AmEPV vector has an

insertion of the selected gene in the proper location.

The vectors and methods provided by the present invention are characterized by several advantages over known vectors and vector systems. Advantageously, such EPV viral vectors of the present invention are not oncogenic or tumorigenic in mammals. Also, the

regulatory signals governing Amsacta moorei

Entomopoxvirus (AmEPV) gene expressions are similar to those of vaccinia. Therefore, it is possible to transfer the strongly expressed spheroidin gene, or the thymidine kinase gene, as an expression cassette, not only in insect cells, but for use in vertebrate poxviruses such as vaccinia and swinepox virus.

Based on reported data with vaccinia, herpes and baculovirus vector systems, which suggest that up to 30 kb can be transferred without disrupting the vector viability, the normal limitation on the amount of

exogenous DNA which can be packaged into a virus is not anticipated to be encountered when using the novel EPV vectors and methods of the subject invention.

Another advantage is that for the novel vectors of the subject invention, the transcription and

translation of foreign proteins is totally cytoplasmic. Still another advantage lies in the expression power of the EPV spheroidin regulatory sequences, which when in operative association with a heterologous gene in a vector of this invention, should produce high levels of heterologous protein expression in the selected host cell.

The EPV vectors of this invention and methods for employing them to express selected heterologous proteins in insect or mammalian cells, as described above, are characterized by the advantage of replication in insect cells, which avoids the use of mammalian viruses, thereby decreasing the possibility of

contamination of the product with mammalian virus. The expression system of this invention is also a helper independent virus expression vector system. These two characteristics are shared by known baculovirus

expression systems. However, as shown in Table 1, the EPV expression vector system (EEVS) using the vectors of this invention has some important distinguishing features compared to the baculovirus expression systems (BEVS). - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Table 1

Differences between EEVs and BEVS

EEVs BEVS

Site of replication: cytoplasm nucleus

Virus family: Poxviridae Baculoviridae

Sites for insertion spheroidin & polyhedrin of foreign genes thymidine & p10

kinase (tk)

Shuttle possibilities yes No mammalian between vertebrate (Orthopoxviruses) counterparts. and insect systems: (Leporipoxviruses) Baculovirus

(Suipoxviruses) is not known

(Avipoxviruses) to contain a tk gene.

Polyhedrin is not found in mammalian systems.

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - The present invention also provides a method for screening recombinant host cells for insertion of heterologous genes is provided by use of the recombinant viral polynucleotide molecules of this invention. The viral molecules containing the selected heterologous gene sequence linked to the polynucleotide sequence encoding less than all of the Entomopoxvirus spheroidin protein. The heterologous gene may be linked to the spheroidin or tk regulatory sequences in the absence of the complete coding sequences, or it may be inserted into the

spheroidin or tk gene coding sequences, thus disrupting the coding sequence. The cell infected with the

recombinant vector is cultured under conditions suitable for expression of the heterologous protein, either unfused or as a fusion protein with a portion of the spheroidin sequence. The absence of occlusion bodies which would ordinarily be formed by the expression of the intact spheroidin protein indicates the integration of the heterologous gene.

If the viral vector similarly contained either incomplete or interrupted EPV tk encoding sequence, the absence of thymidine kinase function (e.g., resistance to methotrexate or an analogue thereof) formed by the

integration of the inactive thymidine kinase sequence indicates the insertion of the heterologous gene.

Alternatively, if a parent virus is deleted of part of its tk or spheroidin gene, and is thereafter mixed with a viral vector containing intact tk or

spheroidin fused to the foreign gene, recombinants would express the methotrexate resistance or produce occlusion bodies, respectively, thus indicating integration of the active tk or spheroidin genes and the foreign gene.

The above-described selection procedures

provide effective and convenient means for selection of recombinant Entomopoxvirus expression vectors. Another embodiment of the present invention involves using novel EPV expression systems of the subject invention for insect control. Control of insect pests can be accomplished by employing the vectors and methods of the invention as described above. For

example, a gene coding for an selected insect toxin may be inserted into the viral expression vector under the control of the spheroidin or tk regulatory sequences or within either of the two genes for purposes of

recombination into a selected virus having homologous flanking regions.

Genes which code for insect toxins are well known to those skilled in the art. An exemplary toxin gene isolated from Bacillus thuringiensis (B.t.) can be used according to the subject invention. B.t. genes are described, for example, in U. S. Patent Nos. 4,775,131 and 4,865,981. Other known insect toxins may also be employed in this method.

The resulting EPV vector containing the toxin gene is applied to the target pest or its surroundings. Advantageously, the viral vector will infect the target pest, and large quantities of the toxin will be produced, thus resulting in the control of the pest. Particularly large quantities of the toxin protein can be produced if the regulatory sequences of the Entomopoxvirus spheroidin gene are used to express the toxin.

Alternatively, the spheroidin gene can be left intact and the toxin gene inserted into a different

Entomopoxvirus gene such as the tk gene. In this

construct, the toxin will be produced by the system and then effectively coated or encapsulated by the natural viral production of spheroidin. This system thus

produces a toxin which will advantageously persist in the environment to prolong the availability to the target pest. In addition to the novel Entomopoxvirus expression vectors and methods for their use described herein, the subject invention pertains to the use of novel regulatory elements from Entomopoxvirus to

construct novel chimeric vaccinia and swinepox vaccines and expression systems which are functional across genera of mammalian poxviruses. The polynucleotide sequences of the invention can also be used with viral vaccines, e.g., known vaccinia virus vaccines, to enhance the

effectiveness of these vaccines. Such vaccines have been described for use in controlling rabies and other

infectious diseases in mammals. Specifically, it is anticipated that the introduction of the EPV spheroidin promoter sequences into known viral vectors which are used to express selected proteins in a mammalian host in vivo may enable the powerful spheroidin promoter to increase expression of the protein in the viral vaccine. This aspect of the invention provides a significant improvement over other expression systems, including the baculovirus expression system (BEVS).

The following examples illustrate the compositions and procedures, including the best mode, for practicing the invention. These examples, should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted. The restriction enzymes disclosed herein can be purchased from Bethesda Research

Laboratories, Gaithersburg, MD, or New England Biolabs, Beverly, MA. The enzymes are used according to the instructions provided by the supplier. Klenow fragment of DNA polymerase, T4 polynucleotide kinase, and T4 DNA ligase were obtained from New England Biolabs and

Promega. EXAMPLE 1; PREPARATION OF AmEPV DNA

The replication of AmEPV has been described previously [R. H. Goodwin et al, J. Invertebr. Pathol., 5(5:190-205 (1990)]. The gypsy moth (Lvmantria dispar) cell line IPLB-LD-652 [Insect Pathology Laboratory, Agricultural Research Service, U.S. Department of

Agriculture, Beltsville, MD] is maintained at 26 to 28°C in EX-CELL 400 [JRH Biosciences, Lenexa, KS] supplemented with 10% fetal bovine serum, 100 U of penicillin, and 100 μg of streptomycin per ml. Other insect cell lines are well known to those skilled in the art and can be used according to the subject invention.

The AmEPV inoculum for cell culturing was from an AmEPV-infected, freeze-dried E. acrea larva stored at -70°C [R. L. Hall et al, Arch. Virol.. 110:77-90

(1990)]. The larva was crushed and macerated in 5 ml of EX-CELL 400 (with penicillin and streptomycin but without fetal bovine serum) to which 0.003 g of cysteine-HCl had been added to prevent melanization. The debris was pelleted at 200 × g for 5 minutes, and the supernatant was passed through a 0.45-μm-pore-size filter.

The gypsy moth cells were infected with AmEPV by addition of the inoculum to a preconfluent monolayer of cells (about 0.1 to 1 PFU per cell), with occasional agitation of the dish during the first day. Infected cells were harvested 5 to 6 days postinfection.

AmEPV DNA was prepared from the infected cells by one of two methods. The first method involved in situ digestion of infected cells embedded within agarose plugs, after which the released cellular and viral DNAs were separated by pulsed-field electrophoresis [Bio-Rad CHEF-II-DR system]. IPLB-LD-652 cells were infected with first-cell-culture-passage AmEPV. Infected cells were harvested 6 days postinfection by centrifugation at 200 × g for 5 minutes, rinsed, and resuspended in modified Hank's phosphate-buffered saline (PBS), which contained 15 g of glucose per liter, but no Ca²⁺ or Mg²⁺.

For embedding of the infected cells in agarose plugs, 1% SeaPlaque GTG agarose (prepared in modified Hank's PBS and equilibrated at 37°C) was mixed 1:1 with infected cells to yield 5 × 10⁶ cells per ml in 0.5% agarose. Digestion to release DNA was done by gentle shaking of the inserts in 1% Sarkosyl-0.5 M EDTA-1 mg of proteinase K per ml at 50°C for 2 days [C. L. Smith et al. Methods Enzymol.. 151:461-489 (1987)]. The CHEF-II- DR parameters for DNA separation were 180 V, a pulse ratio of 1, 50 initial and 90 second final pulse times, and a run time of 20 to 25 hours at 4°C. The separating gel was 1% SeaKem GTG agarose in 0.5x TBE buffer

[Sambrook et al, supra]. Viral DNA bands were visualized by ethidium bromide staining and electroeluted [W. B. Allington et al, Anal. Biochem., 85:188-196 (1978)]. The recovered DNA was used for plasmid cloning following ethanol precipitation.

The second method of viral DNA preparation used the extracellular virus found in the infected-cell- culture supernatant. The supernatant from 10-day- postinfection cell cultures was clarified by

centrifugation at 200 × g for 5 minutes. Virus was collected from the supernatant by centrifugation at

12,000 × g. Viral pellets were resuspended in 6 ml of 1x TE. DNase I and RNase A (10 and 20 μg/ml final

concentrations, respectively) were added, and the mixture was incubated at 37°C for 30 minutes. The mixture was heated to 50°C for 15 minutes. SDS and proteinase K (1% and 200 μg/ml, respectively) were then added. The sample was incubated to 50°C overnight and extracted three times with buffer-saturated phenol and once with SEVAG

[Sambrook et al, supra]. The DNA was ethanol

precipitated and resuspended in 1x TE (pH 8). For routine virus quantitation, 1 ml of an appropriate virus dilution (prepared in unsupplemented EX-CELL 400) was added to a preconfluent monolayer of cells in a 60 mm culture dish, with intermittent

agitation over a 5 hour adsorption period at 26 to 28 °C. The virus inoculum was removed, and 5 ml of a 0.75%

SeaPlaque agarose [FMC BioProducts, Rockland, ME] overlay prepared with 2x EX-CELL 400 and equilibrated at 37°C was added to the monolayer. Plaques were visualized after 5 days of incubation at 26°C by inspection with a

stereomicroscope.

The DNA prepared according to either method was then cut with a variety of restriction endonuclease enzymes, e.g., Bam HI, EcoRI, HindIII, PstI and Xhol. generating the various fragments which appear on the physical map of Fig. 1. Hereafter, reference to each restriction fragment will refer to the enzyme and the applicable letter, e.g., BamHI-A through BamHI-E, EcoRI-A through EcoRI-S, etc. EXAMPLE 2 - ISOLATION OF THE SPHEROIDIN GENE

To localize the spheroidin gene, a purified preparation of occlusion bodies (OBs) from infected caterpillars was solubilized and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [J. K. Laemmli, Nature (London), 122:680-685

(1970)] with a 4% acrylamide stacking gel and a 7.5% separating gel. The acrylamide used to prepare

spheroidin for protein microsequencing was deionized with AG501X8 resin [Bio-Rad, Richmond, CA] . The gels were polymerized overnight at 4°C. For sample preparation, 2x Laemmli sample buffer consisting of 125 mM Tris-HCl (pH 6.8), 4% SDS (w/v), 10% β-mercaptoethanol (v/v), and 20% glycerol (v/v) was used. OB suspension samples were diluted 1:1 with 2x Laemmli sample buffer and boiled for 5 minutes. Several lanes of an OB protein preparation were separated electrophoretically. The spheroidin protein (113 kDa) was the predominant protein of the purified OBs.

Spheroidin within SDS-polyacrylamide gels was tested for glycosylation by periodic acid-Schiff staining [R. M. Zacharius et al. Anal. Biochem.. 30:149-152 (1969)].

Following electrophoretic separation, several lanes in the unstained gel were transferred to an

Immobilon polyvinylidene difluoride (PVDF) membrane with a Bio-Rad TransBlot apparatus at 90 V for 2 hours in a buffer consisting of 10 mM morpholinepropanesulfonic acid (pH 6.0) and 20% methanol. Spheroidin was visualized on the PVDF membrane by Coomassie blue staining.

The region of the PVDF membrane containing spheroidin was excised from the membrane, and direct protein microsequencing was done with an Applied

Biosystems gas-phase sequencer. Microsequencing of the intact protein was unsuccessful, presumably because the N terminus of the protein was blocked.

Cyanogen bromide cleavage was performed on samples of spheroidin eluted from the PVDF membrane to generate internal peptide fragments for sequencing.

Major polypeptides of 15, 9, 8, and 6.2 kDa were

produced.

EXAMPLE 3 - SEQUENCING, HYBRIDIZATIONS

All DNA sequencing was done by the dideoxy chain termination method [F. Sanger et al, Proc. Natl. Acad. Sci. USA. 24:5463-5467 (1977)] with [α-³⁵S]dATP and Sequenase [US Biochemical, Cleveland, OH] . Standard sequencing reactions with Sequenase were carried out in accordance with the instructions of the supplier, US Biochemical. A reliable amino acid sequence was obtained from the 9, 8, and 6.2 kDa polypeptides produced as described in Example 3. The 8 and 9 kDa polypeptides represented overlapping partial CNBr cleavage products which together yielded the longest continuous amino acid sequence: Met-Ala-(Asn or Arg)-Asp-Leu-Val-Ser-Leu-Leu- Phe-Met-(Asn or Arg)-(?)-Tyr-Val-(Asn?)-Ile-Glu-Ile-Asn- Glu-Ala-Val-(?)-(Glu?) [SEQ ID NO: 34]. The amino acid sequence obtained from the 6.2 kDa fragment was Met-Lys- Ile-Thr-Ser-Ser-Thr-Glu-Val-Asp-Pro-Glu-Tyr-Val-(Thr or Ile)-Ser-(Asn?) [SEQ ID NO:35]. A partial sequence for the 15 kDa fragment was also obtained: (Asn?)-Ala-Leu-Phe-(Phe?)(Asn?)-Val-Phe [SEQ ID NO:36]. The question marks in the above sequences indicated undetermined or unconfirmed amino acids. All sequences were ultimately located within the spheroidin gene sequence.

EXAMPLE 4 - PLASMID PRH512

A BglII AmEPV DNA library was prepared by digesting the genomic AmEPV DNA with BglII according to manufacturer's instructions. Plasmid pUC9 [GIBCO;

Bethesda Research Labs] was BamHI-digested and

phosphatase-treated. The genomic BglII cut AmEPV was shotgun cloned into the BamHI site of pUC9. Escherichia coli SURE [Stratagene, La Jolla, CA] was transformed by electroporation with a Bio-Rad Gene Pulser following the instructions provided by the manufacturer with the shotgun ligation, containing a variety of recombinant plasmids. Mini-preparations of plasmids were made by a conventional alkaline lysis procedure [Sambrook et al, supra]. These plasmids were cut with EcoRI-SalI to release the insert and run on a gel. The resulting plasmid DNA was southern blotted to a nylon membrane, producing a number of clones. Among the fragments produced from the

restriction enzyme digestions of the genomic DNA was a 4.4 BglII fragment and an EcoRI-D fragment. In order to locate a desirable clone from among those produced above, the sequence derived from the 6.2 kDa CNBr fragment was used to design a degenerate oligonucleotide for use as a hybridization probe to locate the spheroidin gene in a clone. The nucleotide sequence of this probe called RM58 [SEQ ID NO: 12] was GA5GT7GA6CC7GA5TA6GT, where 5

represents A or G, 6 represents C or T, and 7 represents A, G, C, or T. The peptide sequence of the probe was: Glu-Val-Asp-Pro-Glu-Tyr-Val [SEQ ID NO: 37].

The DNA probe was radiolabeled either with [α- 3²P]dCTP by the random oligonucleotide extension method [A. P. Feinberg et al. Anal. Biochem.. 132:6-13 (1983)] or with [γ-³²P]ATP and T4 polynucleotide kinase [Sambrook et al, supra1. These same procedures were used for all other oligonucleotide probes described below. Both types of probes were purified by passage through spun columns of Sephadex G-50.

Southern transfer was done with Hybond-N

[Amersham]; the transferred DNA was fixed to the membrane by UV cross-linking. Southern hybridization was

performed both with transferred DNA including the

restriction fragments described above, as well as the

BglII library of AmEPV DNA cloned into BamHI-digested plasmid pUC9 as described above. Hybridization with the oligonucleotide probe was done at 37 or 45°C with BLOTTO [Sambrook et al, supra] and was followed by two washes at room temperature with 0.3 M NaCl-0.06 M Tris (pH 8) -2 mM EDTA for 5 minutes.

The RM58 probe [SEQ ID NO: 12] hybridized to the 4.4 kb BglII fragment and the EcoRI-D fragment of AmEPV DNA [See Fig. 1] . A plasmid produced by the shotgun cloning, recombinant pRH512 (a BglII 4.56 kb fragment inserted into the BamHI site of pUC9 which contains about 1.5 kb of the 5' end of the spheroidin gene) was also identified by this hybridization with the RM58

oligonucleotide [SEQ ID NO: 12].

The 4.51 kb pRH512 BglII insert was isolated, radiolabeled as described above, and hybridized back to various AmEPV genomic digests as follows. The DNA-DNA hybridization was done at 65°C with BLOTTO [Sambrook et al, supra] and was followed by two washes at room

temperature with 0.3 M NaCl-0.06 M Tris (pH 8) -2 mM EDTA for 5 minutes, two washes for 15 minutes each at 65°C but with 0.4% SDS added, and two washes at room temperature with 0.03 M NaCl-0.06 M Tris (pH 8) -0.2 mM EDTA.

Hybridization was observed to the BamHI-A, EcoRI-D,

HindIII-G and -J, Pstl-A, and Xhol-B fragments of AmEPV DNA. The results of these hybridizations indicated that the 4.51 kb fragment in pRH512 was substantially

identical to the 4.4 kb fragment produced by BglII digestion of genomic DNA.

The 4.51 kb BglII insert of pRH512 was thereafter sequenced by two procedures. One is the double-stranded plasmid sequencing method [M. Hattori et al. Anal. Biochem.. 152:232-238 (1986)] performed with "miniprep" [Sambrook et al, supra] DNA and 1 pmol of universal, reverse, or custom-designed oligonucleotide primer in each sequencing reaction. Nested exonuclease II deletions [S. Henikoff, Methods Enzvmol.. 155:156-165 (1987) ] were used to sequence plasmid pRH512 according to this method. Deletions were made from the universal primer end. For making these deletions, the DNA was cut with EcoRI. filled in with α-thiophosphate dNTPs [S. D. Putney et al, Proc. Natl. Acad. Sci. USA. 78:7350-7354 (1981) ] by use of the Klenow fragment of E. coli DNA polymerase, cut with Smal, and treated with exonuclease III. Samples were removed every 30 seconds, re-ligated. and used to transform E. coli SURE cells by electroporation. Sequencing reactions were carried out with the universal primer.

When a primer complementary to that sequence was prepared and used to sequence back through the RM58 binding site (bases 3983 to 4002), the generated

sequence, when translated, yielded the amino acid sequence generated from microsequencing the 6.2 kDa CNBr polypeptide fragment.

A second sequencing method was performed using a combination of M12 shotgun sequencing with standard and universal and reverse M13 primers into M13 phage to permit single-stranded sequencing as follows. Plasmid pRH512 was sonicated to produce random fragments,

repaired with bacteriophage T4 DNA polymerase, and these fragments were shotgun cloned into Smal-cut M13mp19

[GIBCO]. Plaque lifts were screened with a radiolabeled probe prepared from the 4.5 kb insert found in pRH512 to identify appropriate clones for shotgun single stranded sequencing [see, e.g., Sambrook et al, supra].

Sequencing of the BglII insert of pRH512 isolated it to nucleotides # 0 to 4505, thus extending the sequence 5' and 3' to the spheroidin gene (Fig. 2).

EXAMPLE 5 - OBTAINING ADDITIONAL AmEPV SEQUENCE

A Dral AmEPV DNA library was prepared by digesting genomic DNA with Dral. These Dral fragments were shotgun cloned into Smal-digested, phosphatase-treated vector M13mpl9. Preparations of M13 virus and DNA were made by standard procedures [J. Sambrook et al, supra]. Ligation and heat shock transformation

procedures were performed conventionally [Sambrook et al, supra.], resulting in the shotgun cloned fragments being transformed into the bacterial strain, E. coli UT481

[University of Tennessee] or the SURE strain. Standard PCR [Innis et al, supra] with 400 ng of genomic AmEPV DNA as a template was used to prepare a probe to identify a 586 bp Dral clone from nitrocellulose filter replicas (plaque lifts) [Micron Separations, Inc.] of the M13 shotgun library of Dral-cut AmEPV fragments. This was done to isolate a clone spanning a central unsequenced region of the spheroidin gene. The standard PCR primers used for this reaction were RM92 [SEQ ID NO: 15] (GCCTGGTTGGGTAACACCTC) and RM118 [SEQ ID NO: 16] (CTGCTAGATTATCTACTCCG) . This sequencing revealed that there was a single HindIII site at base 931 and that the 2 ' end of the spheroidin open reading frame (ORF) was truncated (Fig. 2).

The technique of inverse polymerase chain reaction (PCR) [M. A. Innis et al, PCR protocol, a guide to methods and applications. Academic Press, Inc. San Diego, CA (1990) ] was used with Clal-digested AmEPV DNA fragments which were ligated into a circle, to prepare a probe to identify clones containing a flanking sequence or to verify the absence of an intervening sequence between adjacent clones. The primers used in inverse PCR were RM82 and RM83, which were taken from the pRH512 sequence. The sequence of RM82 [SEQ ID NO: 13] was

TTTCAAATTAACTGGCAACC and that of RM83 [SEQ ID NO: 14] was GGGATGGATTTTAGATTGCG.

The specific PCR reaction conditions for 34 cycles were as follows: 30 seconds at 94°C for

denaturation, 30 seconds at 37°C for annealing, and 1.5 minutes at 72 °C for extension. Finally, the samples were incubated at 72 °C to 8.5 minutes to complete extensions. The concentration of each primer was 1 μM.

The resulting 2.2 kb inverse PCR product was digested with Clal. and a 1.7 kb fragment was gel

purified. The 1.7 kb PCR fragment was sequenced with RM83 as a primer. Additional PCR primers were made to the new sequence as it was identified. The sequencing process employed Sequenase, 5 pmol of each primer, and 10 to 50 ng of template. Prior to being sequenced, the PCR products were chloroform extracted and purified on spun columns [Sambrook et al, supra] of Sephacryl S-400. The DNA sequence was assembled and aligned, and consensus sequence was produced [R. Staden, Nucleic Acids Res..

10:4731-4751 (1982)]. Both strands were completely sequenced; the PCR product sequence was verified by conventional sequence.

The relevant Clal sites of the 1.7 kb PCR fragment are at positions 3485 and 6165. This fragment was radiolabeled and used as a probe to locate additional clones, i.e., pRH827 (307 bp), pRH85 (1.88 kb), and pRH87 (1.88 kb) from the BglII fragment library. Plasmids pRH85 and pRH87 were sequenced using the same nested exonuclease II deletions and sequencing procedure, as described above for pRH512. Sequencing of the inverse PCR products with custom-designed primers confirmed that plasmids pRH85 and pRH87 represented the same 1.88 kb

Bglll DNA insert in opposite orientations, but also revealed a missing 80 bp between pRH827 and pRH85. This 80 bp DNA fragment was identified in the Dral fragment, as extending from bases 4543 to 5128 cloned into M13.

The orientation of the spheroidin ORF on the physical map is shown in Fig. 1. It is interesting to note that the 1.7 kb inverse PCR fragment only hybridized to the AmEPV HindIII-G fragment. The amino acid sequence derived from the 8 and 9 kDa overlapping CnBr-generated polypeptides is found from nucleotide positions 4883 to 4957 [SEQ ID NO: 38]. That derived from the 6.2 kDa polypeptide is found from nucleotides 3962 to 4012 [SEQ ID NO: 39], and that derived from the 15 kDa polypeptide is found from nucleotides 4628 to 4651 [SEQ ID NO: 40].

Therefore, all sequences obtained from protein microsequencing were ultimately found to lie within the spheroidin ORF.

EXAMPLE 6 - SPHEROIDIN GENE TRANSCRIPTION

The start site for spheroidin gene

transcription was determined. A primer complementary to the spheroidin gene sequence beginning 65 bp downstream of the predicted initiating methionine was prepared and used for a series of primer extensions.

A. Preparation of RNA and primer extension reactions.

Six 150 mm dishes of subconfluent cells were prepared. The culture media were aspirated, and 2 ml of viral inoculum was added to each dish. The virus

concentration was about 0.1 to 1 PFU per cell. The dishes were occasionally agitated during a 3 hour

adsorption period. At the end of this period, the cells were rinsed with 5 ml of modified PBS. The media were replaced, and the infected cells were incubated for 72 hours at 27°C. Total RNA from the infected cells was isolated by the guanidinium thiocyanate-cesium chloride procedure [J. M. Chirgwin et al. Biochemistry. 18:5294-5299 (1979)].

Primer extension reactions were carried out with primer RM165 [SEQ ID NO: 17], a 35-base

oligonucleotide (GTTCGAAACAAGTATTTTCATCTTTTAAATAAATC) beginning and ending 100 and 65 bp downstream,

respectively, of the initiating methionine codon found in the TAAATG motif. The primer was end labeled with [γ-³²P]ATP and T4 polynucleotide kinase and purified on a "spun column" [Sambrook et al, supra] . For annealing, 40 μg of total infected-cell RNA and 10⁶ cpm of radiolabeled primer were coprecipitated with ethanol. The pellet was resuspended in 25 μl of hybridization buffer [80%

formamide, 40 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (pH 6.4), 400 mM NaCl, 1 mM EDTA (pH 8.0)], denatured at 72°C for 15 minutes, and incubated at 30°C for 18 hours.

For primer extension, the RNA-primer hybrids were ethanol precipitated, resuspended, and used for five individual reactions. Each reaction contained 8 μg of total infected-cell RNA, 50 mM Tris-HCl, (pH 8.3), 50 mM KC1, 10 mM dithiothreitol, 10 mM MgCl₂, 4 U of avian myeloblastosis virus reverse transcriptase (Life

Sciences), 8 U of RNasin (Promega), 0.25 mM each

deoxynucleoside triphosphate (dNTP), and the appropriate dideoxynucleoside triphosphate (ddNTP), except for a control reaction, which contained no ddNTP. The

dNTP/ddNTP ratios were 4:1, 5:1, 5:1, and 2:1, for the C, T, A, and G reactions, respectively. The reactions were carried out at 42°C for 30 minutes.

One microliter of chase buffer (4 μl of 5 mM dNTP mixture and 1 μl of 20-U/μl reverse transcriptase) was added to each reaction mixture, which was then incubated for an additional 30 minutes at 42°C. Reaction products were separated on a sequencing gel (8%

acrylamide containing 7 M urea) and visualized by

autoradiography. Complementarity was observed until the AAA of the upstream TAAATG motif, indicating that

transcription of the gene initiates within the TAAATG element of the proposed late promoter element.

Immediately upstream is a 5' tract of noncoded poly(A) on the transcripts. The average length of the poly(A) is greater than 6bp. EXAMPLE 7: ANALYSIS OF SPHEROIDIN SEQUENCE

The spheroidin ORF (G5R) was initially identified by sequencing back through the RM58

oligonucleotide primer binding region as described above. Examination of the AmEPV spheroidin gene sequence (ORF G5R) revealed a potential ORF of 3.0 kb capable of encoding 1,003 amino acids or a protein of about 115 kDa. The ORF consists of 29% G+C, in contrast to the 18.5% reported for the entire AmEPV genome [Langridge, W.H.R., R.F. Bozarth, D.W. Roberts [1977] Virology 76:616-620].

Inspection of the 92 bases upstream of the initiating ATG revealed only 7 G or C residues. Also detected was the presence of known vertebrate poxvirus regulatory

sequences within the 92 bp 5' of the spheroidin ORF.

Included are three TTTT TNT early gene termination signals and TAAATG, which presumably represents a late transcription start signal used to initiate transcription and translation of the spheroidin gene. Several adjacent translation termination codons are also present within the 92 bp upstream of the spheroidin ORF.

Analysis of the sequence upstream of the spheroidin gene revealed four additional potential ORFs, GIL [SEQ ID NO:25], G2R [SEQ ID NO:23], G3L [SEQ ID

NO:26], and G4R [SEQ ID NO:24], discussed above. The putative amino acid sequences of these ORFs are reported in Fig. 2 [SEQ ID NO: 2, 3, 4 and 5, respectively]. No significant homologies were found for the small potential polypeptides encoded by ORF G2R [SEQ ID NO: 23] or G3L

[SEQ ID NO:26]. ORF GIL [SEQ ID NO:25], however,

exhibited a significant degree of homology to ORF 17 found within the HindIII-I fragment of vaccinia virus, whose function is unknown. ORF G4R [SEQ ID NO:24] showed homology to ORF HM3 of capripoxvirus. In vaccinia virus, the ORF HM3 homolog was found very near the site of an incomplete ATI gene. The partial G6L ORF [SEQ ID NO: 27] to the right of the spheroidin gene exhibited good

homology to vaccinia virus NTPase I. Much better

homology (78.4% identity over 162 amino acids) was found between the partial G6L ORF [SEQ ID NO: 27] and NPH I of CbEPV [Yuen, L. et al, Virol., 182:403-406 (1991)], another insect poxvirus.

Example 8 - Isolation and Sequencing of the AmEPV EcoRI-O Fragment Containing the tk Gene

Sequencing of the EcoRI-Q fragment of genomic

AmEPV of Example 1 was performed using techniques

described above for spheroidin. The sequencing showed 1511 bp containing two complete and one partial ORF.

Analysis of the DNA sequence of ORF Q2 [SEQ ID NO:28] indicates the sites where the identifying degenerate oligonucleotides (RMO3 SEQ ID NO: 18 and RM04 SEQ ID

NO: 19) might hybridize. Two oligonucleotides, RM03 and RM04, based on different but strongly conserved regions of the tk genes of several poxviruses and vertebrates [C. Upton et al, J. Virol.. .60:920-927 (1986); D. B. Boyle et al. Virology, 156:355-365 (1987)] were prepared by the methods referred to above. RM03 was the 32-fold

degenerate oligonucleotide [SEQ ID NO: 18]

GA(T/C)GA(G/A)GG(G/A)GG(G/A)CA(G/A) TT(C/T)TT

corresponding to the amino acid residues in the vaccinia tk protein from the aspartic acid at position 82 to the phenylalanine at position 87. RM04 [SEQ ID NO: 19] was (GGNCCCATGTT(C/T)TCNGG with 32-fold degeneracy and

corresponded to the region from the glycine at position 11 to the glycine at position 16 in vaccinia. These probes were radiolabeled as described above for the RH58 probe.

The AmEPV thymidine kinase (tk) gene was

identified by hybridization with the degenerate

oligonucleotide probes RMO3 and RMO4 to a Southern blot of the EcoRI-digested EPV DNA. The EcoRI band of

interest (EcoRI-Q) was isolated, purified, and ligated into a pUC18 vector (GIBCO), previously digested with

EcoRI and treated with calf intestinal alkaline phosphatase. Recombinant clones were identified by the size of the insert and by hybridization to the

radioactive labeled oligonucleotide probes.

One such clone was called pMEGtk-1. The recombinant clones containing the EcoRI-Q fragment oriented in both directions relative to the pUC18 vector sequences were used for sequencing. Sequential nested deletions were generated by the method of Henikoff, cited above, as described for pRH512. These clones were used for sequencing the entire EcoRI-Q fragment.

Subsequently, these oligonucleotides and another, RM129 is a non-degenerate oligonucleotide

GGTGCAAAATCTGATATTTC [SEQ ID NO: 20] prepared from the ORF Ql, were employed as sequencing primers to confirm their positioning as indicated in ORF Q2 [SEQ ID NO:28]. ORF Q2 potentially encodes for a protein of 182 amino acids (21.2 kDa) [SEQ ID NO:10]. ORF Q3 potentially encodes a polypeptide of at least 68 amino acids but is incomplete and is transcribed in the opposite direction from ORF Q2. ORF Ql [SEQ ID NO: 31] potentially encodes a small peptide of 66 amino acids (7.75 kDa) [SEQ ID NO:9].

Further analysis of the EcoRI-Q fragment reveals several other points. First, the A+T content is very high (80%). For ORF Q2, the 100 nucleotides

upstream of the start codon for translation are 90% A+T. Some potential poxvirus transcription signals were found between ORFs Q1 and Q2. The five bases immediately preceding the start codon for ORF Q1 are TAAATG which comprise a consensus late poxvirus promoter. A potential poxvirus early transcription termination signal sequence (TTTTTAT) is located 2 nt past the translation stop codon of Q2.

The deduced amino acid sequence for the tk encoded by the ORF Q2 of the EcoRI-Q fragment can be compared to the tk genes for the poxviruses swine pox [W. M. Schnitzlein et al, Virol.. 181:727-732 (1991); J. A. Feller et al, Virol . , 183 : 578-585 (1991) ] ; fowlpox [Boyle et al., supra; M. M. Binns et al, J. Gen. Virol..

69:1275-1283 (1988)]; vaccinia [J. P. Weir et al, J.

Virol.. 46:530-537 (1983); D. E. Hruby et al, Proc. Natl. Acad. Sci. USA. 80:3411-3415 (1983)]; variola and

monkeypox [J. J. Esposito et al, Virol.. 135:561-567 (1984)]; capripoxvirus [P. D. Gershon et al, J. Gen.

Virol.. 70:525-533 (1989)]; Shope fibroma virus [Upton et al., supra]; the cellular thymidine kinases of humans [H.

D. Bradshaw et al, Mol. Cell. Biol., 4:2316-2320 (1984);

E. Flemington et al. Gene, 52:267-277 (1987)]; the tk of mouse [P. F. Lin et al, Mol Cell. Biol.. 5: 3149-3156 (1985)]; the tk of chicken [T. J. Kwoh et al, Nucl. Acids Res., 12:3959-3971 (1984)]; ASF [R. Blasco et al, Virol., 178:301-304 (1990); A. M. Martin Hernandez et al, J.

Virol.. 65:1046-1052 (1991)].

EXAMPLE 9 - EXPRESSION OF THE AmEPV tk GENE IN A VACCINIA VIRUS

The AmEPV tk gene was tested functionally by cloning the gene into a vaccinia virus strain tk mutant, as follows.

The EcoRI-Q fragment of AmEPV, described above, was inserted in both possible orientations into shuttle plasmid pHGN3.1 [D. D. Bloom et al, J. Virol., 65:1530- 1542 (1991)] which had been isolated from bacterial cells by the alkaline lysis method. This EcoRI-Q DNA fragment contains the AmEPV tk open reading frame (ORF). The cloning was performed conventionally. The resulting plasmid was designated pHGN3.1/EcoRI-Q.

The plasmid was transfected by Lipofectin

[GIBCO] as described specifically below into mammalian cells infected with vaccinia virus. The cells were either rat tk, human 143 tk, or CV-1 cell lines onto which the vaccinia virus VSC8 was propagated. These cells were maintained in Eagle's Minimal Essential Medium with Earle's salts [Massung et al, Virol.. 180:347-354 (1991) incorporated by reference herein].

The VSC8 vaccinia strain [Dr. Bernard Moss] contains the β-galactosidase gene driven by the vaccinia P₁₁ promoter (P₁₁-Lac Z cassette) inserted into the viral tk gene. While VSC8 contains an inactive tk gene due to the insertion of the β-galactosidase, portions of the vaccinia tk sequence remain. VSC8 is thus tk- and, upon staining with X-Gal (5-bromo-4-chloro-3-indoyl-ß-D-galactopyranoside), will form blue plaques (ß-galactosidase positive).

Cells were grown to 80% confluence (4 × 10⁶ per 60 mm dish). Lipofectin solution (20 μg of Lipofectin in 50μl of dH₂O) was added to 10μg plasmid DNA (pHGN3.1/AmEPV EcoRI-Q) in 50μl of dH₂O and incubated for 15 minutes at room temperature. After a 2 hour period of viral

adsorption (m.o.i. of 2, 37 °C), the monolayers were washed three times with serum-free OptiMEM. Three milliliters of serum-free OptiMEM was then added to each 60 mm dish. The Lipofectin/DNA mixture was slowly added dropwise with gentle swirling and incubated an additional 12 to 18 hours at 37°C. Fetal bovine serum was then added (10% final) and the infected cells were harvested at 48 hours postinfection.

Recombinant viruses, containing the EcoRI -Q fragment inserted into the hemagglutinin (HA) gene of vaccinia, were identified by hybridization of AmEPV

EcoRI-Q fragments, radioactively labeled by procedures described above, to replicas of nitrocellulose "lifts" of virus plaques from the infected monolayer. Potential recombinants were isolated from replica filters and plaque-purified several times before testing. The tk of AmEPV exhibits some degree of homology with the tk of vaccinia. To confirm that insertion of the AmEPV tk gene was within the HA gene of vaccinia rather than within residual tk sequences remaining in VSC8, the recombinants were examined by a series of Southern hybridizations to HindIII digests of the various viruses. When DNA from wild-type virus was hybridized to a vaccinia virus tk probe, hybridization was observed exclusively within the ≈5 kb HindIII-J fragment of AmEPV.

When either VSC8 or either of the AmEPV tk containing recombinants was examined using the vaccinia tk probe, hybridization occurred instead to an «8 kb fragment consistent with polymerase in the presence of radiolabeled substrates. Extension will terminate at the end of the Pstl-F fragment.

The radiolabeled product was then hybridized to an EcoRI digest of AmEPV DNA. If orientation of the gene is such that the tk ORF reads toward the end of the genome, hybridization would be expected to the EcoRI-E fragment; whereas if the gene is read toward the center of the genome, hybridization would be expected to the

EcoRI-I fragment.

The results indicate hybridization not only to the EcoRI-E fragment, but also to the EcoRI-A fragment. These results infer that the orientation of the tk gene is with reading toward the left end of the genome.

Hybridization of the run-off extension product also to the EcoRI-A fragment is consistent with the presence of an inverted terminal repetition, common in poxviruses, with identical sequences residing in both the EcoRI-A and the EcoRI-E fragments.

The optimal growth temperature for AmEPV in the laboratory is 28°C, whereas that of the vertebrate poxviruses is 37°C. As described herein, when the AmEPV DNA fragment containing the entire tk gene was cloned into the tk strain of vaccinia virus, the recombinant virus was capable of growing at 37°C in the presence of methotrexate [Sigma], indicative of a tk⁺ phenotype.

This example demonstrates that the Entomopoxvirus tk gene can be successfully transferred into mammalian expression systems, and that AmEPV tk is functionally active over a considerable temperature range.

It should be understood that the examples and embodiments described herein are for illustrative

purposes only. Various modifications or changes in light thereof will be suggested to persons skilled in the art by this specification. The subject invention encompasses recombinant polynucleotide sequences, plasmids, vectors, and transformed hosts which are equivalent to those which are specifically exemplified herein in that the

characteristic expression features are retained in said equivalent constructs even if inconsequential

modifications to the DNA sequence have been made. For example, it is within the skill of a person trained in the art to use a fragment of the spheroidin gene's noncoding region which is upstream of the structural gene in order to achieve the desired level of expression. Such fragments of the regulatory sequences fall within the scope of the current invention, so long as the desired level of expression which is characteristic of this system is retained. Furthermore, inconsequential changes to the nucleotide sequences can be made without affecting the disclosed functions of these sequences. Such

modifications also fall within the scope of the current invention and are to be included within the spirit and purview of this application and the scope of the appended claims. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: University of Florida

(ii) TITLE OF INVENTION: Novel Entomopoxvirus Expression System (iii) NUMBER OF SEQUENCES: 40

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: David R. Saliwanchik

(B) STREET: 2421 N.W. 41st Street, Suite A-l

(C) CITY: Gainesville

(D) STATE: Florida

(E) COUNTRY: U.S.A.

(F) ZIP: 32606

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/657,584; US 07/

(B) FILING DATE: 19-FEB-1991; 30-JAN-1992

(Viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Saliwanchik, David R.

(B) REGISTRATION NUMBER: 31,794

(C) REFERENCE/DOCKET NUMBER: UF/S&S-114.C2

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (904) 375-8100

(B) TELEFAX: (904) 372-5800

(2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6768 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Amsacta moorei entomopoxvirus

(ix) FEATURE:

(A) NAME/KEY: CDS*

(B) LOCATION: complement (65..1459}

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1474..2151 ( ix ) FEATURE :

(A) NAME/KEY: CDS

(B) LOCATION: complement (2239..2475)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 2502..2987

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 3080..6091

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: complement (6277..6768)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

AGATCTGATG TTCTATATAT AGTACAAATT TGTATGATTA ATTGATATTT TAAAATTCAA 60

GATATTAAAT ATTAGATTCT AAACTATTCT TCTCATTATC AATATAACTA TCATAATCAT 120

TTTTTATTTT ACTACATACA TTCATAATTC TATTACTATT TTTTTTATAC ATATCTATTA 180

ATTCCATAAA CTTTTTATTT TTTATATTAA ATATTTCTAA TGTATTTTTA AATTCGTCAA 240

TACTATTAAT ATCATATCTA GAAATAAATA ATGCACCTCT ATAACTACTA GCCAATAAAT 300

CACCAATAAA ACTCATAGAA TAATATAATT TTTTAAATTC AAATTTAGAT TTTATGTTGA 360

AATAAACTAT ATAATATAAA AATATTATAT TAAACATACC ACAATCGGGA CTATCATATT 420

GTAATTCAAA AGTATTAAAA AAGTAATAAT TTACATTTTT AAATATATCA TTTAAATATT 480

CTGATAGTAC ATCAATGTAT AAATAAGCAT AATTAGTATT AGGAGTACTA TTGTAGTGTT 540

TATGGCTTTT TATAGTCATA TCAGATTCAA TAAACATATA TTTTTTATTT TGTTTTATAA 600

GTTCTGGTAT ATAACCACTA CTATTAAAAA AGTATGCAGC TTTTTTATCT TTATCAAAGT 660

GTTTATCTAT TACGCAACAA GTAAAATGAT CATTATAAAT TATAGGAAAC ATAAAAAATC 720

TTTTTTTATC ATTCATTAAA AAAAATTTTA CTCTATCTTC AAGTTTATAG CATCTCATAG 780

ATGAAGCTAC TGTAGCAATA TTTTTATCAG TTTTTTCAAA TAAAATCAAA TGAAAATAAT 840

CATAATCTGT ATTAATCATA GTTAATGGAT ATATACAATT ATATATATCT CCCGAACTTA 900

ACCATGTAGA TTTATCATGT TTTCTTGGGT AAGCTTTAGG TTTAGGATTA AATCCCAAAG 960

GCGGTATTCC TATTTGAGCA TCCAAATCAT CATAAATTGT GGCAAATGTA GAAAAATCTC 1020

TTGTTTTGGA TAATTCTGAT TTTAGAAAAG ACTTTCTCAT ATATACTAAT GGAATGCCTT 1080

TATATTTTTT AGATGTAATA AAAGTATTAA TATTTATATT TTTATCTTGT AAATATTTTT 1140

TTATAGTCCA AAATAGAAAA AATTTTCTTT TAATATTATT TTCAAAATTA ATATTATTAA 1200

TATGATTTGG ATCTAAAACT AATTCATTAT ATAATATTTC CAAGTATTTT ATAGGTATAA 1260

ATGTTACTTT ACCTCTTGTT TCATCATCAT CATCTATTTT TTCTAATATA GCTATATTTG 1320

CATTAGTATT ATATTTAATA GGATTTATAA AATATACCAT ATTATCTATT TTACTAAAAA 1380 ATAACATAGA CATAAAATTA ATACCAGATT CTGGCATTTT TAAATTTTTA TTTGGAAATC 1440

TTCTAATTTT ATTATTCATT ATTTATTTAA TAA ATG TTT CTA GTT TAT TTC AAT 1494

Met Phe Leu Val Tyr Phe Asn

1 5

ACA TTT TTA ATA ATA ATT TTA TTA TTT GGT ATT ATA GGT ATT TAT ATA 1542 Thr Phe Leu Ile Ile Ile Leu Leu Phe Gly Ile Ile Gly Ile Tyr Ile

10 15 20

TTA ACA TTT GTG TTT AAT ATA GAT TTT TTA ATA AAT AAT AAT AAA ATA 1590 Leu Thr Phe Val Phe Asn Ile Asp Phe Leu Ile Asn Asn Asn Lys Ile

25 30 35

TAT ATA TTA TCA TAT AAC GCA ACT AAT ATA AAC AAT ATA AAT AAT TTA 1638 Tyr Ile Leu Ser Tyr Asn Ala Thr Asn Ile Asn Asn Ile Asn Asn Leu

40 45 50 55

AAT TTA TAC GAT TAT TCA GAT ATT ATA TTT TTG ACA AAT TTT AAC ATA 1686 Asn Leu Tyr Asp Tyr Ser Asp Ile Ile Phe Leu Thr Asn Phe Asn Ile

60 65 70

AAT AAT AAT CTT TTA GTA ACA CAA GCT AAT AAT TTA CAA GAT ATA CCA 1734 Asn Asn Asn Leu Leu Val Thr Gin Ala Asn Asn Leu Gin Asp Ile Pro

75 80 85

ATA TTT AAT GTA AAT AAT ATT ATA TCT AAT CAA TAT AAT TTT TAT TCA 1782 Ile Phe Asn Val Asn Asn Ile Ile Ser Asn Gin Tyr Asn Phe Tyr Ser

90 95 100

GCG TCT AGT AAT AAT GTA AAT ATA TTA TTA GGA TTA AGA AAA ACA TTA 1830 Ala Ser Ser Asn Asn Val Asn Ile Leu Leu Gly Leu Arg Lys Thr Leu

105 110 115

AAT ATA AAT AGA AAT CCA TTT TTA TTA TTT AGA AAT ACA TCT CTA GCT 1878 Asn Ile Asn Arg Asn Pro Phe Leu Leu Phe Arg Asn Thr Ser Leu Ala

120 125 130 135

ATA GTT TTC AAT AAT AAT GAA ACT TTT CAC TGT TAT ATA AGT TCA AAT 1926 Ile Val Phe Asn Asn Asn Glu Thr Phe His Cys Tyr Ile Ser Ser Asn

140 145 150

CAA AAT AGT GAT GTA TTA GAT ATA GTA TCA CAT ATA GAA TTT ATG AAA 1974 Gin Asn Ser Asp Val Leu Asp Ile Val Ser His Ile Glu Phe Met Lys

155 160 165

TCT AGA TAT AAT AAA TAT GTA ATT ATA GGA GAA ATA CCC GTA AAT AAT 2022 Ser Arg Tyr Asn Lys Tyr Val Ile Ile Gly Glu Ile Pro Val Asn Asn

170 175 180

AAT ATA TCT ATT AAT AAT ATA TTA AAT AAT TTT GCT ATT ATA ACT AAT 2070 Asn Ile Ser Ile Asn Asn Ile Leu Asn Asn Phe Ala Ile Ile Thr Asn

185 190 195

GTG AGA TTA ATA GAT AAA TAT AAC TCT ATA ATA TCA TTT TTA AAT ATC 2118 Val Arg Leu Ile Asp Lys Tyr Asn Ser Ile Ile Ser Phe Leu Asn Ile

200 205 210 215

AAC GTA GGA ACA CTT TTT GTC ATA AAT CCA TAATATTTAG TAATAATCAC 2168 Asn Val Gly Thr Leu Phe Val Ile Asn Pro

220 225

TAACATATTT TTTATTAAAA TGAATAAAAT ATATATTGTT ATTGTCAATA TTTTATATCA 2228 TTTTACAGTC TTATTTTTTT TTTTTGCTTT TAGGTATAAT TTTACCTTCT AAACGTTTAT 2288

CTCCCCAAAC ATCTACAGTA GATGGTTTAT TAGATTCTGT GTTATACACA TCTGCTGGAT 2348

TTGCGGCATT TGTATCCAAA CCATAATATC CAGGTCTATA ATTATCTTTA AAAACTTGGG 2408

ATTGAGATAC TTCTTCAGTT TTTAAATTAT TAAAATATCC AAGATTATTT TTTTTTGATG 2468

AAGACATAAT TGATATTATA ATACTTTATA GAT ATG TCA ATA TTT ATC TAC TAT 2522

Met Ser Ile Phe Ile Tyr Tyr

1 5

ATT TTC AAC AAT AGA TTT TAT ATA TAT AAA AGA ATG AAT ACT GTA CAA 2570 Ile Phe Asn Asn Arg Phe Tyr Ile Tyr Lys Arg Met Asn Thr Val Gin

10 15 20

ATT TTA GTT GTC ATA TTA ATA ACA ACA GCA TTA TCT TTT CTA GTT TTT 2618 Ile Leu Val Val Ile Leu Ile Thr Thr Ala Leu Ser Phe Leu Val Phe

25 30 35

CAA TTA TGG TAT TAT GCC GAA AAT TAC GAA TAT ATA TTA AGA TAT AAT 2666 Gin Leu Trp Tyr Tyr Ala Glu Asn Tyr Glu Tyr Ile Leu Arg Tyr Asn

40 45 50 55

GAT ACA TAT TCA AAT TTA CAA TTT GCG AGA AGC GCA AAT ATA AAT TTT 2714 Asp Thr Tyr Ser Asn Leu Gin Phe Ala Arg Ser Ala Asn Ile Asn Phe

60 65 70

GAT GAT TTA ACT GTT TTT GAT CCC AAC GAT AAT GTT TTT AAT GTT GAA 2762 Asp Asp Leu Thr Val Phe Asp Pro Asn Asp Asn Val Phe Asn Val Glu

75 80 85

GAA AAA TGG CGC TGT GCT TCA ACT AAT AAT AAT ATA TTT TAT GCA GTT 2810 Glu Lys Trp Arg Cys Ala Ser Thr Asn Asn Asn Ile Phe Tyr Ala Val

90 95 100

TCA ACT TTT GGA TTT TTA AGT ACA GAA AGT ACT GGT ATT AAT TTA ACA 2858 Ser Thr Phe Gly Phe Leu Ser Thr Glu Ser Thr Gly Ile Asn Leu Thr

105 110 115

TAT ACA AAT TCT AGA GAT TGT ATT ATA GAT TTA TTT TCT AGA ATT ATA 2906 Tyr Thr Asn Ser Arg Asp Cys Ile Ile Asp Leu Phe Ser Arg Ile Ile

120 125 130 135

AAA ATA GTA TAT GAT CCT TGT ACT GTC GAA ACA TCT AAC GAT TGT AGA 2954 Lys Ile Val Tyr Asp Pro Cys Thr Val Glu Thr Ser Asn Asp Cys Arg

140 145 150

TTA TTA AGA TTA TTG ATG GCC AAT ACA TCA TAAATACATT ATAATATTAT 3004 Leu Leu Arg Leu Leu Met Ala Asn Thr Ser

155 160

TATAATATCA ATCATAATTT TTATATATAT TTTATCTAAA AGGACTTTTT ATTTTTTATA 3064

TATTAATAAT AATAA ATG AGT AAC GTA CCT TTA GCA ACC AAA ACA ATA AGA 3115

Met Ser Asn Val Pro Leu Ala Thr Lys Thr Ile Arg

1 5 10

AAA TTA TCA AAT CGA AAA TAT GAA ATA AAG ATT TAT TTA AAA GAT GAA 3163 Lys Leu Ser Asn Arg Lys Tyr Glu Ile Lys Ile Tyr Leu Lys Asp Glu

15 20 25

AAT ACT TGT TTC GAA CGT GTA GTA GAT ATG GTA GTT CCA TTA TAT GAT 3211 Asn Thr Cys Phe Glu Arg Val Val Asp Met Val Val Pro Leu Tyr Asp 30 35 40

GTG TGT AAT GAA ACT TCT GGT GTT ACT TTA GAA TCA TGT AGT CCA AAT 3259 Val Cys Asn Glu Thr Ser Gly Val Thr Leu Glu Ser Cys Ser Pro Asn

45 50 55 60

ATA GAA GTA ATT GAA TTA GAC AAT ACT CAT GTT AGA ATC AAA GTT CAC 3307 Ile Glu Val Ile Glu Leu Asp Asn Thr His Val Arg Ile Lys Val His

65 70 75

GGC GAT ACA TTA AAA GAA ATG TGT TTT GAA TTA TTG TTC CCG TGT AAT 3355 Gly Asp Thr Leu Lys Glu Met Cys Phe Glu Leu Leu Phe Pro Cys Asn

80 85 90

GTA AAC GAA GCC CAA GTA TGG AAA TAT GTA AGT CGA TTA TTG CTA GAT 3403 Val Asn Glu Ala Gin Val Trp Lys Tyr Val Ser Arg Leu Leu Leu Asp

95 100 105

AAT GTA TCA CAT AAT GAC GTA AAA TAT AAA TTA GCT AAT TTT AGA CTG 3451 Asn Val Ser His Asn Asp Val Lys Tyr Lys Leu Ala Asn Phe Arg Leu

110 115 120

ACT CTT AAT GGA AAA CAT TTA AAA TTA AAA GAA ATC GAT CAA CCG CTA 3499 Thr Leu Asn Gly Lys His Leu Lys Leu Lys Glu Ile Asp Gin Pro Leu

125 130 135 140

TTT ATT TAT TTT GTC GAT GAT TTG GGA AAT TAT GGA TTA ATT ACT AAG 3547 Phe Ile Tyr Phe Val Asp Asp Leu Gly Asn Tyr Gly Leu Ile Thr Lys

145 150 155

GAA AAT ATT CAA AAT AAT AAT TTA CAA GTT AAC AAA GAT GCA TCA TTT 3595 Glu Asn Ile Gin Asn Asn Asn Leu Gin Val Asn Lys Asp Ala Ser Phe

160 165 170

ATT ACT ATA TTT CCA CAA TAT GCG TAT ATT TGT TTA GGT AGA AAA GTA 3643 Ile Thr Ile Phe Pro Gin Tyr Ala Tyr Ile Cys Leu Gly Arg Lys Val

175 180 185

TAT TTA AAT GAA AAA GTA ACT TTT GAT GTA ACT ACA GAT GCA ACT AAT 3691 Tyr Leu Asn Glu Lys Val Thr Phe Asp Val Thr Thr Asp Ala Thr Asn

190 195 200

ATT ACT TTA GAT TTT AAT AAA TCT GTT AAT ATC GCA GTA TCA TTC CTT 3739 Ile Thr Leu Asp Phe Asn Lys Ser Val Asn Ile Ala Val Ser Phe Leu

205 210 215 220

GAT ATA TAT TAC GAA GTT AAT AAT AAT GAA CAA AAA GAT TTA TTA AAA 3787 Asp Ile Tyr Tyr Glu Val Asn Asn Asn Glu Gin Lys Asp Leu Leu Lys

225 230 235

GAT TTA CTT AAG AGA TAC GGT GAA TTT GAA GTC TAT AAC GCA GAT ACT 3835 Asp Leu Leu Lys Arg Tyr Gly Glu Phe Glu Val Tyr Asn Ala Asp Thr

240 245 250

GGA TTA ATT TAT GCT AAA AAT CTA AGT ATT AAA AAT TAT GAT ACT GTG 3883 Gly Leu Ile Tyr Ala Lys Asn Leu Ser Ile Lys Asn Tyr Asp Thr Val

255 260 265

ATT CAA GTA GAA AGG TTG CCA GTT AAT TTG AAA GTT AGA GCA TAT ACT 3931 Ile Gin Val Glu Arg Leu Pro Val Asn Leu Lys Val Arg Ala Tyr Thr

270 275 280 AAG GAT GAA AAT GGT CGC AAT CTA TGT TTG ATG AAA ATA ACA TCT AGT 3979 Lys Asp Glu Asn Gly Arg Asn Leu Cys Leu Met Lys Ile Thr Ser Ser

285 290 295 300

ACA GAA GTA GAC CCC GAG TAT GTA ACT AGT AAT AAT GCT TTA TTG GGT 4027 Thr Glu Val Asp Pro Glu Tyr Val Thr Ser Asn Asn Ala Leu Leu Gly

305 310 315

ACG CTC AGA GTA TAT AAA AAG TTT GAT AAA TCT CAT TTA AAA ATT GTA 4075 Thr Leu Arg Val Tyr Lys Lys Phe Asp Lys Ser His Leu Lys Ile Val

320 325 330

ATG CAT AAC AGA GGA AGT GGT AAT GTA TTT CCA TTA AGA TCA TTA TAT 4123 Met Hie Asn Arg Gly Ser Gly Asn Val Phe Pro Leu Arg Ser Leu Tyr

335 340 345

CTG GAA TTG TCT AAT GTA AAA GGA TAT CCA GTT AAA GCA TCT GAT ACT 4171 Leu Glu Leu Ser Asn Val Lys Gly Tyr Pro Val Lys Ala Ser Asp Thr

350 355 360

TCG AGA TTA GAT GTT GGT ATT TAC AAA TTA AAT AAA ATT TAT GTA GAT 4219 Ser Arg Leu Asp Val Gly Ile Tyr Lys Leu Asn Lys Ile Tyr Val Asp

365 370 375 380

AAC GAC GAA AAT AAA ATT ATA TTG GAA GAA ATT GAA GCA GAA TAT AGA 4267 Asn Asp Glu Asn Lys Ile Ile Leu Glu Glu Ile Glu Ala Glu Tyr Arg

385 390 395

TGC GGA AGA CAA GTA TTC CAC GAA CGT GTA AAA CTT AAT AAA CAC CAA 4315 Cys Gly Arg Gin Val Phe His Glu Arg Val Lys Leu Asn Lys His Gin

400 405 410

TGT AAA TAT ACT CCC AAA TGT CCA TTC CAA TTT GTT GTA AAC AGC CCA 4363 Cys Lys Tyr Thr Pro Lys Cys Pro Phe Gin Phe Val Val Asn Ser Pro

415 420 425

GAT ACT ACG ATT CAC TTA TAT GGT ATT TCT AAT GTT TGT TTA AAA CCT 4411 Asp Thr Thr Ile His Leu Tyr Gly Ile Ser Asn Val Cys Leu Lys Pro

430 435 440

AAA GTA CCC AAA AAT TTA AGA CTT TGG GGA TGG ATT TTA GAT TGC GAT 4459 Lys Val Pro Lys Asn Leu Arg Leu Trp Gly Trp Ile Leu Asp Cys Asp

445 450 455 460

ACT TCT AGA TTT ATT AAA CAT ATG GCT GAT GGA TCT GAT GAT TTA GAT 4507 Thr Ser Arg Phe Ile Lys His Met Ala Asp Gly Ser Asp Asp Leu Asp

465 470 475

CTT GAC GTT AGG CTT AAT AGA AAT GAT ATA TGT TTA AAA CAA GCC ATA 4555 Leu Asp Val Arg Leu Asn Arg Asn Asp Ile Cys Leu Lys Gin Ala Ile

480 485 490

AAA CAA CAT TAT ACT AAT GTA ATT ATA TTA GAG TAC GCA AAT ACA TAT 4603 Lys Gin His Tyr Thr Asn Val Ile Ile Leu Glu Tyr Ala Asn Thr Tyr

495 500 505

CCA AAT TGC ACA TTA TCA TTG GGT AAT AAT AGA TTT AAT AAT GTA TTT 4651 Pro Asn Cys Thr Leu Ser Leu Gly Asn Asn Arg Phe Asn Asn Val Phe

510 515 520

GAT ATG AAT GAT AAC AAA ACT ATA TCT GAG TAT ACT AAC TTT ACA AAA 4699 Asp Met Asn Asp Asn Lys Thr Ile Ser Glu Tyr Thr Asn Phe Thr Lys

525 530 535 540 AGT AGA CAA GAC CTT AAT AAC ATG TCA TGT ATA TTA GGA ATA AAC ATA 4747 Ser Arg Gin Asp Leu Asn Asn Met Ser Cys Ile Leu Gly Ile Asn Ile

545 550 555

GGT AAT TCC GTA AAT ATT AGT AGT TTG CCT GGT TGG GTA ACA CCT CAC 4795 Gly Asn Ser Val Asn Ile Ser Ser Leu Pro Gly Trp Val Thr Pro His

560 565 570

GAA GCT AAA ATT CTA AGA TCT GGT TGT GCT AGA GTT AGA GAA TTT TGT 4843 Glu Ala Lys Ile Leu Arg Ser Gly Cys Ala Arg Val Arg Glu Phe Cys

575 580 585

AAA TCA TTC TGT GAT CTT TCT AAT AAG AGA TTC TAT GCT ATG GCT AGA 4891 Lys Ser Phe Cys Asp Leu Ser Asn Lys Arg Phe Tyr Ala Met Ala Arg

590 595 600

GAT CTC GTA AGT TTA CTA TTT ATG TGT AAC TAT GTT AAT ATT GAA ATT 4939 Asp Leu Val Ser Leu Leu Phe Met Cys Asn Tyr Val Asn Ile Glu Ile

605 610 615 620

AAC GAA GCA GTA TGC GAA TAT CCT GGA TAT GTC ATA TTA TTC GCA AGA 4987 Asn Glu Ala Val Cys Glu Tyr Pro Gly Tyr Val Ile Leu Phe Ala Arg

625 630 635

GCT ATT AAA GTA ATT AAT GAT TTA TTA TTA ATT AAC GGA GTA GAT AAT 5035 Ala Ile Lys Val Ile Asn Asp Leu Leu Leu Ile Asn Gly Val Asp Asn

640 645 650

CTA GCA GGA TAT TCA ATT TCC TTA CCT ATA CAT TAT GGA TCT ACT GAA 5083 Leu Ala Gly Tyr Ser Ile Ser Leu Pro Ile His Tyr Gly Ser Thr Glu

655 660 665

AAG ACT CTA CCA AAT GAA AAG TAT GGT GGT GTT GAT AAG AAA TTT AAA 5131 Lys Thr Leu Pro Asn Glu Lys Tyr Gly Gly Val Asp Lys Lys Phe Lys

670 675 680

TAT CTA TTC TTA AAG AAT AAA CTA AAA GAT TTA ATG CGT GAT GCT GAT 5179 Tyr Leu Phe Leu Lys Asn Lys Leu Lys Asp Leu Met Arg Asp Ala Asp

685 690 695 700

TTT GTC CAA CCT CCA TTA TAT ATT TCT ACT TAC TTT AGA ACT TTA TTG 5227 Phe Val Gin Pro Pro Leu Tyr Ile Ser Thr Tyr Phe Arg Thr Leu Leu

705 710 715

GAT GCT CCA CCA ACT GAT AAT TAT GAA AAA TAT TTG GTT GAT TCG TCC 5275 Asp Ala Pro Pro Thr Asp Asn Tyr Glu Lys Tyr Leu Val Asp Ser Ser

720 725 730

GTA CAA TCA CAA GAT GTT CTA CAG GGT CTG TTG AAT ACA TGT AAT ACT 5323 Val Gin Ser Gin Asp Val Leu Gin Gly Leu Leu Asn Thr Cys Asn Thr

735 740 745

ATT GAT ACT AAT GCT AGA GTT GCA TCA AGT GTT ATT GGA TAT GTT TAT 5371 Ile Asp Thr Asn Ala Arg Val Ala Ser Ser Val Ile Gly Tyr Val Tyr

750 755 760

GAA CCA TGC GGA ACA TCA GAA CAT AAA ATT GGT TCA GAA GCA TTG TGT 5419 Glu Pro Cys Gly Thr Ser Glu His Lys Ile Gly Ser Glu Ala Leu Cys

765 770 775 780

AAA ATG GCT AAA GAA GCA TCT AGA TTA GGA AAT CTA GGT TTA GTA AAT 5467 Lys Met Ala Lys Glu Ala Ser Arg Leu Gly Asn Leu Gly Leu Val Asn

785 790 795 CGT ATT AAT GAA AGT AAT TAC AAC AAA TGT AAT AAA TAT GGT TAT AGA 5515 Arg Ile Asn Glu Ser Asn Tyr Asn Lys Cys Asn Lys Tyr Gly Tyr Arg

800 805 810

GGA GTA TAC GAA AAT AAC AAA CTA AAA ACA AAA TAT TAT AGA GAA ATA 5563 Gly Val Tyr Glu Asn Asn Lys Leu Lys Thr Lys Tyr Tyr Arg Glu Ile

815 820 825

TTT GAT TGT AAT CCT AAT AAT AAT AAT GAA TTA ATA TCC AGA TAT GGA 5611 Phe Asp Cys Asn Pro Asn Asn Asn Asn Glu Leu Ile Ser Arg Tyr Gly

830 835 840

TAT AGA ATA ATG GAT TTA CAT AAA ATT GGA GAA ATT TTT GCA AAT TAC 5659 Tyr Arg Ile Met Asp Leu His Lys Ile Gly Glu Ile Phe Ala Asn Tyr

845 850 855 860

GAT GAA AGT GAA TCT CCT TGC GAA CGA AGA TGT CAT TAC TTG GAA GAT 5707 Asp Glu Ser Glu Ser Pro Cys Glu Arg Arg Cys His Tyr Leu Glu Asp

865 870 875

AGA GGT CTT TTA TAT GGT CCT GAA TAT GTA CAT CAC AGA TAT CAA GAA 5755 Arg Gly Leu Leu Tyr Gly Pro Glu Tyr Val His His Arg Tyr Gin Glu

880 885 890

TCA TGT ACG CCT AAT ACG TTT GGA AAT AAC ACA AAT TGT GTA ACA AGA 5803 Ser Cys Thr Pro Asn Thr Phe Gly Asn Asn Thr Asn Cys Val Thr Arg

895 900 905

AAT GGT GAA CAA CAC GTA TAC GAA AAT AGT TGT GGA GAT AAT GCA ACA 5851 Asn Gly Glu Gin His Val Tyr Glu Asn Ser Cys Gly Asp Asn Ala Thr

910 915 920

TGT GGA AGA AGA ACA GGA TAT GGA AGA AGA AGT AGG GAT GAA TGG AAT 5899 Cys Gly Arg Arg Thr Gly Tyr Gly Arg Arg Ser Arg Asp Glu Trp Asn

925 930 935 940

GAC TAT AGA AAA CCC CAC GTT TAT GAC AAT TGT GCC GAT GCA AAT AGT 5947 Asp Tyr Arg Lys Pro His Val Tyr Asp Asn Cys Ala Asp Ala Asn Ser

945 950 955

TCA TCT TCA GAT AGC TGT TCA GAC AGT AGT AGT AGT AGT GAA TCT GAA 5995 Ser Ser Ser Asp Ser Cys Ser Asp Ser Ser Ser Ser Ser Glu Ser Glu

960 965 970

TCT GAT TCA GAT GGA TGT TGC GAC ACA GAT GCT AGT TTA GAT TCT GAT 6043 Ser Asp Ser Asp Gly Cys Cys Asp Thr Asp Ala Ser Leu Asp Ser Asp

975 980 985

ATT GAA AAT TGT TAT CAA AAT CCA TCA AAA TGT GAT GCA GGA TGC TAAATGAAAT 6098 Ile Glu Asn Cys Tyr Gin Asn Pro Ser Lys Cys Asp Ala Gly Cys

990 995 1000

TTAATATTAT ATAATATTAA CTTACAAGTT ATAAAAATCA TTAAAATGAT TTTTTAAAAT 6158

GATATTATCG ATAGTTGTGA TAATGTGCTC TTTTATTTTA TTAATTGCGA TGATTATAAT 6218

ATTATCTTTT AGATATATTT AATATTAATT ATAAATCGAC TGACAATAAT ATTTATTCCT 6278

ATTCATAATA ATCATCTGCT ATATATATTA ATGTATCATT CTCTATTATA AATATAGGTA 6338

TATTGTCTTT ATCAATCATT AATTTTGCTA CAGCTGTATT ATCTTTATAT ACTATATTTG 6398 TGTCTTTGTT TAATAAACCT TTTAATATAG TGGCTCTATC ATAATCTTTA CAATATGATA 6458

TGGGATATAA TTTTATATTA ATAATAACAT TAGATACGTT CATTTCTTTC ATTCTAGTTT 6518

TACGTATTGT GTCAAAAATT ATTTCATTTT CTGCTGGTTC TATATATTTA TATGTGTTAT 6578

GAATAGATTC GATAGATGAT GATTTTAATA AATCAAATAT AACATTTATT TTACCTTGTT 6638

TATCTTTTAT AATATCTAAT ATTTCTTTAT CTACAGATTT TCTGTTGTTG GTATATGATA 6698

TTAAAAAATG AACGTTAACA TATCTATATT CTTGTGGTAA ATCTTTATGA GAATTTAATC 6758

TTATAGATCT 6768

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 464 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Asn Asn Lys Ile Arg Arg Phe Pro Asn Lys Asn Leu Lys Met Pro

1 5 10 15

Glu Ser Gly Ile Asn Phe Met Ser Met Leu Phe Phe Ser Lys Ile Asp

20 25 30

Asn Met Val Tyr Phe Ile Asn Pro Ile Lys Tyr Asn Thr Asn Ala Asn

35 40 45

Ile Ala Ile Leu Glu Lys Ile Asp Asp Asp Asp Glu Thr Arg Gly Lys

50 55 60

Val Thr Phe Ile Pro Ile Lys Tyr Leu Glu Ile Leu Tyr Asn Glu Leu

65 70 75 80

Val Leu Asp Pro Asn His Ile Asn Asn Ile Asn Phe Glu Asn Asn Ile

85 90 95

Lys Arg Lys Phe Phe Leu Phe Trp Thr Ile Lys Lys Tyr Leu Gin Asp

100 105 110

Lys Asn Ile Asn Ile Asn Thr Phe Ile Thr Ser Lys Lys Tyr Lys Gly

115 120 125

Ile Pro Leu Val Tyr Met Arg Lys Ser Phe Leu Lys Ser Glu Leu Ser

130 135 140

Lys Thr Arg Asp Phe Ser Thr Phe Ala Thr Ile Tyr Asp Asp Leu Asp

145 150 155 160

Ala Gin Ile Gly Ile Pro Pro Leu Gly Phe Asn Pro Lys Pro Lys Ala

165 170 175

Tyr Pro Arg Lys His Asp Lys Ser Thr Trp Leu Ser Ser Gly Asp Ile

180 185 190 Tyr Asn Cys Ile Tyr Pro Leu Thr Met Ile Asn Thr Asp Tyr Asp Tyr 195 200 205

Phe His Leu Ile Leu Phe Glu Lys Thr Asp Lys Asn Ile Ala Thr Val 210 215 220

Ala Ser Ser Met Arg Cys Tyr Lys Leu Glu Asp Arg Val Lys Phe Phe 225 230 235 240

Leu Met Asn Asp Lys Lys Arg Phe Phe Met Phe Pro Ile Ile Tyr Asn

245 250 255

Asp His Phe Thr Cys Cys Val Ile Asp Lys His Phe Asp Lys Asp Lys

260 265 270

Lys Ala Ala Tyr Phe Phe Asn Ser Ser Gly Tyr Ile Pro Glu Leu Ile

275 280 285

Lys Gin Asn Lys Lys Tyr Met Phe Ile Glu Ser Asp Met Thr Ile Lys 290 295 300

Ser His Lys His Tyr Asn Ser Thr Pro Asn Thr Asn Tyr Ala Tyr Leu 305 310 315 320

Tyr Ile Asp Val Leu Ser Glu Tyr Leu Asn Asp Ile Phe Lys Asn Val

325 330 335

Asn Tyr Tyr Phe Phe Asn Thr Phe Glu Leu Gin Tyr Asp Ser Pro Asp

340 345 350

Cys Gly Met Phe Asn Ile Ile Phe Leu Tyr Tyr Ile Val Tyr Phe Asn

355 360 365

Ile Lye Ser Lys Phe Glu Phe Lys Lys Leu Tyr Tyr Ser Met Ser Phe 370 375 380

Ile Gly Asp Leu Leu Ala Ser Ser Tyr Arg Gly Ala Leu Phe Ile Ser 385 390 395 400

Arg Tyr Asp Ile Asn Ser Ile Asp Glu Phe Lys Asn Thr Leu Glu Ile

405 410 415

Phe Asn Ile Lys Asn Lys Lys Phe Met Glu Leu Ile Asp Met Tyr Lys

420 425 430

Lys Asn Ser Asn Arg Ile Met Asn Val Cys Ser Lys Ile Lys Asn Asp

435 440 445

Tyr Asp Ser Tyr Ile Asp Asn Glu Lys Asn Ser Leu Glu Ser Asn Ile 450 455 460

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 226 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Met Phe Leu Val Tyr Phe Asn Thr Phe Leu Ile Ile Ile Leu Leu Phe 1 5 10 15

Gly Ile Ile Gly Ile Tyr Ile Leu Thr Phe Val Phe Asn Ile Asp Phe

20 25 30

Leu Ile Asn Asn Asn Lys Ile Tyr Ile Leu Ser Tyr Asn Ala Thr Asn

35 40 45 Ile Asn Asn Ile Asn Asn Leu Asn Leu Tyr Asp Tyr Ser Asp Ile Ile 50 55 60

Phe Leu Thr Asn Phe Asn Ile Asn Asn Asn Leu Leu Val Thr Gin Ala 65 70 75 80

Asn Asn Leu Gin Asp Ile Pro Ile Phe Asn Val Asn Asn Ile Ile Ser

85 90 95

Asn Gin Tyr Asn Phe Tyr Ser Ala Ser Ser Asn Asn Val Asn Ile Leu

100 105 110

Leu Gly Leu Arg Lys Thr Leu Asn Ile Asn Arg Asn Pro Phe Leu Leu

115 120 125

Phe Arg Asn Thr Ser Leu Ala Ile Val Phe Asn Asn Asn Glu Thr Phe 130 135 140

His Cys Tyr Ile Ser Ser Asn Gin Asn Ser Asp Val Leu Asp Ile Val 145 150 155 160

Ser His Ile Glu Phe Met Lys Ser Arg Tyr Asn Lys Tyr Val Ile Ile

165 170 175

Gly Glu Ile Pro Val Asn Asn Asn Ile Ser Ile Asn Asn Ile Leu Asn

180 185 190

Asn Phe Ala Ile Ile Thr Asn Val Arg Leu Ile Asp Lys Tyr Asn Ser

195 200 205

Ile Ile Ser Phe Leu Asn Ile Asn Val Gly Thr Leu Phe Val Ile Asn 210 215 220

Pro

225

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 78 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Met Ser Ser Ser Lys Lys Asn Asn Leu Gly Tyr Phe Asn Asn Leu Lys 1 5 10 15 Thr Glu Glu Val Ser Gin Ser Gin Val Phe Lys Asp Asn Tyr Arg Pro 20 25 30

Gly Tyr Tyr Gly Leu Asp Thr Asn Ala Ala Asn Pro Ala Asp Val Tyr

35 40 45

Asn Thr Glu Ser Asn Lys Pro Ser Thr Val Asp Val Trp Gly Asp Lys 50 55 60

Arg Leu Glu Gly Lys Ile Ile Pro Lys Ser Lys Lys Lys Lys

65 70 75

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 162 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Met Ser Ile Phe Ile Tyr Tyr Ile Phe Asn Asn Arg Phe Tyr Ile Tyr 1 5 10 15

Lys Arg Met Asn Thr Val Gin Ile Leu Val Val Ile Leu Ile Thr Thr

20 25 30

Ala Leu Ser Phe Leu Val Phe Gin Leu Trp Tyr Tyr Ala Glu Asn Tyr

35 40 45

Glu Tyr Ile Leu Arg Tyr Asn Asp Thr Tyr Ser Asn Leu Gin Phe Ala 50 55 60

Arg Ser Ala Asn Ile Asn Phe Asp Asp Leu Thr Val Phe Asp Pro Asn 65 70 75 80

Asp Asn Val Phe Asn Val Glu Glu Lys Trp Arg Cys Ala Ser Thr Asn

85 90 95

Asn Asn Ile Phe Tyr Ala Val Ser Thr Phe Gly Phe Leu Ser Thr Glu

100 105 110

Ser Thr Gly Ile Asn Leu Thr Tyr Thr Asn Ser Arg Asp Cys Ile Ile

115 120 125

Asp Leu Phe Ser Arg Ile Ile Lys Ile Val Tyr Asp Pro Cys Thr Val 130 135 140

Glu Thr Ser Asn Asp Cys Arg Leu Leu Arg Leu Leu Met Ala Asn Thr 145 150 155 160

Ser

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1003 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Met Ser Asn Val Pro Leu Ala Thr Lys Thr Ile Arg Lys Leu Ser Asn 1 5 10 15

Arg Lys Tyr Glu Ile Lys Ile Tyr Leu Lys Asp Glu Asn Thr Cys Phe

20 25 30

Glu Arg Val Val Asp Met Val Val Pro Leu Tyr Asp Val Cys Asn Glu

35 40 45

Thr Ser Gly Val Thr Leu Glu Ser Cys Ser Pro Asn Ile Glu Val Ile 50 55 60

Glu Leu Asp Asn Thr His Val Arg Ile Lys Val His Gly Asp Thr Leu 65 70 75 80

Lys Glu Met Cys Phe Glu Leu Leu Phe Pro Cys Asn Val Asn Glu Ala

85 90 95

Gin Val Trp Lys Tyr Val Ser Arg Leu Leu Leu Asp Asn Val Ser His

100 105 110

Asn Asp Val Lys Tyr Lys Leu Ala Asn Phe Arg Leu Thr Leu Asn Gly

115 120 125

Lys His Leu Lys Leu Lys Glu Ile Asp Gin Pro Leu Phe Ile Tyr Phe 130 135 140

Val Asp Asp Leu Gly Asn Tyr Gly Leu Ile Thr Lys Glu Asn Ile Gin 145 150 155 160

Asn Asn Asn Leu Gin Val Asn Lys Asp Ala Ser Phe Ile Thr Ile Phe

165 170 175

Pro Gin Tyr Ala Tyr Ile Cys Leu Gly Arg Lys Val Tyr Leu Asn Glu

180 185 190

Lys Val Thr Phe Asp Val Thr Thr Asp Ala Thr Asn Ile Thr Leu Asp

195 200 205

Phe Asn Lys Ser Val Asn Ile Ala Val Ser Phe Leu Asp Ile Tyr Tyr 210 215 220

Glu Val Asn Asn Asn Glu Gin Lys Asp Leu Leu Lys Asp Leu Leu Lys 225 230 235 240

Arg Tyr Gly Glu Phe Glu Val Tyr Asn Ala Asp Thr Gly Leu Ile Tyr

245 250 255

Ala Lys Asn Leu Ser Ile Lys Asn Tyr Asp Thr Val Ile Gin Val Glu

260 265 270

Arg Leu Pro Val Asn Leu Lys Val Arg Ala Tyr Thr Lys Asp Glu Asn

275 280 285

Gly Arg Asn Leu Cys Leu Met Lys Ile Thr Ser Ser Thr Glu Val Asp 290 295 300

Pro Glu Tyr Val Thr Ser Asn Asn Ala Leu Leu Gly Thr Leu Arg Val 305 310 315 320 Tyr Lys Lys Phe Asp Lys Ser His Leu Lys Ile Val Met His Asn Arg 325 330 335

Gly Ser Gly Asn Val Phe Pro Leu Arg Ser Leu Tyr Leu Glu Leu Ser

340 345 350

Asn Val Lys Gly Tyr Pro Val Lys Ala Ser Asp Thr Ser Arg Leu Asp

355 360 365

Val Gly Ile Tyr Lys Leu Asn Lys Ile Tyr Val Asp Asn Asp Glu Asn 370 375 380

Lys Ile Ile Leu Glu Glu Ile Glu Ala Glu Tyr Arg Cyβ Gly Arg Gin 385 390 395 400

Val Phe His Glu Arg Val Lys Leu Asn Lys His Gin Cys Lys Tyr Thr

405 410 415

Pro Lys Cys Pro Phe Gin Phe Val Val Asn Ser Pro Asp Thr Thr Ile

420 425 430

His Leu Tyr Gly Ile Ser Asn Val Cys Leu Lys Pro Lys Val Pro Lys

435 440 445

Asn Leu Arg Leu Trp Gly Trp Ile Leu Asp Cys Asp Thr Ser Arg Phe 450 455 460

Ile Lys His Met Ala Asp Gly Ser Asp Asp Leu Asp Leu Asp Val Arg 465 470 475 480

Leu Asn Arg Asn Asp Ile Cys Leu Lys Gin Ala Ile Lys Gin His Tyr

485 490 495

Thr Asn Val Ile Ile Leu Glu Tyr Ala Asn Thr Tyr Pro Asn Cys Thr

500 505 510

Leu Ser Leu Gly Asn Asn Arg Phe Asn Asn Val Phe Asp Met Asn Asp

515 520 525

Asn Lys Thr Ile Ser Glu Tyr Thr Asn Phe Thr Lys Ser Arg Gin Asp 530 535 540

Leu Asn Asn Met Ser Cys Ile Leu Gly Ile Asn Ile Gly Asn Ser Val 545 550 555 560

Asn Ile Ser Ser Leu Pro Gly Trp Val Thr Pro His Glu Ala Lys Ile

565 570 575

Leu Arg Ser Gly Cys Ala Arg Val Arg Glu Phe Cys Lys Ser Phe Cys

580 585 590

Asp Leu Ser Asn Lys Arg Phe Tyr Ala Met Ala Arg Asp Leu Val Ser

595 600 605

Leu Leu Phe Met Cys Asn Tyr Val Asn Ile Glu Ile Asn Glu Ala Val 610 615 620

Cys Glu Tyr Pro Gly Tyr Val Ile Leu Phe Ala Arg Ala Ile Lys Val 625 630 635 640 Ile Asn Asp Leu Leu Leu Ile Asn Gly Val Asp Asn Leu Ala Gly Tyr

645 650 655 68

Ser Ile Ser Leu Pro Ile His Tyr Gly Ser Thr Glu Lys Thr Leu Pro

660 665 670

Asn Glu Lys Tyr Gly Gly Val Asp Lys Lys Phe Lys Tyr Leu Phe Leu

675 680 685

Lys Asn Lys Leu Lys Asp Leu Met Arg Asp Ala Asp Phe Val Gin Pro 690 695 700

Pro Leu Tyr Ile Ser Thr Tyr Phe Arg Thr Leu Leu Asp Ala Pro Pro 705 710 715 720

Thr Asp Asn Tyr Glu Lys Tyr Leu Val Asp Ser Ser Val Gin Ser Gin

725 730 735

Asp Val Leu Gin Gly Leu Leu Asn Thr Cys Asn Thr Ile Asp Thr Asn

740 745 750

Ala Arg Val Ala Ser Ser Val Ile Gly Tyr Val Tyr Glu Pro Cys Gly

755 760 765

Thr Ser Glu His Lys Ile Gly Ser Glu Ala Leu Cys Lys Met Ala Lys 770 775 780

Glu Ala Ser Arg Leu Gly Asn Leu Gly Leu Val Asn Arg Ile Asn Glu 785 790 795 800

Ser Asn Tyr Asn Lys Cys Asn Lys Tyr Gly Tyr Arg Gly Val Tyr Glu

805 810 815

Asn Asn Lys Leu Lys Thr Lys Tyr Tyr Arg Glu Ile Phe Asp Cys Asn

820 825 830

Pro Asn Asn Asn Asn Glu Leu Ile Ser Arg Tyr Gly Tyr Arg Ile Met

835 840 845

Asp Leu His Lys Ile Gly Glu Ile Phe Ala Asn Tyr Asp Glu Ser Glu 850 855 860

Ser Pro Cys Glu Arg Arg Cys His Tyr Leu Glu Asp Arg Gly Leu Leu 865 870 875 880

Tyr Gly Pro Glu Tyr Val His His Arg Tyr Gin Glu Ser Cys Thr Pro

885 890 895

Asn Thr Phe Gly Asn Asn Thr Asn Cys Val Thr Arg Asn Gly Glu Gin

900 905 910

His Val Tyr Glu Asn Ser Cys Gly Asp Asn Ala Thr Cys Gly Arg Arg

915 920 925

Thr Gly Tyr Gly Arg Arg Ser Arg Asp Glu Trp Asn Asp Tyr Arg Lys 930 935 940

Pro His Val Tyr Asp Asn Cys Ala Asp Ala Asn Ser Ser Ser Ser Asp 945 950 955 960

Ser Cys Ser Asp Ser Ser Ser Ser Ser Glu Ser Glu Ser Asp Ser Asp

965 970 975 Gly Cys Cys Asp Thr Asp Ala Ser Leu Asp Ser Asp Ile Glu Asn Cys 980 985 990

Tyr Gin Asn Pro Ser Lys Cys Asp Ala Gly Cys

995 1000

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 163 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

Arg Ser Ile Arg Leu Asn Ser His Lys Asp Leu Pro Gin Glu Tyr Arg 1 5 10 15

Tyr Val Asn Val His Phe Leu Ile Ser Tyr Thr Asn Asn Arg Lys Ser

20 25 30

Val Asp Lys Glu Ile Leu Asp Ile Ile Lys Asp Lys Gin Gly Lys Ile

35 40 45

Asn Val Ile Phe Asp Leu Leu Lys Ser Ser Ser Ile Glu Ser Ile His 50 55 60

Asn Thr Tyr Lys Tyr Ile Glu Pro Ala Glu Asn Glu Ile Ile Phe Asp 65 70 75 80

Thr Ile Arg Lys Thr Arg Met Lys Glu Met Asn Val Ser Asn Val Ile

85 90 95 Ile Asn Ile Lys Leu Tyr Pro Ile Ser Tyr Cys Lys Asp Tyr Aβp Arg

100 105 110

Ala Thr Ile Leu Lys Gly Leu Leu Asn Lys Asp Thr Asn Ile Val Tyr

115 120 125

Lys Asp Asn Thr Ala Val Ala Lys Leu Met Ile Asp Lys Asp Asn Ile 130 135 140

Pro Ile Phe Ile Ile Glu Asn Asp Thr Leu Ile Tyr Ile Ala Asp Asp 145 150 155 160

Tyr Tyr Glu

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1511 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Amsacta moorei entemopoxvirus (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: complement (18..218)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: complement (234..782)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 852..1511

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

GAATTCAAGT TAAATATTTA TAAACAACAA TCATATTTTT TTAAAGAATC TAATAAATTT 60

TTTAACATTT TATTATTATT TGATAATTGT TTATTTAATT CGTTATTGAT ATTAACAATA 120

TTATTTATCA TTTTACCTAT TTTTTTTTTT CTATCTACTA ACGAAATATC AGATTTTGCA 180

CCTTCAATAT CAGAATAATA ATTATCATTA TTTTGCATTT ATGAATAAAA ATATTAATAT 240

GAATTATTAT AACATAATCT ACACACAGGA ACATATAAAT CTTGTCCACC TATTTCAATT 300

ATTTGATTTT TATTATGTTT TTTAATTGTA AAAGAAGCAT CTTTATAACA AAATTGACAT 360

ATAGCTTGTA ATTTTTTTAT TTTTTCTACT TTAGGAATTA ATTTTGATAT AGAATTAAAT 420

ATATTTCTGT TAAAGTCACA ATTTAATCCA GCAACAATAA CTTTTTTTTT ATTATTAGCC 480

ATTTTATCAC AAAATTGTTC TAAATCATTT TCTTCAAAAA ATTGACACTC ATCTATGCCA 540

ATAATATCAT AATTATCTAC GATATTGATT TCATTAATTA AATTATTTGT TTTAATGTAT 600

AAATATTCTT TATTTAATAT ATTTCCGTCA TGATTTATTA TATTTTTATT TATAAATCTA 660

TTATCTATAT TATGAGTTAT AATTACACAT TTTTGATTAG ATAAAATATA TCTATTAATT 720

TTTCGCATCA ATTCTGTTGT TTTGCCAGAA AACATAGGAC CAATTATTAA TTCTATCGAC 780

ATTTTTTTTT ATTATTTGAT ATATTTTTTC AAAAAAAAAT TAATCAATGA AAAAAAAATA 840

AAATTATCAA A ATG GAT TTA CTA AAT TCT GAT ATA ATT TTA ATA AAT ATT 890

Met Asp Leu Leu Asn Ser Asp Ile Ile Leu Ile Asn Ile

1 5 10

TTA AAA TAT TAT AAT TTA AAA AAA ATA ATA ATA AAC AGA GAT AAT GTT 938 Leu Lys Tyr Tyr Asn Leu Lys Lys Ile Ile Ile Asn Arg Asp Asn Val

15 20 25

ATT AAT ATT AAT ATA TTA AAA AAA TTA GTT AAT TTA GAA GAA TTG CAT 986 Ile Asn Ile Asn Ile Leu Lys Lys Leu Val Asn Leu Glu Glu Leu His

30 35 40 45

ATA ATA TAT TAT GAT AAT AAT ATT TTA AAT AAT ATT CCA GAA AAT ATT 1034 Ile Ile Tyr Tyr Asp Asn Asn Ile Leu Asn Asn Ile Pro Glu Asn Ile

50 55 60

AAA AGT TTA TAT ATT TCA AAT TTA AAT ATT ATT AAT TTA AAT TTT ATA 1082 Lys Ser Leu Tyr Ile Ser Asn Leu Asn Ile Ile Asn Leu Asn Phe Ile

65 70 75 ACA AAA TTA AAA AAT ATA ACA TAT TTA GAT ATA TCT TAT AAC AAA AAT 1130 Thr Lys Leu Lys Asn Ile Thr Tyr Leu Asp Ile Ser Tyr Asn Lys Asn

80 85 90

AGC AAT ATA AGT AAT ATT ATA CTA CCA CAT TCT ATA GAA TTT TTA AAT 1178 Ser Asn Ile Ser Asn Ile Ile Leu Pro His Ser Ile Glu Phe Leu Asn

95 100 105

TGT GAA TCA TGT AAT ATA AAT GAC TAT AAT TTT ATT AAT AAT TTA GTA 1226 Cys Glu Ser Cys Asn Ile Asn Asp Tyr Asn Phe Ile Asn Asn Leu Val

110 115 120 125

AAT TTA AAA AAA TTA ATA ATA TCT AAA AAT AAA TTT GGT AAC TTT AAT 1274 Asn Leu Lys Lys Leu Ile Ile Ser Lys Asn Lys Phe Gly Asn Phe Asn

130 135 140

AAT GTT TTT CCT ATT AGT ATA GTT GAG TTA AAT ATG GAA TCA ATA CAA 1322 Asn Val Phe Pro Ile Ser Ile Val Glu Leu Asn Met Glu Ser Ile Gin

145 150 155

ATA AAA GAT TAT AAA TTT ATA GAA AAA TTA ATT AAT TTA AAA AAA TTA 1370 Ile Lys Asp Tyr Lys Phe Ile Glu Lys Leu Ile Asn Leu Lys Lys Leu

160 165 170

GAT ATA TCT TTC AAT GTT AAA AAA AAT AAT ATA CAT TTG ATA AAA TTT 1418 Asp Ile Ser Phe Asn Val Lys Lys Asn Asn Ile His Leu Ile Lys Phe

175 180 185

CCA AAA AGT ATA ACT CAT TTA TGT GAT TAT CAA TCA TAT AAA GAA AAT 1466 Pro Lys Ser Ile Thr His Leu Cys Asp Tyr Gin Ser Tyr Lys Glu Asn

190 195 200 205

TAT AAT TAT TTA AAA AAT TTA TCA AAT ATA ATT GAA TAT GAA TTC 1511

Tyr Asn Tyr Leu Lys Asn Leu Ser Asn Ile Ile Glu Tyr Glu Phe

210 215 220

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 67 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Met Gin Asn Asn Asp Asn Tyr Tyr Ser Asp Ile Glu Gly Ala Lys Ser

1 5 10 15

Asp Ile Ser Leu Val Asp Arg Lys Lys Lys Ile Gly Lys Met Ile Asn

20 25 30

Asn Ile Val Asn Ile Asn Asn Glu Leu Asn Lys Gin Leu Ser Asn Asn

35 40 45

Asn Lys Met Leu Lys Asn Leu Leu Asp Ser Leu Lys Lys Tyr Asp Cys

50 55 60

Cys Leu

65 (2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 183 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Met Ser Ile Glu Leu Ile Ile Gly Pre Met Phe Ser βly Ly« *hr »hr 1 5 10 15

Glu Leu Met Arg Lys Ile Asn Arg Tyr Ile Leu Ser Asn Gin Lys Cys

20 25 30

Val Ile Ile Thr His Asn Ile Asp Asn Arg Phe Ile Asn Lys Asn Ile

35 40 45 Ile Asn His Asp Gly Asn Ile Leu Asn Lys Glu Tyr Leu Tyr Ile Lys 50 55 60

Thr Asn Asn Leu Ile Asn Glu Ile Asn Ile Val Asp Asn Tyr Asp Ile 65 70 75 80 Ile Gly Ile Asp Glu Cys Gin Phe Phe Glu Glu Asn Asp Leu Glu Gin

85 90 95

Phe Cys Asp Lys Met Ala Asn Asn Lys Lys Lys Val Ile Val Ala Gly

100 105 110

Leu Asn Cys Asp Phe Asn Arg Asn Ile Phe Asn Ser Ile Ser Lys Leu

115 120 125

Ile Pro Lys Val Glu Lys Ile Lys Lys Leu Gin Ala Ile Cys Gin Phe 130 135 140

Cys Tyr Lys Asp Ala Ser Phe Thr Ile Lys Lys His Asn Lys Asn Gin 145 150 155 160 Ile Ile Glu Ile Gly Gly Gin Asp Leu Tyr Val Pro Val Cys Arg Leu

165 170 175

Cys Tyr Asn Asn Ser Tyr

180

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 220 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

Met Asp Leu Leu Asn Ser Asp Ile Ile Leu Ile Asn Ile Leu Lys Tyr 1 5 10 15 Tyr Asn Leu Lys Lys Ile Ile Ile Asn Arg Asp Asn Val Ile Asn Ile

20 25 30

Asn Ile Leu Lys Lys Leu Val Asn Leu Glu Glu Leu His Ile Ile Tyr

35 40 45

Tyr Asp Asn Asn Ile Leu Asn Asn Ile Pro Glu Asn Ile Lys Ser Leu

50 55 60

Tyr Ile Ser Asn Leu Asn Ile Ile Asn Leu Asn Phe Ile Thr Lys Leu

65 70 75 80

Lys Asn Ile Thr Tyr Leu Asp Ile Ser Tyr Asn Lys Asn Ser Asn Ile

85 90 95

Ser Asn Ile Ile Leu Pro His Ser Ile Glu Phe Leu Asn Cys Glu Ser

100 105 110

Cys Asn Ile Asn Asp Tyr Asn Phe Ile Asn Asn Leu Val Asn Leu Lys

115 120 125

Lys Leu Ile Ile Ser Lys Asn Lys Phe Gly Asn Phe Asn Asn Val Phe

130 135 140

Pro Ile Ser Ile Val Glu Leu Asn Met Glu Ser Ile Gin Ile Lys Asp

145 150 155 160

Tyr Lys Phe Ile Glu Lys Leu Ile Asn Leu Lys Lys Leu Asp Ile Ser

165 170 175

Phe Asn Val Lys Lys Asn Asn Ile His Leu Ile Lys Phe Pro Lys Ser

180 185 190

Ile Thr His Leu Cys Asp Tyr Gin Ser Tyr Lys Glu Asn Tyr Asn Tyr

195 200 205

Leu Lys Asn Leu Ser Asn Ile Ile Glu Tyr Glu Phe

210 215 220

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

GARGTNGAYC CNGARTAYGT 20

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

TTTCAAATTA ACTGGCAACC 20

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

GGGATGGATT TTAGATTGCG 20

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

GCCTGGTTGG GTAACACCTC 20

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

CTGCTAGATT ATCTACTCCG 20

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

GTTCGAAACA AGTATTTTCA TCTTTTAAAT AAATC 35

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

GAYGARGGRG GRCARTTYTT 20

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

GGNCCCATGT TYTCNGG 17

(2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

GGTGCAAAAT CTGATATTTC 20

(2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3012 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

ATGAGTAACG TACCTTTAGC AACCAAAACA ATAAGAAAAT TATCAAATCG AAAATATGAA 60

ATAAAGATTT ATTTAAAAGA TGAAAATACT TGTTTCGAAC GTGTAGTAGA TATGGTAGTT 120

CCATTATATG ATGTGTGTAA TGAAACTTCT GGTGTTACTT TAGAATCATG TAGTCCAAAT 180

ATAGAAGTAA TTGAATTAGA CAATACTCAT GTTAGAATCA AAGTTCACGG CGATACATTA 240

AAAGAAATGT GTTTTGAATT ATTGTTCCCG TGTAATGTAA ACGAAGCCCA AGTATGGAAA 300

TATGTAAGTC GATTATTGCT AGATAATGTA TCACATAATG ACGTAAAATA TAAATTAGCT 360

AATTTTAGAC TGACTCTTAA TGGAAAACAT TTAAAATTAA AAGAAATCGA TCAACCGCTA 420

TTTATTTATT TTGTCGATGA TTTGGGAAAT TATGGATTAA TTACTAAGGA AAATATTCAA 480

AATAATAATT TACAAGTTAA CAAAGATGCA TCATTTATTA CTATATTTCC ACAATATGCG 540

TATATTTGTT TAGGTAGAAA AGTATATTTA AATGAAAAAG TAACTTTTGA TGTAACTACA 600

GATGCAACTA ATATTACTTT AGATTTTAAT AAATCTGTTA ATATCGCAGT ATCATTCCTT 660

GATATATATT ACGAAGTTAA TAATAATGAA CAAAAAGATT TATTAAAAGA TTTACTTAAG 720

AGATACGGTG AATTTGAAGT CTATAACGCA GATACTGGAT TAATTTATGC TAAAAATCTA 780

AGTATTAAAA ATTATGATAC TGTGATTCAA GTAGAAAGGT TGCCAGTTAA TTTGAAAGTT 840

AGAGCATATA CTAAGGATGA AAATGGTCGC AATCTATGTT TGATGAAAAT AACATCTAGT 900

ACAGAAGTAG ACCCCGAGTA TGTAACTAGT AATAATGCTT TATTGGGTAC GCTCAGAGTA 960

TATAAAAAGT TTGATAAATC TCATTTAAAA ATTGTAATGC ATAACAGAGG AAGTGGTAAT 1020

GTATTTCCAT TAAGATCATT ATATCTGGAA TTGTCTAATG TAAAAGGATA TCCAGTTAAA 1080

GCATCTGATA CTTCGAGATT AGATGTTGGT ATTTACAAAT TAAATAAAAT TTATGTAGAT 1140

AACGACGAAA ATAAAATTAT ATTGGAAGAA ATTGAAGCAG AATATAGATG CGGAAGACAA 1200

GTATTCCACG AACGTGTAAA ACTTAATAAA CACCAATGTA AATATACTCC CAAATGTCCA 1260

TTCCAATTTG TTGTAAACAG CCCAGATACT ACGATTCACT TATATGGTAT TTCTAATGTT 1320

TGTTTAAAAC CTAAAGTACC CAAAAATTTA AGACTTTGGG GATGGATTTT AGATTGCGAT 1380

ACTTCTAGAT TTATTAAACA TATGGCTGAT GGATCTGATG ATTTAGATCT TGACGTTAGG 1440

CTTAATAGAA ATGATATATG TTTAAAACAA GCCATAAAAC AACATTATAC TAATGTAATT 1500

ATATTAGAGT ACGCAAATAC ATATCCAAAT TGCACATTAT CATTGGGTAA TAATAGATTT 1560

AATAATGTAT TTGATATGAA TGATAACAAA ACTATATCTG AGTATACTAA CTTTACAAAA 1620

AGTAGACAAG ACCTTAATAA CATGTCATGT ATATTAGGAA TAAACATAGG TAATTCCGTA 1680

AATATTAGTA GTTTGCCTGG TTGGGTAACA CCTCACGAAG CTAAAATTCT AAGATCTGGT 1740

TGTGCTAGAG TTAGAGAATT TTGTAAATCA TTCTGTGATC TTTCTAATAA GAGATTCTAT 1800

GCTATGGCTA GAGATCTCGT AAGTTTACTA TTTATGTGTA ACTATGTTAA TATTGAAATT 1860 AACGAAGCAG TATGCGAATA TCCTGGATAT GTCATATTAT TCGCAAGAGC TATTAAAGTA 1920

ATTAATGATT TATTATTAAT TAACGGAGTA GATAATCTAG CAGGATATTC AATTTCCTTA 1980

CCTATACATT ATGGATCTAC TGAAAAGACT CTACCAAATG AAAAGTATGG TGGTGTTGAT 2040

AAGAAATTTA AATATCTATT CTTAAAGAAT AAACTAAAAG ATTTAATGCG TGATGCTGAT 2100

TTTGTCCAAC CTCCATTATA TATTTCTACT TACTTTAGAA CTTTATTGGA TGCTCCACCA 2160

ACTGATAATT ATGAAAAATA TTTGGTTGAT TCGTCCGTAC AATCACAAGA TGTTCTACAG 2220

GGTCTGTTGA ATACATGTAA TACTATTGAT ACTAATGCTA GAGTTGCATC AAGTGTTATT 2280

GGATATGTTT ATGAACCATG CGGAACATCA GAACATAAAA TTGGTTCAGA AGCATTGTGT 2340

AAAATGGCTA AAGAAGCATC TAGATTAGGA AATCTAGGTT TAGTAAATCG TATTAATGAA 2400

AGTAATTACA ACAAATGTAA TAAATATGGT TATAGAGGAG TATACGAAAA TAACAAACTA 2460

AAAACAAAAT ATTATAGAGA AATATTTGAT TGTAATCCTA ATAATAATAA TGAATTAATA 2520

TCCAGATATG GATATAGAAT AATGGATTTA CATAAAATTG GAGAAATTTT TGCAAATTAC 2580

GATGAAAGTG AATCTCCTTG CGAACGAAGA TGTCATTACT TGGAAGATAG AGGTCTTTTA 2640

TATGGTCCTG AATATGTACA TCACAGATAT CAAGAATCAT GTACGCCTAA TACGTTTGGA 2700

AATAACACAA ATTGTGTAAC AAGAAATGGT GAACAACACG TATACGAAAA TAGTTGTGGA 2760

GATAATGCAA CATGTGGAAG AAGAACAGGA TATGGAAGAA GAAGTAGGGA TGAATGGAAT 2820

GACTATAGAA AACCCCACGT TTATGACAAT TGTGCCGATG CAAATAGTTC ATCTTCAGAT 2880

AGCTGTTCAG ACAGTAGTAG TAGTAGTGAA TCTGAATCTG ATTCAGATGG ATGTTGCGAC 2940

ACAGATGCTA GTTTAGATTC TGATATTGAA AATTGTTATC AAAATCCATC AAAATGTGAT 3000

GCAGGATGCT AA 3012

(2) INFORMATION FOR SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 419 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:

TCAACTAATA ATAATATATT TTATGCAGTT TCAACTTTTG GATTTTTAAG TACAGAAAGT 60 ACTGGTATTA ATTTAACATA TACAAATTCT AGAGATTGTA TTATAGATTT ATTTTCTAGA 120 ATTATAAAAA TAGTATATGA TCCTTGTACT GTCGAAACAT CTAACGATTG TAGATTATTA 180 AGATTATTGA TGGCCAATAC ATCATAAATA CATTATAATA TTATTATAAT ATCAATCATA 240 ATTTTTATAT ATATTTTATC TAAAAGGACT TTTTATTTTT TATATATTAA TAATAATAAA 300 TGAGTAACGT ACCTTTAGCA ACCAAAACAA TAAGAAAATT ATCAAATCGA AAATATGAAA 360 TAAAGATTTA TTTAAAAGAT GAAAATACTT GTTTCGAACG TGTAGTAGAT ATGGTAGTT 419

(2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 678 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

ATGTTTCTAG TTTATTTCAA TACATTTTTA ATAATAATTT TATTATTTGG TATTATAGGT 60

ATTTATATAT TAACATTTGT GTTTAATATA GATTTTTTAA TAAATAATAA TAAAATATAT 120

ATATTATCAT ATAACGCAAC TAATATAAAC AATATAAATA ATTTAAATTT ATACGATTAT 180

TCAGATATTA TATTTTTGAC AAATTTTAAC ATAAATAATA ATCTTTTAGT AACACAAGCT 240

AATAATTTAC AAGATATACC AATATTTAAT GTAAATAATA TTATATCTAA TCAATATAAT 300

TTTTATTCAG CGTCTAGTAA TAATGTAAAT ATATTATTAG GATTAAGAAA AACATTAAAT 360

ATAAATAGAA ATCCATTTTT ATTATTTAGA AATACATCTC TAGCTATAGT TTTCAATAAT 420

AATGAAACTT TTCACTGTTA TATAAGTTCA AATCAAAATA GTGATGTATT AGATATAGTA 480

TCACATATAG AATTTATGAA ATCTAGATAT AATAAATATG TAATTATAGG AGAAATACCC 540

GTAAATAATA ATATATCTAT TAATAATATA TTAAATAATT TTGCTATTAT AACTAATGTG 600

AGATTAATAG ATAAATATAA CTCTATAATA TCATTTTTAA ATATCAACGT AGGAACACTT 660

TTTGTCATAA ATCCATAA 678

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 486 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

ATGTCAATAT TTATCTACTA TATTTTCAAC AATAGATTTT ATATATATAA AAGAATGAAT 60

ACTGTACAAA TTTTAGTTGT CATATTAATA ACAACAGCAT TATCTTTTCT AGTTTTTCAA 120

TTATGGTATT ATGCCGAAAA TTACGAATAT ATATTAAGAT ATAATGATAC ATATTCAAAT 180

TTACAATTTG CGAGAAGCGC AAATATAAAT TTTGATGATT TAACTGTTTT TGATCCCAAC 240 GATAATGTTT TTAATGTTGA AGAAAAATGG CGCTGTGCTT CAACTAATAA TAATATATTT 300

TATGCAGTTT CAACTTTTGG ATTTTTAAGT ACAGAAAGTA CTGGTATTAA TTTAACATAT 360

ACAAATTCTA GAGATTGTAT TATAGATTTA TTTTCTAGAA TTATAAAAAT AGTATATGAT 420

CCTTGTACTG TCGAAACATC TAACGATTGT AGATTATTAA GATTATTGAT GGCCAATACA 480

TCATAA 486

(2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1395 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25

TTAAATATTA GATTCTAAAC TATTCTTCTC ATTATCAATA TAACTATCAT AATCATTTTT 60 TATTTTACTA CATACATTCA TAATTCTATT ACTATTTTTT TTATACATAT CTATTAATTC 120 CATAAACTTT TTATTTTTTA TATTAAATAT TTCTAATGTA TTTTTAAATT CGTCAATACT 180 ATTAATATCA TATCTAGAAA TAAATAATGC ACCTCTATAA CTACTAGCCA ATAAATCACC 240 AATAAAACTC ATAGAATAAT ATAATTTTTT AAATTCAAAT TTAGATTTTA TGTTGAAATA 300 AACTATATAA TATAAAAATA TTATATTAAA CATACCACAA TCGGGACTAT CATATTGTAA 360 TTCAAAAGTA TTAAAAAAGT AATAATTTAC ATTTTTAAAT ATATCATTTA AATATTCTGA 420 TAGTACATCA ATGTATAAAT AAGCATAATT AGTATTAGGA GTACTATTGT AGTGTTTATG 480 GCTTTTTATA GTCATATCAG ATTCAATAAA CATATATTTT TTATTTTGTT TTATAAGTTC 540 TGGTATATAA CCACTACTAT TAAAAAAGTA TGCAGCTTTT TTATCTTTAT CAAAGTGTTT 600 ATCTATTACG CAACAAGTAA AATGATCATT ATAAATTATA GGAAACATAA AAAATCTTTT 660 TTTATCATTC ATTAAAAAAA ATTTTACTCT ATCTTCAAGT TTATAGCATC TCATAGATGA 720 AGCTACTGTA GCAATATTTT TATCAGTTTT TTCAAATAAA ATCAAATGAA AATAATCATA 780 ATCTGTATTA ATCATAGTTA ATGGATATAT ACAATTATAT ATATCTCCCG AACTTAACCA 840 TGTAGATTTA TCATGTTTTC TTGGGTAAGC TTTAGGTTTA GGATTAAATC CCAAAGGCGG 900 TATTCCTATT TGAGCATCCA AATCATCATA AATTGTGGCA AATGTAGAAA AATCTCTTGT 960 TTTGGATAAT TCTGATTTTA GAAAAGACTT TCTCATATAT ACTAATGGAA TGCCTTTATA 1020 TTTTTTAGAT GTAATAAAAG TATTAATATT TATATTTTTA TCTTGTAAAT ATTTTTTTAT 1080 AGTCCAAAAT AGAAAAAATT TTCTTTTAAT ATTATTTTCA AAATTAATAT TATTAATATG 1140 ATTTGGATCT AAAACTAATT CATTATATAA TATTTCCAAG TATTTTATAG GTATAAATGT 1200 TACTTTACCT CTTGTTTCAT CATCATCATC TATTTTTTCT AATATAGCTA TATTTGCATT 1260

AGTATTATAT TTAATAGGAT TTATAAAATA TACCATATTA TCTATTTTAC TAAAAAATAA 1320

CATAGACATA AAATTAATAC CAGATTCTGG CATTTTTAAA TTTTTATTTG GAAATCTTCT 1380

AATTTTATTA TTCAT 1395

(2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 237 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

TTATTTTTTT TTTTTGCTTT TAGGTATAAT TTTACCTTCT AAACGTTTAT CTCCCCAAAC 60 ATCTACAGTA GATGGTTTAT TAGATTCTGT GTTATACACA TCTGCTGGAT TTGCGGCATT 120 TGTATCCAAA CCATAATATC CAGGTCTATA ATTATCTTTA AAAACTTGGG ATTGAGATAC 180 TTCTTCAGTT TTTAAATTAT TAAAATATCC AAGATTATTT TTTTTTGATG AAGACAT 237

(2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 492 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:

CTATTCATAA TAATCATCTG CTATATATAT TAATGTATCA TTCTCTATTA TAAATATAGG 60

TATATTGTCT TTATCAATCA TTAATTTTGC TACAGCTGTA TTATCTTTAT ATACTATATT 120

TGTGTCTTTG TTTAATAAAC CTTTTAATAT AGTGGCTCTA TCATAATCTT TACAATATGA 180

TATGGGATAT AATTTTATAT TAATAATAAC ATTAGATACG TTCATTTCTT TCATTCTAGT 240

TTTACGTATT GTGTCAAAAA TTATTTCATT TTCTGCTGGT TCTATATATT TATATGTGTT 300

ATGAATAGAT TCGATAGATG ATGATTTTAA TAAATCAAAT ATAACATTTA TTTTACCTTG 360

TTTATCTTTT ATAATATCTA ATATTTCTTT ATCTACAGAT TTTCTGTTGT TGGTATATGA 420

TATTAAAAAA TGAACGTTAA CATATCTATA TTCTTGTGGT AAATCTTTAT GAGAATTTAA 480

TCTTATAGAT CT 492 (2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 549 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

TTAATATGAA TTATTATAAC ATAATCTACA CACAGGAACA TATAAATCTT GTCCACCTAT 60

TTCAATTATT TGATTTTTAT TATGTTTTTT AATTGTAAAA GAAGCATCTT TATAACAAAA 120

TTGACATATA GCTTGTAATT TTTTTATTTT TTCTACTTTA GGAATTAATT TTGATATAGA 180

ATTAAATATA TTTCTGTTAA AGTCACAATT TAATCCAGCA ACAATAACTT TTTTTTTATT 240

ATTAGCCATT TTATCACAAA ATTGTTCTAA ATCATTTTCT TCAAAAAATT GACACTCATC 300

TATGCCAATA ATATCATAAT TATCTACGAT ATTGATTTCA TTAATTAAAT TATTTGTTTT 360

AATGTATAAA TATTCTTTAT TTAATATATT TCCGTCATGA TTTATTATAT TTTTATTTAT 420

AAATCTATTA TCTATATTAT GAGTTATAAT TACACATTTT TGATTAGATA AAATATATCT 480

ATTAATTTTT CGCATCAATT CTGTTGTTTT GCCAGAAAAC ATAGGACCAA TTATTAATTC 540

TATCGACAT 549

(2) INFORMATION FOR SEQ ID NO: 29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 69 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

TTTTTTTTAT TATTTGATAT ATTTTTTCAA AAAAAAATTA ATCAATGAAA AAAAAATAAA 60 ATTATCAAA 69

(2) INFORMATION FOR SEQ ID NO: 30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 141 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

AAACATAGGA CCAATTATTA ATTCTATCGA CATTTTTTTT TATTATTTGA TATATTTTTT 60

CAAAAAAAAA TTAATCAATG AAAAAAAAAT AAAATTATCA AAATGGATTT ACTAAATTCT 120

GATATAATTT TAATAAATAT T 141

(2) INFORMATION FOR SEQ ID NO:31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 201 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:

TTATAAACAA CAATCATATT TTTTTAAAGA ATCTAATAAA TTTTTTAACA TTTTATTATT 60

ATTTGATAAT TGTTTATTTA ATTCGTTATT GATATTAACA ATATTATTTA TCATTTTACC 120

TATTTTTTTT TTTCTATCTA CTAACGAAAT ATCAGATTTT GCACCTTCAA TATCAGAATA 180

ATAATTATCA TTATTTTGCA T 201

(2) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 660 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

ATGGATTTAC TAAATTCTGA TATAATTTTA ATAAATATTT TAAAATATTA TAATTTAAAA 60

AAAATAATAA TAAACAGAGA TAATGTTATT AATATTAATA TATTAAAAAA ATTAGTTAAT 120

TTAGAAGAAT TGCATATAAT ATATTATGAT AATAATATTT TAAATAATAT TCCAGAAAAT 180

ATTAAAAGTT TATATATTTC AAATTTAAAT ATTATTAATT TAAATTTTAT AACAAAATTA 240

AAAAATATAA CATATTTAGA TATATCTTAT AACAAAAATA GCAATATAAG TAATATTATA 300

CTACCACATT CTATAGAATT TTTAAATTGT GAATCATGTA ATATAAATGA CTATAATTTT 360

ATTAATAATT TAGTAAATTT AAAAAAATTA ATAATATCTA AAAATAAATT TGGTAACTTT 420

AATAATGTTT TTCCTATTAG TATAGTTGAG TTAAATATGG AATCAATACA AATAAAAGAT 480

TATAAATTTA TAGAAAAATT AATTAATTTA AAAAAATTAG ATATATCTTT CAATGTTAAA 540 AAAAATAATA TACATTTGAT AAAATTTCCA AAAAGTATAA CTCATTTATG TGATTATCAA 600 TCATATAAAG AAAATTATAA TTATTTAAAA AATTTATCAA ATATAATTGA ATATGAATTC 660

(2) INFORMATION FOR SEQ ID NO:33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3907 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:

TTCTAAACGT TTATCTCCCC AAACATCTAC AGTAGATGGT TTATTAGATT CTGTGTTATA 60

CACATCTGCT GGATTTGCGG CATTTGTATC CAAACCATAA TATCCAGGTC TATAATTATC 120

TTTAAAAACT TGGGATTGAG ATACTTCTTC AGTTTTTAAA TTATTAAAAT ATCCAAGATT 180

ATTTTTTTTT GATGAAGACA TAATTGATAT TATAATACTT TATAGATATG TCAATATTTA 240

TCTACTATAT TTTCAACAAT AGATTTTATA TATATAAAAG AATGAATACT GTACAAATTT 300

TAGTTGTCAT ATTAATAACA ACAGCATTAT CTTTTCTAGT TTTTCAATTA TGGTATTATG 360

CCGAAAATTA CGAATATATA TTAAGATATA ATGATACATA TTCAAATTTA CAATTTGCGA 420

GAAGCGCAAA TATAAATTTT GATGATTTAA CTGTTTTTGA TCCCAACGAT AATGTTTTTA 480

ATGTTGAAGA AAAATGGCGC TGTGCTTCAA CTAATAATAA TATATTTTAT GCAGTTTCAA 540

CTTTTGGATT TTTAAGTACA GAAAGTACTG GTATTAATTT AACATATACA AATTCTAGAG 600

ATTGTATTAT AGATTTATTT TCTAGAATTA TAAAAATAGT ATATGATCCT TGTACTGTCG 660

AAACATCTAA CGATTGTAGA TTATTAAGAT TATTGATGGC CAATACATCA TAAATACATT 720

ATAATATTAT TATAATATCA ATCATAATTT TTATATATAT TTTATCTAAA AGGACTTTTT 780

ATTTTTTATA TATTAATAAT AATAAATGAG TAACGTACCT TTAGCAACCA AAACAATAAG 840

AAAATTATCA AATCGAAAAT ATGAAATAAA GATTTATTTA AAAGATGAAA ATACTTGTTT 900

CGAACGTGTA GTAGATATGG TAGTTCCATT ATATGATGTG TGTAATGAAA CTTCTGGTGT 960

TACTTTAGAA TCATGTAGTC CAAATATAGA AGTAATTGAA TTAGACAATA CTCATGTTAG 1020

AATCAAAGTT CACGGCGATA CATTAAAAGA AATGTGTTTT GAATTATTGT TCCCGTGTAA 1080

TGTAAACGAA GCCCAAGTAT GGAAATATGT AAGTCGATTA TTGCTAGATA ATGTATCACA 1140

TAATGACGTA AAATATAAAT TAGCTAATTT TAGACTGACT CTTAATGGAA AACATTTAAA 1200

ATTAAAAGAA ATCGATCAAC CGCTATTTAT TTATTTTGTC GATGATTTGG GAAATTATGG 1260

ATTAATTACT AAGGAAAATA TTCAAAATAA TAATTTACAA GTTAACAAAG ATGCATCATT 1320

TATTACTATA TTTCCACAAT ATGCGTATAT TTGTTTAGGT AGAAAAGTAT ATTTAAATGA 1380 AAAAGTAACT TTTGATGTAA CTACAGATGC AACTAATATT ACTTTAGATT TTAATAAATC 1440

TGTTAATATC GCAGTATCAT TCCTTGATAT ATATTACGAA GTTAATAATA ATGAACAAAA 1500

AGATTTATTA AAAGATTTAC TTAAGAGATA CGGTGAATTT GAAGTCTATA ACGCAGATAC 1560

TGGATTAATT TATGCTAAAA ATCTAAGTAT TAAAAATTAT GATACTGTGA TTCAAGTAGA 1620

AAGGTTGCCA GTTAATTTGA AAGTTAGAGC ATATACTAAG GATGAAAATG GTCGCAATCT 1680

ATGTTTGATG AAAATAACAT CTAGTACAGA AGTAGACCCC GAGTATGTAA CTAGTAATAA 1740

TGCTTTATTG GGTACGCTCA GAGTATATAA AAAGTTTGAT AAATCTCATT TAAAAATTGT 1800

AATGCATAAC AGAGGAAGTG GTAATGTATT TCCATTAAGA TCATTATATC TGGAATTGTC 1860

TAATGTAAAA GGATATCCAG TTAAAGCATC TGATACTTCG AGATTAGATG TTGGTATTTA 1920

CAAATTAAAT AAAATTTATG TAGATAACGA CGAAAATAAA ATTATATTGG AAGAAATTGA 1980

AGCAGAATAT AGATGCGGAA GACAAGTATT CCACGAACGT GTAAAACTTA ATAAACACCA 2040

ATGTAAATAT ACTCCCAAAT GTCCATTCCA ATTTGTTGTA AACAGCCCAG ATACTACGAT 2100

TCACTTATAT GGTATTTCTA ATGTTTGTTT AAAACCTAAA GTACCCAAAA ATTTAAGACT 2160

TTGGGGATGG ATTTTAGATT GCGATACTTC TAGATTTATT AAACATATGG CTGATGGATC 2220

TGATGATTTA GATCTTGACG TTAGGCTTAA TAGAAATGAT ATATGTTTAA AACAAGCCAT 2280

AAAACAACAT TATACTAATG TAATTATATT AGAGTACGCA AATACATATC CAAATTGCAC 2340

ATTATCATTG GGTAATAATA GATTTAATAA TGTATTTGAT ATGAATGATA ACAAAACTAT 2400

ATCTGAGTAT ACTAACTTTA CAAAAAGTAG ACAAGACCTT AATAACATGT CATGTATATT 2460

AGGAATAAAC ATAGGTAATT CCGTAAATAT TAGTAGTTTG CCTGGTTGGG TAACACCTCA 2520

CGAAGCTAAA ATTCTAAGAT CTGGTTGTGC TAGAGTTAGA GAATTTTGTA AATCATTCTG 2580

TGATCTTTCT AATAAGAGAT TCTATGCTAT GGCTAGAGAT CTCGTAAGTT TACTATTTAT 2640

GTGTAACTAT GTTAATATTG AAATTAACGA AGCAGTATGC GAATATCCTG GATATGTCAT 2700

ATTATTCGCA AGAGCTATTA AAGTAATTAA TGATTTATTA TTAATTAACG GAGTAGATAA 2760

TCTAGCAGGA TATTCAATTT CCTTACCTAT ACATTATGGA TCTACTGAAA AGACTCTACC 2820

AAATGAAAAG TATGGTGGTG TTGATAAGAA ATTTAAATAT CTATTCTTAA AGAATAAACT 2880

AAAAGATTTA ATGCGTGATG CTGATTTTGT CCAACCTCCA TTATATATTT CTACTTACTT 2940

TAGAACTTTA TTGGATGCTC CACCAACTGA TAATTATGAA AAATATTTGG TTGATTCGTC 3000

CGTACAATCA CAAGATGTTC TACAGGGTCT GTTGAATACA TGTAATACTA TTGATACTAA 3060

TGCTAGAGTT GCATCAAGTG TTATTGGATA TGTTTATGAA CCATGCGGAA CATCAGAACA 3120

TAAAATTGGT TCAGAAGCAT TGTGTAAAAT GGCTAAAGAA GCATCTAGAT TAGGAAATCT 3180

AGGTTTAGTA AATCGTATTA ATGAAAGTAA TTACAACAAA TGTAATAAAT ATGGTTATAG 3240

AGGAGTATAC GAAAATAACA AACTAAAAAC AAAATATTAT AGAGAAATAT TTGATTGTAA 3300 TCCTAATAAT AATAATGAAT TAATATCCAG ATATGGATAT AGAATAATGG ATTTACATAA 3360

AATTGGAGAA ATTTTTGCAA ATTACGATGA AAGTGAATCT CCTTGCGAAC GAAGATGTCA 3420

TTACTTGGAA GATAGAGGTC TTTTATATGG TCCTGAATAT GTACATCACA GATATCAAGA 3480

ATCATGTACG CCTAATACGT TTGGAAATAA CACAAATTGT GTAACAAGAA ATGGTGAACA 3540

ACACGTATAC GAAAATAGTT GTGGAGATAA TGCAACATGT GGAAGAAGAA CAGGATATGG 3600

AAGAAGAAGT AGGGATGAAT GGAATGACTA TAGAAAACCC CACGTTTATG ACAATTGTGC 3660

CGATGCAAAT AGTTCATCTT CAGATAGCTG TTCAGACAGT AGTAGTAGTA GTGAATCTGA 3720

ATCTGATTCA GATGGATGTT GCGACACAGA TGCTAGTTTA GATTCTGATA TTGAAAATTG 3780

TTATCAAAAT CCATCAAAAT GTGATGCAGG ATGCTAAATG AAATTTAATA TTATATAATA 3840

TTAACTTACA AGTTATAAAA ATCATTAAAA TGATTTTTTA AAATGATATT ATCGATAGTT 3900

GTGATAA 3907

(2) INFORMATION FOR SEQ ID NO:34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 3

(D) OTHER INFORMATION: /note= "This amino acid may be either Asn or Arg."

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 12

(D) OTHER INFORMATION: /note= "This amino acid may be either Asn or Arg."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:

Met Ala Xaa Asp Leu Val Ser Leu Leu Phe Met Xaa Xaa Tyr Val Asn 1 5 10 15

Ile Glu Ile Asn Glu Ala Val Xaa Glu

20 25

(2) INFORMATION FOR SEQ ID NO:35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide (ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 15

(D) OTHER INFORMATION: /note= "This amino acid may be

either Thr or Ile."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:

Met Lys Ile Thr Ser Ser Thr Glu Val Asp Pro Glu Tyr Val Xaa Ser 1 5 10 15

Asn

(2) INFORMATION FOR SEQ ID NO:36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:

Asn Ala Leu Phe Phe Asn Val Phe

1 5

(2) INFORMATION FOR SEQ ID NO:37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:

Glu Val Asp Pro Glu Tyr Val

1 5

(2) INFORMATION FOR SEQ ID NO:38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 66 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:

ATGGCTAGAG ATCTCGTAAG TTTACTATTT ATGTGTAACT ATGTTAATAT TGAAATTAAC 60 GAAGCA 66 (2) INFORMATION FOR SEQ ID NO:39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 51 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:

ATGAAAATAA CATCTAGTAC AGAAGTAGAC CCCGAGTATG TAACTAGTAA T 51

(2) INFORMATION FOR SEQ ID NO:40:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:

AATAATAGAT TTAATAATGT ATTT 24

Claims

1. An Entomopoxvirus spheroidin gene polynucleotide sequence free from association with other viral nucleotide sequences with which it is associated in nature.

2. The sequence according to claim 1 selected from the group consisting of one or more of a spheroidin gene coding sequence, a spheroidin gene regulatory sequence, a spheroidin gene promoter sequence, an allelic variant or fragment thereof.

3. The sequence according to claim 1 wherein said polynucleotide sequence is a DNA sequence.

4. The sequence according to claim 1 wherein said sequence is derived from the Amsacta moorei

Entomopoxvirus.

5. The sequence according to claim 4 selected from the group consisting of the sequence SEQ ID NO:1 spanning nucleotide #1 through #6768 of Fig. 2, the sequence spanning nucleotide SEQ ID NO: 21 #3080 through #6091 of Fig. 2, an allelic variant, analog or fragment thereof.

6. The sequence according to claim 2 characterized by the ability to direct the expression of a heterologous gene to which said sequence or fragment is operably linked in a selected host cell.

7. A polynucleotide sequence comprising a first polynucleotide sequence comprising an

Entomopoxvirus spheroidin gene polynucleotide sequence, an allelic variant or a fragment thereof associated with a second polynucleotide sequence encoding a heterologous gene.

8. An Entomopoxvirus thymidine kinase gene polynucleotide sequence free from association with other viral nucleotide sequences with which it is associated in nature.

9. The sequence according to claim 1 selected from the group consisting of one or more of a thymidine kinase gene coding sequence, a thymidine kinase gene regulatory sequence, a thymidine kinase gene promoter sequence, an allelic variant or fragment thereof.

10. The sequence according to claim 8 wherein said polynucleotide sequence is a DNA sequence.

11. The sequence according to claim 8 wherein said sequence is derived from the Amsacta moorei

Entomopoxvirus.

12. The sequence according to claim 11

selected from the group consisting of the sequence SEQ ID NO:8 spanning nucleotide #1 through #1511 of Fig. 3, SEQ ID NO:28 nucleotide #234 through #782 of Fig. 3, SEQ ID NO: 29 nucleotide #783 through #851 of Fig. 3, an allelic variant, a or a fragment thereof.

13. The sequence according to claim 9 characterized by the ability to direct the expression of a heterologous gene to which said sequence or fragment is operably linked in a selected host cell.

14. A polynucleotide sequence comprising a first polynucleotide sequence comprising an

Entomopoxvirus thymidine kinase gene polynucleotide sequence, an allelic variant or a fragment thereof associated with a second polynucleotide sequence encoding a heterologous gene.

15. An Entomopoxvirus spheroidin polypeptide, a fragment thereof, or an analog thereof.

16. The polypeptide according to claim 15 fused to a heterologous protein or peptide.

17. An Entomopoxvirus thymidine kinase

polypeptide, a fragment thereof, or an analog thereof.

18. The polypeptide according to claim 17, fused to a heterologous protein or peptide.

19. A recombinant polynucleotide molecule comprising a polynucleotide sequence encoding the

Entomopoxvirus spheroidin promoter sequence, an allelic variant or a fragment thereof, wherein said promoter sequence is operably linked to a selected heterologous gene sequence, said promoter sequence being capable of directing the replication and expression of said gene in a selected host cell or virus.

20. A recombinant polynucleotide molecule comprising a polynucleotide sequence encoding the

Entomopoxvirus thymidine kinase promoter sequence, an allelic variant, or a fragment thereof, wherein said promoter sequence is operably linked to a selected heterologous gene sequence, said promoter sequence being capable of directing the replication and expression of said gene in a selected host cell.

21. A recombinant molecule comprising a polynucleotide sequence encoding the Entomopoxvirus spheroidin gene, an allelic variant, or a fragment thereof, linked in frame to a polynucleotide sequence encoding a selected heterologous gene sequence.

22. A recombinant molecule comprising a polynucleotide sequence encoding the Entomopoxvirus thymidine kinase gene, an allelic variant, or a fragment thereof, linked in frame to a polynucleotide sequence encoding a selected heterologous gene sequence.

23. A recombinant molecule comprising an Entomopoxvirus spheroidin gene polynucleotide sequence, an allelic variant, or a fragment thereof, into which a selected heterologous gene sequence has been inserted.

24. A recombinant molecule comprising an

Entomopoxvirus thymidine kinase gene polynucleotide sequence, an allelic variant, or a fragment thereof, into which a selected heterologous gene sequence has been inserted.

25. A recombinant virus comprising a polynucleotide sequence comprising an Entomopoxvirus spheroidin gene polynucleotide sequence, an allelic variant or a fragment thereof, optionally linked to a selected heterologous gene sequence.

26. The virus according to claim 25, which is a poxvirus selected from the group consisting of a vertebrate poxvirus, orthopoxvirus, suipoxvirus, vaccinia virus and entomopoxvirus.

27. A recombinant virus comprising a polynucleotide sequence comprising an Entomopoxvirus thymidine kinase gene polynucleotide sequence, an allelic variant or a fragment thereof, optionally linked to a selected heterologous gene sequence.

28. The virus according to claim 27, which is a poxvirus selected from the group consisting of a vertebrate poxvirus, orthopoxvirus, suipoxvirus, vaccinia virus and entomopoxvirus.

29. A cell infected with a recombinant virus comprising an Entomopoxvirus spheroidin gene

polynucleotide sequence, an allelic variant, or a

fragment thereof, optionally linked to a selected

heterologous gene sequence.

30. The cell according to claim 29 selected from the group consisting of insect cells and mammalian cells.

31. A cell infected with a recombinant virus comprising an Entomopoxvirus thymidine kinase gene polynucleotide sequence, an allelic variant, or a

fragment thereof, optionally linked to a selected

heterologous gene sequence.

32. The cell according to claim 31 selected from the group consisting of insect cells and mammalian cells.

33. A method for producing a selected polypeptide comprising culturing a selected host cell infected with a recombinant virus comprising an

Entomopoxvirus thymidine kinase gene polynucleotide sequence, an allelic variant, or a fragment thereof, operably linked to a heterologous gene sequence encoding said selected polypeptide, and recovering said

polypeptide from the culture medium.

34. A method for producing a selected

polypeptide comprising culturing a selected host cell infected with a recombinant virus comprising an

Entomopoxvirus spheroidin gene polynucleotide sequence, an allelic variant, or a fragment thereof, operably linked to a selected heterologous gene sequence encoding said selected polypeptide, and recovering said

polypeptide from the culture medium.

35. A method for screening recombinant host cells for insertion of heterologous genes comprising transforming said cells with a polynucleotide molecule comprising the selected heterologous gene sequence inserted into the polynucleotide sequence encoding entomopox spheroidin, wherein the absence of occlusion bodies normally formed by the expression of the

spheroidin protein indicates the integration of the heterologous gene.

36. A method for screening recombinant host cells for insertion of heterologous genes comprising infecting said cells with a polynucleotide molecule comprising the selected heterologous gene sequence inserted into the polynucleotide sequence encoding entomopox thymidine kinase, wherein the presence of thymidine kinase function formed by the integration of the inactive thymidine kinase sequence indicates the insertion of the heterologous gene.