AU663709B2 - Entomopoxvirus expression system comprising spheroidin or thymidine-kinase sequences - Google Patents

Description

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ANNOUNCEMENT OF THE LATER PUBUCATION OF INTERNATIONAL SEARCH REPORT INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATEN 1 CUUtbRATION TREATY (PCT) (51) International Patent Classification 5 (1i) International Publication Number: WO 92/14818 C12N 15/00 A3 (43) International Pubiication Date: 3 September 1992 (03.09.92) (21) International Application Number: PCT/US92/00855 (74) Agents: SALIWANCHIK, David, R. et al.; Saliwanchik Saliwanchik, 2421 N.W. 41st Street, Suite A-1, Gaines- (22) International Filing Date: 12 February 1992 (12.02.92) ville, FL 32606 (US).

Priority data: (81) Designated States: AT (European patent), AU, BE (Euro- 657,584 19 February 1991 (19.02.91) US pean patent), CA; CH (European patent), DE (Euro- 827,685 30 January 1992 (30.01.92) US pean patent), OK (European patent), ES (European patent), FR (European patent), GB (European patent), GR (European patent), IT (European patent), JP, KR, LU (71) Applicant (for all designated States except US): UNIVERSI- (European patent), MC (European patent), NL (Euro- TY OF FLORIDA [US/US]; 186 Grinter Hall, Gaines- pean patent), PL, SE (European patent), US.

ville, FL 32611 (US).

(72) Inventors; and Published Inventors/Applicants (for US only) MOYER, Richard, W. With international secrch report.

[US/US]; 1225 N.W. 23rd Terrace, Gainesville, FL Before the expiration of the time limit for amending the 32605 HALL, Richard, L. [US/US]; 3611 S.W. claims and to be republished in the event of the receipt of 34th Street, Apt. 178, Gainesville, FL 32608 amendments.

GRUIDL, Michael, E. [US/US]; 211 Alhambra, Columbia, MO 65203 (88) Date of publication of the international search report: December 1992 (10.12.92) (54)Title: ENTOMOPOXVIRUS EXPRESSION SYSTEM COMPRISING SPHEROIDIN OR THYMIDINE-KINASE SE-

QUENCES

(57) Abstract The subject invention pertains to 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 provided herein, as well as methods for use thereof in the expression of heterologous proteins in both insect and mammalian host cells.

AmEPV Hind I11 map: C I B A F IIJ G D HI E 0.5 kb 3 H B BB S1 II

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I-1 307 bp 80 bp 1.88 kb E 4504 4811 4691 G1L G2R G4R 1.4 kb 0.7 kb 0.5kb 0.2kb G5R 3.0 kb Spheroidin G6L Vaccinia INTPase I Vaccinia ORF 17 Capripoxvirus HM3 Amino Acid Homologies r- (sl(IIIIY*XI3rl i WO 92/14818 PCT/US92/00855 1 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, 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. Granados et al, "Replication of Amsacta moorei Entomopoxvirus and Autographa californica ruclear Polyhedrosis Virus in Hemocyte Cell Lines from Estigmene acrea", in Invertebrate Tissue Culture Applications in Medicine, Biology, and Agriculture, E. Kurstak and K. Maramorosch 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.

SUBSTITUTE SHEET WO 92/14818 PCI/US92/00855 2 Mitsuhashi 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) H. R. Langridge et al, Virology, 76:616- 620 (1977)]. Recently, a series of restriction maps for AmEPV were published L. Hall et al, Arch. Virol., 110:77-90 (1990)]. No DNA homology to vaccinia has been detected 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 4 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 SUBSTITUTE

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f p.- WO 92/14818 PCT/US92/00855 3 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 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, 88:5364-5368 (1982)]. Numerous viral antigens have been expressed using vaccinia virus vectors Paoletti et al, Proc. Natl. Acad. Sci. USA, 81:193- 197 (1984); A. Piccine et al, BioEssavs, 5:248-252 (1986)] including, among others, HBsAg, rabies G protein Sand the gpl20/gp41 of human immunodeficiency virus (HIV).

Regulatory sequences from the spruce budworm EPV have been used previously with vaccinia Yuen et al, Viroloqy, 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 cellmediated 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 SUBSTITUTE SHEET Ar~ WO 92/14818 PCT/US92/00855 4 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 SUBSTITUTE

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WO 92/14818 PCT/US92/00855 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 4 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 SUBSTITUTE

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L, t r WO 92/1L h s 81, PCT/US92/00855 eterologous gene is flanked on both termini by tk equences.

ILl 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, 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 30 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 3 SUBSTITUTE SHEET WO 92/14818 PCT/US92/00855 7 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, 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 HindIII-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:ll], 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 N0:17], RM03 [SEQ ID NO:18], RM04 [SEQ ID NO:19], and RM129 [SEQ ID SUBSTITUTE

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r WO 92/14818 PC/US92/00855 8 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 Sfrom 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.

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i i WO 92/14818 PCT/US92/00855 9 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 spheroidir 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 sphe;roidin. 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 througn 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 Lhe 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 (I7) 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 fragmei.t which relates to the AmEPV G6L ORF [SEQ ID NO:7] S. Broyles et al, J. Virol., 61:1738-1742 (1987); and J. F.

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WO 92/14818 PCT/US92/00855 SRodriguez 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 L;ale, 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. 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 SUBSTITUTE SHEET h 1 WO 92/14818 PCT/US92/00855 11 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 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, 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 SUBSTITUTE

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i I WO 92/14818 PCT/US92/00855 12 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, 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 (naturallyoccurring 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 Iigs. 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.

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nj :Liri k 113 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, 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 C activity and preferably have a homology to Fig. 2 cr 3, respectively, of at least 80%, more preferably 90%, and most preferably 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 SUBSTITUTE

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I i i. 1-.:11 i ii ii !i I WO 92/14818 PCT/US92/00855 14 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 The relatedness of the vertebrate tk proteins to AmEPV is still lower (39.3 to while African Swine Fever (ASF) showed the least homology of all the tk proteins tested 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 AmiPV 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.

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WO 92/14818 PCT/US92/00855 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: acidic aspartate, glutamate; basic lysine, arginine, histidine; non-polar alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and 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 3.eucine 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 '1 SUBSTITUTE

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Sknown in the art, both natural. 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, in vaccinia vectors. Methods for the construction of expression systems, in general, and the components SUBSTITUTE SHEET WO 92/14818 PCT/US92/00855 17 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.

SUBSTITUTE SHEET ill iii-I-il-..'-i--liil^i r l 'r WO 92/14818 PCT/US92/00855 18 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 gele, 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.

SUBSTITUTE SHEET L I WO 92/14818 PCT/US92/00855 19 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 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, 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 SUBSTITUTE

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p 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 (Stratagene) pMEG-tkl ATCC 68532 26 February 1991 E. coli SURE strain (Stratagene) pRH512 ATCC 68533 26 February 1991 E. coli pRH7 ATCC 68902 28 January 1992 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 IG:\WPUSER\LIBVVIO 1 26:GSA 20 of 2

L~

WO 92/14818 PCT/US92/00855 21 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, 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 SUBSTITUTE SHEET

L

r P i -L SWO 92/14818 PCT/US92/00855 22 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, 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.

SUBSTITUTE SHEET L WO 92/14818 PCT/US92/00855 23 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.

SUBSTITUTE SHEET i i WO 92/14818 PCT/US92/00855 24 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 s:itably 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 1 or EMV tk gene, or a fragment thereof, is linked to a Sheterologous gene. The polynucleotide sequence further contains a flanking region on either side of the spheroidir-heterologous gene or tk-heterologous gene to enable ready transfer into a selected virus. This resulting construct is termed a cassette. Such a SUBSTITUTE

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III I WO 92/14818 PCT/US92/00855 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 4 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 SUBSTITUTE

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WO 92/14818 PCT/US92/00855 26 or a portion of the EMV sphercidin 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 SUBSTITUTE

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n WO 92/14818 PCT/US92/00855 ii 27 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 piopagated 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 exampl-, 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 spi.iroidin 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 f-interferon (IFN-0) synthesis as the heterologous gene. A DNA fragment containing the IFN-0 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.

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I O .r WO 92/14818 PCT/US92/00855 28 The insertion of the IFN-3 gene produces a hybrid or fused spheroidin-IFN-0 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-0 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- gene. The hybrid spheroidin-IFN-0 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 -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 SApproach, 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 wildtype AmEPV by homologous recombination between the recombinant shuttle vector and AmEPV DNA. Accordingly, a SUBSTITUTE

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_J i i ^B i^ ^B B I^ WO 92/14818 PCT/US92/00855 29 mixture is produced comprising wild-type, nonrecombinant EPVs and recombinant EPVs capable of expressing the IFN-0 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-f 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 0-galactosidase gene to facilitate color selection. This procedure involves the incorporation of the E. coli 0-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 SUBSTITUTE

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r I-

I

i •1 i WO 92/14818 PCr/US92/00855 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 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 SUBSTITUTE

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I

WO 92/14818 PCr/US92/00855 31 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: Virus family: Sites for insertion of foreign genes Shuttle possibilities between vertebrate and insect systems: cytoplasm nucleus Poxviridae spheroidin thymidine kinase (tk) yes (Orthopoxviruses) (Leporipoxviruses) (Suipoxviruses) (Avipoxviruses) Baculoviridae polyhedrin pl0 No mammalian counterparts.

Baculovirus is not known to contain a tk gene.

Polyhedrin is not found in mammalian systems.

SUBSTITUTE SHEET WO 92/14818 PCT/US92/00855 32 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 containi'ij 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 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.

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WO 92/14818 PCT/US92/00855 33 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 em ,loying 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 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.

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WO 92/14818 PCT/US92/00855 34 In addition to the novel Entomopoxvirus expression vectors and methods for their use described herein, the subject invention pertains to the u:;e 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, SBeverly, 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.

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7 WO 92/14818 PCT/US92/00855 EXAMPLE 1: PREPARATION OF AmEPV DNA The replication of AmEPV has been described previously H. Goodwin et al, J. Invertebr. Pathol., 56:190-205 (1990)]. The gypsy moth (Lymantria 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 0

C

in EX-CELL 400 [JRH Biosciences, Lenexa, KS] supplemented with 10% fetal bovine serum, 100 U of penicillin, and 100 ug 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 -700C 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 x g for 5 minutes, and the supernatant was passed through a 0.45-Am-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 x g for 5 minutes, rinsed, and resuspended in modified SUBSTITUTE SHEET WO 92/14818 PCT/US92/00855 36 Hank's phosphate-buffered saline (PBS), which contained g of glucose per liter, but no Ca 2 or Mg".

For embedding of the infected cells in agarose plugs, 1% SeaPlaque GTG agarose (prepared in modified Hank's PBS and equilibrated at 37 0 C) was mixed 1:1 with infected cells to yield 5 x 106 cells per ml in 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 500C for 2 days L. Smith et al, Methods Enzvmol., 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 40C. 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 B.

7llington 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-cellculture supernatant. The supernatant from postinfection cell cultures was clarified by centrifugation at 200 x g for 5 minutes. Virus was collected from the supernatant by centrifugation at 12,000 x g. Viral pellets were resuspended in 6 ml of Ix TE. DNase I and RNase A (10 and 20 Ag/ml final concentrations, respectively) were added, and the mixture Swas incubated at 37 0 C for 30 minutes. The mixture was heated to 50 0 C for 15 minutes. SDS and proteinase K (1% Sand 200 Ag/ml, respectively) were then added. The sample was incubated to 50 0 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 Ix TE (pH 8).

SUBSTITUTE SHEET h WO 92/14818 PCT/US92/00855 37 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 0

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 0 C was added to the monolayer. Plaques were visualized after days of incubation at 26 0 C by inspection with a stereomicroscope.

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

EXAMPLE 2 ISOLATION OF THE SPHEROTIIN 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) K. Laemmli, Nature (London), 227:680-685 (1970)] with a 4% acrylamide stacking gel and a 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 0 C. For sample preparation, 2x Laemmli sample buffer consisting of 125 mM Tris-HCl (pH 4% SDS 10% /-mercaptoethanol and glycerol was used.

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i f I WO 92/14818 PCT/US92/00855 38 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 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 d.Lfluoride (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 Sanger et al, Proc. Natl.

Acad. Sci. USA, 74:5463-5467 (1977)] with [a- 35 S]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.

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SI1 WO 92/14818 PCT/US92/00855 39 SA 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 BqlII 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 BclII 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.

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WO 92/14818 PCT/US92/00855 Among the fragments produced from the restriction enzyme digestions of the genomic DNA was a I 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 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 [a- 2 P]dCTP by the random oligonucleotide extension method P. Feinberg et al, Anal. Biochem., 132:6-13 (1983)] or with [y- 32 P]ATP and T4 polynucleotide kinase [Sambrook et al, supra]. 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 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 0 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 BclII fragment and the EcoRI-D fragment of AmEPV DNA [See Fig. A plasmid produced by the shotgun cloning, recombinant pRH512 (a BqlII 4.56 kb fragment SUBSTITUTE

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c WO 92/14818 PCT/US92/00855 41 inserted into the BamHI site of pUC9 which contains about 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 -k to various AmEPV genomic digests as follows. The DNA-DNA hybridization was done at 65 0 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 0 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 PstI-A, and XhoI-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 3glII 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 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 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 a-thiophosphate dNTPs 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 SmaI, and treated with exonuclease III. Samples were removed every 30 seconds, re-ligated, SUBSTITUTE SHEET iI SWO 92/14818 PCT/US92/00855 42 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 SmaI-cut M13mpl9 [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, Sambrook et al, supra].

Sequencing of the BqlII insert of pRH512 isolated it to nucleotides 0 to 4505, thus extending the sequence 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 SmaI-digested, phosphatasetreated vector M13mpl9. Preparations of M13 virus and DNA were made by standard r:-ocedures 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.

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7f WO 92/14818 PCT/US92/00855 43 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 DraI clone from nitrocellulose filter replicas (plaque lifts) [Micron Separations, Inc.] of the M13 shotgun library of DraI-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 (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) 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 0 C for denaturation, 30 seconds at 37 0 C for annealing, and minutes at 72 0 C for extension. Finally, the samples were incubated at 72 0 C to 8.5 minutes to complete extensions.

The concentration of each primer was 1 AM.

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 SUBSTITUTE

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WO 92/14818 PCr/US92/00855

I

I

the new sequence as it was identified. The sequencing process employed Sequenase, 5 pmol of each primer, and 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 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, pRH827 (307 bp), pRH85 (1.88 kb), and pRH87 (1.88 kb) from the BglII fragment library. Plasmids 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 BqlII DNA insert in opposite orientations, but also revealed a missing 80 bp between pRH827 and pRH85. This 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 Therefore, all sequences obtained from protein SUBSTITUTE

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1 WO 92/14818 PCT/US92/00855 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 0 C. Total RNA from the infected cells was isolated by the guanidinium thiocyanate-cesium chloride procedure M. Chirgwin et al, Biochemistry, 18:5294- 5299 (1979)].

Primer extension reactions were carried out with primer RM165 [SEQ ID NO:17], a 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 [7- "P]ATP and T4 polynucleotide kinase and purified on a "spun column" [Sambrook et al, supra]. For annealing, gg of total infected-cell RNA and 106 cpm of radiolabeled primer were coprecipitated with ethanol. The pellet was resuspended in 25 Al of hybridization buffer formamide, 40 mM piperazine-N,N'-bis(2-ethanesulfonic SUBSTITUTE

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WO 92/14818 PCT/US92/00855 46 acid) (pH 400 mM NaCl, 1 mM EDTA (pH denatured at 72 0 C for 15 minutes, and incubated at 300C for 18 hours.

For primer extension, the RNA-primer hybrids were ethanol precipitated, resuspended, and used for five individual reactions. Each reaction contained 8 Ag of total infected-cell RNA, 50 mM Tris-HCl, (pH 50 mM KC1, 10 mM dithiothreitol, 10 mM MgCl 2 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 pl of 5 mM dNTP mixture and 1 1l of 20-U/gl reverse transcriptase) was added to each reaction mixture, which was then incubated for an additional 30 minutes at 420C. 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. i 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 SUBSTITUTE

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SWO 92/14818 PCT/US92/00855 47 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 Spolypeptides encoded by ORF G2R [SEQ ID NO:23] or G3L [SEQ ID NO:26]. ORF G1L [SEQ ID N0:25], 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 SUBSTITUTE

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i iiiiiiii__-_ WO 92/14818 PCT/US92/00855 48 CbEPV [Yuen, L. et al, Virol., 182:403-406 (1991)], another insect poxvirus.

Example 8 Isolation and Sequencing of the AmEPV EcoRI-Q 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 (RM03 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, Viroloqv, 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 RM03 and RM04 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 SUBSTITUTE SHEET

S*

WO 92/14818 PCT/US92/00855 49 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-l. 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 Q1 [SEQ ID NO:31] potentially ancodes 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 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.

SUBSTITUTE SHEET -q WO 92/14818 PCT/US92/00855 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 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. Esposito et al, Virol., 135:561-567 (1984)]; capripoxvirus 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 F. Lin et al, Mol Cell. Biol., 5:3149-3156 (1985)]; the tk of chicken J. Kwoh et al, Nucl. Acids Res., 12:3959-3971 (1984)]; ASF 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. 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 J SUBSTITUTE SHEET 1 o WO 92/14818 PCT/US92/00855 51 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 B-galactosidase gene driven by the vaccinia P, promoter (P,-Lac Z cassette) inserted into the viral tk gene. While VSC8 contains an inactive tk gene due to the insertion of the B-galactosidase, portions of the vaccinia tk sequence remain. VSC8 is thus tk- and, upon staining with X-Gal (5-bromo-4-chloro-3-indoyl-B-Dgalactopyranoside), will form blue plaques (8galactosidase positive).

Cells were grown to 80% confluence (4 x 106 per 60 mm dish). Lipofectin solution (20 pg of Lipofectin in of dHO) was added to 10ig plasmid DNA (pHGN3.1/AmEPV EcoRI-Q) in 501 of dHO and incubated for 15 minutes at room temperature. After a 2 hour period of viral adsorption of 2, 37 0 the monolayers were washed three times with serum-free OptiMEM. Three milliliters of serum-free OptiMEM was then added to each mm dish. The Lipofectin/DNA mixture was slowly added dropwise with gentle swirling and incubated an additional 12 to 18 hours at 37 0 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.

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WO 92/14818 PCT/US92/00855 52 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 PstI-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 arowth temperature for AmEPV in the laboratory is 28 0 C, whereas that of the vertebrate poxviruses is 37 0 C. As described-herein, when the AmEPV SUBSTITUTE SHEET WO 92/14818 PCT/US92/00855 53 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 0 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.

SUBSTITUTE SHEET WO 92/14818 PCT/US92/00855 54 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: University of Florida (ii) TITLE OF INVENTION: Novel Entomopoxvirus Expression System (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: David R. Saliwanchik STREET: 2421 N.W. 41st Street, Suite A-1 CITY: Gainesville STATE: Florida E) COUNTRY: U.S.A.

ZIPt 32606 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 07/657,584; US 07/ FILING DATE: 19-FEB-1991; 30-JAN-1992 (viii) ATTORNEY/AGENT INFORMATION: NAME: Saliwanchik, David R.

REGISTRATION NUMBER: 31,794 REFERENCE/DOCKET NUMBER: UF/S&S-114.C2 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (904) 375-8100 TELEFAX: (904) 372-5800 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 6768 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: ORGANISM: Amsacta moorei entomopoxvirus (ix) FEATURE: NAME/KEY: CDS- LOCATION: complement (65..1459) (ix) FEATURE: NAME/KEY: CDS LOCATION: 1474..2151 SUBSTITUTE SHEET r

I

f WYO 92/14818 PCr/US92/00855 (ix) FEATURE:

NAME/KEY:

LOCATION:

(ix) FEATURE:

NAME/KEY:

LOCATION:

(ix) FEATURE:

NAME/KEY:

LOCATION:

(ix) FEATURE:

NAME/KEY:

LOCATION:

CDS

complement CD S 2502. .2987

CDS

3080. .6091

CDS

complement (2239. .2475) (6277. .6768) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

AGATCTGATG

GATATTAAAT

TTTTTATTTT

ATTCCATAAA

TACTATTAAT

CACCAATAAA

AATAAA'ITAT

GTAATTCAAA

CTGATAGTAC

TATGGCTTTT

GTTCTGGTAT

GTTTATCTAT

TTTTTTTATC

ATGAAGCTAC

CATAATCTGT

ACCATGTAGA

GCGGTATTCC

TTGTTTTGGA

TATATTTTTT

TTATAGTCCA

TATGATTTGG

ATGTTACTTT

CATTAGTATT

TTCTATATAT

ATTAGATTCT

ACTACATACA

CTTTTTATTT

ATCATATCTA

ACTCATAGAA.

ATAATATAAA

AGTATTAAAA

ATCAATGTAT

TATAGTCATA

ATAACCACTA

TACGCAACAA

ATTCATTAAA

TGTAGCAATA

ATTAATCATA

TTTATCATGT

TATTTGAGCA

TAATTCTGAT

AGATGTAATA

AAATAGAAAA

ATCTAAAACT

ACCTCTTGTT

ATATTTAATA

AGTACAAATT

AAACTATTCT

TTCATAATTC

TTTATATTAA

GAAATAAATA

TAATATAATT

AATATTATAT

XAGTAATAAT

AAATAAGCAT

TCAGATTCAA

CTATTAAAAA

GTAAAATGAT

AAAAATTTTA

TTTTTATCAG

GTTAATGGAT

TTTCTTGGGT

TCCAAATCAT

TTTAGAAAAG

AAAGTATTAA

AATTTTCTTT

AATTCATTAT

TCATCATCAT

GGATTTATAA

TGTATGATTA

TCTCATTATC

TATTACTATT

ATATTTCTAA

ATGCACCTCT

TTTTAAATTC

TAAACATACC

TTACATTTTT

AATTAGTATT

TAAACATATA

AGTATGCAGC

CATTATAAAT

CTCTATCTTC

TTTTTTCAAA

ATATACAATT

AAGCTTTAGG

CATAAATTGT

ACTTTCTCAT

TATTTATATT

TAATATTATT

ATAATATTTC

CATCTATTTT

AATATACCAT

ATTGATATTT

AATATAACTA

TTTTTTATAC

TGTATTTTTA

ATAACTACTA

AA.ATTTAGAT

ACAATCGGGA

AAATATATCA

AGGAGTACTA

TTTTTTATTT

TTTTTTATCT

TATAGGA.AAC

AAGTTTATAG

TAAAATCAAA

ATATATATCT

TTTAGGATTA

GGCAAATGTA

ATATACTAAT

TTTATCTTGT

TTCAAAATTA

CAAGTATTTT

TTCTAATATA

ATTATCTATT

TAAAATTCAA

TCATAATCAT

ATATOTATTA

AATTCGTCAA.

GCCAATAAAT

TTTATGTTGA

CIATCATATT

TTTAAATATT

TTGTAGTGTT

TGTTTTATAA

TTATCAAAGT

ATAAAAAATC

CATCTCATAG

TGAAAATAAT

CCCGAACTTA

AATCCCAAAG

GAAAAATCTC

GGAATGCCTT

AAATATTTTT

ATATTATTAA

ATAGGTATAA

GCTATATTTG

TTACTAAAAA

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 SUBSTITUTE

SHEET

~1

F

WO 92/14818 PCT/US92/00855 ATAACATAGA CATAAAATTA ATACCAGATT CTGGCATTTT TAAATTTTTA TTTGGAAATC TTCTAATTTT ATTATTCATT ATTTATTTAA TAA ATG TTT CTA GTT TAT TTC AAT Met Phe Leu Val Tyr Phe Asn 1'

I

ACA

Thr

TTA

Leu

TAT

Tyr

AAT

Asn

AAT

Asri

ATA

Ile

GCG

Ala

AAT

Asn 120

ATA

Ile

CAA

Gin

TCT

Ser

AAT

Asn

GTG

Val 200

AAC

TTT

Phe

ACA

Thr

ATA

Ile

TTA

Leu

AAT

Asn

TTT

Phe

TCT

Ser 105

ATA

Ile

GTT

Val

AAT

Asn

AGA

Arg

ATA

Ile 185

AGA

Arg

GTA

TTA

Leu

TTT

Phe

TTA

Leu

TAC

Tyr

AAT

Asn

AAT

Asn

AGT

Ser

AAT

Asn

TTC

Phe

AGT

Ser

TAT

Tyr 170

TCT

Ser

TTA

Leu

GGA

ATA

Ile

GTG

Val

TCA

Ser

GAT

Asp

CTT

Leu

GTA

Vai

AAT

Asn

AGA

Arg

AAT

Asn

GAT

Asp 155

AAT

Asn

ATT

Ile

ATA

Ile

ACA

TTA

Leu

ATA

Ile

GCA

Ala

GAT

Asp

ACA

Thr

ATT

Ile

AAT

Asn 110

TTT

Phe

GAA

Glu

GAT

Asp

GTA

Val

ATA

Ile 190

TAT

Tyr

GTC

TTA

Leu

GAT

Asp

ACT

Thr

ATT

Ile

CAA

Gin

ATA

Ile

ATA

Ile

TTA

Leu

ACT

Thr

ATA

Ile

ATT

Ile 175

TTA

Leu

AAC

Asn

ATA

Gly

TTA

Leu

ATA

Ile

TTT

Phe

AAT

Asn

AAT

Asn

TTA

Leu

TTT

Phe

CAC

His 145

TCA

Ser

GGA

Gly

AAT

Asn

ATA

Ile

CCA

Ile

AAT

Asn

AAT

Asn

TTT

Phe

GAT

Asp

TTT

Phe

AAA

Lys

TCT

Ser

AGT

Ser

TTT

Phe 165

GTA

Vai

ATA

Ile

TTA

Ile

ATA

11ie

TTA

Leu

ATA

Ile

CCA

Pro

TCA

Ser

TTA

Leu

GCT

Aia 135

AAT

Asn

AAA

Lys

AAT

Asn

AAT

Asn

ATC

GGT ATT ATA GGT ATT TAT ATA 1440 1494 1542 1590 1638 1686 1734 1782 1830 1878 1926 1974 2022 2070 2118 2168 2228 Ile Ser Phe Leu Asn Ile 210 215 TAATATTTAG TAA'±AATCAC Asn Val Gly Thr Leu Phe Vai Ile Asn Pro TAACATATTT TTTATTAAJ TGAATAAAAT ATATATTGTT ATTGTCAATA TTTTATATCA SUBSTITUTE SHEET WO 92/14818 1 WO 9214818PCr/US92/00855 TTTTACAGTC TTATTTTTTT TTTTTGCTTT TAOOTATAAT TTTACCTTCT AAACGTTTAT CTCCCCAAAC ATCTACAGTA GATGGTTTAT TAGATTCTGT OTTATACACA TCTOCTGGAT TTGCGGCATT TGTATCCAAA CCATAATATC CAGGTCTATA ATTATCTTTA AAAACTTGGG ATTOAGATAC TTCTTCAGTT TTTAAATTAT TAAAATATCC AAGATTATTT TTTTTTGATG AAGACATAAT TOATATTATA ATACTTTATA OAT ATO TCA ATA TTT ATC TAC TAT Met Ser Ile Phe Ile Tyr Tyr 1

ATT

Ile

ATT

Ile

CAA

Gin

GAT

Asp

GAT

Asp

GA.A

Giu

TCA

Ser

TAT

Tyr 120

AAA

Lys

TTA

TTC

Phe

TTA

Leu

TTA

Leu

ACA

Thr

GAT

Asp

AAA

Lys

ACT

Thr 105

ACA

Thr

ATA

Ile

TTA

AAC

Asn

OTT

Val

TGG

Trp

TAT

Tyr

TTA

Leu

TOO

Trp

TTT

Phe

AAT

Asn

GTA

Vali

AGA

TTT

Phe

TTA

Leu

GCC

Ala 45

TTA

Leu

TTT

Phe

OCT

Ala

TTA

Leu

OAT

Asp 125

CCT

Pro

ATO

TAT

Tyr

ATA

Ile

OAA

Glu

CAA

Gin

OAT

Asp

TCA

Ser

ACT

Ser 110

TOT

Cys

TOT

Cys 0CC

ATA

Ile 15

ACA

Thr

AAT

As n

TTT

Phe

CCC

Pro

ACT

Thr 95

ACA

Thr

ATT

Ile

ACT

Thr

AAT

TAT

Tyr

ACA

Thr

TAC

Tyr

OCO

Ala

AAC

Asn

AAT

Asn

OAA

Oiu

ATA

Ile

OTC

Val

ACA

AAA

Lys

OCA

Al a

OAA

Oiu

AGA

Arg

OAT

Asp

AAT

Asn

ACT

Ser

OAT

Asp

OA.A

Oiu 145

TCA

ATO AAT ACT OTA CAA Asn

TTT

Phe

TTA

Leu

AAT

Asn

TTT

Phe

TTT

Phe 100

ATT

Ile

TCT

S er

AAC

Thr

CTA

Leu

AGA

Arg

ATA

Ile

AAT

Asn

TAT

Tyr

AAT

Asn

AGA

Arg

OAT

Val

OTT

Val

TAT

Tyr

AAT

Asn

OTT

Val

OCA

Ala

TTA

Leu

ATT

Ile

TOT

Gin

TTT

Phe

AAT

Asn

TTT

Phe

OAA

Oiu

OTT

Val

ACA

Thr

ATA

Ile 135

AGA

2288 2348 2408 2468 2522 2570 2618 2666 2714 2762 2810 2858 2906 2954 3004 3064 3115 3163 3211 Ser Asn Asp Cys Arg 150 TAAATACATT ATAATATTAT Leu Leu Arg Leu Leu Met Ala Asn Thr Ser TATAATATCA ATCATAATTT TTATATATAT TTTATCTAAA AGGOTTTTT ATTTTTTATA TATTAATAAT AATAA ATO ACT AAC OTA CCT TTA GCA ACC AAA ACA ATA AGA Met Ser Asn Val Pro Leu Ala Thr Lys Thr Ile Arg 1 5 AAA TTA TCA AAT COA AAA TAT OAA ATA AAO ATT TAT TTA AAA OAT OAA Lys Leu Ser Asn Arg Lys Tyr Oiu Ile Lys Ile Tyr Leu Lys Asp Oiu 20 AAT ACT TOT TTC OAA COT OTA OTA OAT ATO OTA OTT CCA TTA TAT OAT SUBSTITUTE SHEET f i: i i L~ i WO 92/14818 PC/US92/00855 Asn

GTC

Val

ATA

Ile

GGC

Gly

GTA

Val

AAT

Asn

ACT

Thr 125

TTT

Phe

GAA

Glu

ATT

Ile

TAT

Tyr

ATT

Ile 205

GAT

Asp

GAT

Asp

GGA

Gly

ATT

Ile Thr

TGT

Cys

GAA

Glu

GAT

Asp

AAC

Asn

GTA

Vai 110

CTT

Lev

ATT

Ile

AAT

Asn

ACT

Thr

TTA

Leu 190

ACT

Thr

ATA

Ile

TTA

Leu

TTA

Leu

CAA

Gin 270 Phe

GAA

Glu

ATT

Ile

TTA

Leu

GCC

Ala

CAT

His

GGA

Cly

TTT

Phe

CAA

Gin 160

TTT

Phe

GAA

Glu

GAT

Asp

TAC

Tyr

AAG

Lys 240

TAT

Tyr

GAA

Glu Glu

ACT

Thr

GAA

Glu

AAA

Lys

CAA

Gin

AAT

Asn

AAA

Lys

GTC

Vai 145

AAT

Asn

CCA

Pro

AAA

Lys

TTT

Phe

GAA

Glu 225

AGA

Arg

GCT

Ala

AGG

Arg Val

TCA

Ser

AGA

Arg

TTG

Leu

CGA

Arg

GCT

Ala 120

ATC

Ile

CGA

Cly

AAA

Lys

TTA

Leu

ACA

Thr 200

GCA

Ala

AAA

Lys

TAT

Tyr

AAT

Asn

GTT

Val 280 Leu

AGT

Ser

AAA

Lys

CCG

Pro

TTG

Leu

TTT

Phe

CAA

Gin

ATT

Ile

GCA

Ala 170

AGA

Arg

GCA

Ala

TCA

Ser

TTA

Leu

GCA

Ala 250

GAT

Asp

GCA

Ala Tyr

CCA

Pro

GTT

Val

TGT

Cys

CTA

Leu

AGA

Arg

CCC

Pro

ACT

Thr 155

TCA

Ser

AAA

Lys

ACT

Thr

TTC

Phe

TTA

Leu 235

GAT

Asp

ACT

Thr

TAT

Tyr Asp

AAT

Asr

CAC

His

AAT

Asn

GAT

Asp

CTG

Leu

CTA

Leu 140

AAC

Lys

TTT

Phe

CTA

Val

AAT

Asn

CTT

Leu 220

AAA

Lys

ACT

Thr

GTG

Val

ACT

Thr 3259 3307 3355 3403 3451 3499 3547 3595 3643 3691 3739 3787 3835 3883 3931 SUBSTITUTE SHEET 1 1

I

i i. WO 92/14818 PCT/US92/00855

AAG

Lys 285

ACA

Thr

ACG

Thr

ATG

Met

CTG

Leu

TCG

Ser 365

AAC

Asn

TGC

Cys

TGT

CyB

GAT

Asp

AAA

Lys 445

ACT

Thr

CTT

Leu

AAA

Lys

CCA

Pro

GAT

Asp 525

;AT

%sp

GAA

lu

CTC

Leu

CAT

His

GAA

Glu 350

AGA

Arg

GAC

Asp

GGA

Gly

AAA

Lys

ACT

Thr 430

GTA

Val

TCT

Ser

GAC

Asp

CAA

Gin

AAT

Asn 510

ATG

Met

GAA

Glu

GTA

Val

AGA

Arg

AAC

Aen 335

TTG

Leu

TTA

Leu

OAA

Glu

AGA

Arg

TAT

Tyr 415

ACG

Thr

CCC

Pro

AGA

Arg

GTT

Val

CAT

His 495

TGC

Cys

AAT

Asn

AAT

Asn

GAC

Asp

GTA

Val 320

AGA

Arg

TCT

Ser

GAT

Asp

AAT

Asn

CAA

Gin 400

ACT

Thr

ATT

Ile

AAA

Lys

TTT

Phe

AGG

Arg 480

TAT

Tyr

ACA

Thr

GAT

Asp

GGT

Gly

CCC

Pro 305

TAT

Tyr

GGA

Gly

AAT

Asn

OTT

Val

AAA

Lys 385

GTA

Val

CCC

Pro

CAC

His

AAT

Asn

ATT

Ile 465

CTT

Leu

ACT

Thr

TTA

Leu

AAC

Asn

CGC

Arg 290

GAG

Glu

AAA

Lys

AGT

Ser

OTA

Vai

GGT

Gly 370

ATT

Ile

TTC

Phe

AAA

Lye

TTA

Leu

TTA

Leu 450

AAA

Lye

AAT

Asn

AAT

Asn

TCA

Ser

AAA

Lys 530

AAT

Asn

TAT

Tyr

AAG

Lye

GGT

Gly

AAA

Lye 355

ATT

Ile

ATA

Ile

CAC

His

TGT

Cys

TAT

Tyr 435

AGA

Arg

CAT

His

AGA

Arg

GTA

Val

TTG

Leu 515

ACT

Thr

CTA

Leu

GTA

Val

TTT

Phe

AAT

Asn 340

GGA

Gly

TAC

Tyr

TTG

Leu

GAA

Glu

CCA

Pro 420

GGT

Gly

CTT

Leu

ATG

Met

AAT

Asn

ATT

Ile 500

GGT

Gly

ATA

Ile

TGT

Cys

ACT

Ihr

OAT

Asp 325

GTA

Vai

TAT

Tyr

AAA

Lye

GAA

Glu

CGT

Arg 405

TTC

Phe

ATT

Ile

TGG

Trp

OCT

Ala

OAT

Asp 485

ATA

Ile

AAT

Asn

TCT

Ser

TTO

Leu

AGT

Ser 310

AAA

Lys

TTT

Phe

CCA

Pro

ITA

Leu

OAA

Glu 390

OTA

Val

CAA

Gin

TCI

Ser

GGA

Gly

OAT

Asp 470

ATA

Ile

TTA

Leu

AAT

Asn

GAO

Glu

ATO

Met 295

AAT

Asn

TCT

Ser

CCA

Pro

OTT

Val

AAT

Aen 375

ATI

Ile

AAA

Lys

TTT

Phe

AAT

Asn

TGG

Trp 455

GGA

Gly

TGT

Cys

GAO

Glu

AGA

Arg

TAT

Tyr 535

AAA

Lys

AAT

Asn

CAT

His

TTA

Leu

AAA

Lys 360

AAA

Lys

GAA

Glu

CTT

Leu

OTT

Val

OTT

Val 440

ATT

Ile

TCT

Ser

TTA

Leu

TAC

Tyr

TTT

Phe 520

ACT

Thr ATA ACA Ile Thr OCT TTA Ala Leu TTA AAA Leu Lys 330 AGA TCA Arg Ser 345 OCA ICT Ala Ser ATT TAT Ile Tyr OCA GAA Ala Glu AAT AAA Asn Lys 410 OTA AAC Val Asn 425 TGT TTA Cys Leu TTA OAT Leu Asp OAT OAT Asp Asp AAA CAA Lys Gin 490 OCA AAT Ala Asn 505 AAT AAT Asn Asn AAC TTT Asn Phe

TCT

Ser

TTG

Leu 315

ATT

Ile

TTA

Leu

OAT

Asp

OTA

Vai

TAT

Tyr 395

CAC

His

AGC

Ser

AAA

Lys

TGC

Cys

TTA

Leu 475

GCC

Ala

ACA

Thr

OTA

Val

ACA

Thr

AGT

Ser 300

GGT

Gly

OTA

Val

TAT

Tyr

ACT

Thr

OAT

Asp 380

AGA

Arg

CAA

Gin

CCA

Pro

CCT

Pro

OAT

Asp 460

OAT

Asp

ATA

Ile

TAT

Tyr

TTT

Phe

AAA

Lys 540 3979 4027 4075 4123 4171 4219 4267 4315 4363 4411 4459 4507 4555 4603 4651 4699 SUBSTITUTE

SHEET

i 1 WO 92/14818 PCT/US92/00855

AGT

Ser

GGT

Gly

GAA

Glu

AAA

Lye

GAT

Asp 605

AAC

Asn

GCT

Ala

CTA

Leu

AAG

Lye

TAT

Tyr 685

TTT

Phe

GAT

Asp

GTA

Val

ATT

Ile

GAA

Glu 765

AAA

Lye

AGA

Arg

AAT

Asn

GCT

Ala

TCA

Ser 590

CTC

Leu

GAA

Glu

ATT

Ile

GCA

Ala

ACT

Thr 670

CTA

Leu

GTC

Val

GCT

Ala

CAA

Gin

CAT

Asp 750

CCA

Pro

ATG

Met

CAA

Gin

TCC

Ser

AAA

Lye 575

TTC

Phe

GTA

Vai

GCA

Ala

AAA

Lye

GGA

Gly 655

CTA

Leu

TTC

Phe

CAA

Gin

CCA

Pro

TCA

Ser 735

ACT

Thr

TGC

Cys

GCT

Ala

CTT

Leu 545

AAT

Asn

CTA

Leu

GAT

Asp

TTA

Leu

TGC

Cys 625

ATT

Ile

TCA

Ser

AAT

Asn

AAG

Lye

CCA

Pro 705

ACT

Thr

GAT

Asp

OCT

Ala

ACA

Thr

GAA

Glu 785

AAT

Aen

ATT

Ile

AGA

Arg

CTT

Leu

CTA

Leu 610

GAA

Glu

AAT

Asn

ATT

Ile

GAA

Glu kAT Aen 690

TTA

Leu

GAT

Asp

GTT

Val

AGA

Arg

TCA

Ser 770

GCA

Ala

TGT

Cys

GGA

Gly

TTA

Leu 645

CCT

Pro

GGT

Gly

AAA

Lye

TCT

Ser

GAA

Glu 725

GGT

Cly

TCA

Ser

AAA

Lye

TTA

Leu TTA GGA Leu Gly TGC GTA Trp Val GTT AGA Val Arg 585 TAT OCT Tyr Ala 600 OTT AAT Val Asn ATA TTA Ile Leu AAC GGA Asn Gly TAT GGA Tyr Gly 665 CAT AAG Asp Lye 680 ATO CGT Met Arg TTT AGA Phe Arg TTO GTT Leu VaI.

AAT ACA Asn Thr 745 ATT GGA Ile Gly 760 TCA GAA Ser Glu CTA GGT Leu Gly 4747 4795 4843 4891 4939 4987 5035 5083 5131 5179 5227 5275 5323 5371 5419 5467 SUBSTITUTE

SHEET

E.

WO 92/14818 PCI/US92/00855 COT ATT AAT GAA ACT AAT TAC AAC AAA TGT AAT AAA TAT GGT TAT AGA 5515 Arg

GGA

Cly

TTT

Phe

TAT

Tyr 845

GAT

Asp

AGA

Arg

TCA

Ser

AAT

Asn

TGT

Cys 925

GAC

Asp

TCA

Ser

TCT

Ser

ATT

Ile

GTA

Val

GAT

Asp 830

AGA

Arg

GAA

Giu

OCT

Gly

TGT

Cys

GGT

Gly 910

GGA

Gly

TAT

Tyr

TCT

Ser

GAT

Asp

GAA

Asn

TAC

Tyr 815

TOT

Cys

ATA

Ile

AGT

Ser

CTT

Leu

ACG

Thr 895

GAA

Giu

AGA

Arg

AGA

Arg

TCA

Ser

TCA

Ser 975

AAT

Giu 800

GAA

Giu

AAT

Asn

ATG

Met

GAA

Giu

TTA

Leu 880

CCT

Pro

CAA

Gin

AGA

Arg

AAA

Lys

GAT

Asp 960

GAT

Asp Ser

AAT

Asn

CCT

Pro

OAT

Asp

TCT

Ser 865

TAT

Tyr

AAT

Asn

CAC

His

ACA

Thr ccc Pro 945

AC

Ser

OGA

Giy Asn

AAC

Asn

AAT

Asn

TTA

Leu 850

CCT

Pro

COT

Gly

ACO

Thr

OTA

Val1

GGA

Gly 930

CAC

His

TOT

Cys

TOT

Cys Tyr

AAA

Lys

AAT

Asn 835

CAT

His

TOC

Cys

CCT

Pro

TTT

Phe

TAC

Tyr 915

TAT

Tyr

OTT

Val

TCA

Ser

TOC

Cy s Asn Lys Tyr Gly Tyr Arg 810 TAT TAT AGA GAA Tyr Tyr Arg Giu 825 ATA TCC AGA TAT Ile Ser Arg Tyr 840 ATT TTT OCA AAT Ile Phe Ala Aen CAT TAC TTO GAA His Tyr Leu Oiu 875 CAC AGA TAT CAA His Arg Tyr Gin 890 AAT TOT OTA ACA Asn Cys Vai Thr 905 OGA OAT AAT GCJ.

Gly Asp Asn Ala 920 AGO CAT GAA TG Arg Asp Giu Trp 0CC GAT GCA AAT Ala Asp Ala Asn 955 ACT ACT OAA TCT Ser Ser Giu Ser 970 ACT TTA CAT TCT Ser Leu Asp Ser 985 GAT OCA GGA TGC Asp Ala Gly Cys 1000 ATA 5563 Ile OGA 5611 Cly TAC 5659 Tyr 860 OAT 5707 Asp GAA 5755 O lu AGA 5803 Arg ACA 5851 Thr AAT 5899 Asn 940 ACT 5947 Ser GAA 5995 Oiu GAT 6043 Asp TAAATGAAAT 6098 TOT TAT CAA AAT Ile Giu Asn Cys Tyr Gin Asn Pro Ser Lys Cys 990 995 TTAATATTAT ATAATATTAA CTTACAAGTT CATATTATCO ATAGTTCTOA TAATGTGCTC ATTATCTTTT AOATATATTT AATATTAATT ATTCATAATA ATCATCTCCT ATATATATTA TATTGTCTTT ATCAATCATT AATTTTGCTA

ATAAAAATCA

TTTTATTTTA

ATAAATCGAC

ATOTATCATT

CACCTGTATT

TTAAAATGAT

TTAATTGCGA

TGACAATAAT

CTCTATTATA

ATCTTTATAT

TTTTTAAAAT

TGATTATAAT

ATTTATTCCT

AATATAGGTA

ACTATATTTG

6158 6218 6278 6338 6398 SUBSTITUTE SHEET 1

F

WO 92/14818 PCT/US92/00855

TGTCTTTGTT

TGGGATATAA

TACGTATTGT

GAATAGATTC

TATCTTTTAT

TTAAAAAATG

TTATAGATCT

TAATAAACCT

TTTTATATTA

GTCAAAAATT

GATAGATGAT

AATATCTAAT

AACGTTAACA

TTTAATATAG

ATAATAACAT

ATTTCATTTT

GATTTTAATA

ATTTCTTTAT

TATCTATATT

TGGCTCTATC

TAGATACGTT

CTGCTGGTTC

AATCAAATAT

CTACAGATTT

CTTGTGGTAA.

ATAATCTTTA

CATTTCTTTC

TATATATTTA

AACATTTATT

TCTGTTGTTG

ATCTTTATGA

CAATATGATA

ATTCTAGTTT

TATGTGTTAT

TTACCTTGTT

GTATATGATA

GAATTTAATC

6458 6518 6578 6638 6698 6758 6768 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 464 amino acids TYPE: amino acid TOPOLOGY: linear (ii) (xi) Asn Asr Ser Gly

MOLECULE

SEQUENCE

Lys Ile 5 Ile Asn TYPE: protein DESCRIPTION: SEQ ID NO:2: Met

I

G iu Arg Arg Phe Pro Phe Met Ser Met Lys Asn Leu Lys Met Pro Leu Phe Phe Ser 25 Ile Lys Tyr Asn Asn Met Val Ile Ala Ile Tyr Phe Ile Asn Lys Ile Asp Asn Ala Asn Arg Giy Lys Leu Glu Lys Asp Asp Asp Asp Vai Thr Phe Ile Pro Tyr Leu Glu Tyr Asn Giu Leu Asp Pro Ile Asn Asn Phe Glu Asn Asn Ile Lys Arg Lys Lys An Ile 115 Ile Pro Leu Leu Phe Trp Lys Lys Tyr Ile Asn Thr Thr Ser Lys Leu Gin Asp 110 Tyr Lys Gly Giu Leu Ser Val Tyr Met Lys Ser Phe Leu 130 Lvs Thr Arg Asp Phe Phe Ala Thr Asp Asp Leu Gin Ile Gly Pro Leu Gly Pro Lys Pro Lys Ala 175 Asp Ile Tyr Pro Arg Asp Lys Ser Trp Leu Ser Ser SUBSTITUTE SHEET

I

WO 92/14818 WO 9214818PCT/US92/00855 Tyr Phe Ala 225 Leu Asp Lys Ly s Ser 305 Tyr Asn Cy s Ile Ile 385 Arg Phe Lys Tyr Leu G iu 215 Tyr Arg Val1 Asn Met 295 Ser Glu Thr Ile Phe 375 Ser Ile Lys Met Asn 455 Met Thr Leu Phe Asp 265 Ser Ile Pro Leu 0 iu 345 Leu Lys Tyr G iu Met 425 Val Lys Ile Asp Giu Met 250 Lys Gly Giu Asn Asn 330 Leu Tyr Leu Arg Phe 410 Giu Cys Asn Thr Asn 220 Arg Pro Phe Ile Asp 300 Asn Ile Tyr Ile Tyr 380 Ala Asn Ile Lys Leu 460 Asp Thr Phe Tyr 255 Asp Leu Ile Tyr Asn 335 Pro Phe Ser Ile Giu 415 Tyr Asn Asn Tyr Val Phe 240 Asn Lys Ile Lys Leu 320 Vai Asp Asn" Phe Ser 400 Ile Lys Asp Ile INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 226 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein SUBSTITUTE SHEET WO 92/14818 64 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Met Phe Leu Val Tyr Phe Asn Thr Phe Leu Ile Ile Ile Leu PCT/US92/00855 G ly Leu Ile Phe Asn Asn Leu Phe His 145 Ser Giy Asn Ile Pro 225 Ile Ile Asn Leu Asn Gin Giy Arg 130 Cys His Giu Phe lie 210 Phe Val Phe Ser Asp Leu 75 Vai Asn Arg Asn Asp 155 Asn Ile Ile Leu Phe Asp Phe Thr Asn Ile Iil.' Gin Ala Ile Ser Ile Leu Leu Leu Thr Phe Ile Vai 160 Ile Ile 175 Leu Asn Asn Ser Aen Vai Giy Thr Leu Phe Vai Ile Asn INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 78 amino acids TYPE: amino acid TOPOLOGY: iinear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Ser Ser Ser Lys Lys Asn Asn Leu Gly Tyr Phe Asn Aen Leu Lys 10 SUBSTITUTE SHEET 1 i.i- FI1~ WO 92/14818 PC/US92/00855 Thr Gly Asn Arg (2) Glu Glu Val Ser Gin Ser Gin Val Phe Lys Asp Asn Tyr Arg Pro 25 Tyr Tyr Gly Leu Asp Thr Asn Ala Ala Asn Pro Ala Asp Val Tyr 40 Thr Glu Ser Asn Lys Pro Ser Thr Val Asp Val Trp Gly Asp Lys 55 Leu Glu Gly Lys Ile Ile Pro Lys Ser Lys Lys Lys Lys 70 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 162 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein Met 1 Lys Ala Glu Arg Asp Asn Ser Asp Glu 145 Ser (xi) SEQUENCE Ser Ile Phe Ile 5 Arg Met Asn Thr Leu Ser Phe Leu Tyr Ile Leu Arg Ser Ala Asn Ile Asn Val Phe Asn Asn Ile Phe Tyr 100 Thr Gly Ile Asn 115 Leu Phe Ser Arg 130 Thr Ser Asn Asp DESCRIPTION: SEQ ID Tyr Tyr Ile Phe Asn Asn Arg Phe Tyr Ile Tyr 10 Val Gln Ile Leu Val Val Ile Leu Ile Thr Thr 25 Val Phe Gin Leu Trp Tyr Tyr Ala Glu Asn Tyr 40 Tyr Asn Asp Thr Tyr Ser Asn Leu Gin Phe Ala 55 an Phe Asp Asp Leu Thr Val Phe Asp Pro Asn o0 75 Val Glu Glu Lys Trp Arg Cys Ala Ser Thr Asn 90 Ala Val Ser Thr Phe Gly Phe Leu Ser Thr Glu 105 110 Leu Thr Tyr Thr Asn Ser Arg Asp Cys Ile Ile 120 125 Ile Ile Lys Ile Val Tyr Asp Pro Cys Thr Val 135 140 Cys Arg Leu Leu Arg Leu Leu Met Ala Asn Thr INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 1003 amino acids TYPE: amino acid TOPOLOGY: linear SUBSTITUTE SHEET WO 92/14818 WO 9214818PCT/U3S92/00855 (ii) MOLECULE (xi) SEQUENCE TYPE: prrc tein DESCRIPTION: SEQ ID NO:6: Met Arg G iu Thr Giu Lys Gin Asn Lys Val 145 Asn Pro Lys Phe G iu 225 Arg Ala Arg Gly Ser Asn Val Lys Arg Ser Leu Giu Val Asp His 130 Asp Aen Gin Val As n 210 Val1 Tyr Lye Leu Arg 290 Tyr Val1 Gly Asp Met Trp Vai 115 Leu Asp Asn Tyr Thr 195 Lye Asn Gly Aen Pro 275 Asn Pro Ile Asp Thr Thr Phe 8b Tyr Tyr Leu G ly Gin 165 Tyr Asp Vai As n Phe 245 Ser Aen Cys Leu Ala Thr Lys Lys Met Leu His 70 Giu Val Lye Lye Aen 150 Val Ile Val Aen G iu 230 Giu Ile Leu Leu Ser 310 Thr 10 Lye Leu Ser Lye Pro 90 Leu Phe Gin Ile Aila 170 Arg Aia Ser Leu Aia 250 Asp Ala Thr Leu Asp Tyr Pro Val 75 Cys Leu Arg Pro Thr 155 Ser Lye Thr Phe Leu 235 Asp Thr Tyr Ser Leu 315 Ile Arg Lye Leu Thr Cys Giu Asp Asn Val Leu Ile Aen Thr Leu 190 Thr Ile Leu Leu Gin 270 Asp Giu Leu Ser Cys Asn Val Thr Giu Ser Asn Tyr Ile Ile 175 Asn Leu Tyr Leu Ile 255 Vai Giu Val Ar g Aen Phe G iu Ile Leu Ala His G ly Phe Gin 160 Phe Giu Asp Tyr Lye 240 Tyr Glu Aen Asp Val 320 Giu Tyr Val Thr SUBSTITUTE

SHEET

WO 92/14818 WO 9214818PCr/US92/00855 Asp 325 Val Tyr Lys Giu Arg 405 Phe Ile Trp Al a Asp 485 Ile Asn Ser Ser Leu 565 Cys Lys Cys Gly Leu 645 67 Ltys 330 Ser Ser Tyr Giu Lys 410 Asn Leu Asp Asp Gin 490 Asn Asn Phe Ile Thr 570 Giu Met Ile Phe Val1 650 Ile Leu Asp Vai Tyr 395 His Ser Lys Cys Leu 475 Ala Thr Val Thr Asn 555 Pro Phe Ala Giu Ala 635 Asp Val Tyr Thr Asp 380 Arg Gin Pro Pro Asp 460 Asp Ile Tyr Phe Lys 540 Ile His Cys Arg Ile 620 Arg Asn Met Leu Ser 365 Asn Cys Cys Asp Lys5 445 Thr Leu Lys Pro Asp 525 Ser Gly Giu Lys Asp 605 Aen Ala Leu His Aen 335 Giu Leu 350 Arg Leu Asp Giu Gly Arg Lys Tyr 415 Thr Thr 430 Val Pro Ser Arg Asp Val Gin His 495 Asn Cys 510 Met Asn Arg Gin Asn Ser Ala Lys 575 Ser Phe 590 Leu Val Giu Ala Ile Lye Ala Gly 655 Arg Ser Asp Asn Gin 400 Thr Ile Lys Phe Arg 480 Tyr Thr Asp Asp Val 560 Ile Cys Ser Val Val 640 Tyr SUBSTITUTE

SHEET

WO 92/14818 WO 9214818PCIF/US92/0085$ Ser Asn LyB Pro 705 Thr Asp Ala Thr C lu 785 Ser Asn Pro Asp Ser 865 Tyr Asn His Thr Pro 945 Ile Giu Asn 690 Leu Asp Val Arg Ser 770 Ala Asn Asn Asn Leu 850 Pro Gly Thr Val Gly 930 His Ser Lys 675 Lys Tyr Aen Leu Val 755 Glu Ser Tyr Lye Asn 835 His Cys Pro Phe Tyr 915 Tyr Val Leu 660 Tyr Leu Ile Tyr Gln 740 Ala His Arg Asn Leu 820 Asn Lys Glu Glu Gly 900 Giu Gly Tyr Thr Phe Al a Leu 715 Ser Asn Val Leu Val 795 Tyr Glu Tyr Asn G lu 875 Gin Thr Al a Trp Asn 955 Glu Lye Lye Tyr 685 Asp Phe 700 Leu Asp Ser Val Thr Ile Tyr Glu 765 Cys Lye 780 Aen Arg Arg Gly Ile Phe Gly Tyr 845 Tyr Asp 860 Asp Arg Glu Ser Arg Aen Thr Cys 925 Asn Asp 940 Ser Ser Leu Phe Gin Pro Ser 735 Thr Cys Ala Asn Tyr 815 Cys Ile Ser Leu Thr 895 G lu Arg Arg Ser Pro Leu Pro Pro 720 Gin Asn Gly Lys Glu 800 Glu Aen Met Glu Leu 880 Pro Gin Arg Lye Asp 960 Ser Cys Ser Asp Ser Ser Ser Ser Ser Giu Ser Giu Ser Asp Ser Asp SUBSTITUTE

SHEET

h1 i i; WO 92/14818 PCT/US92/00855 Gly Tyr Cye Cys Asp Thr Asp Ala Ser Leu Asp Ser Asp Ile Glu Asn Cys 980 985 990 Gin Asn Pro Ser Lys Cys Asp Ala Gly Cys 995 1000 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 163 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENC Ser Ile Arg Le

I

Arg 1 Tyr Val Asn Asn Thr Ile Ala Lys Pro 145 Tyr Val Asp Val Thr Ile Asn Thr Asp 130 Ile Tyr ;E DESCRIPTION: SEQ ID iu Asn Ser His Lys Asp 5 Ls Phe Leu Ile Ser Tyr Le Leu Asp Ile Ile Lys 3p Leu Leu Lys Ser Ser 55 (r Ile Glu Pro Ala Glu 70 ir Arg Met Lys Glu Met au Tyr Pro Ile Ser Tyr 105 fs Gly Leu Leu Asn Lys 120 La Val Ala Lys Leu Met 135 Le Glu Asn Asp Thr Leu 150 NO:7: Leu Pro Thr Asn Asp Lys Ser Ile Asn Glu 75 Asn Val Cys Lys Asp Thr Ile Asp 140 Ile Tyr 155 Glu Arg Gly Ser Ile Asn Tyr 110 Ile Asp Ala Arg Ser Ile His Asp Ile Arg Tyr Ile Asp 160 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 1511 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: ORGANISM: Amsacta moorei entemopoxvirus SUBSTITUTE

SHEET

r WO092/14818 PCI'/US92/00855, (ix) FEATURE: NAME/KEY: CDS LOCATION: complement (18. .218) (ix) FEATURE: NAME/KEY: CDS LOCATION: complement (234. .782) (ix) FEATURE: NAME/KEY: CDS LOCATION: 852.. 1511 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

GAATTCAAGT

TTTAACATTT

TTATTTATCA

CCTTCAATAT

GAATTATTAT

ATTTGATTTT

ATAGCTTGTA

ATATTTCTGT

ATTTTATCAC

ATAATATCAT

AAATATTCTT

TTATCTATAT

TTTCGCATCA

TAAATATTTA

TATTATTATT

TTTTACCTAT

CAGAATAATA

AACATAATCT

TATTATGTTT

ATTTTTTTAT

TAAAGTCACA

AAAhTTGTTC

AATTATCTAC

TATTTAATAT

TATGAGTTAT

ATTCTGTTGT

TAAACAACAA

TGATAATTGT

TTTTTTTTTT

ATTATCATTA

ACACACAGGA

TTTAATTGTA

TTTTTCTACT

ATTTAATCCA

TAAATCATTT

GATATTGATT

ATTTCCGTCA

AATZ ACACAT

TTTGCCAGAA

TCATATTTTT

TTATTTAATT

CTATCTACTA

TTTTGCATTT

ACATATAAAT

AAAGAAG CAT

TTAGGAATTA

GCAACAATAA

TCTTCAAAAA

TCATTAATTA

TGATTTATTA

TTTTGATTAG

AACATAGGAC

TTAAAGAATC

CGTTATTGAT

ACGAAATATC

ATGAATAAAA

CTTGTCCACC

CTTTATAACA

ATTTTGATAT

CTTTTTTTTT

ATTGACACTC

AATTATTTGT

TATTTTTATT

ATAAAATATA

CAATTATTAA

TAATCAATGA

TAATAAATTT

ATTAACAATA

AGATTTTGCA

ATATTAATAT

TATTTCAATT

AAATTGACAT

AGAATTAAAT

ATTATTAGCC

ATCTATGCCA

TTTAATGTAT

TATAAATCTA

TCTATTAATT

TTCTATCGAC

AAAAAAATA

ATTTTTTTTT ATTATTTGAT ATATTTTTTC AAAAAAAAAT AAATTATCAA A ATG GAT TTA CTA AAT Met Asp Leu Leu Asn 1 5 TCT GAT ATA ATT TTA ATA AAT ATT Ser Asp Ile Ile Leu Ile Asn Ile TTA AAA TAT TAT AAT TTA AAA AAA ATA ATA ATA AAC AGA GAT AAT GTT Leu Lys Tyr Tyr Asn Leu Lys Lye Ile Ile Ile Asn Arg Asp Asn Val 20 ATT AAT ATT AAT ATA TTA AAA AAA TTA GTT AAT TTA GAA GAA TTG CAT Ile Asn Ile Asn Ile Leu Lys Lys Leu Val. Asn Leu Glu Giu Leu His 35 40 ATA ATA TAT TAT GAT AAT AAT ATT TTA AAT AAT ATT CCA GAA AAT ATT Ile Ile Tyr Tyr Asp Asn Aen Ile Leu Asn Aen Ile Pro Glu Asn Ile 55 AAA AGT TTA TAT ATT TCA AAT TTA AAT ATT ATT AAT TTA AAT TTT ATA Lys Ser Leu Tyr Ile Ser Asn Leu Asn Ile Ile Aen Leu Aen Phe Ile 70 SUBSTITUTE SHEET 938 986 1034 1082 I WO 92/14818 PCT/US92/00855

GAT

Asp

CAT

His

AAT

Asn

AAT

Asn 135

TTA

Leu

TTA

Leu

AAT

Asn

TAT

Tyr

ATA

Ile 215

ATA

Ile

TCT

Ser

TTT

Phe 120

AAA

Lys

AAT

Asn

ATT

Ile

ATA

Ile

CAA

Gin 200

ATT

Ile

TCT

Ser

ATA

Ile 105

ATT

Ile

TTT

Phe

ATG

Met

AAT

Asn

CAT

His 185

TCA

Ser

GAA

Glu

TAT

Tyr

GAA

Glu

AAT

Asn

GGT

Gly

GAA

Glu

TTA

Leu 170

TTG

Leu

TAT

Tyr

TAT

Tyr

AAA

Lys

TTA

Leu

TTA

Leu

TTT

Phe 140

ATA

Ile

AAA

Lys

AAA

Lys

GAA

Glu

TTC

Phe 220 1130 1178 1226 1274 1322 1370 1418 1466 1511 INFORMATION FOR SEQ ID NO:9: (i) (ii) (xi) Met Gin Asr 1 Asp Ile Ser Asn Ile Val Asn Lys Met Cys Leu SEQUENCE CHARACTERISTICS: LENGTH: 67 amino acids TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: protein SEQUENCE DESCRIPTION: SEQ ID SAsn Asp Asn Tyr Tyr Ser Asp 5 10 Leu Val Asp Arg Lys Lys Lys 25 Asn Ile Asn Asn Glu Leu Asn 40 SLeu Lys Asn Leu Leu Asp Ser 55 NO:9: Ile Glu Gly Ala Lys Ser Ile Gly Lye Met Ile Asn Lys Gin Leu Ser Asn Asn Leu Lys Lys Tyr Asp Cys SUBSTITUTE

SHEET

il-L...I:L C. -YC"U--~s WO 92/14818 PCT/US92/00855 72 INFORMATION FOR SEQ ID NC '0: SEQUENCE CHARACTERISTICS: LENGTH: 183 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ Met Ser Ile Olu Leu l1e 11e Gly Pro Leu Ile Asn Asn Gly Cye Asn Pro 130 Tyr Ile Met Ile His Asn Ile Asp Cys 115 Lys Lys Glu Lys His Gly Ile Glu Met Phe Glu Ala Gly 165 Ser ID Met Phe Ser Ile Leu Ser Arg Phe Ile Lys Glu Tyr Ile Val Asp Glu Glu Asn 90 Lys Lys Val Phe Asn Ser Leu Gin Ala 140 Lys Lys His 155 Tyr Val Pro 170 Thr Lys Asn Ile Asp Glu Ala Lys Gin Asn Arg 175 Tyr Asn Asn 180 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 220 amino acids TYPE: amino acid 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 SUBSTITUTE

SHEET

r c ,WO 92/14818 PCT/US92/00855 Tyr Asn Tyr Tyr Lys Ser Cys Lys Pro 145 Tyr Phe Ile Leu Asn Ile Asp Ile Asn Asn Asn Leu 130 Ile Lys Asn Thr Lys 210 Asn Leu Asn Phe Lys Leu Leu 0 Phe 140 Ile Lye Lys Glu Phe 220 Val His Ile Ile Asn Asn Val 125 Asn Gin Leu Phe Asn 205 Ile Asn Ile Ile Lys Ser Thr Lys Ser Asn Cys Glu 110 Asn Leu Asn Val Ile Lys Asp Ile 175 Pro Lye 190 Tyr Asn Ile Tyr Leu Leu Ile Ser Lys Phe Asp 160 Ser Ser Tyr INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GARGTNGAYC CNGARTAYGT INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown SUBSTITUTE SHEET WO 92/14818 PCT/US92/00855 74 (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: TTTCAAATTA ACTGGCAACC INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GGGATGGATT TTAGATTGCG INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID GCCTGGTTGG GTAACACCTC INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: CTGCTAGATT ATCTACTCCG INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) SUBSTITUTE

SHEET

SWO 92/14818 PCT/US92/00855 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GTTCGAAACA AGTATTTTCA TCTTTTAAAT AAATC INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: GAYGARGGRG GRCARTTYTT INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: GGNCCCATGT TYTCNGG 17 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID GGTGCAAAAT CTGATATTTC INFORMATION FOR SEQ ID NO:21: S(i) SEQUENCE CHARACTERISTICS: LENGTH: 3012 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) SUBSTITUTE

SHEET

.i.

WO 92/14818 76 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: PCr/US92/00855

ATGAGTAACG

ATAAAGATTT

CCATTATATG

ATAGAAGTAA

AAAGAAATGT

TATGTAAGTC

AATTTTAGAC

TTTATTTATT

AATAATAATT

TATATTTGTT

GATGCAACTA

GATATATATT

AGATACGGTG

AGTATTAAAA

AGAGCATATA

ACAGAAGTAG

TATAAAAAGT

GTATTTCCAT

GCATCTGATA

AACGACGAAA

GTATTCCACG

TTCCAATTTG

TGTTTAAAAC

ACTTCTAGAT

CTTAATAGAA

ATATTAGAGT

AATAATG TAT

AGTAGACAAG

AATATTAGTA

TGTGCTAGAG

GCTATGGCTA

TACCTTTAGC

ATTTAAAAGA

ATGTGTGTAA

TTGAATTAGA

GTTTTGAATT

GATTATTGCT

TGACTCTTAA

TTGTCGATGA

TACAAGTTAA

TAGGTAGAAA

ATATTACTTT

ACGAAGTTAA

AATTTGAAGT

ATTATGATAC

CTAAGGATGA

ACCCCGAGTA

TTGATAAATC

TAAGATCATT

CTTCGAGATT

ATAAAATTAT

AACGTGTAAA

TTGTAAACAG

CTAAAGTACC

TTATTAAACA

ATGATATATG

ACGCAAATAC

TTGATATGAA

ACCTTAATAA

GTTTGCCTGG

TTAGAGAATT

GAGATCTCGT

AACCAAAACA

TGAAAATACT

TGAAACTTCT

CAATACTCAT

ATTGTTCCCG

AGATAATGTA

TGGAAAACAT

TTTGGGAAAT

CAAAGATGCA

AGTATATTTA

AGATTTTAAT

TAATAATGAA

CTATAACGCA

TGTGATTCAA

AAATGGTCGC

TGTAACTAGT

TCATTTAAAA

ATATCTGGAA

AGATGTTGGT

ATTGGALAGAA

ACTTPAATAAA

CCCAGATACT

CAAAAATTTA

TATGGCTGAT

TTTAAAACAA

ATATCCAAAT

TGATAACAAA

CATGTCATGT

TTGGGTALACA

TTGTAAATCA

AAGTTTACTA

ATAAGAAA.AT

TGTTTCGALAC

GGTGTTACTT

GTTAGAATCA

TGTAATGTAA

TCACATAATG

TTAAAATTAA

TATGGATTAA

TCATTTATTA

AATGAAAAAG

AAATCTGTTA

CAAAAAGATT

GATACTGGAT

GTAGAAAGGT

AATCTATGTT

AATAATGCTT

ATTGTAATGC

TTGTCTAATG

ATTTACAAAT

ATTGAAGCAG

CACCAATGTA

ACGATTCACT

AGACTTTGGG

GGATCTGATG

GCCATAAAAC

TGCACATTAT

ACTATATCTG

ATATTAGGAA

CCTCACGAAG

TTCTGTGATC

TTTATGTGTA

TATCAAATCG

GTGTAGTAGA

TAGAATCATG

AAGTTCACGG

ACGAAGCCCA

ACGTAAAATA

AAGAAATCGA

TTACTAAGGA

CTATATTTCC

TAACTTTTGA

ATATCGCAGT

TAT TAAAAGA

TAATTTATGC

TGCCAGTTAA

TGATGAAA.AT

TATTGGGTAC

ATAACAGAGG

TAAAAGGATA

TAAATAAAAT

AATATAGATG

AATATACTCC

TATATGGTAT

GATGGATTTT

ATTTAGATCT

AACATTATAC

CATTGGGTAA

AGTATACTAA

TAAACATAGG

CTAAAATTCT

TTTCTAATAA

ACTATGTTALA

AAAATATGAA

TATGGTAGTT

TAGTCCAAAT

CGATACATTA

AGTATGGAAA

TAAATTAGCT

TCAACCGCTA

AAATATTCAA

ACAATATGCG

TGTAACTACA

ATCATTCCTT

TTTACTTAAG

TAAAAATCTA

TTTGAAAGTT

AACATCTAGT

GCTCAGAGTA

AAGTGGTAAT

TCCAGTTAAA

TTATGTAGAT

CGGAAGACAA

CAAATGTCCA

TTCTAATGTT

AGATTGCGAT

TGACGTTAGG

TAATGTAATT

TAATAGATTT

CTTTACAAAA

TAATTCCGTA

AAGATCTGGT

GAGATTCTAT

TATTGAAATT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 SUBSTITUTE SHEET -i WO 92/14918 PCI'/US92/00855 AACGAAGCAG TATGCGAATA TCCTGGATAT GTCATATTAT ATTAATGATT TATTATTAAT CCTATACATT ATGGATCTAC AAGAAATTTA AATATCTATT TTTGTCCAAC CTCCATTATA ACTGATAATT ATGAAAAATA GGTCTGTTGA ATACATGTAA GGATATGTTT ATGAACCATG AAAATGGCTA AAGAAGCATC AGTAATTACA ACAAATGTAA AAAACAAAAT ATTATAGAGA TCCAGATATG GATATAGAAT GATGAAAGTG AATCTCCTTG TATGGTCCTG AATATGTACA AATAACACAA ATTGTGTAAC GATAATGCAA CATGTGGAAG GACTATAGAA, AACCCCACGT AGCTGTTCAG ACAGTAGTAG ACAGATGCTA GTTTAGATTC GCAGGATGCT AA

TAACGGAGTA

TGAAAAGACT

CTTAAAGAAT

TATTTCTACT

TTTGGTTGAT

TACTATTGAT

CGGAACATCA

TAGATTAGGA

TAAATATGGT

AATATTTGAT

AATGGATTTA

CGAACGAAGA

TCACAGATAT

AAGAAATGGT

AAGAACAGGA

TTATGACAAT

TAGTAGTGAA

TGATATTGAA

GATAATCTAG

CTACCAAATG

AAACTAAAAG

TACTTTAGAA

TCGTCCGTAC

ACTAATGCTA

GAACATAAAA

AATCTAGGTT

TATAGAGGAG

TGTAATCCTA

CATAAAATTG

TGTCATTACT

CAAGAAT CAT

GAACAACACG

TATGGAAGAA

TGTGCCGATG

TCTGAATCTG

AATTGTTATC

TCGCAAGAGC

CAGGATATTC

AAAAGTATGG

ATTTAATGCG

CTTTATTGGA

AATCACAAGA

GAGTTGCATC

TTGGTTCAGA

TAGTAAATCG

TATACGAAAA

ATAAVhATAA GAG1RAATTTT

TGGAAGATAG

GTACGCCTAA

TATACGAAAA

GAAGTAGGGA

CAAATAGTTC

ATTCAGATGG

AAAATCCATC

TATTAAAGTA

AATTTCCTTA

TGGTGTTGAT

TGATGCTGAT

TGCTCCACCA

TGTTCTACAG

AAGTGTTATT

AGCATTGTGT

TAT TAATGAA

TAACAAACTA

TGAATTAATA

TGCAAATTAC

AGGTCTTTTA

TACGTTTGGA

TAGTTGTGGA

TGA.ATGGAAT

ATCTTCAGAT

ATGTTGCGAC

AAAATGTGAT

1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3012 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 419 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genoniic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: TCAACTAATA ATAATATATT TTATGCAGTT TCAACTTTTG GATTTTTAAG ACTGGTATTA ATTTAACATA TACAAATTCT AGAGATTGTA TTATAGATTT ATTATAAAAA TAGTATATGA TCCTTGTACT GTCGAAACAT CTAACGATTG AGATTATTGA TGGCCKNTAC ATCATAAATA CATTATAATA TTATTATAAT ATTTTTATAT ATATTTTATC TAAAAGGACT TTTTATTTTT TATATATTAA

TACAGAAAGT

ATTTTCTAGA

TAGATTATTA

ATCAATCATA

TAATAATAAA

120 180 240 300 SUBSTITUTE SHEET

I

r -7 i WO 92/14818 PCr/US92/00855.

TGAGTAACGT ACCTTTAGCA ACCAAAACAA TAAGAAAATT ATCAAATCGA AAATATGAAA TAAAGATTTA TTTAAAAGAT GAAAATACTT GTTTCGAACG TGTAGTAGAT ATGGTAGTT INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 678 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) I JLE TYPE: DNA (genonic) (xi) JENCE DESCRIPTION: SEQ ID NO:23:

ATGTTTCTAG

ATTTATATAT

ATATTATCAT

TCAGATATTA

AATAATTTAC

TTTTATTCAG

ATAAATAGAA

AATGAAACTT

TCACATATAG

GTAAATAATA

AGATTAATAG

TTTGTCATAA

TTTATTTCAA

TAACATTTGT

ATAACGCAAC

TATTTTTGAC

AAGATATACC

CGTCTAGTAA

ATCCATTTTT

TTCACTGTTA

AATTTATGAA

ATATATCTAT

ATAAATATAA

ATCCATAA

TACATTTTTA

GTTTAATATA

TAATATAAAC

AAATTTTAAC

AATATTTAAT

TAATGTAAAT

ATTATTTAGA

TATAAGTTCA

ATCTAGATAT

TAATAATATA

CTCTATAATA

ATAATAATTT

GATTTTTTAA

AATATAAATA

ATAAATAATA

GTAAATAATA

ATATTATTAG

AATACATCTC

AATCAAAATA

AATAAATATG

TTAAATAATT

TCATTTTTAA

TATTATTTGG

TAAATAATAA

ATTTAAATTT

ATCTTTTAGT

TTATATCTAA

GATTAAGAAA

TAGCTATAGT

GTGATGTATT

TAATTATAGG

TTGCTATTAT

ATATCAACGT

TATTATAGGT

TAAAATATAT

ATACGATTAT

AACACAAGCT

TCAATATAAT

AACATTAAAT

TTTCAATAAT

AGATATAGTA

AGAAATACCC

AACTAATGTG

AGGAACACTT

INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 486 base pairs TYPE: nucleic P-cid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: ATGTCAATAT TTATCTACTA TATTTTCAAC AATAGATTTT ATATATATAA AAGAATGAAT ACTGTACAAA TTTTAGTTGT CATATTAATA ACAACAGCAT TATCTTTTCT AGTTTTTCAA TTATGGTATT ATGCCGAAAA TTACGAATAT ATATTAAGAT ATAATGATAC ATATTCAAAT TTACAATTTG CGAGAAGCGC AAATATAAAT TTTGATGATT TAACTGTTTT TGATCCCAAC SUBSTITUTE SHEET i -1 I F i:i- -_I WO092/14818 PCU/US92/00855 79 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 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1395 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID

TTAAATATTA

TATTTTACTA

CATAAACTTT

ATTAATATCA

AATAAAACTC

AACTATATAA

TTCAAAAGTA

TAGTACATCA

GCTTTTTATA

TGGTATATAA

ATCTATTACG

TTTATCATTC

AGCTACTGTA

ATCTGTATTA

TGTAGATTTA

TATTCCTATT

TTTGGATAAT

TTTTTTAGAT

AGTCCAAAAT

ATTTGGATCT

GATTCTAAAC

CATACATTCA

TTATTTTTTA

TATCTAGAAA

ATAGA.ATAAT

TATAAAAATA

TTAAAAAAGT

ATGTA'IAAAT

GTCATATCAG

CCACTACTAT

CAACAAGTAA

ATTAAAAAAA

GCAATATTTT

ATCATAGTTA

TCATGTTTTC

TGAGCATCCA

TCTGATTTTA

GTAATAAAAr2

AGAAAAAATT

AAAACTAATT

TATTCTTCTC

TAATTCTATT

TATTAAATAT

TAAATAATGC

ATAATTTTTT

TTATATTAAA

AATAATTTAC

AAGCATA.ATT

ATTCAATAAA

TAAAAAAGTA

AATGATCATT

ATTTTACTCT

TATCAGTTTT

ATGGATATAT

TTGGGTAAGC

AATCATCATA

GAAAAGACTT

TATTAATATT

TTCTTTTAAT

CATTATATAA

ATTATCAATA

ACTATTTTTT

TTCTrAATGTA

ACCTCTATAA

AAATTCAAAT

CATACCACAA

ATTTTTAAAT

AGTATTAGGA

CATATATTTT

TGCAGCTTTT

ATAAATTATA

ATCTTCAAGT

TTCAAATAAA

ACAATTATAT

TTTAGGTTTA

AATTGTGGCA

TCTCATATAT

TATATTTTTA

ATTATTTTCA

TATTTCCAAG

TRACTATCAT

TTATACATAT

TTTTTAAATT

CTACTAGCCA

TTAGATTTTA

TCGGGACTAT

ATATCATTTA

GTA TTATTGT

TTATTTTGTT

TTATCTTTAT

GGAAACATAA

TTATAGCATC

ATCAAATGXA

ATATCTCCCG

GGATTAAATC

AATGTAGAAA

ACTAATGGAA

TCTTGTAAAT

AAATTAATAT

TATTTTATAG

AATCATTTTT

CTATTAATTC

CGTCAATACT

ATAAATCACC

TGTTGAA.ATA

CATATTGTAA

AATATTCTGA

AGTGTTTATG

TTATAAGTTC

CAAAGTGTTT

AAAATCTTTT

TCATAGATGA

AATAATCATA

AACTTAACCA

CCAAAGGCGG

AATCTCTTGT

TGCCTTTATA

ATTTTTTTAT

TATTAATATG

GTATAAATGT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 SUBSTITUTE SHEET

I

4

F,

i i l-C l~ ~.~Eli~FU t I WO 92/14818 pcri TACTTTACCT CTTGTTTCAT CATCATCATC TATTTTTTCT AATATAGCTA TATTTGCATT AGTATTATAT TTAATAGGAT TTATAAAATA TACCATATTA TCTATTTTAC TAAAAAATAA CATAGACATA AAATTAATAC CAGATTCTGG CATTTTTAAA TTTTTATTTG GAAATCTTCT AATTTTATTA TTCAT INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 237 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: TTATTTTTTT TTTTTGCTTT TAGGTATAAT TTTACCTTCT AAACGTTTAT CTCCCCAAAC ATCTACAGTA GATGGTTTAT TAGATTCTGT GTTATACACA TCTGCTGGAT TTGCGGCATT TGTATCCAAA CCATAATATC CAGGTCTATA ATTATCTTTA AAAACTTGGG ATTGAGATAC TTCTTCAGTT TTTTAATTAT TAAAATATCC AAGATTATTT TTTTTTGATG AAGACAT US92/00855 1260 1320 1380 1395 INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 492 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: CTATTCATAA TAATCATCTG CTATATATAT TAATGTATCA TTCTCTATTA TATATTGTCT TTATCAATCA TTAATTTTGC TACAGCTGTA TTATCTTTAT TGTGTCTTTG TTTAATAAAC CTTTTAATAT AGTGGCTCTA TCATAATCTT TATGGGATAT AATTTTATAT TAATAATAAC ATTAGATACG TTCATTTCTT TTTACGTATT GTGTCAAAAA TTATTTCATT TTCTGCTGGT TCTATATATT ATGAATAGAT TCGATAGATG ATGATTTTAA TAAATCAAAT ATAACATTTA TTTATCTTTT ATAATATCTA ATATTTCTTT ATCTACAGAT TTTCTGTTGT TATTAAAAAA TGAACGTTAA CATATCTATA TTCTTGTGGT AAATCTTTAT TCTTATAGAT CT

TAAATATAGG

ATACTATATT

TACAATATGA

TCATTCTAGT

TATATGTGTT

TTTTACCTTG

TGGTATATGA

GAGAATTTAA

120 180 240 300 360 420 480 SUBSTITUTE SHEET fii-~a: ll:s~ *Ilpr~ WO 92/14818 PCT/US92/00855 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 549 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

TTAATATGAA

TTCAATTATT

TTGACATATA

ATTAAATATA

ATTAGCCATT

TATGCCAATA

AATGTATAAA

AAATCTATTA

TTATTATAAC

TGATTTTTAT

GCTTGTAATT

TTTCTGTTAA

TTATCACAAA

ATATCATAAT

TATTCTTTAT

TCTATATTAT

ATAATCTACA

TATGTTTTTT

TTTTTATTTT

AGTCACAATT

ATTGTTCTAA

TATCTACGAT

TTAATATATT

GAGTTATAAT

CACAGGAACA

AATTGTAAAA

TTCTACTTTA

TAATCCAGCA

ATCATTTTCT

ATTGATTTCA

TCCGTCATGA

TACACATTTT

TATAAATCTT

GAAGCATCTT

GGAATTAATT

ACAATAACTT

TCAAAAAATT

TTAATTAAAT

TTTATTATAT

TGATTAGATA

ATAGGACCALS

GTCCACCTAT

TATAACAAAA

TTGATATAGA

TTTTTTTATT

GACACTCATC

TATTTGTTTT

TTTTATTTAT

AAATATATCT

TTATTAATTC ATTAATTTTT CGCATCAATT CTGTTGTTTT GCCAGAAAAC

TATCGACAT

INFORMATION FOR SEQ ID N0!29: SEQUENCE CHARACTERISTICS: LENGTH: 69 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NOz29: TTTTTTTTAT TATTTGATAT ATTTTTTCAA AAAAAAATTA ATCAATGAAA AAAAAATAAA

ATTATCAAA

INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 141 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) SUBSTITUTE SHEET WO 92/14818 PCr/!US92/00855, (xi) SEQUENCE DESCRIPTION: SEQ ID AAACATAGGA CCAATTATTA ATTrCTATCGA CATTTTTTTT TATTATTTGA TATATTTTTT CAAAAAAAAA TTAATCAATG AAAAAAAAAT AAAATTATCA AAATGGATTT ACTAAATTCT GATATAATTT TAATAAATAT T INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 20L base! pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: TTATAAACAA CAATCATATT TTTTTAAAGA ATCTAATAAA TTTTTTAACA TTTTATTATT ATTTGATAAT TGTTTATTTA ATTCGTTATT GATATTAACA ATATTATTTA TCATTTTACC TATTTTTTTT TTTCTATCTA CTAACGAAAT ATCAGATTTT GCACCTTCA.A TATCAGAATA ATAATTATCA TTATTTTGCA T INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 660 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

ATGGATTTAC

AAAATAATAA

TTAGAAGAAT

ATTAAAAGTT

AAAAATATAA

CTACCACATT

ATTAATAATT

AATAATGTTT

TATAAATTTA

TAAATTCTGA

TAAACAGAGA

TGCATATAAT

TATATATTTC

CATATTTAGA

CTATAGAATT

TAGTAAATTT

TTCCTATTAG

TAGAAAAATT

TATAATTTTA ATAAATATTT

TAATGTTATT

ATATTATGAT

AAr,TTTAAAT

TATATCTTAT

TTTAAATTGT

AAAAAAATTA

TATAGTTGAG

AATTAATTTA

AATATTAATA

AATAATATTT

ATTATTAATT

AACAAAAATA

GAATCATGTA

ATAATATCTA

TTAAATATGG

AAAAAATTAG

TAAAATATTA

TATTAAAAAA

TAAATAATAT

TAAATTTTAT

GCAATATA.AG

ATATAAATGA

AAAATAAATT

AATCAATACA

ATATATCTTT

TAATTTAAAA

ATTAGTTAAT

TCCAGAAAAT

AACAAAATTA

TAATATTATA

CTATAATTTT

TGGTAACTTT

AATAAAAGAT

CAATGTTAAA

SUBSTITUTE SHEET WO 92/14818 PCr/US92/00855 AAAAATAATA TACATTTGAT AAAATTTCCA AAAAGTATAA CTCATTTATG TGATTATCAA TCATATAAAG AAAATTATAA TTATTTAAAA AATTTATCAA ATATAATTGA ATATGAATTC INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 3907 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:

TTCTAAACGT

CACATCTGCT

TTTAAAAACT

ATTTTTTTTT

TCTACTATAT

TAGTTGTCAT

CCGAAAATTA

GAAGCGCAAA

ATGTTGAAGA

CTTTTGGATT

ATTGTATTAT

AAACATCTAA

ATAATATTAT

ATTTTTTATA

AAAATTATCA

CGAACGTGTA

TACTTTAGAA

AATCAAAGTT

TGTAAACGAA

TAATGACGTA

ATTAAAAGAA

ATTAATTACT

TATTACTATA

TTATCTCCCC

GGATTTGCGG

TGGGATTGAG

GATGAAGACA

TTTCAACAAkT

ATTAATAACA

CGAATATATA

TATAAATTTT

AAAATGGCGCC

TTTAAGTACA

AGATTTATTT

CGATTGTAGA

TATAATATCA

TATTAATAAT

AATCGAAAAT

GTAGATATGG

TCATGTAGTC

CACGGCGATA

GCCCAAGTAT

AAATATAAAT

ATCGATCAAC

AAGGAAAATA

TTTCCACAAT

AAACATCTAC

CATTTGTATC

ATACTTCTTC

TAATTGATAT

AGATTTTATA

ACAGCATTAT

TTAAGATATA

GATGATTTAA

TGTGCTTCAA

GAAAGTACTG

TCTAGAATTA

TTATTAAGAT

ATCATAATTT

AATAAATGAG

ATGAAATAAA

TAGTTCCATT

CAAATATAGA

CATTAAAAGA

GGAAATATGT

TAGCTAATTT

CGCTATTTAT

TTCAAAATAA

ATGCGTATAT

AGTAGATGGT

CAAACCATAA

AGTTTTTAAA

TATAATACTT

TATATAAAAG

CTTTTCTAGT

ATGATACATA

CTGTTTTTGA

CTAATAATAA

GTATTAATTT

TAAAAATAGT

TATTGATGC

TTATATATAT

TAACGTACCT

GATTTATTTA

ATATGATGTG

AGTAATTGAA

AATGTGTTTT

AAGTCGATTA

TAGACTGACT

TTATTTTGTC

TA"TTTACAA

TTGTTTAGGT

TTATTAGATT CTGTGTTATA TATCCAGGTC TATAATTATC TTATTAAAAT ATCCAAGATT TATAGATATG TCAATATTTA AATGAATACT GTACAAATTT TTTTCAATTA TGGTATTATG TTCAAATTTA CAATTTGCGA TCCCAACGAT AATGTTTTTA TATATTTTAT GCAGTTTCAA AACATATACA AATTCTAGAG ATATGATCCT TGTACTGTCG CAATACATCA TAAATACATT TTTATCTAAA AGGACTTTTT TTAGCAACCA AAACAATAAG AAAGATGAAA ATACTTGTTT TGTAATGAAA CTTCTGGTGT TTAGRCAATA CTCATGTTAG GAATTATTGT TCCCGTGTAA TTGCTAGATA ATGTATCACA CTTAATGGAA AACATTTAAA GATGATTTGG GAAATTATGG GTTAACAAAG ATGCATCATT AGAAAAGTAT ATTTAAATGA 120 180 240 300 360 420 480 540 600 660 720 760 840 900 960 1020 1080 1140 1200 1260 1320 1380 SUBSTITUTE SHEET WO 92/14818 PCT/US92/00855,

AAAAGTAACT

TGTTAATATC

AGATTTATTA

TGGATTAATT

AAGGTTGCCA

ATGTTTGATG

TGCTTTATTG

AATGCATAAC

T.AATGTAAAA

CAAATTAAAT

AGCAGAATAT

ATGTAAATAT

TCACTTATAT

TTGGGGATGG

TGATGATTTA

AA.AACAACAT

ATTATCATTG

ATCTGAGTAT

AGGAATAAAC

CGAAGCTAAA

TGATCTTTCT

GTGTAACTAT

ATTATTCGCA

TCTAGCAGGA

AAATGAAAAG

AAAAGATTTA

TAGAACTTTA

CGTACAATCA

TGCTAGAGTT

TAAAATTGGT

AGGTTTAGTA

AGGAGTATAC

TTTGATGTAA

GCAGTATCAT

AAAGATTTAC

TATGCTAAAA

GTTAATTTGA

AAAATAACAT

GGTACGCTCA

AGAGGAAGTG

GGATATCCAG

AAAATTTATG

AGATGCGGAA

ACTCCCAAAT

GGTATTTCTA

ATTTTAGATT

GATCTTGACG

TATACTAATG

GGTAATAATA

ACTAACTTTA

ATAGGTAATT

ATTCTAAGAT

AATAAGAGAT

GTTAATATTG

AGAGCTATTA

TATTCAATTT

TATGGTGGTG

ATGCGTGATG

TTGGATGCTC

CAAGATGTTC

GCATCAAGTG

TCAGAAGCAT

AATCGTATTA

GAAAATALACA

CTACAGATGC

TCCTTGATAT

TTAAGA.GATA

ATCTAAGTAT

AAGTTAGAGC

CTAGTACAGA

GAGTATATAA

GTAATGTATT

TT"AAGCATC

TAGATAACGA

GACAAGTATT

GTCCATTCCA

ATGTTTGTTT

GCGATACTTC

TTAGGCTTA

TAATTATATT

GATTTAATAA

CAAAAAGTAG

CCGTAAATAT

CTGGTTGTGC

TCTATGCTAT

AAATTAACGA

AAGTAATTAA

CCTTACCTAT

TTGATAAGAA

CTGATTTTGT

CACCAACTGA

TACAGGGTCT

TTATTGGATA

TGTGTAAAAT

ATGAAAGTAA

AACTAAAAAC

AACTAATATT

ATATTACGAA

CGGTGAATTT

TAAAAATTAT

ATATACTAAG

AGTAGACCCC

AAAGTTTGAT

TCCATTAAGA

TGATACTTCG

CGAAAATAAA

CCACGAACGT

ATTTTTGTA

AAAACCTAAA

TAATTTATT

TAGAAATGAT

AGAGTACGCA

TGTATTTGAT

ACAAGACCTT

TAGTAGTTTG

TAGAGTTAGA

GGCTAGAGAT

AG CAGTATCC

TGATTTATTA

ACATTATGGA

ATTTAAATAT

CCAACCTCCA

TAATTATGAA

GTTGAATACA

TGTTTATGAA

GGCTAAAGAA

TTACAACAAA

AAAATATTAT

ACTTTAGATT

GTTAATAATA

GAAGTCTATA

GATACTGTGA

GATGAAAATG

GAGTATGTAA

AAATCTCATT

TCATTATATC

AGATTAGATG

ATTATATTGG

GTAAAACTTA

AACAGCCCAG

GTACCCAAAA

AAACATATGG

ATATGTTTAA

AATACATATC

ATGAATGATA

AATAACATGT

CCTGGTTGGG

GAATTTTGTA

CTCGTAAGTT

GAATATCCTG

TTAATTAACG

TCTACTGAAA

CTATTCTTAA

TTATATATTT

AAATATTTGG

TGTAATACTA

CCATGCGGAA

GCATCTAGAT

TGTAATAALAT

AGAGAAATAT

TTAATAAATC

ATGAACAAAA

ACGCAGATAC

TTCALAGTAGA

GTCGCAATCT

CTAGTAATAA

TAAAAATTGT

TGGAATTGTC

TTGGTATTTA

AAGAAATTGA

ATAAACACCA

ATACTACGAT

ATTTAAGACT

CTGATGGATC

AACAAGCCAT

CAAATTGCAC

ACAAAACTAT

CATGTATATT

TAACACCTCA

AATCATTCTG

TACTATTTAT

GATATGTCAT

GAGTAGATAA

AGACTCTACC

AGAATAAACT

CTACTTACTT

TTGATTCGTC

TTGATACTAA

CATCAGAACA

TAGGAAATCT

ATGGTTATAG

TTGATTGTAA

1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 SUBSTITUTE

SHEET

n 1'~

F

WO092/14818 PCr/US92/00855

TCCTAATAAT

AATTGGAGAA

TTACTTGGAA

ATCATGTACG

ACACGTATAC

AAGAAGAAGT

CGPATGCAAAT

ATCTGATTCA

TTATCAAAAT

TTAACTTACA

GTGATAA

AATAATGAAT

ATTTTTGCAA

GATAGAGGTC

CCTAATACGT

GAAAATAGTT

AGGGATGAAT

AGTTCATCTT

GATGGATGTT

CCATCAAAAT

AGTTATAAAA

TAATATCCAG

ATTACGATGA

TTTTATATGG

TTGGAAATAA

GTGGAGATAA

GGAATGACTA

CAGATAGCTG

GCGACACAGA

GTGATGCAGG

ATCATTAAAA

ATATGGATAT

AAGTGAATCT

TCCTGAATAT

CACAAATTGT

TGCAACATGT

TAGAAAACCC

TTCACACAGT

TGCTAGTTTA

ATGCTAAATG

TGATTTTTTA

AGAATAATGG

CCTTGCGAAC

GTACATCACA

GTAACAAGAA

GGAAGAAGAA

CACGTTTATG

AGTAGTAGTA

GATTCTGATA

AAATTTAATA

AAATGATATT

ATTTACATAA

GAAGATGTCA

GATATCAAGA

ATGGTGAACA

CAGGATATGG

ACAATTGTGC

GTGAATCTGA

TTGAAAATTG

TTATATAATA

ATCGATAGTT

3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3907

I

I

INFORMATION FOR SEQ ID NO:34: SEQUENCE CIHARACTERISTICS: LENGTH: 25 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Region LOCATION: 3 OTHER INFORMATION: /note= either Asn or Arg."1 (ix) FEATURE: NAME/KEY: R~egion LOCATION: 12 OTHER INFORMATION: /note= either Asn or Arg."1 "This amino acid may be "This amino acid may be (xi) Met 1 Ile SEQUENCE DESCRIPTION: SEQ ID NO:34: Ala Xaa Asp Leu Val. Ser Leu Leu Phe Met Xaa Xaa Tyr Val Asn 5 10 Glu Ile Asn Glu Ala Val Xaa Glu INFORM.1TION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 17 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide SUB3STITUTE

SHEET

-I

I

SWO 92/14818 PCT/US92/00855 86 (ix) FEATURE: NAME/KEY: Region LOCATION: OTHER INFORMATION: /note= "This amino acid may be either Thr or Ile." (xi) SEQUENCE DESCRIPTION: SEQ ID Met Lys Ile Thr Ser Ser Thr Glu Val Asp Pro Glu Tyr Val Xaa Ser 1 5 10 Asn INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: Asn Ala Leu Phe Phe Asn Val Phe 1 INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: Glu Val Asp Pro Glu Tyr Val 1 INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 66 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: ATGGCTAGAG ATCTCGTAAG TTTACTATTT ATGTGTAACT ATGTTAATAT TGAAATTAAC GAAGCA 66 SUBSTITUTE

SHEET

LI

WO 92/14818 87 INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO ATGAAAATAA CATCTAGTAC AGAAGTAGAC CCCGAGTi INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown PCT/US92/00855 :39: ATG TAACTAGTAA T (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID AATAATAGAT TTAATAATGT ATTT SUBSTITUTE

SHEET

1 fi WO 92/14818 PCT/US92/00855 87A American Type Culture Collection 12101 rarklIwn Drive Rock, llc, M D 20152 USA Telephone: (301)231.5520 Tiles: 198-05 ATCCNORTH FAXi 301-770-2517 BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF TIE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE INTERNA TIONAL FORM RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT ISSUED PURSUANTTO RULE 7.3 AND VIABILITY STATEMENT ISSUED PURSUANTTO RULE 10.2 To: (Name and Address of epositor or Attorney) University of Florida Attention: Richard W. Moyer, Ph.D.

Department of Immunology and Medical Microbiology College of Medicine Box J-266 J. Hillis Miller Health Center Galnesville, FL 32610-0266 Deposited on Behalf of: University of Florida Identification Reference by Depositor: ATCC Designation Escherichia coli SURE strain Stratagene, pMEG-tkl 68532 Escherlchia coil (Stratagene SURE strain), pRH512 68533 The deposits were accompanied by: a scientific description X a proposed taxonomic description Indicated above.

The deposits were received February 26. 1991 by this International Depository Authority and have been accepted.

AT YOUR REQUEST: X We will inform you of requests for the strains for 30 years.

The strains will be made available if a patent office signatory to the Budapest Treaty certifies one's right to receive, or if a U.S. Patent is issued citing the strains.

If the cultures should die or be destroyed during the effective term of the deposit, it shall be your responsibility to replace them with living cultures of the same.

The strains will be maintained for a period of at least 30 years after the date of deposit, and for a period of at least five years after the most recent request for a samole. The United States and many other countries are signatory to the Budapest Treaty.

The viability of the cultures cited above was tested March 11, 1 91. On that date, the cultures Swere viable.

International Depository Authority: American Type Culture Collection, Rockville, Md. 20852 USA Signature of person having authority to represent ATCC: bblA. PH a P Date: March 13. 1991 Bobble A. Brandon, Head, ATCC Patent Depository cc: Roman Saliwanchik Bruce Clary SUBSTITUTE SHEET

Claims (17)

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 being any 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 which specifically characterises said Entomopoxvirus spheroidin gene polynucleotide sequence.

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. The sequence according to claim 4 being any one 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 which specifically characterises said Entomopoxvirus spheroidin gene polynucleotide sequence.

6. The sequence according to any one of the preceding claims 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 fragment thereof which specifically characterises said Entomopoxvirus spheroidin gene polynucleotide sequence, associated with a second polynucleotide sequence encoding a heterologous gene.

8. An Entomopoxvirus spheroidin polypeptide, an analog or fragment thereof which specifically characterises said Entomopoxvirus spheroidin gene polynucleotide sequence. i 9. The polypeptide according to claim 8, fused to a heterologous protein or S peptide. S 30 10. A recombinant polynucleotide molecule comprising a polynucleotide sequence encoding the Entomopoxvirus spheroidin promoter sequence, an allelic variant or fragment thereof which specifically characterises said Entomopoxvirus spheroidin gene polynucleotide sequence, 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.

11. A recombinant molecule comprising a polynucleotide sequence encoding the Entomopoxvirus spheroidin gene, an allelic variant or fragment thereof which specifically characterises said Entomopoxvirus spheroidin gene polynucleotide sequence, linked in frame to a polynucleotide sequence encoding a selected heterologous gene sequence. (N:\LIBAAI00272:LMM I 89

12. A recombinant molecule comprising an Entomopoxvirus spheroidin gene polynucleotide sequence, in allelic variant or fragment thereof which specifically characterises said Entomopoxvirus spheroidin gene polynucleotide sequence, into which a heterologous gene sequence has been inserted.

13. A recombinant virus comprising a polynucleotide sequence comprising an Entomopoxvirus spheroidin gene polynucleotide sequence, an allelic variant or fragment thereof which specifically characterises said Entomopoxvirus spheroidin gene polynucleotide sequence, optionally linked to a selected heterolcgous gene sequence.

14. The virus according to claim 13, which is a poxvirus selected from the group consisting of a vertebrate poxvirus, orthopoxvirus, suipoxvirus, vaccinia virus and entomopoxvirus. A cell infected with a recombinant virus comprising an Entomopoxvirus spheriodin gene polynucleotide sequence, an allelic variant or fragment thereof which specifically characterises said Entomopoxvirus spheroidin gene polynucleotide sequence, optionally linked to a selected heterologous gene sequence. 2 16. The cell according to claim 15 consisting of insect cells or mammalian cells. ,17. 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 fragment thereof which specifically characterises said Entomopoxvirus spheroidin gene polynucleotide sequence, operably linked to a selected heterologous gene sequence encoding said polypeptide, and recovering said polypeptide from the culture medium.

18. 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. S..e 19. An Entomopoxvirus spheroidin gene polynucleotide sequence free from S association with other viral nucleotide sequences with which it is associated in nature substantially as herein described with reference to any one of Examples 1 to 7. An Entomopoxvirus spheroidin polypeptide, an analog or fragment thereof which specifically characterises said Entomopoxvirus spheroidin gene polynucleotide sequence, substantially as herein described with reference to Example 3.

21. A recombinant polynucleotide molecule substantially as herein described with reference to any one of Examples 1 to 9.

22. A recombinant molecule substantially as herein described with refe: to any one of Examples 1 to 9.

23. A recombinant virus comprising a polynucleotide sequence of claim 19, optionally linked to a selected heterologous gene sequence. IN:\LIBAA100272:LMM tP rr~trsrer;.~i2?3i=-~:

24. A cell infected with a recombinant virus according to claim 23. A method for producing a selected polypeptide which method is substantially as herein described with reference to any one of Examples 1 to 9.

26. The selected polypeptide produced by the method according to claim 17 or claim Dated 22 August, 1995 University of Florida Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON I S [N:\LIBAA10027.' '.M