Technical Field

This invention relates to providing plants with resistance to maize chlorotic dwarf virus (MCDV) and viruses to which MCDV infection or resistance provides cross-resistance, including maize dwarf mosaic virus strain A (MDMV-A). Background of the Invention

Virus-induced diseases in agronomically important crops have cost farmers a great loss of income due to reduced yields. Traditionally, virus diseases have been controlled by breeding for host plant resistance or by controlling insects that transmit diseases. Chemical means of protection are not generally possible for most viruses, and where possible are not generally practical. It has been known for many years that viral symptoms can be reduced in virus-infected plants by prior inoculation with a mild strain of the same virus, a phenomena known as cross- protection, as described by Sequeira, L., Trends in Biotechnology. 2, 25 (1984). Cross-protection is considered successful if the disease symptoms of the superinfecting (the more virulent) virus can be delayed or suppressed. There are several disadvantages to applying this type of cross-protection to the field situation: 1) application of the mild strain virus to entire fields is usually not practical, 2) the mild strain might undergo mutation to a more highly virulent strain,

3) the protecting strain might interact synergistically with a non-related virus causing a severe pathogenic infection,

4) a protecting virus in one crop may be a severe pathogen in another crop, and

5) a protective strain may cause a significant loss of yield in itself.

One proposed solution to these disadvantages has been to introduce a single viral gene into the host plant genome to cross-protect, rather than infect-_with an intact virus. This single gene cross-protection strategy has already been proven successful using the coat protein gene from tobacco mosaic virus (TMV-CP). As

reported by Abel, P.P., et.al., Science. 232, 738 (1986), transgenic tobacco plants, expressing TMV mRNA and coat protein (CP),demonstrated delayed or suppressed symptom development upon infection with TMV. TMV-CP transgenic tomato plants have been described by Nelson, R.S., e al., Bio /Technology. 6, 403 (1988), to show evidence of protection from TMV as well as three strains of tomato mosaic virus (ToMV). Other approaches using DNA clones of viruses to engineer resistance include positive interference, as described by Golemboski et al. Proc. Natl. Acad. Sci. USA.87, 6311 (1990) and Carr and Zaitlin, Mol. PI. .Microbe Inter.. 4, 579 (1991); and antisense RNA, as described by Powell et al., Proc. Natl. Acad. Sci. USA. 86, 6949 (1989).

Numerous viruses exist for which resistance is desired. Maize chlorotic dwarf virus causes a somewhat variable mosaic or yellow streaking and occasional stunting in maize. Early infections can result in severe symptoms including premature death. The virus is spread by the blackfaced leafhopper (Graminella nigrifons). MCDV can overwinter in Johnsongrass (Sorghum halepense) and as a result has become a recurrent problem in areas where Johnsongrass is a common weed. Combined infections with maize dwarf mosaic virus can cause more severe symptoms although the syndrome is less well characterized than Corn Lethal Necrosis. Only limited success has been obtained to date in developing MCDV-resistant maize lines, due to the difficulties of selecting efficiently for resistance to an obligately insect transmitted virus, as well as a lack of usable sources of resistance in agronomically useful maize lines. Thus, there is a continuing need for genes, plant transformation vectors, and transformed plant materials providing resistances to pathogenic viruses such as MCDV. Unfortunately, while certain plant viruses, such as tobacco mosaic virus, have coat protein genes that are found on subgenomic RNA and are therefore relatively easy to identify and clone for use in engineered cross-protection, maize chlorotic dwarf virus belongs to a completely separate group, the only other (tentatively assigned) member of which is the spherical virus of the rice tungro disease (RTSV). In addition, MCDV has a number of unusual biological properties which make identification of an appropriate gene difficult. For example, all attempts to mechanically transmit MCDV have been unsuccessful. As another example, MCDV appears to be a phloem-restricted virus. MCDV also has three coat proteins, and it was not known whether expression of one protein would be sufficient to confer immunity or whether all three would need to be expressed. Nor was it known

which protein would be the appropriate one to express if only one could be expressed. Further, the genome of MCDV has an unusual genome organization to provide for the expression of multiple coat proteins. Brief Description of the Drawing Figures Figure 1 is a schematic illustration of the manner in which the nucleic acid sequence of MCDV-type strain was obtained by sequencing overlapping cDNA clones.

Figure 2 is an a schematic illustration of the unusual organization of the MCDV genome. Disclosure of the Invention

In the present invention, methods and materials are provided to isolate any or all of the three coat protein genes from maize chlorotic dwarf virus (MCDV). One or more of these genes (MCDV-CP , where x is 1, 2, or 3) is then incorporated in an expression cassette designed for suitable expression in a plant cell system. The resulting transformation vector is then introduced into maize callus to provide cross-protection to MCDV-related viral infections. MCDV has a single, long RNA core having the sequence shown in SEQUENCE I.D. No. 4. Description of the Preferred Embodiments

The present invention provides cDNA clones from the RNA genome of maize chlorotic dwarf virus which code substantially solely for the coat protein of the virus. These clones are incorporated into an expression cassette in which the cDNA clone is operably linked to plant or bacterial regulatory sequences which cause the expression of the cDNA clone in living plant or bacterial cells, respectively. It is important that the cloned gene have a start codon in the correct reading frame for the structural sequence. The resulting bacterial vectors can be readily inserted into bacteria for expression and characterization of the sequence. Accordingly, the present invention also provides bacterial cells containing as a foreign plasmid at least one copy of the foregoing bacterial expression cassette. In addition, the plant expression cassette preferably includes a strong constitutive promoter sequence at one end to cause the gene to be transcribed at a high level and a poly-A recognition sequence at the other end for proper processing and transport of the messenger RNA. .An example of such a preferred (empty) expression cassette into which the cDNA of the present invention can be inserted is the pPHI414 plasmid developed by Beach et al. of Pioneer Hi-Bred International, Inc., Johnston, IA, as disclosed in U.S. Patent Application No. 07/785,648, filed October 31, 1991. Highly preferred

plant expression cassettes will be designed to include one or more selectable marker genes, such as kanamycin resistance or herbicide tolerance genes. The plant expression vectors of this invention can be inserted, using any convenient technique, including electroporation (in protoplasts), microprojectile bombardment, and microinjection, into cells from monocotyledonous or dicotyledonous plants, in cell or tissue culture, to provide transformed plant cells containing as foreign DNA at least one copy of the DNA sequence of the plant expression cassette. Preferably, the monocotyledonous species will be selected from maize, sorghum, wheat and rice, and the dicotyledonous species will be selected from soybean, alfalfa, tobacco and tomato. Using known techniques, protoplasts can be regenerated and cell or tissue culture can be regenerated to form whole fertile plants which carry and express the desired cDNA clone for MCDV coat protein. Accordingly, a highly preferred embodiment of the present invention is a transformed maize plant, the cells of which contain as foreign DNA at least one copy of the DNA sequence of an expression cassette of this invention.

Finally, this invention provides methods of imparting resistance to maize chlorotic dwarf virus to plants of a MCDV susceptible taxon, comprising the steps of: a) culturing cells or tissues from at least one plant from the taxon, b) introducing into the cells of the cell culture or tissue culture at least one copy of an expression cassette comprising a cDNA clone from the RNA genome of MCDV which codes substantially solely for the coat protein of the virus, operably linked to plant regulatory sequences which cause the expression of the cDNA clone in the cells, and c) regenerating MCDV-resistant whole plants from the cell or tissue culture. Once whole plants have been obtained, they can be sexually or clonally reproduced in such manner that at least one copy of the sequence provided by the expression cassette is present in the cells of progeny of the reproduction.

.Alternatively, once a single transformed plant has been obtained by the foregoing recombinant DNA method, conventional plant breeding methods can be used to transfer the coat protein gene and associated regulatory sequence via crossing and backcrossing. Such intermediate methods will comprise the further steps of a) sexually crossing the MCDV resistant plant with a plant from the MCDV susceptible taxon;

b) recovering reproductive material from the progeny of the cross; and c) growing resistant plants from the reproductive material. Where desirable or necessary, the characteristics of the susceptible taxon can be substantially preserved by expanding this method to include the further steps of repetitively: a) backcrossing the MCDV resistant progeny with MCDV susceptible plants from the susceptible taxon; and b) selecting for expression of MCDV resistance among the progeny of the backcross, until the desired percentage of the characteristics of the susceptible taxon are present in the progeny along with the gene imparting MCDV resistance.

By the term "taxon" herein is meant a unit of botanical classification of genus or lower. It thus includes genus, species, cultivars, varieties, variants, and other minor taxonomic groups which lack a consistent nomenclature. It will also be appreciated by those of ordinary skill that the plant vectors provided herein can be incorporated into Agrobacterium tumefaciens or Agrobacterium rhizogenes, which can then be used to transfer the vector into susceptible plant cells, primarily from dicotyledonous species. Thus, this invention provides a method for imparting MCDV resistance in Agrobacterium-susceptible dicotyledonous plants in which the expression cassette is introduced into the cells by infecting the cells with Agrobacterium tumefaciens, a plasmid of which has been modified to include the plant expression cassette of this invention. The following description further exemplifies the compositions of this invention and the methods of making and using them. However, it will be understood that other methods, known by those of ordinary skill in the art to be equivalent, can also be employed.

1. Isolation and cloning of MCDV cDNA

The type strain of MCDV was maintained in the maize inbred Oh28 by transmission with the leafhopper G. nigrifrons and viral particles were isolated as previously described (Hunt et al., Phytopathology 78, 449 (1-988)). MCDV particles were suspended in NETS (10 mM Tris, pH 7.5; 100 mM NaCl; 1 mM Na ₂ EDTA; 0.5% SDS) and extracted with 1:1 chloroform:ρhenol to isolate MCDV RNA.

First and second strand cDNA synthesis were by the method of Gubler and Hoffman, Gene 25, 263 (1983) utilizing cDNA synthesis kits (Amersham, Arlington Heights, IL). For the initial cDNA libraries, double-stranded cDNA was treated

with EcoRI methylase, ligated to GGAATTCC EcoRI linkers, digested with EcoRI and separated from linkers by column fractionation. The cDNA was ligated to EcoRI-cleaved _gtl0 and EcoRI-deaved, phosphatased (CIP) _gtll phage arms. After packaging, the _gtlO phage were plated on bacterial strain NM514 and screened for MCDV-spedfic inserts by filter plaque hybridization (Benton and

32 Davis, Science 196, 180 (1977)), using P-labeled cDNA's random-primed from the

MCDV genomic RNA. MCDV-positive phage were purified and the cDNA inserts subdoned into ρUC119 (Vieira and Messing, Meth. Enzymol. 153, 3 (1987)) for further analysis. Hybridization positive dones from the initial gtlO library induded: p3-13, p36-45, pH9, pKl, pGl, pC5 (Figure 1). After packaging, the _gtll phage were plated on bacterial strain Y1090 ^r" and screened with antisera to either intact MCDV virions or isolated, individual MCDV capsid proteins (Maroon, MS Thesis, Ohio State University (1989)) as described by Mierendorf, et al. Meth. Enzymol. 152,458 (1987). Positive phage dones were identified with antisera spedfic to either cpl or cp2, and cDNA inserts from these phage were subdoned into pUC119. The anti-cpl-speάfic cDNA done, p7C5, and the anti-cp2-spedfic cDNA dones, p7E6 and p7D7, (Figure 1) were chosen for study. .Analysis of initial cDNAs revealed that a number of dones terminated at identical EcoRI sites which were shown to be present in the viral sequence. This result indicated that the methylation of the initial cDNAs was incomplete. To obtain cDNAs to the rest of MCDV and to overlap the initial dones, two additional cDNA libraries were prepared, one primed with oligo-dT(12-18) and one random-primed. Double-stranded cDNA prepared as above was ligated to a 20/24 nt. blunt end /EcoRI adaptor (Amersham), and adaptor cDNAs were kinased and ligated to EcoRI-deaved/phosphatased ρUC119. Plasmid done ρdT2 (Figure 1) was derived from the dT-primed library and plasmids pL142, pL221, and ρL411 (Figure 1) were derived from the random-primed library. 2. Sequendng of MCDV cDNA Single-stranded DNA templates for sequencing were derived by superinfection with M13K07 of bacterial strain MV1190 containing the pUC119 based cDNAs (Figure 1), doned in both orientations, as described by McMullen et al-, Nuc. Acids Res.. 14, 4953 (1986) and Vieira and Messing, Meth. Enzymol. 153, 3 (1987). Ordered deletions from the full-length single-stranded templates were prepared by the method of Dale et al., Plasmid 13, 31 (1985). Dideoxynudeotide sequencing reactions with the Klenow fragment of Pol. I or Sequenase (U.S.

Biochemicals, Cleveland, OH) were performed using 35 S-dATP. Greater than 99% of the total sequence was obtained from both strands and the majority was read from three or more templates. The 5' sequence not contained on cDNA was obtained by direct RNA sequendng, using the sequencing primer S'-GGTCTACTCACGGCACGCCA-S" (SEQUENCE I.D. NO. 3) with an RNA sequendng kit (Boehringer Mannhein, Indianapolis, IN) as recommended except that tailing of reaction products with dTTP by terminal deoxynudeotidyl transferase using the method of DeBorde et al., Anal. Biochem. 157,275 (1986) was added to improve resolution of final bases. To obtain the amino-terminal protein sequence of MCDV capsid proteins,

MCDV partides were disrupted in Laemmli loading buffer and the individual capsid protein separated on a 12.5%-4% Laemmli slab gel (Laemmli, Nature 227, 680 (1970)). The proteins were electrotransfered to Immobilon-P membrane (Millipore, Bedford, MA) using a 10 mM CAPS, pH 11.0; 10% MeOH transfer buffer, stained. with Coomassie Blue R-250 for visualization and exdsed. Automated amino-terminal protein sequendng was performed by the Iowa State University Biochemistry Instrumentation Center (.Ames, IA).

DNA and protein sequence analysis was performed using the IntelliGenetics (Mountain View, CA) molecular biology software on a Digital VAX 8250 located at the USDA-ARS-ASRR (Agricultural Systems Research Resource) Beltsville, MD.

The nudeic add sequence of MCDV-type strain was obtained by sequendng overlapping cDNA dones (Figure 1) that covered all but 13 nudeotides at the 5' terminus of MCDV. The 5' end sequence was obtained by direct RNA sequendng. Despite repeated attempts and the use of terminal transferase in the manner of DeBorde et ak, -Anal. Biochem.. 157, 275 (1986) the first nudeotide could not be definitely determined. In part for this reason, the expressions "coding substantially for" and "coding substantially solely for" are used herein, and with regard to the use of the word "substantially" refer to sequences which code for no more than a few (five or less) amino adds greater or lesser on either end of the desired protein or proteins, or which have an equivalent number of nudeotide-bases more or less than the native sequence.

The genomic RNA of MCDV-type (SEQUENCE I.D. NO. 4) was determined to be 11785 nudeotides long, exdusive of the poly-A tail at the 3' terminus. This sequence permits the construction of a DNA molecule which codes for the entire maize chlorotic dwarf virus, or any portion or functional unit thereof which is

useful in conferring resistance to the virus when expressed in plant cells. Such resistance can readily be evaluated using routine testing methods such as those disclosed herein. Computer anlaysis of the sequence indicated a long open reading frame from nudeotide 456 to nudeotide 10826. The translation of this open reading frame would result in a protein of 3457 amino adds with a derived molecular weight of 388,890 daltons. The open reading frame begins with two AUG triplets, neither of which is in a particularly favorable context for initiation of translation when compared with the analyses of translation start sequences by Lutcke et al., EMBO T. 6, 43 (1987); and Kosak, T. Cell Biol. 108, 229 (1989) by the scanning model. In addition, there are 13 AUG triplets preceding the double AUG that starts the open reading frame. A long untranslated 3' leader containing multiple AUG triplets before the beginning of a very long reading frame is similar to the animal picornaviruses as described by Stanway, T. Gen. Virol. 71, 2483 (1990). Internal initiation at the AUG for the long open reading frame has been demonstrated to occur for a number of the animal picornaviruses as seen in Pelletier and Sonenberg, Nature. 334, 320 (1988) and Jang ej al., L Virol.. 63, 1651 (1989). The mechanism for initiation of translation for MCDV has not been characterized.

The derived amino add sequence of MCDV-type was compared to the Protein Identification Resource, Version 32 and the University of Geneva, Version 22, protein data banks for sequence similarity using the IFIND (IntelliGenetics) program based on the algorithm of Wilber and Lipman, Proc. Natl. Acad. Sci. USA. 80, 726 (1983). The highest similarity score was with the comovirus, cowpea mosaic virus (CPMV) as reported by Lomonossoff and Shanks, EMBO T.. 2, 2253 (1983) and the second highest score was with the nepovirus, grapevine fanleaf virus (GFLV) as reported by Ritzenthaler et al., T. Gen. Virol.. 72, 2357 (1991). For both viruses the region of similarity preceeded and induded the first conserved motif of RNA-dependent RNA polymerases as defined by Poch et al. EMBO T.. 8, 3867 (1989). The IFIND program identified weaker similarity with additional nepoviruses and some of the animal picornaviruses. The conservation of protein sequence and gene order for the plant comoviruses, nepoviruses and potyviruses, and the animal picornaviruses is well documented by, inter alia, Agros et al., Nuc. Adds Res.. 12, 7251 (1984); Goldbach, Ann. Rev. Phytopath.. 24, 289 (1986); and Domier et al., Virology. 158, 20 (1987) and has led to the proposal- of the picornavirus-like "supergroup". Two additional conserved protein regions involved in genome replication for picorna-like viruses are the NTP bind g/helicase region,

as described by Agros et al., above, and Gorbalenya et al., Nuc. Acids Res.. 17, 4713 (1989) and the C-terminal region, cysteine active site of the 3C-like proteases, as also described by Agros et al., above, and by Grief et al., . Gen. Virol.. 69, 1517 (1988).

The electrophoresis of MCDV virions on denaturing protein gels reveals three structural proteins, designated cpl, cp2 and cp3 with molecular weights of 32.5 kd, 27 kd, and 24.5 kd; respectively. Antiserum spedfic to cpl was used to screen a _gtll library to isolate the done p7C5, and antiserum spedfic to cp2 was used to identify the cDNAs p7E6 and p7D7 (Figure 1). This result indicated that an antigenic region of cpl was located between 4063-4903 and an antigenic region of cp2 was located between 1815-2941. Automated amino-terminal sequendng was performed on each of the MCDV capsid proteins. The amino-terminus of cp2 was apparently blocked as no sequence was obtained. The 15 amino acids at the NH2"terminus of cp3 were determined to be LQVASLTDIGELSSV, as shown in SEQUENCE I.D. NO. 2 and SEQUENCE I.D. NO. 6. This sequence is an exact match to the derived protein sequence encoded by nudeotides 3144-3188. Likewise, the 15 amino addes at the NH ₂ -terminus of cpl, VSLGRSFENGVLIGS, as shown in SEQUENCE I.D. NO. 5 and SEQUENCE I.D. NO. 7, are an exact match to the derived protein sequence encoded by nudeotides 3750-3794. Both proteins must be derived by proteolytic deavage of the large polyprotein. The Gin/Leu deavage at the NH _^ -terminus of cρ3 and Gln/Val deavage at the N^-terminus of cpl are dipeptide deavage sites that may be used by animal picornavirus 3C proteases, according to Krausslich and Wimmer, Ann. Rev. Biochem., 57, 754 (1988), which could indicate that the 3C-similar region of the MCDV may function in capsid protein processing. Assuming that cp3 begins with the Leu at the Gin/Leu cleavage and ends with the Gin at the Gln/Val deavage for cpl, cp3 would have a derived MW of 21,933, a little less than the 24.5 kd MW determined by SDS gel electrophoresis. Although protein sequence was not obtained for cp2, the position of dones p7E6 and p7D7, and the finding that protein fusions expressed from the pEX vector for the PstI fragments 2076-2619 and 2613-3149 reacted positively with cp2-specific antiserum (McMullen, unpublished), is consistent with cp2 preceding cp3 in the polyprotein similar to the order of vp2-vp3-vpl for the animal picornaviruses. However, it is still not known if the coding region for cp2 immediately preceds cp3.

The overall genome structure of MCDV-type strain is shown in Figure 2. MCDV genome organization resembled that of the animal picornaviruses, a single

large polyprotein in which the capsid proteins are encoded 5' of the proteins presumed to be involved in genome replication. Depending on the exact location of cp2, the MCDV genome can encode up to 78 kd of protein 5' of the capsid proteins for which there are no corresponding animal picornavirus protein. This region may encode plant virus spedfic functions such as cell-to-cell movement or helper protein for insect transmission. Because MCDV is a phloem restricted virus, there is no evidence for a virus-encoded cell-to-cell movement protein. However, there is evidence for the presence of an insect transmission helper component in MCDV-infected plants according to Hunt et al., Phytopathology. 78, 449 (1988). The presence of plant-virus-spedfic proteins at the NH -terminus of the polyprotein would allow addition of these proteins without disruption of the cp proteins-replication functions genome structure typical of picornaviruses.

3. Design of the plasmid vector.

The gene MCDV coat protein 3 was placed under control of tandem cauliflower mosaic virus 35S promoters isolated from the 1841 strain of the virus, and a polyadenylation signal sequence obtained from the potato proteinase inhibitor II (Pin II) gene that exhibits enhancer-like activity. The chimeric gene also induded a 79 bp sequence Ω' from the 5' leader region of tobacco mosaic virus

(TMV) that functions as a translational enhancer; and a Zea mays alcohol dehydrogenase 1, intron 1 fragment (ADH) spanning nudeotides 119-672, trimmed to 557 bp with Bal 31 nudease, which has been shown to function as an enhancer of gene expression in monocots. The plasmids were grown in E. coli and purified by the known polyethylene glycol precipitation method of Sambrook et al., Molecular

Cloning. 1, 40 (1989). Purity was confirmed by electrophoretic analysis of the DNA fragments obtained after digestion with restriction endonudeases. The plasmid was designated ρPHI1406 and the sequence is shown in SEQUENCE I.D. No. 1.

4. Preparation of the redpient organism.

Separately, an embryogenic cell suspension line 54-68-5 was established from immature embryos obtained from a cross between a line derived from the public inbred corn line B73 and a WX 1-9 translocation stock of public inbred corn line W23.

5. Transformation

Suspension cells from (4) were bombarded with 1 ul aliquots of-a 30 ul mixture containing 10 μg of purified plasmid DNA (5 μg of the MCDV plasmid pPHI1406 (SEQUENCE I.D. No. 1), and 5 μg of the same plasmid in which the BAR

(Basta resistance) gene was substituted for the MCDV cp3 gene) pre pitated onto 1 um tungsten particles as described by numerous artides induding Klein, T.M., et al., 1988 (May) Bio /Technology 6:559-563; Klein, T. M., et al., 1988 (June) Proc. Natl. Acad. Sci. USA 85:4305-4309; T. M. Klein, et al., "Stable Genetic Transformation of Intact Applicant Nicotiana Cells by the Partide Bombardment Process", Proc. Natl. Acad. Sci. USA. Vol. 85, November 1988, pp. 8502-8505; D. T. Tomes, et al., "Transgenic Tobacco Plants and their Progeny Derived by Microprojectile Bombardment of Tobacco Leaves", Plant Molecular Biology. Vol. 14, No. 2, February, 1990, pp. 261-268, Kluwer Academic Publishers, BE; and M. C. Ross, et al., "Transient and Stable Transgenic Cells and Calli of Tobacco and Maize Following Microprojectile Bombardment", T. Cell. Biochem.. Suppl. 13D, 27th March - April 1989, P. 268, Abstract No. M. 149, Alan R. Liss, Inc. New York, US; and plated onto selective medium containing 5 ppb phosphinothriαn (Basta™).

Following a prolonged period of selection and callus growth, regeneration was initiated by placing callus on a Murashige & Skoog medium modified by addition of 0.5 mg/1 2,4-D and 5 ppb Basta. Embryogenic callus was selected and transferred to medium lacking 2,4-D and kept in a lighted growth room. Germinated plantlets were placed in culture tubes and finally planted out into soil in pots in the greenhouse. More than 150 RΛ (recombinant) plants were obtained, representing twenty independent transformation events. Transformation was confirmed by PCR amplification of a DNA fragment spanning part of the MCDV coat protein gene and the CaMV promoter. Genomic DNA samples, in which a fragment of the expected size was successfully amplified were presumed to be transformed. These plants were pollinated with pollen from non-transgenic B73 plants and the resulting It, seed was planted in a field trial under USDA supervision. The resulting plants exhibited a virus resistant phenotype, i.e., they survived and set seed under virus infection conditions in which non-transgenic plants died prematurely, as seen in the following table: Field Test Results

Transgenic* Control

Number of Plants 379 32

Number of Harvestable Ears 52 0--

% Harvested vs. Total 13.7% 0%

The screening was performed in a manner to insure maximum infection levels and severity. Thus, the level of resistance seen in this extreme test corresponds to effective, usable virus tolerance when the transformants of this invention are used under normal farming conditions. The MCDV resistance is a simply inherited, dominant trait and can, if desired, be introduced into other maize varieties by simple crossing or backcrossing. In addition to providing resistance to MCDV, this invention is also capable of conferring resistance to viruses to which plants obtain cross-resistance through infection by MCDV. In the field test described above, resistance to maize dwarf mosaic virus strain A (MDMV-A) was also observed. Accordingly, this invention provides resistance to that virus as well.

SEQUENCE LISTING (1) GENERAL INFORMATION:

(i) APPLICANT: McMullen, Michael D. ; Roth, Bradley A.; Townsend, Rod (ii) TITLE OF INVENTION: MAIZE CHLOROTIC DWARF VIRUS RESISTANCE (iii) NUMBER OF SEQUENCES: 7 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Pioneer Hi-Bred International, Inc.

(B) STREET: 700 Capital Square, 400 Locust Street

(C) CITY: Des Moines

(D) STATE: Iowa

(E) COUNTRY: United States

(F) ZIP: 50309 (V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette, 3.5 inch, 1.44 Mb storage

(B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: MS-DOS, Microsoft Windows

(D) SOFTWARE: Microsoft Windows Notepad (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Roth, Michael J.

(B) REGISTRATION NUMBER: 29,342 (C) REFERENCE/DOCKET NUMBER: 0235 US (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (515) 245-3594

(B) TELEFAX: (515) 245-3634

(2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH : 5033 base pairs

(B) TYPE : nudeotide

(C) STRANDEDNESS : double

(D) TOPOLOGY : linear (ii) MOLECULE TYPE: synthetic DNA

(A) DESCRIPTION : transformation plasmid pPHI1406 ( iii) HYPOTHETICAL: No

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 : TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG 50 GAGACGGTCA CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG 100 TCAGGGCGCG TCAGCGGGTG TTGGCGGGTG TCGGGGCTGG CTTAACTATG 150 CGGCATCAGA GCAGATTGTA CTGAGAGTGC ACCATATGCG GTGTGAAATA 200 CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGGCGCC ATTCGCCATT 250 CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTAT 300 TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA 350 ACGCCAGGGT TTTCCCAGTC ACGACGTTGT AAAACGACGG CCAGTGCCAA 400 GCTCAGATCT GAGCTTCTAG AAATCCGTCA ACATGGTGGA GCACGACACT 450 CTCGTCTACT CCAAGAATAT CAAAGATACA GTCTCAGAAG ACCAAAGGGC 500 TATTGAGACT TTTCAACAAA GGGTAATATC GGGAAACCTC CTCGGATTCC 550 ATTGCCCAGC TATCTGTCAC TTCATCAAAA GGACAGTAGA AAAGGAAGGT 600 GGCACCTACA AATGCCATCA TTGCGATAAA GGAAAGGCTA TCGTTCAAGA 650 TGCCTCTGCC GACAGTGGTC CCAAAGATGG ACCCCCACCC ACGAGGAGCA 700 TCGTGGAAAA AGAAGACGTT CCAACCACGT CTTCAAAGCA AGTGGATTGA 750 TGTGATGCTC TAGAAATCCG TCAACATGGT GGAGCACGAC ACTCTCGTCT 800 ACTCCAAGAA TATCAAAGAT ACAGTCTCAG AAGACCAAAG GGCTATTGAG 850 ACTTTTCAAC AAAGGGTAAT ATCGGGAAAC CTCCTCGGAT TCCATTGCCC 900 AGCTATCTGT CACTTCATCA AAAGGACAGT AGAAAAGGAA GGTGGCACCT 950 ACAAATGCCA TCATTGCGAT AAAGGAAAGG CTATCGTTCA AGATGCCTCT 1000 GCCGACAGTG GTCCCAAAGA TGGACCCCCA CCCACGAGGA GCATCGTGGA 1050 AAAAGAAGAC GTTCCAACCA CGTCTTCAAA GCAAGTGGAT TGATGTGATA 1100 TCTCCACTGA CGTAAGGGAT GACGCACAAT CCCACTATCC TTCGCAAGAC 1150 CCTTCCTCTA TATAAGGAAG TTCATTTCAT TTGGAGAGGA CGAGCTGCAG 1200 CTTATTTTTA CAACAATTAC CAACAACAAC AAACAACAAA CAACATTACA 1-250 ATTACTATTT ACAATTACAG TCGACGGATC AAGTGCAAAG GTCCGCCTTG 1300 TTTCTCCTCT GTCTCTTGAT CTGACTAATC TTGGTTTATG ATTCGTTGAG 1350

TAATTTTGGG GAAAGCTTCG TCCACAGTTT TTTTTTCGAT GAACAGTGCC 1400

GCAGTGGCGC TGATCTTGTA TGCTATCCTG CAATCGTGGT GAACTTATGT 1450

CTTTTATATC CTTCACTACC ATGAAAAGAC TAGTAATCTT TCTCGATGTA 1500

ACATCGTCCA GCACTGCTAT TACCGTGTGG TCCATCCGAC AGTCTGGCTG 1550 AACACATCAT ACGATATTGA GCAAAGATCG ATCTATCTTC CCTGTTCTTT 1600

AATGAAAGAC GTCATTTTCA TCAGTATGAT CTAAGAATGT TGCAACTTGC 1650

AAGGAGGCGT TTCTTTCTTT GAATTTAACT AACTCGTTGA GTGGCCCTGT 1700

TTCTCGGACG TAAGGCCTTT GCTGCTCCAC ACATGTCCAT TCGAATTTTA 1750

CCGTGTTTAG CAAGGGCGAA AAGTTTGCAT CTTGATGATT TAGCTTGACT 1800 ATGCGATTGC TTTCCTGGAC CCGTGCAGCT GCGGACGGAT CCACCATGGC 1850

ACTGCAGGTG GCATCTCTTA CAGACATAGG AGAATTGAGC AGTGTGGTTG 1900

CTACTGGTTC TTGGTCTACT ACCTCGGCTA CTAATTTGAT GGAATTAAAC 1950

ATTCATCCCA CCTCCTGTGC TATTCAGAAC GGATTGATAA CACAGACACC 2000

ATTGAGTGTT TTAGCTCATG CTTTTGCAAG GTGGAGAGGA TCGTTGAAAA 2050 TTTCCATCAT TTTCGGAGCG AGTTTGTTTA CCCGAGGACG AATCTTAGCC 2100 GCTGCTGTGC CCGTTGCTAA GCGCAAAGGT ACCATGAGCC TTGACGAGAT 2150 TAGTGGGTAT CATAATGTTT GCTGCTTATT GAATGGTCAG CAAACTACAT 2200 TTGAATTGGA AATCCCATAT TATTCTGTGG GCCAAGATTC TTTCGTGTAC 2250 CGTGATGCTC TTTTTGATAT CTCTGCGCAC GATGGGAATT TTATGATTAC 2300 TCGCTTGCAT CTCGTGATAC TGGATAAATT GGTAATGAGC GCTAATGCGA 2350 GCAACAGCAT AAATTTTTCC GTGACTCTTG GACCAGGTTC TGATTTGGAA 2400 TTGAAATATC TTGCAGGAGT ACATGGGCAG CGCATAGTCC GCGAGTTGAA 2450 GATGCAGTGA TCAACCTAGA CTTGTCCATC TTCTGGATTG GCCAACTTAA 2500 TTAATGTATG AAATAAAAGG ATGCACACAT AGTGACATGC TAATCACTAT 2550 AATGTGGGCA TCAAAGTTGT GTGTTATGTG TAATTACTAG TTATCTGAAT 2600 AAAAGAGAAA GAGATCATCC ATATTTCTTA TCCTAAATGA ATGTCACGTG 2650 TCTTTATAAT TCTTTGATGA ACCAGATGCA TTTCATTAAC CAAATCCATA 2700 TACATATAAA TATTAATCAT ATATAATTAA TATCAATTGG GTTAGCAAAA 2750 CAAATCTAGT CTAGGTGTGT TTTGCGAATT GCGGCCGCGA TCTGGGGAAT 2800 TCGTAATCAT GGTCATAGCT GTTTCCTGTG TGAAATTGTT ATCCGCTCAC 2850 AATTCCACAC AACATACGAG CCGGAAGCAT AAAGTGTAAA GCCTGGGGTG 2900 CCTAATGAGT GAGCTAACTC ACATTAATTG CGTTGCGCTC ACTGCCCGCT 2950 TTCCAGTCGG GAAACCTGTC GTGCCAGCTG CATTAATGAA TCGGCCAACG 3000 CGCGGGGAGA GGCGGTTTGC GTATTGGGCG CTCTTCCGCT TCCTCGCTCA 3050 CTGACTCGCT GCGCTCGGTC GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC 3100

TCAAAGGCGG TAATACGGTT ATCCACAGAA TCAGGGGATA ACGCAGGAAA 3 150 GAACATGTGA GCAAAAGGCC AGCAAAAGGC CAGGAACCGT AAAAAGGCCG 3200 CGTTGCTGGC GTTTTTCCAT AGGCTCCGCC CCCCTGACGA GCATCACAAA 3250 AATCGACGCT CAAGTCAGAG GTGGCGAAAC CCGACAGGAC TATAAAGATA 3300 CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT GTTCCGACCC 3350 TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG 3400 CTTTCTCATA GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG 3450 CTCCAAGCTG GGCTGTGTGC ACGAACCCCC CGTTCAGCCC GACCGCTGCG 3500 CCTTATCCGG TAACTATCGT CTTGAGTCCA ACCCGGTAAG ACACGACTTA 3550 TCGCCACTGG CAGCAGCCAC TGGTAACAGG ATTAGCAGAG CGAGGTATGT 3600 AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GCCTAACTAC GGCTACACTA 3650 GAAGGACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA 3700 AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA CAAACCACCG CTGGTAGCGG 3750 TGGTTTTTTT GTTTGCAAGC AGCAGATTAC GCGCAGAAAA AAAGGATCTC 3800 AAGAAGATCC TTTGATCTTT TCTACGGGGT CTGACGCTCA GTGGAACGAA 3850 AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCAAAAA GGATCTTCAC 3900 CTAGATCCTT TTAAATTAAA AATGAAGTTT TAAATCAATC TAAAGTATAT 3950 ATGAGTAAAC TTGGTCTGAC AGTTACCAAT GCTTAATCAG TGAGGCACCT 4000 ATCTCAGCGA TCTGTCTATT TCGTTCATCC ATAGTTGCCT GACTCCCCGT 4050 CGTGTAGATA ACTACGATAC GGGAGGGCTT ACCATCTGGC CCCAGTGCTG 4100 CAATGATACC GCGAGACCCA CGCTCACCGG CTCCAGATTT ATCAGCAATA 4150 AACCAGCCAG CCGGAAGGGC CGAGCGCAGA AGTGGTCCTG CAACTTTATC 4200 CGCCTCCATC CAGTCTATTA ATTGTTGCCG GGAAGCTAGA GTAAGTAGTT 4250 CGCCAGTTAA TAGTTTGCGC AACGTTGTTG CCATTGCTAC AGGCATCGTG 4300 GTGTCACGCT CGTCGTTTGG TATGGCTTCA TTCAGCTCCG GTTCCCAACG 4350 ATCAAGGCGA GTTACATGAT CCCCCATGTT GTGCAAAAAA GCGGTTAGCT 4400 CCTTCGGTCC TCCGATCGTT GTCAGAAGTA AGTTGGCCGC AGTGTTATCA 4450 CTCATGGTTA TGGCAGCACT GCATAATTCT CTTACTGTCA TGCCATCCGT 4500 AAGATGCTTT TCTGTGACTG GTGAGTACTC AACCAAGTCA TTCTGAGAAT 4550 AGTGTATGCG GCGACCGAGT TGCTCTTGCC CGGCGTCAAT ACGGGATAAT 4600 ACCGCGCCAC ATAGCAGAAC TTTAAAAGTG CTCATCATTG GAAAACGTTC 4650 TTCGGGGCGA AAACTCTCAA GGATCTTACC GCTGTTGAGA TCCAGTTCGA 4700 TGTAACCCAC TCGTGCACCC AACTGATCTT CAGCATCTTT TACTTTCACC 4750 AGCGTTTCTG GGTGAGCAAA AACAGGAAGG CAAAATGCCG CAAAAAAGGG 4800 AATAAGGGCG ACACGGAAAT GTTGAATACT CATACTCTTC CTTTTTCAAT 4850

ATTATTGAAG CATTTATCAG GGTTATTGTC TCATGAGCGG ATACATATTT 4900 GAATGTATTT AGAAAAATAA ACAAATAGGG GTTCCGCGCA CATTTCCCCG 4950 AAAAGTGCCA CCTGACGTCT AAGAAACCAT TATTATCATG ACATTAACCT 5000 ATAAAAATAG GCGTATCACG AGGCCCTTTC GTC 5033 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 bases

(B) TYPE: nudeotide

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: viral RNA

(A) DESCRIPTION: RNA codons for first 15 amino acids at

5' end of MCDV coat protein 3 (CP3)

(iii) HYPOTHETICAL: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

CUG CAG GUG GCA UCU CUU ACA GAC AUA GGA GAA UUG AGC AGU GUG 45

Leu Gin Val Ala Ser Leu Thr Asp lie Gly Asp Leu Ser Ser Val

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 bases

(B) TYPE: nudeotide

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: synthetic DNA (A) DESCRIPTION: sequencing primer

(iii) HYPOTHETICAL: No

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

GGTCTACTCA CGGCACGCCA 20

(2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11785 bases

(B) TYPE: nudeotide

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: viral RNA

(iii) HYPOTHETICAL: No

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

XUGAAAAGGA GGGUAUAGAG AUACCCUUCA UAUAUUCUGC GGAUGGCGUG 5 CCGUGAGUAG ACCUCGCGAC GUUUCCCAGA GGAAAAUGGA AAUGGUCCAU 10 GUAACACCAG AUAUUUAUCU GGUUGAGGAA CAUGGUUUAG UGGUAGAGAU 15 AAACUCAACU UUGUGUUGGA CCCCGAUGCU GUGAAAAGUA AAUAAAGACA 20 AGGCCACUUA GCGAAGGAUA UUCGAAGUAG UGAUGAAAGG AAGUGCAAUA 25 AGUCAUGCCG UAAGUCGCAA UGCGCUAUAA GUCAUGCCGU AAGCCGCGUC 30 GCCUGGAUUU GCUAUUAGAA UGUCCCUAGC CGGUGAUAAC CUUGAGUCCC 35 CGUCAUAGGA CUACUUUUGU UUGCUUAGUA AUACAUUGGG ACCACCCGCA 40 UGGAGCUCUG AGCCUACCAU ACAUAGUACA UUUUCCGAGG GAUUGUCUUU 45 UGAUA AUG AUG CAG ACA AAC AAC AAC CAA AAU CCC 48

Met Met Gin Thr Asn Asn Asn Gin Asn Pro ACU CAA GGA AGC AUU CCU GAG AAC UCC UCA CAA GAU CGC AAC UUA 530 Thr Gin Gly Ser lie Pro Glu Asn Ser Ser Gin Asp Arg Asn Leu

GGA GUG CCC GCU GGA UAU UCU UUA AGC GUU GAG GAC CCC UUC GGG 575 Gly Val Pro Ala Gly Tyr Ser Leu Ser Val Glu Asp Pro Phe Gly AAC CGG UCU GAC UUU CAU AUC CCA GUG CAC CAA AUC AUU CGG GAA 620 Asn Arg Ser Asp Phe His lie Pro Val His Gin lie lie Arg Glu GAG AUU GAU CGU CCA AAU UGG GUU CCU AUA UGU UCA AAC GAU UUU 665 Glu lie Asp Arg Pro Asn Trp Val Pro lie Cys Ser Asn Asp Phe CAU CUU AAC AGU GAG GAU UAU UGU GAG GAG UGC GAA UCU GAA CGG 710 His Leu Asn Ser Glu Asp Tyr Cys Glu Glu Cys Asp Ser Asp Arg AUC AAA AAU UUC GAA AUA UUC AGA UCA CAG AAU UUG AUU GAC CAA 755 lie Lys Asn Phe Asp lie Phe Arg Ser Gin Asn Leu lie Asp Gin

CAC CUA AAU CUC UGU ACU GAU UCA AAG GAU UGU GAU CAU UUU UCU 800 His Leu Asn Leu Cys Thr Asp Ser Lys Asp Cys Asp His Phe Ser UGU UUU UCC ACG AGU ACA AGU UGC.AGA UUU UGC CCU UUU UGC UUA 845 Cys Phe Ser Thr Ser Thr Ser Cys Arg Phe Cys Pro Phe Cys Leu UUC AUU UUU AAU UUG GAU AAA UUU UAC AAA CAA AAU CUA UAU UUG 890 Phe lie Phe Asn Leu Asp Lys Phe Tyr Lys Gin Asn Leu Tyr Leu AUU AGU CGU CAG GCU CUA GCU AGA UUG UUC CAC GGA AGC GCC GAA 935 lie Ser Arg Gin Ala Leu Ala Arg Leu Phe His Gly Ser Ala-Asp GAG UUA CUC AGU AGA GCG AUU UUC UUU ACG UAU AAU AUU UGU AUU 980 Glu Leu Leu Ser Arg Ala lie Phe Phe Thr Tyr Asn lie Cys lie

GAU GCA GAG GUG GUU GCU AAU AAU AGG AUU GGC UGU GAA UAU GUU 1025 Asp Ala Glu Val Val Ala Asn Asn Arg lie Gly Cys Asp Tyr Val AAG UUG UUU CAU CCA GAC CUU AGG CCU AGU AUU ACG UCU CCC CCU 1070 Lys Leu Phe His Pro Asp Leu Arg Pro Ser lie Thr Ser Pro Pro UAU GCU AGU GAU UGG GUU AUG UGU GAU AAU GCU AAA CAU CUU UUU 1115 Tyr Ala Ser Asp Trp Val Met Cys Asp Asn Ala Lys His Leu Phe GAG UGU CUU GGC CUU GGU GAC ACG ACC AGA GGA CAC CUA UAU GGA 1160 Glu Cys Leu Gly Leu Gly Asp Thr Thr Arg Gly His Leu Tyr Gly CUU AUU AGC GAG AAU GCA UAU UGG AAC GCC ACG UGC UCA AAA UGC 1205 Leu lie Ser Glu Asn Ala Tyr Trp Asn Ala Thr Cys Ser Lys Cys

GGA GCC UGU UGU CAG GGA GCA AAU GCC CGU ACG GCG AUA CCG AUA 1250 Gly Ala Cys Cys Gin Gly Ala Asn Ala Arg Thr Ala lie Pro lie GUG AUG GCG UUG CAG UAC UGC AGG GUG GAU GUG UAU UAU AGU GAG 1295 Val Met Ala Leu Gin Tyr Cys Arg Val Asp Val Tyr Tyr Ser Glu UAC UAU UUA UAC CAC AUC UAC GCU CCG GAA GAG AGA AUG AAG AUU 1340 Tyr Tyr Leu Tyr His lie Tyr Ala Pro Asp Glu Arg Met Lys lie GAU CAA CAG ACA GCA CAC UUG CUA CAC AGU AUA AUC CGA GGA GCA 1385 Asp Gin Gin Thr Ala His Leu Leu His Ser lie lie Arg Gly Ala CCA GCA GUG GAU UGC UCU GAG UUA UCU CAG GAG CCA AUU CAC AGG 1430 Pro Ala Val Asp Cys Ser Glu Leu Ser Gin Glu Pro lie His Arg

AUG GUA AUG GAU AGC UCA AAG UUA GUG GCA CUG GAU UCG ACA AUC 1475 Met Val Met Asp Ser Ser Lys Leu Val Ala Leu Asp Ser Thr lie AGG CAU CCU AAG AGC CAA GGA AGU UUG CUC GAU UCA GAA UGC GAU 1520 Arg His Pro Lys Ser Gin Gly Ser Leu Leu Asp Ser Asp Cys Asp CAU GAG UUU AUU CUA AGA ACG UCC CAU GGU AUC AAA AUA CCG AUG 1565 His Glu Phe lie Leu Arg Thr Ser His Gly lie Lys lie Pro Met AGU AAG UCU UUA UUU AUA UCA UUU CUU ACC AUG GGA GCU UAU CAU 1610 Ser Lys Ser Leu Phe lie Ser Phe Leu Thr Met Gly Ala Tyr His GGG UAU GCU CAU GAU GAU CAG CAG GAG CAA AAU GCG AUA AUA UCU 1655 Gly Tyr Ala His Asp Asp Gin Gin Glu Gin Asn Ala lie lie Ser

UUU GGU GGG AUG CCC GGA GUC AAU UUG GCU UGU AAC AAA AAU UUC 1700 Phe Gly Gly Met Pro Gly Val Asn Leu Ala Cys Asn Lys Asn Phe CUG AGA AUG CAU AAG UUG UUU UAU UCU GGA AGU UUU AGG CGC-AGA 1745 Leu Arg Met His Lys Leu Phe Tyr Ser Gly Ser Phe Arg Arg Arg CCC CUG UUU AUG AGC CAA AUU CCC UCU ACG AAU GCC ACC GCU CAG 1790

Pro Leu Phe Met Ser Gin lie Pro Ser Thr Asn Ala Thr Ala Gin UCC GGU UUU AAU GAU GAA GAA UUC GAA AGA UUG AUG GCU GAA GAG 1835 Ser Gly Phe Asn Asp Asp Asp Phe Asp Arg Leu Met Ala Asp Glu GGU GUG CAU GUC AAA GUC GAG CGU CCA AUA GCA GAG AGG UUU GAU 1880 Gly Val His Val Lys Val Glu Arg Pro lie Ala Glu Arg Phe Asp

UAU GAG GAC GUU AUU GAU AUU UAC GAU GAG ACC GAC CAC GAC AGG 1925 Tyr Glu Asp Val lie Asp lie Tyr Asp Glu Thr Asp His Asp Arg ACA CGA GCU CUA GGC CUU GGC CAA GUA UUC GGA GGU UUG CUC AAA 1970 Thr Arg Ala Leu Gly Leu Gly Gin Val Phe Gly Gly Leu Leu Lys GGA AUU UCU CAU UGU GUA GAU AGC CUA CAU AAG GUA UUU GAU UUC 2015 Gly lie Ser His Cys Val Asp Ser Leu His Lys Val Phe Asp Phe CCU CUG GAC CUG GCC AUA GAA GCA GCU CAG AAA ACU GGU GAU UGG 2060 Pro Leu Asp Leu Ala lie Asp Ala Ala Gin Lys Thr Gly Asp Trp CUU GAA GGA AAU AAA GCU GCA GUA GAU GAA ACU AAA AUU UGU GUG 2105 Leu Asp Gly Asn Lys Ala Ala Val Asp Asp Thr Lys lie Cys Val

GGC UGU CCC GAG AUU CAA AAA GAU AUG AUC AGU UUC CAG AAU GAA 2150 Gly Cys Pro Glu lie Gin Lys Asp Met lie Ser Phe Gin Asn Asp ACA AAA GAA GCU UUU GAA UUA AUA CGA UCA AGU AUA AAG AAG CUU 2195 Thr Lys Asp Ala Phe Asp Leu lie Arg Ser Ser lie Lys Lys Leu UCC GAG GGC AUU GAC AAA AUC ACG AAG AUG AAU GCU ACG AAC UUU 2240 Ser Glu Gly lie Asp Lys lie Thr Lys Met Asn Ala Thr Asn Phe GAA CGA AUC CUA GAC GGG AUU AAA CCA AUC GAG AGC AGG UUG ACA 2285 Asp Arg lie Leu Asp Gly lie Lys Pro lie Glu Ser Arg Leu Thr GAA CUU GAG AAC AAG GCA CCC GCU UCA GAC AGC AAA GCC AUG GAA 2330 Asp Leu Glu Asn Lys Ala Pro Ala Ser Asp Ser Lys Ala Met Asp

GCU CUG GUC CAG GCC GUG AAA GAC UUG AAA AUC AUG AAA GAG GCG 2375 Ala Leu Val Gin Ala Val Lys Asp Leu Lys lie Met Lys Glu Ala AUG CUC GAU CUA AAU CGA AGA CUG AGC AAG CUG GAA GGA AAG AAA 2420 Met Leu Asp Leu Asn Arg Arg Leu Ser Lys Leu Asp Gly Lys Lys AGU GAU GGC CAG ACU ACU GAA GGG ACA GCG GGA GAG CAA CAA CCG 2465 Ser Asp Gly Gin Thr Thr Asp Gly Thr Ala Gly Glu Gin Gin Pro AUC CCU AAG ACU CCA ACU CGA GUG AAG GCA AGA CCA GUU GUG AAG 2510 lie Pro Lys Thr Pro Thr Arg Val Lys Ala Arg Pro Val Val Lys CAA UCA GGA ACG AUA AUG GUA AAC GAA GAG AGC ACA GAA ACU UUC 2555 Gin Ser Gly Thr lie Met Val Asn Asp Glu Ser Thr Asp Thr Phe

AGG GAU AAU GAG AGU CGA GUG ACU GAC CCU AAC AGG AGC GAU AUG 2600 Arg Asp Asn Glu Ser Arg Val Thr Asp Pro Asn Arg Ser Asp Met UUU GCU GCU GUU ACU GCA GAA UAC UUA GUU AAA UCG UUU ACA UGG 2645 Phe Ala Ala Val Thr Ala Asp Tyr Leu Val Lys Ser Phe Thr Trp AAA GUU UCU GAU GGA CAA GAU AAA GUU UUG GCU GAC CUU GAU UUA 2690 Lys Val Ser Asp Gly Gin Asp Lys Val Leu Ala Asp Leu Asp Leu CCU CAA GAC UUA UGG AAA UCC AAU UCC CGA UUG AGU GAU AUC AUG 2735 Pro Gin Asp Leu Trp Lys Ser Asn Ser Arg Leu Ser Asp lie Met GGG UAU UUC CAA UAU UAU GAU GCA ACC GGA AUC ACU UUU CGC AUA 2780 Gly Tyr Phe Gin Tyr Tyr Asp Ala Thr Gly lie Thr Phe Arg lie

ACG ACA ACA UGU GUU CCU AUG CAC GGU GGU ACU UUA UGU GCU GCU 2825 Thr Thr Thr Cys Val Pro Met His Gly Gly Thr Leu Cys Ala Ala UGG GAU GCU AAU GGU UGC GCU ACA CGA CAA GGU AUA GCC ACA ACG 2870 Trp Asp Ala Asn Gly Cys Ala Thr Arg Gin Gly lie Ala Thr Thr GUU CAG CUG ACU GGU UUG CCC AAA ACA UUU AUU GAA GCU CAC AGC 2915 Val Gin Leu Thr Gly Leu Pro Lys Thr Phe lie Asp Ala His Ser UCA UCA GAA ACG AUA AUC GUG GUA AAG AAU UCC AAU AUA CAA UCC 2960 Ser Ser Asp Thr lie lie Val Val Lys Asn Ser Asn lie Gin Ser GCG AUU UGU CUA AGU GGA AGU GAG CAC UCG UUU GGG AGA AUG GGA 3005 Ala lie Cys Leu Ser Gly Ser Glu His Ser Phe Gly Arg Met Gly

AUC CUG AAG AUC UGU UGC UUG AAU ACG UUG AAU GCG CCA AAG GAA 3050 lie Leu Lys lie Cys Cys Leu Asn Thr Leu Asn Ala Pro Lys Asp GCU ACA CAG CAA GUG GCU GUG AAC GUC UGG AUU AAG UUU GAC GGA 3095 Ala Thr Gin Gin Val Ala Val Asn Val Trp lie Lys Phe Asp Gly GUU AAA UUU CAC GUU UAU UCU UUA AGG AAA AAU CCA GUC GUU UCG 3140 Val Lys Phe His Val Tyr Ser Leu Arg Lys Asn Pro Val Val Ser CAA CUG CAG GUG GCA UCU CUU ACA GAC AUA GGA GAA UUG AGC AGU 3185 Gin Leu Gin Val Ala Ser Leu Thr Asp lie Gly Asp Leu Ser Ser GUG GUU GCU ACU GGU UCU UGG UCU ACU ACC UCG GCU ACU AAU UUG 3230 Val Val Ala Thr Gly Ser Trp Ser Thr Thr Ser Ala Thr Asn Leu

AUG GAA UUA AAC AUU CAU CCC ACC UCC UGU GCU AUU CAG AAC GGA 3275 Met Asp Leu Asn lie His Pro Thr Ser Cys Ala lie Gin Asn Gly UUG AUA ACA CAG ACA CCA UUG AGU GUU UUA GCU CAU GCU UUU GCA 3320 Leu lie Thr Gin Thr Pro Leu Ser Val Leu Ala His Ala Phe Ala AGG UGG AGA GGA UCG UUG AAA AUU UCC AUC AUU UUC GGA GCG AGU 3365

Arg Trp Arg Gly Ser Leu Lys lie Ser lie lie Phe Gly Ala Ser UUG UUU ACC CGA GGA CGA AUC UUA GCC GCU GCU GUG CCC GUU GCU 3410 Leu Phe Thr Arg Gly Arg lie Leu Ala Ala Ala Val Pro Val Ala AAG CGC AAA GGU ACC AUG AGC CUU GAC GAG AUU AGU GGG UAU CAU 3455 Lys Arg Lys Gly Thr Met Ser Leu Asp Glu lie Ser Gly Tyr His

AAU GUU UGC UGC UUA UUG AAU GGU CAG CAA ACU ACA UUU GAA UUG 3500 Asn Val Cys Cys Leu Leu Asn Gly Gin Gin Thr Thr Phe Asp Leu GAA AUC CCA UAU UAU UCU GUG GGC CAA GAU UCU UUC GUG UAC CGU 3545 Asp lie Pro Tyr Tyr Ser Val Gly Gin Asp Ser Phe Val Tyr Arg GAU GCU CUU UUU GAU AUC UCU GCG CAC GAU GGG AAU UUU AUG AUU 3590 Asp Ala Leu Phe Asp lie Ser Ala His Asp Gly Asn Phe Met lie ACU CGC UUG CAU CUC GUG AUA CUG GAU AAA UUG GUA AUG AGC GCU 3635 Thr Arg Leu His Leu Val lie Leu Asp Lys Leu Val Met Ser Ala AAU GCG AGC AAC AGC AUA AAU UUU UCC GUG ACU CUU GGA CCA GGU 3680 Asn Ala Ser Asn Ser lie Asn Phe Ser Val Thr Leu Gly Pro Gly

UCU GAU UUG GAA UUG AAA UAU CUU GCA GGA GUA CAU GGG CAG CGC 3725 Ser Asp Leu Asp Leu Lys Tyr Leu Ala Gly Val His Gly Gin Arg AUA GUC CGC GAG UUG AAG AUG CAG GUU UCA UUG GGU CGG UCA UUU 3770 lie Val Arg Glu Leu Lys Met Gin Val Ser Leu Gly Arg Ser Phe GAG AAU GGA GUG CUU AUU GGU AGU GGC UUC GAC GAC UUG CUA CAA 3815 Glu Asn Gly Val Leu lie Gly Ser Gly Phe Asp Asp Leu Leu Gin AGA UGG AGU CAU UUG GUG UCC AUG CCU UUU AAU GCA AAA GGA GAC 3860 Arg Trp Ser His Leu Val Ser Met Pro Phe Asn Ala Lys Gly Asp AGC GAU GAG AUC CAA GUC UUU GGC UAU AUC AUG ACU GUU GCC CCG 3905 Ser Asp Glu lie Gin Val Phe Gly Tyr lie Met Thr Val Ala Pro

GCG UAU CGU UCC CUU CCA GUC CAC UGC ACG CUG CUA AGU UGG UUU 3950 Ala Tyr Arg Ser Leu Pro Val His Cys Thr Leu Leu Ser Trp Phe UCA CAA UUA UUC GUG CAG UGG AAA GGU GGU AUA AAG UAU AGA CUA 3995 Ser Gin Leu Phe Val Gin Trp Lys Gly Gly lie Lys Tyr Arg Leu CAC AUU GAU UCA GAA GAG CGC AGA UGG GGU GGA UUC AUC AAA GUU 4040 His lie Asp Ser Asp Glu Arg Arg Trp Gly Gly Phe lie Lys Val UGG CAU GAC CCA AAU GGC UCU UUG GAU GAA GGG AAA GAA UUU GCU 4085 Trp His Asp Pro Asn Gly Ser Leu Asp Asp Gly Lys Asp Phe-Ala AAA GCG GAU AUU CUA UCG CCA CCA GCC GGA GCU AUG GUU CGU UAU 4130 Lys Ala Asp lie Leu Ser Pro Pro Ala Gly Ala Met Val Arg Tyr

UGG AAC UAU UUA AAU GGA GAC UUG GAG UUU ACA GUA CCA UUU UGU 4175 Trp Asn Tyr Leu Asn Gly Asp Leu Glu Phe Thr Val Pro Phe Cys GCU AGA ACC AGU ACG CUG UUC AUA CCA AAA GCU AUG AUU GCC ACC 4220 Ala Arg Thr Ser Thr Leu Phe lie Pro Lys Ala Met lie Ala Thr GAU UCA AAG UCA UGG AUU CUG AAC UAC AAC GGU ACA UUG AAU UUC 4265 Asp Ser Lys Ser Trp lie Leu Asn Tyr Asn Gly Thr Leu Asn Phe GCG UAC CAA GGA GUA GAU GAC UUC ACA AUU ACA GUG GAA ACA AGU 4310 Ala Tyr Gin Gly Val Asp Asp Phe Thr lie Thr Val Asp Thr Ser GCA GCC GAC GAC UUU GAA UUU CAC GUU CGA ACA GUU GCA CCC CGC 4355 Ala Ala Asp Asp Phe Asp Phe His Val Arg Thr Val Ala Pro Arg

GCU GGA AAG GUC AAC GAA GCU UUU GCC AAA UUG GAG UAC GCU UCU 4400 Ala Gly Lys Val Asn Asp Ala Phe Ala Lys Leu Glu Tyr Ala Ser GAU UUA AAG GAU AUC AAA GAA UCU CUG ACA UCU UCC ACU CGU UUG 4445 Asp Leu Lys Asp lie Lys Asp Ser Leu Thr Ser Ser Thr Arg Leu AAA GGG CCU CAU UAU AAA ACG AAA AUU ACC UCA AUA GAG CCA AAU 4490 Lys Gly Pro His Tyr Lys Thr Lys lie Thr Ser lie Glu Pro Asn AAA AUU GAU GAA AAU GAG UCC UCA CGU GGU AAA GAU AAC AAG UCA 4535 Lys lie Asp Asp Asn Glu Ser Ser Arg Gly Lys Asp Asn Lys Ser AAU UCG AAA UUU GAG GAC UUA CUC AAU GCA ACA GCU CAG AUG GAU 4580 Asn Ser Lys Phe Glu Asp Leu Leu Asn Ala Thr Ala Gin Met Asp

UUU GAU CGA GCC ACA GCG AAC GUU GGG UGU GUG CCA UUC UCC AUU 4625 Phe Asp Arg Ala Thr Ala Asn Val Gly Cys Val Pro Phe Ser lie GCA AAG ACA GCA AAG GUG CUU UCG GAA CGC GAG ACG UGU AAG AAG 4670 Ala Lys Thr Ala Lys Val Leu Ser Asp Arg Glu Thr Cys Lys Lys AUG GCA GAU GUG UUA GAU UUC ACA CAC UCA UGU UUG AAC UUA GAC 4715 Met Ala Asp Val Leu Asp Phe Thr His Ser Cys Leu Asn Leu Asp AGU CAA CCU GCG GCG GCA AGA UUA GCA GCG GCC AUU UCU CAA AUA 4760 Ser Gin Pro Ala Ala Ala Arg Leu Ala Ala Ala lie Ser Gin lie GCA CCU AUU AUG GAG AGC AUC GGU AGA ACC ACU CAA AGC GUA GAG 4805 Ala Pro lie Met Glu Ser lie Gly Arg Thr Thr Gin Ser Val Glu

GAA AAA UUG GCU UCU GUG GAU ACA UUU AGG GAC AAA AUC AUG GCU 4850 Asp Lys Leu Ala Ser Val Asp Thr Phe Arg Asp Lys lie Met Ala CUA AUU UCA AAC GUG CUU GGG GAU ACU CUA CCU GGA CUG GCC-AUU 4895 Leu lie Ser Asn Val Leu Gly Asp Thr Leu Pro Gly Leu Ala lie GCU GAC UUC AAA AAA GGA AAA UAU GUG UGG GCC UCG UUC CUG ACA 4940

Ala Asp Phe Lys Lys Gly Lys Tyr Val Trp Ala Ser Phe Leu Thr AUG AUA GCC GCU UGC GUA GUA GCU UGG GCU GCC ACU AGC AAG AAA 4985 Met lie Ala Ala Cys Val Val Ala Trp Ala Ala Thr Ser Lys Lys AGC UUC UUG AAA AGA UUU GCA GUG GUA GCU AUG AUA AUU UGG AGC 5030 Ser Phe Leu Lys Arg Phe Ala Val Val Ala Met lie lie Trp Ser

CCA UUU CUC GCA AGU AAA AUA UGG GCG CUU GGU ACA UGG AUU AGG 5075 Pro Phe Leu Ala Ser Lys lie Trp Ala Leu Gly Thr Trp lie Arg AAG AGC UGG AGU AAG CUU UGG CCU AAG UCA GAC UCA UGC CGA CAA 5120 Lys Ser Trp Ser Lys Leu Trp Pro Lys Ser Asp Ser Cys Arg Gin CAC UCU UUG GCA GGC CUG UGU GAA AGU GUG UUC ACA UCA UUC AAG 5165 His Ser Leu Ala Gly Leu Cys Asp Ser Val Phe Thr Ser Phe Lys GAU UUC CCU GAC UGG UUU AAA UCA GGA GGA AUC ACG AUU GUG ACG 5210 Asp Phe Pro Asp Trp Phe Lys Ser Gly Gly lie Thr lie Val Thr CAA GUU UGC ACA GUA UUA CUG ACG AUA GUG AGU CUG AUU ACA CUU 5255 Gin Val Cys Thr Val Leu Leu Thr lie Val Ser Leu lie Thr Leu

GGA ACU AUA CCA AGC ACG AAA CAA AAU GCU ACG UUC GCA GAC AAA 5300 Gly Thr lie Pro Ser Thr Lys Gin Asn Ala Thr Phe Ala Asp Lys UUU AAA GAA UUU GGU AAC AUG AGC AGA GCU ACA ACG UCA AUA GCU 5345 Phe Lys Asp Phe Gly Asn Met Ser Arg Ala Thr Thr Ser lie Ala GCA GGU UAC AAG ACG AUA UCA GAG CUG UGU UCG AAA UUC ACC AAU 5390 Ala Gly Tyr Lys Thr lie Ser Glu Leu Cys Ser Lys Phe Thr Asn UAC UUG GCU GUA ACC UUC UUU GGG GCG CAA GUU GAU GAC GAU GCU 5435 Tyr Leu Ala Val Thr Phe Phe Gly Ala Gin Val Asp Asp Asp Ala UUC AAG GGU UUG GUA GCG UUC AAC GUU AAG GAA UGG AUU CUU GAA 5480 Phe Lys Gly Leu Val Ala Phe Asn Val Lys Asp Trp lie Leu Asp

GUG AAA AAC CUG UCU CUU GAG GAA AAC AAA UUU AGU GGU UUU GGU 5525 Val Lys Asn Leu Ser Leu Glu Asp Asn Lys Phe Ser Gly Phe Gly GGU GAU GAG CAU CUU GUC AAG GUU AGA CAU UUA UAU GAU AAA UCU 5570 Gly Asp Glu His Leu Val Lys Val Arg His Leu Tyr Asp Lys Ser GUG GAA AUA ACC UAU AAG UUG CUC CAG AAA AAU CGA GUU CCC AUU 5615 Val Asp lie Thr Tyr Lys Leu Leu Gin Lys Asn Arg Val Pro lie GCU AUG CUU CCU AUC AUC CGA GAC ACG UGU AAG AAG UGC GAG GAU 5660 Ala Met Leu Pro lie lie Arg Asp Thr Cys Lys Lys Cys Glu—Asp UUG CUA AAC GAG AGU UAU ACU UAC AAA GGU AUG AAA ACU CCG CGC 5705 Leu Leu Asn Glu Ser Tyr Thr Tyr Lys Gly Met Lys Thr Pro Arg

GUG GAC CCA UUC UAU AUA UGC CUU UUU GGA GCA CCU GGA GUU GGC 5750 Val Asp Pro Phe Tyr lie Cys Leu Phe Gly Ala Pro Gly Val Gly AAG UCC ACA GUG GCA UCG AUG AUU GUU GAC GAU UUG UUG GAU GCU 5795 Lys Ser Thr Val Ala Ser Met lie Val Asp Asp Leu Leu Asp Ala AUG GGC GAA CCU AAG GUU GAU AGG AUC UAU ACG CGA UGC UGU UCU 5840 Met Gly Asp Pro Lys Val Asp Arg lie Tyr Thr Arg Cys Cys Ser GAU CAA UAU UGG AGC AAU UAU CAC CAC GAG CCA GUU AUU UGU UAU 5885 Asp Gin Tyr Trp Ser Asn Tyr His His Glu Pro Val lie Cys Tyr GAC GAC UUG GGG GCA AUC AGC AGA CCA GCG AGU UUA UCA GAC UAU 5930 Asp Asp Leu Gly Ala lie Ser Arg Pro Ala Ser Leu Ser Asp Tyr

GGG GAG AUA AUG GGA AUC AAA UCG AAC AGA CCA UAC UCC CUA CCU 5975 Gly Glu lie Met Gly lie Lys Ser Asn Arg Pro Tyr Ser Leu Pro AUG GCU GCU GUU GAU GAG AAA GGA AGG CAU UGU UUA UCG CGA UAC 6020 Met Ala Ala Val Asp Glu Lys Gly Arg His Cys Leu Ser Arg Tyr CUC AUU GCU UGU ACA AAU UUA ACC CAU CUG GAC GAU ACG GGC GAU 6065 Leu lie Ala Cys Thr Asn Leu Thr His Leu Asp Asp Thr Gly Asp GUG AAA ACA AAG GAU GCC UAC UAU CGC AGA AUC AAU GUC CCA GUG 6110 Val Lys Thr Lys Asp Ala Tyr Tyr Arg Arg lie Asn Val Pro Val ACA GUG ACG AGA GAA GUA ACC GCC AUG AUG AAC CCC GAG GAC CCA 6155 Thr Val Thr Arg Asp Val Thr Ala Met Met Asn Pro Glu Asp Pro

ACU GAU GGA CUA CGU UUC ACC GUG GAG CAA GUG CUU GAU GGA GGU 6200 Thr Asp Gly Leu Arg Phe Thr Val Glu Gin Val Leu Asp Gly Gly AGA UGG AUU AAU GUU ACU GAA AGC CGU CUC CUC AAU GGA AGG AUG 6245 Arg Trp lie Asn Val Thr Asp Ser Arg Leu Leu Asn Gly Arg Met CCA UUC AGG GCU GAA GAU CUC AUG AAC AUG AAC UAC AGU UAC UUU 6290 Pro Phe Arg Ala Asp Asp Leu Met Asn Met Asn Tyr Ser Tyr Phe AUG GAG UUU CUC AAG AUG UAU GCU GCU UUA UAU AUG GAA AAU CAA 6335 Met Glu Phe Leu Lys Met Tyr Ala Ala Leu Tyr Met Asp Asn Gin AAC AUG UUG GUG GCA AAA UUG AGA GGA ACA GAG AUC CCA GAA UCA 6380 Asn Met Leu Val Ala Lys Leu Arg Gly Thr Glu He Pro Asp Ser

CGU AGU UCA GAG AAU GAA GAA CUU.GAA UUC GAU UAU UUG GCU ACA 6425 Arg Ser Ser Glu Asn Asp Asp Leu Asp Phe Asp Tyr Leu Ala Thr GCU CAG AUG GAC CAU ACA GUG ACA UUU GGG GAA CUA GUU ACC AAA 6470 Ala Gin Met Asp His Thr Val Thr Phe Gly Asp Leu Val Thr Lys UUC AAC UCG UAU AAG CUU ACU GGG AAA CAA UGG AAC AAG AGG CUC 6515

Phe Asn Ser Tyr Lys Leu Thr Gly Lys Gin Trp Asn Lys Arg Leu UGU GAA CUU GGA UGG ACA UCU CUA GAC GGA UGG AAC ACG AAC AAG 6560 Cys Asp Leu Gly Trp Thr Ser Leu Asp Gly Trp Asn Thr Asn Lys AUU AUG AGA UUC GAC GAU CUA GUU GCC GGA UUC UGU GGU UGC UCA 6605 He Met Arg Phe Asp Asp Leu Val Ala Gly Phe Cys Gly Cys Ser

AGG AAU GAG AAU UGC AAU UUU GAC UUC UAU CAU CAG AGA CUU CAA 6650 Arg Asn Glu Asn Cys Asn Phe Asp Phe Tyr His Gin Arg Leu Gin GCA UGU UUG AAC AAG AAA GGG UUU GCU CCC GCA UAU CAA UAU UUC 6695 Ala Cys Leu Asn Lys Lys Gly Phe Ala Pro Ala Tyr Gin Tyr Phe AAC CUU CAC AAG UUG AAU UCA GAC ACC CAG AAG ACA GAG CUC AAG 6740 Asn Leu His Lys Leu Asn Ser Asp Thr Gin Lys Thr Glu Leu Lys CUU AAA UGC GGG ACA ACU GCU GAA GAU UUA UUC AGA CAA GCU GAC 6785 Leu Lys Cys Gly Thr Thr Ala Asp Asp Leu Phe Arg Gin Ala Asp UUG AUG GUC AUA UUC UCC UAC CUC UUA UUU GUU GCG AGA AUU GGG 6830 Leu Met Val He Phe Ser Tyr Leu Leu Phe Val Ala Arg He Gly

GUG AGU GGA UCU CAU GUG UGU CUG UCA UAU AAC AUG UUG AAC GUC 6875 Val Ser Gly Ser His Val Cys Leu Ser Tyr Asn Met Leu Asn Val AAG GAU GUC AAG GAU UUU GAG AUA UGC AGG GAG AAC GUU CUU GAU 6920 Lys Asp Val Lys Asp Phe Glu He Cys Arg Glu Asn Val Leu Asp UUG UCC AGA AAA ACU ACA AUC GAC GGU GAA GAA UGC UAU AUC UGG 6965 Leu Ser Arg Lys Thr Thr He Asp Gly Asp Asp Cys Tyr He Trp AAU UUU AUU UCU GAU AUC UUC CCA CGC AUU GUG GCU AAG UAC AAC 7010 Asn Phe He Ser Asp He Phe Pro Arg He Val Ala Lys Tyr Asn UGU GUU GUG CUU AAC GAC GGA GAG AAG AGA UAC AUC UUC GUG ACU 7055 Cys Val Val Leu Asn Asp Gly Glu Lys Arg Tyr He Phe Val Thr

GAC AGC GCG CCC ACU AGG AUC UUU CCC GAU UUG GCU UGG UCA GAU 7100 Asp Ser Ala Pro Thr Arg He Phe Pro Asp Leu Ala Trp Ser Asp CUU AUU UCC GGC AAG CAA GUU GUG AGU CCA AAC AUU AUC AAA GUG 7145 Leu He Ser Gly Lys Gin Val Val Ser Pro Asn He He Lys Val GCU GGA GAA ACC AAG UCG AAA ACC AUU GCC CCU GUG CUA GCA GAU 7190 Ala Gly Asp Thr Lys Ser Lys Thr He Ala Pro Leu Leu Ala Asp UCC UAC AAG GUU UUC AAG GAU CCG AAG GCA UGG CUU GAG AGG AAC 7235 Ser Tyr Lys Val Phe Lys Asp Pro Lys Ala Trp Leu Glu Arg-Asn AAA GAA UUG AAA GCA GCU CUA GAA ACA GAA GAA UAU AUC GCU CUC 7280 Lys Asp Leu Lys Ala Ala Leu Asp Thr Asp Asp Tyr He Ala Leu

CUC UUU GCU GUU GCA UGU GAA GCU GGU AGA UUC ACU CAA AUU UUA 732 Leu Phe Ala Val Ala Cys Asp Ala Gly Arg Phe Thr Gin He Leu GAC AAA CCU CCC AGU AGA CGC AAG AUU UUA AAU AUG UCC GAA AGG 737 Asp Lys Pro Pro Ser Arg Arg Lys He Leu Asn Met Ser Asp Arg UAU AAU GCA UAU AUU GAA CAG GAA AAA GGG CUG AUU GGG AGA CUU 7415 Tyr Asn Ala Tyr He Asp Gin Asp Lys Gly Leu He Gly Arg Leu UCU AAA CCA GCA AAG AUA UGC UUA GCC AUA GGA ACU GGA GUU GCG 7460 Ser Lys Pro Ala Lys He Cys Leu Ala He Gly Thr Gly Val Ala AUC UUU GGG GCC CUA GCA GGC AUU GGA GUG GGU UUG UUU AAG CUG 7505 He Phe Gly Ala Leu Ala Gly He Gly Val Gly Leu Phe Lys Leu

AUA GCU CAC UUC AAC AAA GAU GAA GAA GAG GUA GAC GAA AUU GAA 7550 He Ala His Phe Asn Lys Asp Asp Asp Glu Val Asp Asp He Asp UUU GAU AUA CUC UCC CCA GAG AUG AGC GGU UCG CAC GAA UCC GGC 7595 Phe Asp He Leu Ser Pro Glu Met Ser Gly Ser His Asp Ser Gly CAA CAU ACC ACG AGG UAC GUC ACG AAG GAG CGA GUU CCA UCC AAA 7640 Gin His Thr Thr Arg Tyr Val Thr Lys Glu Arg Val Pro Ser Lys CCA GCA AGG AGG CAA CAU GAA UUU GAU CUA AUG UUC GAU AAU CUA 7685 Pro Ala Arg Arg Gin His Asp Phe Asp Leu Met Phe Asp Asn Leu CCC ACU CCA CAA GUU GAA GAG CUA AAG AGU GAG AUG ACC UGC GCC 7730 Pro Thr Pro Gin Val Asp Glu Leu Lys Ser Glu Met Thr Cys Ala

AGU GCC AGU GAU GAG CAU AAG ACU CAG UAU GUU AAA AGA AGA GUG 7775 Ser Ala Ser Asp Glu His Lys Thr Gin Tyr Val Lys Arg Arg Val GGA CCU GUA AGC AAA CGU AAG GAU GCU UCG GUA GCA GAA AUU AGU 7820 Gly Pro Val Ser Lys Arg Lys Asp Ala Ser Val Ala Asp He Ser GGA GCU CAU GCG AGU GAU CAG CAU CAU ACA GAA UAC UUG AAA GCA 7865 Gly Ala His Ala Ser Asp Gin His His Thr Asp Tyr Leu Lys Ala CGC GUU CCA CUC AUG AAA AGA AUA GCU ACC AAA GAG AGC UAU GUU 7910 Arg Val Pro Leu Met Lys Arg He Ala Thr Lys Glu Ser Tyr Val GUA ACU UAC GAU GAC GAA CCC AGC UCU CAU AUU UCC CUA GUU CGC 7955 Val Thr Tyr Asp Asp Asp Pro Ser Ser His lie Ser Leu Val Arg

AGG AUC CGA CGU ACA CGA CUG GCA AGA GCC AUC AAG CAA AUG GCA 8000 Arg He Arg Arg Thr Arg Leu Ala Arg Ala He Lys Gin Met Ala GUC CUG GAG GAC UUC CCA UCU ACC UUG GAA GAG AUA CGA CUU UGG 8045 Val Leu Glu Asp Phe Pro Ser Thr Leu Asp Glu He Arg Leu Trp AGA CAA AAC GCU GCA AAU AAA GGG GUU AUU GUU CCG AAG UAC UCA 8090

Arg Gin Asn Ala Ala Asn Lys Gly Val He Val Pro Lys Tyr Ser ACA AGU GGG AAA UUC UUC AGU GGC UUG UUG GAU GAU GAA GAA GAA 8135 Thr Ser Gly Lys Phe Phe Ser Gly Leu Leu Asp Asp Asp Asp Asp GAA CCU CAG AAU GUG AAU AUG UUG AAC GAA GAG GAC AUU GAG GUA 8180 Asp Pro Gin Asn Val Asn Met Leu Asn Asp Glu Asp He Glu Val

GAU AAG CGA AUG UUU GAG AAG AUU UCU GAG GUU AUA AGC GUG AUU 8225 Asp Lys Arg Met Phe Glu Lys He Ser Glu Val He Ser Val lie CAA CCC AGA AAG AAU GAG CUG GAA AGA AUG AUU GAG GAA GGC GUA 8270 Gin Pro Arg Lys Asn Glu Leu Asp Arg Met He Glu Asp Gly Val CAC CAC AAG GUC GUA AAG CAG GCA AGG GUU AAC GAC AAG GGC UUA 8315 His His Lys Val Val Lys Gin Ala Arg Val Asn Asp Lys Gly Leu GCC AAA GAC CCC AAC AUG GUG ACU AUC UUG ACG GAC AAA UUA AUU 8360 Ala Lys Asp Pro Asn Met Val Thr He Leu Thr Asp Lys Leu lie AAU AUU AGU GCG GUG AUC GUC AAU UUA ACG CCG ACA CGC CGG GCA 8405 Asn He Ser Ala Val He Val Asn Leu Thr Pro Thr Arg Arg Ala

UAC AUG AAC GUG GUA CGU CUU AUA GGC ACU AUA GUU GUU UGC CCA 8450 Tyr Met Asn Val Val Arg Leu He Gly Thr He Val Val Cys Pro GCC CAC UAC UUG GAA GCU UUA GAG GAA GGA GAU GAG CUG UAU UUC 8495 Ala His Tyr Leu Asp Ala Leu Glu Asp Gly Asp Glu Leu Tyr Phe AUU UGC UUC UCA UUG GUU AUC AAG CUC ACU UUU GAU CCA AGU AGA 8540 He Cys Phe Ser Leu Val He Lys Leu Thr Phe Asp Pro Ser Arg GUG ACU CUC GUG AAU AGC CAG CAG GAU UUG AUG GUU UGG GAU CUU 8585 Val Thr Leu Val Asn Ser Gin Gin Asp Leu Met Val Trp Asp Leu GGG AAC AUG GUA CCA CCC UCA AUU GAU ACU CUU AAA AUG AUA CCU 8630 Gly Asn Met Val Pro Pro Ser He Asp Thr Leu Lys Met He Pro

ACG CUU GAA GAC UGG GAU CAC UUU CAG GAU GGA CCA GGA GCC UUU 8675 Thr Leu Asp Asp Trp Asp His Phe Gin Asp Gly Pro Gly Ala Phe GCU GUU ACG AAA UAU AAC UCG AAA UUC CCA ACC AAU UAU AUC AAC 8720 Ala Val Thr Lys Tyr Asn Ser Lys Phe Pro Thr Asn Tyr He Asn ACA CUG ACU AUG AUU GAG AGG AUU AGG GCA AAU ACU CAG AAU CCC 8765 Thr Leu Thr Met He Glu Arg He Arg Ala Asn Thr Gin Asn Pro ACG GGU UGU UAU UCC AUG AUG GGC UCC CAA CAU ACA AUC ACC ACA 8810 Thr Gly Cys Tyr Ser Met Met Gly Ser Gin His Thr He Thr -Thr GGA UUG CGA UAU CAA AUG UUC UCU CUU GAU GGA UUC UGC GGU GGG 8855 Gly Leu Arg Tyr Gin Met Phe Ser Leu Asp Gly Phe Cys Gly Gly

UUA AUC CUG AGA GCC AGC ACA AAC AUG GUG AGA AAG GUC GUC GGG 890 Leu He Leu Arg Ala Ser Thr Asn Met Val Arg Lys Val Val Gly AUC CAC GUU GCU GGA AGC CAG AAU CAC GCU AUG GGA UAU GCA GAG 894 He His Val Ala Gly Ser Gin Asn His Ala Met Gly Tyr Ala Glu UGC CUU AUU GCA GAA GAU UUA CGG GCU GCA GUG GCG AGA UUG GCG 899 Cys Leu He Ala Asp Asp Leu Arg Ala Ala Val Ala Arg Leu Ala CUA GAU CCU AGA AGC ACC AUC CAG GCA AGU CUG AAA GGU AGG AUU 903 Leu Asp Pro Arg Ser Thr He Gin Ala Ser Leu Lys Gly Arg He GAU GCU GUU UCU AAA CAA UGU GGU UUA GAC AGA GCU CUG GGU ACG 9080 Asp Ala Val Ser Lys Gin Cys Gly Leu Asp Arg Ala Leu Gly Thr

AUA GGA UGU CAC GGG AAA GUU GCC UCU GAA GAU AUU ACA AGU GCC 9125 He Gly Cys His Gly Lys Val Ala Ser Asp Asp He Thr Ser Ala GCC ACG AAA ACU UCC AUA AGA AAG UCA AGA AUA CAU GGU CUA GUG 9170 Ala Thr Lys Thr Ser He Arg Lys Ser Arg He His Gly Leu Val GGU GAG AUU AGA ACU GAG CCU UCA AUU UUA CAC GCU CAU GAU CCC 9215 Gly Glu He Arg Thr Glu Pro Ser He Leu His Ala His Asp Pro CGA CUG CCU AAA GAC AAG AUU GGG AAA UGG GAC CCG GUU AUU GAG 9260 Arg Leu Pro Lys Asp Lys He Gly Lys Trp Asp Pro Val He Glu GCA UCA AUG AAG UAU GGU UCG AGA AUC ACA CCG UUC CCU GUA GAC 9305 Ala Ser Met Lys Tyr Gly Ser Arg He Thr Pro Phe Pro Val Asp

CAA AUU CUG GAA GUG GAG GAU CAU CUU UCU AAA AUG UUG GCC AAU 9350 Gin He Leu Asp Val Glu Asp His Leu Ser Lys Met Leu Ala Asn UGU GAG AAU UCA AAA AAC AAG CGG CAG GUU AAU AAU CUA GAA AUA 9395 Cys Glu Asn Ser Lys Asn Lys Arg Gin Val Asn Asn Leu Asp He GGG AUU AAU GGA AUU GAC CAG UCG GAU UAU UGG CAA CAG AUA GAA 9440 Gly He Asn Gly He Asp Gin Ser Asp Tyr Trp Gin Gin He Asp AUG GAU ACU UCA AGU GGU UGG CCA UAC GCU AAG CGU AAA CCU GUU 9485 Met Asp Thr Ser Ser Gly Trp Pro Tyr Ala Lys Arg Lys Pro Val GGG GCA GCU GGA AAG AAA UGG CUA UUC GAG CAA GAC GGC ACA UAU 9530 Gly Ala Ala Gly Lys Lys Trp Leu Phe Glu Gin Asp Gly Thr Tyr

CCC UCC GGA AAA CCU CGA UAU GUA UUU GGA GAU GCC GGG UUG AUU 9575 Pro Ser Gly Lys Pro Arg Tyr Val Phe Gly Asp Ala Gly Leu He GAG AGC UAU AAC UCG AUG CUU GGU GAG GCG AAG CAA GGC AUU-AGU 9620 Glu Ser Tyr Asn Ser Met Leu Gly Glu Ala Lys Gin Gly He Ser CCC ACU GUC GUC ACA AUU GAG UGC GCA AAA GAU GAG AGG CGG AAG 9665

Pro Thr Val Val Thr He Glu Cys Ala Lys Asp Glu Arg Arg Lys CUU AAU AAG AUA UAU GAG AAA CCC GCC ACU CGG ACG UUC ACC AUA 971 Leu Asn Lys He Tyr Glu Lys Pro Ala Thr Arg Thr Phe Thr He CUG CCA CCU GAG AUU AAU AUU UUA UUC AGG CAG UAU UUC GGA GAU 975 Leu Pro Pro Glu He Asn He Leu Phe Arg Gin Tyr Phe Gly Asp

UUU GCA GCG AUG GUA AUG ACA UGU AGA GCC AAG CUU UUC UGU CAA 980 Phe Ala Ala Met Val Met Thr Cys Arg Ala Lys Leu Phe Cys Gin GUU GGC AUC AAC CCA GAG UCA AUG GAG UGG GGU GAU CUC AUG CUA 984 Val Gly He Asn Pro Glu Ser Met Glu Trp Gly Asp Leu Met Leu GGU CUA AAG GAG AAA UCA ACU AAG GGA UUU GCA GGA GAU UAU UCG 989 Gly Leu Lys Glu Lys Ser Thr Lys Gly Phe Ala Gly Asp Tyr Ser AAG UUC GAU GGA AUC GGA GAC CCC CAG AUU UAU CAU UCA AUU ACC 9935 Lys Phe Asp Gly He Gly Asp Pro Gin He Tyr His Ser He Thr CAA GUA GUC AAC AAC UGG UAU AAC GAU GGG GAA GAA AAU GCG ACU 9980 Gin Val Val Asn Asn Trp Tyr Asn Asp Gly Asp Asp Asn Ala Thr

AUC AGG CAU GCU CUG AUA AGU AGC AUU AUA CAC AGG CGG GGC AUU 1002 He Arg His Ala Leu He Ser Ser He He His Arg Arg Gly He GUG AAA GAA UAU UUG UUC CAG UAU UGC CAG GGU AUG CCA UCA GGG 1007 Val Lys Asp Tyr Leu Phe Gin Tyr Cys Gin Gly Met Pro Ser Gly UUC GCC AUG ACA GUG AUA UUC AAU UCG UUU AUG AAC UAU UAU UAU 1011 Phe Ala Met Thr Val He Phe Asn Ser Phe Met Asn Tyr Tyr Tyr CUG UCU UUG GCC UGG AUG AAU CUG AUA AGU GCA UCC CCC CUU AGU 1016 Leu Ser Leu Ala Trp Met Asn Leu He Ser Ala Ser Pro Leu Ser CCA CAA GCU UCU UUG AGA UAU UUU GAU GAG UAU UGU AAG GUC AUU 1020 Pro Gin Ala Ser Leu Arg Tyr Phe Asp Glu Tyr Cys Lys Val He

GUU UAC GGU GAU GAU AAU AUU GUU GCC GUC AAC GAA GAA UUC UUA 1025 Val Tyr Gly Asp Asp Asn He Val Ala Val Asn Asp Asp Phe Leu GAG UAC UAU AAC UUG AGG CUU GUG GCA GGC UAU CUU .AGU CAA UUU 1029 Glu Tyr Tyr Asn Leu Arg Leu Val Ala Gly Tyr Leu Ser Gin Phe GGA GUA AGC UAC ACU GAU GAC GCC AAG AAC CCA AUA GAG AAG AGC 1034 Gly Val Ser Tyr Thr Asp Asp Ala Lys Asn Pro He Glu Lys Ser GAA CGA UAU GUG AAG AUA GAA GAC GUU ACG UUC UUA AAA CGG CGA 1038 Asp Arg Tyr Val Lys He Asp Asp Val Thr Phe Leu Lys Arg-Arg UGG GUG AGU CUU GGC GGU AGA GCU UCG AUG CUG UAC AAA GCU CCG 1043 Trp Val Ser Leu Gly Gly Arg Ala Ser Met Leu Tyr Lys Ala Pro

CUU GAC AAG GUU AGC AUU GAG GAA AGG CUU AAC UGG AUC AGA GAG 104 Leu Asp Lys Val Ser He Glu Asp Arg Leu Asn Trp He Arg Glu UGU GAC GAU GGG GAA CUA GCU CUG GUG CAG AAC AUU GAA AGU GCU 105 Cys Asp Asp Gly Asp Leu Ala Leu Val Gin Asn He Asp Ser Ala CUG UAC GAA GCU AGU AUU CAU GGC CAC ACA UAU UUU GGA GAG CUU 105 Leu Tyr Asp Ala Ser He His Gly His Thr Tyr Phe Gly Glu Leu AAA GAU AAA AUU GCU AAA GCC UGU GAU GCA GUC AUG AUA ACU AUG 106 Lys Asp Lys He Ala Lys Ala Cys Asp Ala Val Met He Thr Met CCA AAU AUA AGA UAU AUU GAC UGC CAG AGA CGA UGG UGG ACC UCC 1065 Pro Asn He Arg Tyr He Asp Cys Gin Arg Arg Trp Trp Thr Ser AUG ACU GGU GGG UAU CUU GAG CCG UCU GAU GUC ACC AAA CUU GUA 1070 Met Thr Gly Gly Tyr Leu Glu Pro Ser Asp Val Thr Lys Leu Val AGG CUU GUU GAG AAA GGA CUA CUA GAC CCG AAA UCA GUA UGG AAA 1074 Arg Leu Val Glu Lys Gly Leu Leu Asp Pro Lys Ser Val Trp Lys GAC CCA UUG UAC AGA ACC AAC AAG UUG CUA UUC GAC CUA UUG AGG 1079 Asp Pro Leu Tyr Arg Thr Asn Lys Leu Leu Phe Asp Leu Leu Arg GAG GUU AAG GCA GCA CCC CUG GCC GCA UUU GUG GUC UAA 1082 Glu Val Lys Ala Ala Pro Leu Ala Ala Phe Val Val Stop GUUACCCUUC UGACAAAAGG GCCUUGAACG GUUAUGGUUG AACAGAACUG 1087 UAAAAGGUGA GGACUAUAUA AGUUGUAGUA CGGAUGAGAU UGAAAGAAAA 1092 UUGGGUCACU CCCAUUCCUU UAUUAGGAAG GAGUGAUACC UUUUGUGUAG 1097 AUCUCUACCC CGAAACUCUU GAACCCUCAC ACGUUUUGGA GUAACCAGUA 1102 CACCCUUUUA GGUGGACCCU CGACUAUAGA UCGAGACCAA GUAUUGACUU 1107 GGUGUUCACG UCUUGCCGGA CGCAAAAUGG CACCCUUGUU UAGUGAUAUC 1112 AAGGUUACAA AUGUCACGCC CCACUAGUAA AAGUUUUGGU AUAUACGCAU 1117 UCGAACCGCC AAUGUAUACG UGUUUUCCCU UUUACUUUUU GUAUGUCGUC 1122 GUGGUGACGA GAUGCACGCC UGGUCAGCGG GGAAUAAGUU CACUAUAUGA 1127 ACAGACUCCG GCGAGCGAGA CACGCUGUCG GCCUCGGGAG AGGGAACUAG 1132 CUCCAGGCAC UUAAAUCCUG AAGUGUUAGA ACUAAGCGUU UGAUCCUCCU 1137 CCGGGGGAAA GAGAACGCCA GUUCUUUAAG CCAUAACUCU AGUGAGUUGA 1142 AUCCUAUUCA UCCUUCUUAG GAUUAAGGAU UUCUGAAGUC UAUCAUGAAA 1147 AGUAGAUAGA AAGCAACACG UCAAUAACGU GGAACCUUUU CCGAGGAAGU 1152 AGGGUGCUUG UUCGAAAAUC AUGGUAGAUU CGGAAACAAU UUGCUUAGAG - 1157 UGUGUCUUUU CGCGUUGGUA GUUCAACCGU UAGGGCUAGG CACACUUCUC 1162 CACGGGUUUG UGCUGCAGUA UUAAAUAUCA UUAAGGUACU GUGCUAUAGC 1167

GGAGAAAUUA CAAAGCGUUG AACACAUUGA CGAUGGGGCC CAAUGCGCAC 11729 CCGGAUGUGU UACGCACCGU UUUUCUCUGU GUCACUAUAG AUAAAAGUGG 11779 GGUAGC-polyA 11785

(2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 bases

(B) TYPE: nudeotide

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: viral RNA

(A) DESCRIPTION: RNA codons for first 15 amino acids at 5 ' end of

MCDV coat protein 1 (CP1)

(iii) HYPOTHETICAL: No

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GUU UCA UUG GGU CGG UCA UUU GAG AAU GGA GUG CUU AUU GGU AGU 45

Val Ser Leu Gly Arg Ser Phe Glu Asn Gly Val Leu He Gly Ser

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(A) DESCRIPTION: first 15 amino acids of MCDV coat protein 3 (iii) HYPOTHETICAL: No

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

Leu Gin Val Ala Ser Leu Thr Asp He Gly Asp Leu Ser Ser Val 15 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (A) DESCRIPTION: first 15 amino acids of MCDV coat protein 1

(iii) HYPOTHETICAL: No

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Val Ser Leu Gly Arg Ser Phe Glu Asn Gly Val Leu He Gly Ser