WO1998003652A2

WO1998003652A2 - P300/cbp-associated transcriptional co-factor p/caf and uses thereof

Info

Publication number: WO1998003652A2
Application number: PCT/US1997/012877
Authority: WO
Inventors: Yoshihiro Nakatani; Bruce H. Howard
Original assignee: The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services
Priority date: 1996-07-23
Filing date: 1997-07-23
Publication date: 1998-01-29
Also published as: AU4043897A; WO1998003652A3

Abstract

The present invention provides a purified protein designated P/CAF having a molecular weight of about 93,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions and which acetylates histones and which also binds to the p300/CBP cellular protein. The present invention further provides a nucleic acid encoding the P/CAF protein as well as a vector containing the nucleic acid and a host for the vector. A purified antibody which specifically binds the P/CAF protein is also provided. Also provided are methods of screening for compounds that inhibit or stimulate the transcription modulating and histone acetyltransferase activity of P/CAF and p300/CBP.

Description

P300/CBP-ASSOCIATED TRANSCRIPTIONAL CO-FACTOR P/CAF AND USES THEREOF

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention provides a transcriptional co-factor, p300/CBP-associated factor (P/CAF), which modulates transcription through binding to the cellular transcription co-factors p300 and CBP and through acetylation of histones. Also provided are methods for screening for the presence of P/CAF and for substances which alter the transcription modulating effect and growth regulatory activity of P/CAF.

Background Art

Cellular proteins p300 and CBP are global transcriptional coactivators that are involved in the regulation of various DNA-binding transcriptional factors (Janknecht and Hunter, 1 96). Recently, p300 was found to be very closely related to CBP, a factor that binds selectively to the protein kinase A-phosphorylated form of CREB (3-5). Cellular factors p300 and CBP exhibit strong amino acid sequence similarity and share the capacity to bind both CREB and El A (6-8). Although neither p300 nor CBP by itself binds to DNA, each can be recruited to promoter elements via interaction with sequence-specific activators and functions to be a transcriptional adaptor. For simplicity, p300 and CBP will be termed p300/CBP in the context of discussing their shared functional properties.

p300/CBP is a large protein consisting of over 2,400 amino acids, known to interact with a variety of DNA-binding transcriptional factors including nuclear hormone receptors (13,57), CREB (3,4, 7), c-Jun/v-Jun (9,l l), YY1 (10), c-Myb/v-Myb (12,58), Sap-la (59), c-Fos (11) and MyoD (60). DNA-binding factors recruit p300/CBP not only by direct but also indirect interactions through cofactors; for example, nuclear hormone receptors recruit p300/CBP directly as well as through indirect interactions, via SRC-1, which stimulates transcription by binding to various nuclear hormone receptors (13,61). The transforming proteins encoded by adenovirus and several other small DNA tumor viruses disturb host cell growth control by interacting with cellular factors that normally function to repress cell proliferation. One of the most intensively studied of these viral proteins, the product of the adenovirus ElA gene, is itself sufficient for transformation (1). El A transforming activity resides in two distinct domains, the targets of which include p300/CBP and products of the retinoblastoma (RB) susceptibility gene family (1,2). Interactions of El A with p300/CBP and RB are thought to influence functionally distinct growth regulatory pathways, allowing the two domains to contribute additively to transformation (1).

The paradigm for how ElA and functionally related viral proteins perturb cell growth regulation derives in large part from studies on their interactions with RB (1,2) The molecular function of El A is based on its capacity to interfere with cellular protein- protein interactions. Since both ElA and various cellular targets bind to a site in RB termed the pocket domain (2), ElA can competitively disrupt the complex formation between RB and its cellular targets.

The second cellular factor implicated in El A-dependent transformation, p300, is believed to inhibit G0/G1 exit, to activate certain enhancers, and to stimulate differentiation (1,2). ElA inhibits the p300/CBP-mediated transcriptional activation of many promoters (14). In one case that has been examined, the complex of p300 and YY1, ElA inhibits transcription without disrupting the complex (10).

The present invention provides a cellular protein designated P/CAF which binds to p300/CBP and plays an important role in both transcription and cell cycle regulation associated with a histone acetyhransferase activity. The present invention also provides a histone acetyhransferase activity in the p300/CBP cellular protein, thus providing targets for modulating transcription and cell cycle regulation in cells. SUMMARY OF THE INVENTION

The present invention provides a purified protein designated P/CAF having a molecular weight of about 93,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions and which acetylates histones and which also binds to the p300/CBP cellular protein.

The present invention further provides a nucleic acid encoding the P/CAF protein as well as a vector containing the nucleic acid and a host for the vector A purified antibody which specifically binds the P/CAF protein is also provided.

In addition, also provided is a bioassay for screening substances for the ability to inhibit the transcription modulating activity of P/CAF and/or histone acetyhransferase activity, comprising contacting the substance with a system in which histone acetylation by P/CAF can be determined; determining the amount of histone acetylation by P/CAF in the presence of the substance; and comparing the amount of histone acetylation by P/CAF in the presence of the substance with the amount of histone acetylation by P/CAF in the absence of the substance, a decreased amount of histone acetylation by P/CAF in the presence of the substance indicating a substance that can inhibit the transcription modulating activity and/or histone acetyhransferase activity of P/CAF.

Furthermore, the present invention provides a bioassay for screening substances for the ability to inhibit the transcription modulating activity and/or histone acetyhransferase activity of P/CAF comprising contacting the substance with a system in which the p300 binding of P/CAF can be determined; determining the amount of p300 binding of P/CAF in the presence of the substance; and comparing the amount of p300 binding of P/CAF in the presence of the substance with the amount of p300 binding of P/CAF in the absence of the substance, a decreased amount of p300 binding of P/CAF in the presence of the substance indicating a substance that can inhibit the transcription modulating activity and or histone acetyhransferase activity of P/CAF. Also provided is a method for determining the amount of P/CAF in a biological sample comprising contacting the biological sample with a polypeptide comprising the amino acid sequence of SEQ ID NO:3 under conditions whereby a P/CAF/p300 complex can be formed; and determining the amount of the P/CAF/p300 complex, the amount of the complex indicating the amount of P/CAF in the sample.

The present invention additionally provides a method for determining the amount of P/CAF in a biological sample comprising contacting the biological sample with an antibody which specifically binds P/CAF under conditions whereby a P/CAF/antibody complex can be formed; and determining the amount of the P/CAF/antibody complex, the amount of the complex indicating the amount of P/CAF in the sample.

Also provided herein is an assay for screening substances for the ability to inhibit or stimulate the histone acetyhransferase activity of P/CAF, comprising: contacting the substance with a system in which histone acetylation by P/CAF can be determined; determining the amount of histone acetylation by P/CAF in the presence of the substance; and comparing the amount of histone acetylation by P/CAF in the presence of the substance with the amount of histone acetylation by P/CAF in the absence of the substance, a decreased or increased amount of histone acetylation by P/CAF in the presence of the substance indicating a substance that can inhibit or stimulate, respectively, the histone acetyhransferase activity of P/CAF.

The present invention further provides an assay for screening substances for the ability to inhibit binding of P/CAF to p300/CBP comprising: contacting the substance with a system in which the P/CAF binding of P300/CBP can be determined; determining the amount of P/CAF binding of p300/CBP in the presence of the substance, and comparing the amount of binding of P/CAF to p300/CBP in the presence of the substance with the amount of binding of P/CAF to p300/CBP in the absence of the substance, a decreased amount of binding of P/CAF to p300/CBP in the presence of the substance indicating a substance that can inhibit the ability to inhibit binding of P/CAF to p300/CBP. In addition, an assay is provided for screening substances for the ability to inhibit or stimulate the histone acetyhransferase activity of p300/CBP, comprising: contacting the substance with a system in which histone acetylation by p300/CBP can be determined; determining the amount of histone acetylation by p300/CBP in the presence of the substance; and comparing the amount of histone acetylation by p300/CBP in the presence of the substance with the amount of histone acetylation by p300/CBP in the absence of the substance, a decreased or increased amount of histone acetylation by p300/CBP in the presence of the substance indicating a substance that can inhibit or stimulate, respectively, the histone acetyhransferase activity of p300/CBP.

Furthermore, the present invention provides an assay for screening substances for the ability to inhibit binding of a DNA-binding transcription factor to p300/CBP comprising: contacting the substance with a system in which the DNA-binding transcription factor binding of P300/CBP can be determined; determining the amount of DNA-binding transcription factor binding of p300/CBP in the presence of the substance; and comparing the amount of binding of DNA-binding transcription factor to p300/CBP in the presence of the substance with the amount of binding of DNA-binding transcription factor to p300/CBP in the absence of the substance, a decreased amount of binding of DNA-binding transcription factor to p300/CBP in the presence of the substance indicating a substance that can inhibit the ability to inhibit binding of DNA- binding transcription factor to p300/CBP.

A method is also provided for inhibiting the transcription modulating activity of P/CAF in a subject, comprising administering to the subject a transcription modulating activity inhibiting amount of a substance in a pharmaceutically acceptable carrier.

Also provided in the present invention is a method for stimulating the transcription modulating activity of P/CAF in a subject, comprising administering to the subject a transcription modulating activity stimulating amount of a substance in a pharmaceutically acceptable carrier. Furthermore, the present invention provides a method for inhibiting the histone acetyhransferase activity of p300/CBP in a subject, comprising administering to the subject a histone acetyhransferase activity inhibiting amount of a substance in a pharmaceutically acceptable carrier.

Finally, the present invention additionally provides a method for stimulating the histone acetyhransferase activity of p300/CBP in a subject, comprising administering to the subject a histone acetyhransferase activity stimulating amount of a substance in a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE FIGURES

Figs. 1A-B. Fig 1 A: P/CAF-p300/CBP interaction in vivo. Cell extract was immunoprecipitated with rabbit anti-P/CAF (lanes 1, 4, and 7), rabbit anti-CBP (lanes 2 and 5), and mouse anti-p300 (lane 9) antibodies. For controls, cell extract was precipitated with rabbit control IgG (lanes 3, 6, and 8) or mouse anti-HA monoclonal antibody (lane 10). The precipitates were analyzed by immunoblotting with anti-P/CAF (lanes 1-3), anti-CBP (lanes 4-6), and anti-p300 (lanes 7-10) antibodies. The positions of non-specific bands are indicated by asterisks. Fig. IB: ElA inhibits the P/CAF-p300 interaction in vivo. Osteosarcoma cells were transfected with either control vector (lanes 1 and 4) or ElA- (lanes 2 and 5) or El AΔN- (lanes 3 and 6) expression vectors. Extract from the transfected subpopulation was immunoprecipitated with anti-P/CAF (lanes 1-3) or control (lanes 4-6) IgG. The precipitates were analyzed by immunoblotting with anti-p300 and anti-P/CAF.

Figs. 2A-F. P/CAF and ElA mediate antagonistic effects on cell cycle progression. HeLa cells (ATCC accession number CCL 2) were transfected by electroporation with 7 μg of P/CAF-expression plasmid and/or 3 μg of the full-length or the N-terminally deleted (Δ2-36) ElA 12S-expression plasmid as indicated in the figure. These plasmids were constructed by subcloning FLAG-P/CAF and ElA cDN s into pCX (34) and pcDNAI (Invitrogen), respectively. All samples, in addition, contained 1 μg of sorting plasmid (pCMN-IL2R) (31) and carrier plasmid (pCX) to normalize the total amount of DΝA to 11 μg. After transfection, cells were incubated in Dulbecco's modified Eagle's medium with 10% fetal bovine calf serum for 12 hours and subsequently labeled in medium containing 10 μM bromo-deoxyuridine (BrdU) for 30 min. Subsequently, the transfected subpopulation was purified by magnetic affinity cell sorting and nuclei were analyzed by dual parameter flow cytometry as described (32) Histograms show percentages of cells in Gl and S phases. Abscissa values represent fluorescence intensity of bound anti-BrdU antibodies in log scale.

Fig. 3. Histone acetyhransferase activity of P/CAF. Activity of hGCΝ5 (lanes 1 and 4) and P/CAF (lanes 2 and 5) that acetylates free histones (lanes 1-3) or histones in the nucleosome core particle (35) (lanes 4-6) was measured as described (36). Each reaction contains 0.3 pmol of affinity purified FLAG-hGCN5 or FLAG-P/CAF, 4 pmol of the histone octamer or the nucleosome core particle and 10 pmol of [ 1 -¹⁴C]acetyl- CoA. Note that the histone octamer dissociates into dimers or tetramers under assay conditions. Acetylated histones were detected by autoradiography after separation by SDS-PAGE. The bands corresponding to acetylated histones H3 and H4 are indicated by arrows.

DETAILED DESCRIPTION OF THE INVENTION

As used in the specification and in the claims, "a" can mean one or more, depending upon the context in which it is used.

P/CAF protein and fragments

The present invention provides a purified protein designated P/CAF having a molecular weight of about 93,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions and which acetylates histones. The P/CAF protein can also bind to the amino acid region of SEQ ED NO:3 (ammo acid (aa) residues 1753 - 1966) of the cellular transcriptional factor, p300 (which has the complete amino acid sequence of SEQ ID NO 6 and the nucleotide sequence of SEQ ID NO 12), and the amino acid region of SEQ ID NO 6 (amino acid residues 1805 - 1854) of the cellular transcriptional factor, CBP (which has the complete amino acid sequence of SEQ ID NO 7 and the nucleotide sequence of SEQ ID NO 13) The P/CAF protein can be defined by any one or more of the typically used parameters Examples of these parameters include, but are not limited to molecular weight (calculated or empirically determined), isoelectπc focusing point, specific epιtope(s), complete ammo acid sequence, sequence of a specific region (e g , N-terπunus) of the amino acid sequence and the like

For example, The P/CAF protein can consist of the amino acid sequence of SEQ ID NO 1 or the P/CAF protein can comprise the ammo acid sequence of SEQ ID NO 2 which represents the carboxy terminal end of the P/CAF protein and contains the histone acetyhransferase activity, or the amino acid sequence of SEQ ID NO 4, which represents the amino terminal end of the P/CAF protein, containing the binding site for p300/CBP Because the amino-terminal region is specific for P/CAF it can be used to define and identify P/CAF

As used herein, "purified" refers to a protein (polypeptide, peptide, etc ) that is sufficiently free of contaminants or cell components with which it normally occurs to distinguish it from the contaminants or other components of its natural environment The purified protein need not be homogeneous, but must be sufficiently free of contaminants to be useful in a clinical or research setting, for example, in an assay for detecting antibodies to the protein Greater levels of puπty can be obtained using methods derived from well known protocols Specific methods for purifying P/CAF proteins are known in the art

As will be appreciated by those skilled in the art, the invention also includes those P/CAF polypeptides having slight vaπations in amino acid sequence which yield polypeptides equivalent to the P/CAF protein defined herein Such variations may aπse naturally as allelic variations (e.g., due to genetic polymorphism) or may be produced by human intervention (e.g., by mutagenesis of cloned DNA sequences), such as induced point, deletion, insertion and substitution mutants. Minor changes in amino acid sequence are generally preferred, such as conservative amino acid replacements, small internal deletions or insertions, and additions or deletions at the ends of the molecules. Substitutions may be designed based on, for example, the model of Dayhoff, et al. (37). These modifications can result in changes in the amino acid sequence, provide silent mutations, modify a restriction site, or provide other specific mutations.

Modifications to any of the P/CAF proteins or fragments can be made, while preserving the specificity and activity (function) of the native protein or fragment thereof. As used herein, "native" describes a protein that occurs in nature. The modifications contemplated herein can be conservative amino acid substitutions, for example, the substitution of a basic amino acid for a different basic amino acid. Modifications can also include creation of fusion proteins with epitope tags or known recombinant proteins or genes encoding them created by subcloning into commercial or non-commercial vectors (e.g., polyhistidine tags, flag tags, myc tag, glutathione-S- transferase [GST] fusion protein, xylE fusion reporter construct). Furthermore, the modifications can be such as do not affect the function of the protein or the way the protein accomplishes that function (e.g., its secondary structure or the ultimate result of the protein's activity). These products are equivalent to the P/CAF protein. The means for determining the function, way and result parameters are well known.

Having provided an example of a purified P/CAF protein, the invention also enables the purification of P/CAF homologs from other species and allelic variants from individuals within a species. For example, an antibody raised against the exemplary human P/CAF protein can be used routinely to screen preparations from different humans for allelic variants of the P/CAF protein that react with the P/CAF protein- specific antibody. Similarly, an antibody raised against an epitope, for example, from a conserved amino acid region of the human P/CAF protein can be used to routinely screen for homologs of the P/CAF protein in other species. A P/CAF protein can be routinely identified in and obtained from other species and from individuals within a species using the methods taught herein and others known in the art. For example, given the present sequence, the DNA encoding a conserved amino acid sequence can be used to probe genomic DNA or DNA libraries of an organism to predictably obtain the P/CAF gene for that organism. The gene can then be cloned and expressed as the P/CAF protein and purified according to any of a number of routine, predictable methods. An example of the routine protein purification methods available in the art can be found in Pei et al. (38).

A purified polypeptide fragment of the P/CAF protein is also provided. The term "fragment" as used herein regarding a P/CAF protein, means a molecule of at least five contiguous amino acids of P/CAF protein that has at least one function shared by P/CAF protein or a region thereof. These functions can include antigenicity, binding capacity, acetyhransferase activity and structural roles, among others. The P/CAF fragment can be specific for a recited source. As used herein to describe an amino acid sequence (protein, polypeptide, peptide, etc.), "specific" means that the amino acid sequence is not found identically in any other source. The determination of specificity is made routine by the availability of computerized amino acid sequence databases and sequence comparison programs, wherein an amino acid sequence of almost any length can be quickly and reliably checked for the existence of identical sequences. If an identical sequence is not found, the protein is "specific" for the recited source. For example, a P/CAF fragment can be species- specific (e.g., found in the P/CAF protein of humans, but not of other species).

A fragment of the P/CAF protein having histone acetyhransferase activity can consist of the amino acid sequence of SEQ ID NO:2. A fragment of the P/CAF protein which binds to the amino acid sequence of SEQ ID NO: 3 on p300 and the amino acid sequence of SEQ ID NO: 9 on CBP can consist of the amino acid sequence of SEQ ID NO:4. To the extent that these fragments are specific for P/CAF, they can be used to identify and define P/CAF . An antigenic fragment of P/CAF protein is provided. An antigenic fragment has an amino acid sequence of at least about five consecutive amino acids of a P/CAF protein amino acid sequence and binds an antibody or elicits an immune response in an animal. An antigenic fragment can be selected by applying the routine technique of epitope mapping to P/CAF protein to determine the regions of the proteins that contain epitopes reactive with antibodies or are capable of eliciting an immune response in an animal Once the epitope is selected, an antigenic polypeptide containing the epitope can be synthesized directly, or produced recombinantly by cloning nucleic acids encoding the antigenic polypeptide in an expression system, according to standard methods.

Alternatively, an antigenic fragment of the antigen can be isolated from the whole P/CAF protein or a larger fragment of the P/CAF protein by chemical or mechanical disruption. Fragments can also be randomly chosen from a known P/CAF protein sequence and synthesized. The purified fragments thus obtained can be tested to determine their antigenicity and specificity by routine methods.

Nucleic Acids Encoding P/CAF Protein

An isolated nucleic acid that encodes a P/CAF protein is also provided. As used herein, the term "isolated" means a nucleic acid separated or substantially free from at least some of the other components of the naturally occurring organism, for example, the cell structural components commonly found associated with nucleic acids in a cellular environment and/or other nucleic acids. The isolation of nucleic acids can therefore be accomplished by techniques such as cell lysis followed by phenol plus chloroform extraction, followed by ethanol precipitation of the nucleic acids (39). It is not contemplated that the isolated nucleic acids are necessarily totally free of all non- nucleic acid components or all other nucleic acids, but that the isolated nucleic acids are isolated to a degree of purification to be useful in clinical, diagnostic, experimental, or other procedures such as, for example, gel electrophoresis, Southern, Northern or dot blot hybridization, or polymerase chain reaction (PCR). A skilled artisan in the field will readily appreciate that there are a multitude of procedures which may be used to isolate the nucleic acids prior to their use in other procedures These include, but are not limited to, lysis of the cell followed by gel filtration or anion exchange chromatography, binding DNA to silica in the form of glass beads, filters or diatoms in the presence of high concentrations of chaotropic salts, or ethanol precipitation of the nucleic acids

The nucleic acids of the present invention can include positive and negative strand RNA as well as DNA and can include genomic and subgenomic nucleic acids found in the naturally occurring organism The nucleic acids contemplated by the present invention include double stranded and single stranded DNA of the genome, complementary positive stranded cRNA and mRNA, and complementary cDNA produced therefrom and any nucleic acid which can selectively or specifically hybridize to the isolated nucleic acids provided herein Stringent conditions (further described below) are used to distinguish selectively or specifically hybridizing nucleic acids from non-selectively and non-specifically hybridizing nucleic acids

An isolated nucleic acid that encodes a P/CAF protein can be species-specific (i.e , does not encode the P/CAF protein of other species and does not occur in other species) Examples of the nucleic acids contemplated herein include the nucleic acid of SEQ ID NO.10 as well as the nucleic acids that encode each of the P/CAF proteins or fragments thereof described herein P/CAF proteins and protein fragments can be routinely obtained as described herein and their structure (sequence) determined by routine means including the methods as used herein

P/CAF protein-encoding nucleic acids can be isolated from an organism in which they are normally found (e.g , humans), using any of the routine techniques For example, a genomic DNA or cDNA library can be constructed and screened for the presence of the nucleic acid of interest using one of the present P/CAF protein-encoding nucleic acids as a probe Methods of constructing and screening such libranes are well known in the art and kits for performing the construction and screening steps are commercially available (for example, Stratagene Cloning Systems, La Jolla, CA). Once isolated, the nucleic acid can be directly cloned into an appropriate vector, or if necessary, be modified to facilitate the subsequent cloning steps. Such modification steps are routine, an example of which is the addition of oligonucleotide linkers, which contain restriction sites, to the termini of the nucleic acid (See, for example, ref. 39).

P/CAF protein-encoding nucleic acids can also be synthesized. For example, a method of obtaining a DNA molecule encoding a specific P/CAF protein is to synthesize a recombinant DNA molecule which encodes the P/CAF protein. For example, nucleic acid synthesis procedures are routine in the art and oligonucleotides coding for a particular protein region are readily obtainable through automated DNA synthesis. A nucleic acid for one strand of a double-stranded molecule can be synthesized and hybridized to its complementary strand. One can design these oligonucleotides such that the resulting double-stranded molecule has either internal restriction sites or appropriate 5' or 3' overhangs at the termini for cloning into an appropriate vector.

Oligonucleotides complementary to or identical with the P/CAF protein- encoding nucleic acid sequence can be synthesized as primers for amplification reactions, such as PCR, or as probes to detect P/CAF protein encoding nucleic acids by various hybridization protocols (e.g., Northern blot; Southern blot; dot blot, colony screening, etc.).

Double-stranded molecules coding for relatively large proteins can readily be synthesized by first constructing several different double-stranded molecules that code for particular regions of the protein, followed by ligating these DNA molecules together For example, Cunningham, et al. (40), have constructed a synthetic gene encoding the human growth hormone by first constructing overlapping and complementary synthetic oligonucleotides and ligating these fragments together. See also, Ferretti, et al. (41), wherein synthesis of a 1057 base pair synthetic bovine rhodopsin gene from synthetic oligonucleotides is disclosed. By constructing a P/CAF protein-encoding nucleic acid in this manner, one skilled in the art can readily obtain any particular P/CAF protein with modifications at any particular position or positions See also, U.S Patent No 5,503,995 which describes an enzyme template reaction method of making synthetic genes Techniques such as this are routine in the art and are well documented DNA encoding the P/CAF protein or P/CAF protein fragments can then be expressed in vivo or in vitro

The nucleic acid encoding the P/CAF protein can be any nucleic acid that functionally encodes the P/CAF protein To functionally encode the protein (i e , allow the nucleic acid to be expressed), the nucleic acid can include, but is not limited to, expression control sequences, such as an origin of replication, a promoter, regions upstream or downstream of the promoter, such as enhancers that may regulate the transcriptional activity of the promoter, appropriate restriction sites to facilitate cloning of inserts adjacent to the promoter, antibiotic resistance genes or other markers which can serve to select for cells containing the vector or the vector containing the insert, and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites and transcription termination sequences as well as any other sequence which may facilitate the expression of the inserted nucleic acid

Preferred expression control sequences are promoters derived from metallothionine genes, actin genes, immunoglobulin genes, CMV, SV40, adenovirus, bovine papilloma virus, etc A nucleic acid encoding a P/CAF protein can readily be determined based upon the genetic code for the amino acid sequence of the P/CAF protein and many nucleic acid sequences will encode a P/CAF protein Modifications in the nucleic acid sequence encoding the P/CAF protein are also contemplated Modifications that can be useful are modifications to the sequences controlling expression of the P/CAF protein to make production of P/CAF protein inducible or repressible as controlled by the appropriate inducer or repressor Such means are standard in the art (.see, e.g., ref 39) The nucleic acids can be generated by means standard in the art, such as by recombinant nucleic acid techniques, as exemplified in the examples herein, and by synthetic nucleic acid synthesis or in vitro enzymatic synthesis After a nucleic acid encoding a particular P/CAF protein of interest, or a region of that nucleic acid, is constructed, modified, or isolated, that nucleic acid can then be cloned into an appropriate vector, which can direct the in vivo or in vitro synthesis of that wild-type and/or modified P/CAF protein The vector is contemplated to have the necessary functional elements that direct and regulate transcription of the inserted nucleic acid, as described above The vector containing the P/CAF nucleic acid or nucleic acid fragment can be in a host (e g , cell or transgenic animal) for expressing the nucleic acid The P/CAF protein or fragment thereof can thus be produced in a host system containing the expression vector and its functional activity as described herein can be demonstrated according to methods well known in the art

There are numerous E. coli (Escherwhia coli) expression vectors known to one of ordinary skill in the art useful for the expression of proteins. Other microbial hosts suitable for use include bacilli, such as Bacillus subtihs, and other enterobacteria, such as Salmonella, Serratia, as well as various Pseudomonas species. These prokaryotic hosts can support expression vectors which will typically contain expression control sequences compatible with the host cell (e.g , an origin of replication). In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (Tip) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda The promoters will typically control expression, optionally with an operator sequence and have ribosome binding site sequences, for example, for initiating and completing transcription and translation If necessary, an amino terminal methionine can be provided by insertion of a Met codon 5' and in-frame with the gene sequence. Also, the carboxy-terminal extension of the protein can be removed using standard oligonucleotide mutagenesis procedures

Additionally, yeast expression can be used There are several advantages to yeast expression systems First, evidence exists that proteins produced in yeast secretion systems exhibit correct disulfide pairing. Second, post-translational glycosylation is efficiently carried out by yeast secretory systems The Saccharomyces cerevisiae pre- pro-alpha-factor leader region (encoded by the MFa-1 gene) is routinely used to direct protein secretion from yeast (42) The leader region of pre-pro-alpha-factor contains a signal peptide and a pro-segment which includes a recognition sequence for a yeast protease encoded by the KEX2 gene This enzyme cleaves the precursor protein on the carboxyl side of a Lys-Arg dipeptide cleavage-signal sequence The polypeptide coding sequence can be fused in-frame to the pre-pro-alpha-factor leader region This construct is then put under the control of a strong transcription promoter, such as the alcohol dehydrogenase I promoter or a glycolytic promoter The protein coding sequence is followed by a translation termination codon which is followed by transcription termination signals Alternatively, the polypeptide encoding sequence of interest can be fused to a second protein coding sequence, such as Sj26 or β-galactosidase, used to facilitate purification of the resultant fusion protein by affinity chromatography The insertion of protease cleavage sites to separate the components of the fusion protein is applicable to constructs used for expression in yeast

Efficient post-translational glycosylation and expression of recombinant proteins can also be achieved in Baculovirus expression systems in insect cells.

Mammalian cells permit the expression of proteins in an environment that favors important post-translational modifications such as folding and cysteine pairing, addition of complex carbohydrate structures and secretion of active protein Vectors useful for the expression of proteins in mammalian cells are characterized by insertion of the protein encoding sequence between a strong viral promoter and a polyadenylation signal The vectors can contain genes conferring either gentamicin or methotrexate resistance for use as selectable markers For example, the antigen and immunoreactive fragment coding sequence can be introduced into a Chinese hamster ovary (CHO) cell line using a methotrexate resistance-encoding vector Presence of the vector RNA in transformed cells can be confirmed by Northern blot analysis and production of a cDNA or opposite strand RNA corresponding to the protein encoding sequence can be confirmed by Southern and Northern blot analysis, respectively A number of other suitable host cell lines capable of secreting intact proteins have been developed in the art and include the CHO cell lines, HeLa cells, myeloma cell lines, Jurkat cells, and the like Expression vectors for these cells can include expression control sequences, as described above. The vectors containing the nucleic acid sequences of interest can be transferred into the host cell by well-known methods, which vary depending on the type of cell host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cell hosts.

Alternative vectors for the expression of protein in mammalian cells, similar to those developed for the expression of human gamma-interferon, tissue plasminogen activator, clotting Factor VIII, hepatitis B virus surface antigen, protease Nexin 1, and eosinophil major basic protein, can be employed. Further, the vector can include CMV promoter sequences and a polyadenylation signal available for expression of inserted nucleic acid in mammalian cells (such as COS7).

The nucleic acid sequences can be expressed in hosts after the sequences have been positioned to ensure the functioning of an expression control sequence. These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors can contain selection markers, e.g., tetracycline resistance or hygromycin resistance, to permit detection and/or selection of those cells transformed with the desired nucleic acid sequences (see, e.g., U.S. Patent 4,704,362).

The nucleic acids produced as described above can also be expressed in a host which is a non-human animal to create a transgenic animal, containing, in a germ or somatic cell, a nucleic acid comprising the coding sequence for all or a portion of the P/CAF protein, as well as all of the other regulatory elements required for expression of the P/CAF protein-encoding sequence. The animal will express the P/CAF gene or portion thereof to produce the P/CAF protein or protein fragment and such expression can be detected by determination of a particular phenotype unique to the transgenic animal expressing the transferred nucleic acid. The nucleic acid can be the nucleic acid of SEQ ED NO: 10, a nucleic acid having a nucleotide sequence which encodes the P/CAF protein, a nucleic acid having a nucleotide sequence which encodes the protein of SEQ ID NO: 1, as well as the nucleic acids that encode the proteins comprising the fragments of SEQ ID NOS: 2 and 4.

The nucleic acids of the invention can contain substitutions or deletions which provide a particular phenotype of interest. For example, various deletions or base substitutions can be introduced into the nucleic acid encoding the P/CAF protein for the purpose of studying the effects of these particular deletions or substitutions on the transcription modulation activity of the P/CAF protein. These effects can be monitored by observation of such characteristics as growth and development of the animal, the ability to develop tumors, survival rates and the like. The gene construct introduced into the animal cells to produce the transgenic animal can contain any of the regulatory elements described above to modulate expression of the foreign genes. As used herein, the term "phenotype" includes morphology, biochemical profiles, changes in tumor formation and other parameters that are affected by the presence of the P/CAF protein

The transgenic animals of the invention can also be used in a method for determining the effectiveness of administering a nucleic acid encoding a functional P/CAF protein to a subject in need of a functional P/CAF protein. First, a nucleic acid encoding a nonfunctional P/CAF protein can be introduced into the animal's cells and expressed to yield a characteristic phenotype. Then, using standard gene therapy techniques, a nucleic acid encoding a functional P/CAF protein can be introduced into the animal's cells and the effects on the animal's phenotypic characteristics can be determined.

Having provided and taught how to obtain a nucleic acid that encodes a P/CAF protein, an isolated nucleic acid that encodes a fragment of P/CAF protein is also provided. The nucleic acid encoding the fragment can be obtained using any of the methods applicable to the nucleic acid encoding the entire P/CAF protein. The nucleic acid fragment can encode a species-specific P/CAF protein fragment (e.g., found in the P/CAF protein of humans, but not in the P/CAF proteins of other species). Nucleic acids encoding species-specific fragments of P/CAF protein are themselves species- specific or allele-specific fragments of the P/CAF gene.

Examples of fragments of a nucleic acid encoding a fragment of the P/CAF protein can include the nucleic acid sequences which encode the amino acid sequences of the fragments of SEQ ID NOS:2 or 4. The same routine computer analyses used to select these examples of fragments can be routinely used to obtain others. Fragments of P/CAF-encoding nucleic acids can be primers for PCR or probes, which can be species- specific, gene-specific or allele-specific. P/CAF-encoding nucleic acid fragments can encode antigenic or immunogenic fragments of P/CAF protein that can be used in therapeutic assays or screening protocols. P/CAF gene fragments can encode fragments of P/CAF protein having histone acetylase activity and/or p300/CBP binding activity as described above, as well as other uses that may become apparent.

An isolated nucleic acid of at least ten nucleotides that selectively hybridizes with the nucleic acid of SEQ ID NO: 10 under selected conditions is provided. For example, the conditions can be PCR amplification conditions and the hybridizing nucleic acid can be a primer consisting of a specific fragment of the reference sequence or a nearly identical nucleic acid that hybridizes only to the exemplified P/CAF-encoding nucleic acid or allelic variants thereof.

The invention provides an isolated nucleic acid that selectively hybridizes with the P/CAF-encoding nucleic acid sequence of SEQ ID NO: 10 under stringent conditions. The hybridizing nucleic acid can be a probe that hybridizes only to the exemplified P/CAF-encoding nucleic acid sequence. Thus, the hybridizing nucleic acid can be a naturally occurring species-specific allelic variant of the exemplified P/CAF gene. The hybridizing nucleic acid can also include insubstantial base substitutions that do not prevent hybridization under the stated stringent conditions or affect either the function of the encoded protein, the way the protein accomplishes that function (e.g., its secondary structure) or the ultimate result of the protein's activity. The means for determining these parameters are well known.

As used herein to describe nucleic acids, the term "selectively hybridizes" excludes the occasional randomly hybridizing nucleic acids as well as nucleic acids that encode other known homologs of the P/CAF protein. The selectively hybridizing nucleic acids of the invention can have at least 70%, 73%, 78%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementarity with the segment and strand of the sequence to which it hybridizes. This list is not intended to exclude percent complementarity values between these values. The nucleic acids can be at least 10, 15, 16, 17, 18, 20, 21, 23, 24, 25, 30, 35, 40, 50, 100, 150, 200, 300, 500, 550, 750, 900, 950, or 1000 nucleotides in length or any intervening length, depending on whether the nucleic acid is to be used as a primer, probe or for protein expression. The hybridizing nucleic acid can comprise a region of at least ten nucleotides (up to full length) that is completely complementary to a unique region of the nucleic acid to which it hybridizes.

The nucleic acid can be an alternative coding sequence for the P/CAF protein, or can be used as a probe or primer for detecting the presence of or obtaining the P/CAF protein. If used as primers, the invention provides compositions including at least two nucleic acids which selectively hybridize with different regions of the nucleic acid so as to amplify a desired region. Depending on the length of the probe or primer, it can range between 70% complementary bases and full complementarity and still hybridize under stringent conditions.

For example, for the purpose of obtaining or determining the presence of a nucleic acid encoding the P/CAF protein, the degree of complementarity between the hybridizing nucleic acid (probe or primer) and the sequence to which it hybridizes (P/CAF DNA in a sample) should be at least enough to exclude hybridization with a nucleic acid from another species. The invention provides examples of these nucleic acids of P/CAF, so that the degree of complementarity required to distinguish selectively hybridizing from nonselectively hybridizing nucleic acids under stringent conditions can be clearly determined for each nucleic acid. It should also be clear that the hybridizing nucleic acids of the invention will not hybridize with nucleic acids encoding unrelated proteins (hybridization is selective) under stringent conditions.

"Stringent conditions" refers to the washing conditions used in a hybridization protocol. In general, the washing conditions should be a combination of temperature and salt concentration chosen so that the denaturation temperature is approximately 5- 20 °C below the calculated T_m of the nucleic acid hybrid under study. The temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to the probe or protein encoding nucleic acid of interest and then washed under conditions of different stringencies. For example, the nucleic acid sequence of SEQ ID NO: 10 was used as a specific radiolabeled probe for the detection of messenger RNA transcribed from the P/CAF gene by performing hybridizations under stringent conditions. The T_m of such an oligonucleotide can be estimated by allowing 2°C for each A or T nucleotide, and 4°C for each G or C. For example, an 18 nucleotide probe of 50% G+C would, therefore, have an approximate T_m of 54° C.

The invention provides an isolated nucleic acid that selectively hybridizes with the P/CAF gene shown in the sequence set forth as SEQ ID NO: 10 under stringent conditions. The invention further provides an isolated nucleic acid complementary to the nucleotide sequence set forth in SEQ ID NO: 10.

Antibodies to the P/CAF protein

A purified antibody and an antiserum containing polyclonal antibodies that specifically bind the P/CAF protein or antigenic fragment are also provided. The term "bind" means the well understood antigen/antibody binding as well as other nonrandom association with an antigen. "Specifically bind" as used herein describes an antibody or other ligand that does not cross react substantially with any antigen other than the one specified, in this case, an antigen of the P/CAF protein. Antibodies can be made as described in Harlow and Lane (33) Briefly, purified P/CAF protein or an antigenic fragment thereof can be injected into an animal in an amount and in intervals sufficient to elicit a humoral immune response Serum polyclonal antibodies can be purified directly, or spleen cells from the animal can be fused with an immortal cell line and screened for monoclonal antibody secretion, according to procedures well known in the art Purified monospecific polyclonal antibodies that specifically bind the P/CAF antigen are also within the scope of the present invention The antibodies of the present invention can bind the protein of claim 1, the protein of claim 2, the protein of claim 3 and/or the protein of claim 4, as well as any other proteins of the present invention

A ligand that specifically binds the antigen is also contemplated The ligand can be a fragment of an antibody, such as , for example, an Fab fragment which retains P/CAF binding activity, or a smaller molecule designed to bind an epitope of the P/CAF antigen The antibody or ligand can be bound to a substrate or labeled with a detectable moiety or both bound and labeled The detectable moieties contemplated within the compositions of the present invention include those listed above in the description of the diagnostic methods, including fluorescent, enzymatic and radioactive markers

The antibody can be bound to a solid support substrate or conjugated with a detectable moiety or therapeutic compound or both bound and conjugated Such conjugation techniques are well known in the art For example, conjugation of fluorescent, radioactive or enzymatic moieties can be performed as described in the art (33,43). The detectable moieties contemplated in the present invention can include fluorescent, radioactive and enzymatic markers and the like Therapeutic drugs contemplated with the present invention can include cytotoxic moieties such as ricin A chain, diphtheria toxin, pseudomonas exotoxin and other chemotherapeutic compounds

It is well understood by one of skill in the art that all of the above discussion regarding antibodies to P/CAF can also be applied with regard to production, characterization and use of antibodies which bind the p300/CBP protein or any of the DNA-binding transcription factors of this invention Measuring the P/CAF protein in a sample

The present invention also provides a method for determining the presence and thus the amount of P/CAF protein in a biological sample. As used herein, a biological sample includes any tissue or cell which would contain the P/CAF protein. Examples of cells include tissues taken from surgical biopsies, isolated from a body fluid or prepared in an in vitro tissue culture environment.

One example of determining the amount of P/CAF in a biological sample can comprise contacting the biological sample with a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 under conditions whereby a P/CAF/p300 complex can be formed; and determining the amount of the P/CAF/p300 complex, the amount of the complex indicating the amount of P/CAF in the sample. Determination of the amount of P/CAF/p300 complex can be accomplished through techniques standard in the art. For example, the complex may be precipitated out of a solution and detected by the addition of a detectable moiety conjugated to the p300 protein or by the detection of an antibody which binds p300 or the P/CAF protein, as taught in the Examples herein. Antibodies which bind p300 or the P/CAF protein can be either monoclonal or polyclonal antibodies and can be obtained as described herein. Detection of P/CAF/p300 complexes by the detection of the binding of antibodies reactive with p300 or the P/CAF protein can be accomplished using various immunoassays as are available in the art, as described below.

Alternatively, determination of the amount of P/CAF in a biological sample can comprise contacting the biological sample with a polypeptide comprising the amino acid sequence of SEQ ID NO: 9 under conditions whereby a P/CAF/CBP complex can be formed; and determining the amount of the P/CAF/CBP complex, the amount of the complex indicating the amount of P/CAF in the sample. Determination of the amount of P/CAF/CBP complex can be accomplished through techniques standard in the art. For example, the complex may be precipitated out of a solution and detected by the addition of a detectable moiety conjugated to the CBP protein or by the detection of an antibody which binds either CBP or the P/CAF protein, as taught in the Examples herein. Antibodies which bind CBP or the P/CAF protein can be either monoclonal or polyclonal antibodies and can be obtained as described herein. Detection of P/CAF/CBP complexes by the detection of the binding of antibodies reactive with CBP or the P/CAF protein can be accomplished using various immunoassays as are available in the art, as described below.

Another example of determining the amount of P/CAF in a biological sample comprises contacting the biological sample with an antibody which specifically binds P/CAF under conditions whereby a P/CAF/ antibody complex can be formed and determining the amount of the P/CAF/antibody complex, the amount of the complex indicating the amount of P/CAF in the sample. Antibodies which bind P/CAF can be either monoclonal or polyclonal antibodies and can be obtained as described herein Determination of P/CAF/antibody complexes can be accomplished using various immunoassays as are available in the art, as described below.

Immunoassays such as immunofluorescence assays, radioimmunoassays (RIA), immunoblotting and enzyme linked immunosorbent assays (ELISA) can be readily adapted for detection and measurement of P/CAF in a biological sample. Both polyclonal and monoclonal antibodies can be used in the assays. Available immunoassays are well known in the art and are extensively described in the patent scientific literature. See, for example, U.S. Patent Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074, 3,984,533; 3,996,345; 4,034,074; and 4,098,876.

Screening assays for P/CAF

The present invention also provides a bioassay for screening substances for the ability to inhibit the histone acetyhransferase activity of P/CAF comprising contacting a system, in which histone acetylation by P/CAF can be determined, with the substance under conditions whereby histone acetylation by P/CAF can occur; determining the amount of histone acetylation by P/CAF in the presence of the substance; and comparing the amount of histone acetylation by P/CAF in the presence of the substance with the amount of histone acetylation by P/CAF in the absence of the substance, a decreased amount of histone acetylation by P/CAF in the presence of the substance indicating a substance that can inhibit the histone acetyhransferase activity of P/CAF. The acetylation of histones by P/CAF can be determined in a system including, for example, either core histones (histones H2A, H2B, H3 and H4) or the nucleosome core particles (146 base pairs of DNA wrapped around the octamer of core histones) as substrates, the P/CAF protein and radiolabeled acetyl-CoA (e.g., [l-¹⁴C]acetyl CoA). The presence of acetylated histones can be detected by autoradiography after separation by SDS-PAGE as described herein in the Examples. Thus, the compound to be tested for the ability to inhibit the histone acetyhransferase activity of P/CAF can be added to this system and assayed for inhibiting ability.

The present invention also provides a bioassay for screening substances for the ability to inhibit the transcription modulating activity of P/CAF, comprising contacting a system, in which histone acetylation by P/CAF can be determined, with the substance under conditions whereby histone acetylation by P/CAF can occur; determining the amount of histone acetylation by P/CAF in the presence of the substance; and comparing the amount of histone acetylation by P/CAF in the presence of the substance with the amount of histone acetylation by P/CAF in the absence of the substance, a decreased amount of histone acetylation by P/CAF in the presence of the substance indicating a substance that can inhibit the transcription modulating activity and cell cycle progression suppressing activity of P/CAF. The acetylation of histones by P/CAF can be determined in a system including, for example, either core histones (histones H2A, H2B, H3 and H4) or the nucleosome core particles (146 base pairs of DNA wrapped around the octamer of core histones) as substrates, the P/CAF protein and radiolabeled acetyl-CoA (e.g., [l-¹⁴C]acetyl CoA). The presence of acetylated histones can be detected by autoradiography after separation by SDS-PAGE as described herein in the Examples. Thus, the compound to be tested for the ability to inhibit the transcription modulating activity of P/CAF by interfering with the histone acetyhransferase activity of P/CAF can be added to this system and assayed for inhibiting ability. Also provided in the present invention is a bioassay for screening substances for the ability to inhibit the binding of p300 to P/CAF, comprising contacting a system in which the binding of p300 to P/CAF can be determined, with the substance under conditions whereby the binding of p300 and P/CAF can occur; determining the amount of p300 binding to P/CAF in the presence of the substance; and comparing the amount of p300 binding to P/CAF in the presence of the substance with the amount of p300 binding to P/CAF in the absence of the substance, a decreased amount of p300 binding to P/CAF in the presence of the substance indicating a substance that can inhibit the binding of p300 to P/CAF. The binding of p300 to P/CAF can be determined in a system, for example, which can include a cell free reaction mixture comprising a fragment of the p300 protein comprising the amino acid sequence of SEQ ID NO: 3 and P/CAF. Alternatively, the system can comprise a cell extract produced from cells producing both p300 and P/CAF. Determination of the binding of p300 to P/CAF can be carried out as taught herein.

Additionally provided in the present invention is a bioassay for screening substances for the ability to inhibit the binding of CBP to P/CAF, comprising contacting a system in which the binding of CBP to P/CAF can be determined, with the substance under conditions whereby the binding of CBP to P/CAF can occur; determining the amount of CBP binding to P/CAF in the presence of the substance; and comparing the amount of CBP binding to P/CAF in the presence of the substance with the amount of CBP binding to P/CAF in the absence of the substance, a decreased amount of CBP binding to P/CAF in the presence of the substance indicating a substance that can inhibit the binding of CBP to P/CAF. The binding of CBP to P/CAF can be determined in a system, for example, which can include a cell free reaction mixture comprising a fragment of the CBP protein comprising the amino acid sequence of SEQ ID NO: 9 and P/CAF. Alternatively, the system can comprise a cell extract produced from cells producing both CBP and P/CAF. Determination of the binding of CBP to P/CAF can be carried out as taught herein. The present invention further contemplates a bioassay for screening substances for the ability to stimulate the histone acetyhransferase activity of P/CAF comprising contacting a system, in which histone acetylation by P/CAF can be determined, with the substance; determining the amount of histone acetylation by P/CAF in the presence of the substance, and comparing the amount of histone acetylation by P/CAF in the presence of the substance with the amount of histone acetylation by P/CAF in the absence of the substance, an increased amount of histone acetylation by P/CAF in the presence of the substance indicating a substance that can stimulate the histone acetyhransferase activity of P/CAF. The acetylation of histones by P/CAF can be determined in a system including, for example, either core histones (histones H2A, H2B, H3 and H4) or the nucleosome core particles (146 base pairs of DNA wrapped around the octamer of core histones) as substrates, the P/CAF protein and radiolabeled acetyl- CoA (e.g., [l-¹⁴C]acetyl CoA). The presence of acetylated histones can be detected by autoradiography after separation by SDS-PAGE as described herein in the Examples. Thus, the compound to be tested for the ability to stimulate the histone acetyhransferase activity of P/CAF can be added to this system and assayed for stimulating ability.

The present invention further contemplates a bioassay for screening substances for the ability to stimulate the transcription modulating activity of P/CAF comprising contacting a system, in which histone acetylation by P/CAF can be determined, with the substance; determining the amount of histone acetylation by P/CAF in the presence of the substance; and comparing the amount of histone acetylation by P/CAF in the presence of the substance with the amount of histone acetylation by P/CAF in the absence of the substance, an increased amount of histone acetylation by P/CAF in the presence of the substance indicating a substance that can stimulate the transcription modulating activity of P/CAF. The acetylation of histones by P/CAF can be determined in a system including, for example, either core histones (histones H2A, H2B, H3 and H4) or the nucleosome core particles (146 base pairs of DNA wrapped around the octamer of core histones) as substrates, the P/CAF protein and radiolabeled acetyl-CoA (e.g., [l-¹⁴C]acetyl CoA). The presence of acetylated histones can be detected by autoradiography after separation by SDS-PAGE as described herein in the Examples. Thus, the compound to be tested for the ability to stimulate the transcription modulating activity of P/CAF by increasing the histone acetyhransferase activity of P/CAF can be added to this system and assayed for stimulating ability.

The present invention further provides a bioassay for screening substances for the ability to stimulate binding of p300 to P/CAF, comprising contacting a system in which the binding of p300 to P/CAF can be determined, with the substance under conditions whereby the binding of p300 to P/CAF can occur; determining the amount of p300 binding to P/CAF in the presence of the substance; and comparing the amount of p300 binding to P/CAF in the presence of the substance with the amount of p300 binding to P/CAF in the absence of the substance, an increased amount of p300 binding to P/CAF in the presence of the substance indicating a substance that can stimulate the binding of p300 to P/CAF. The binding of p300 to P/CAF can be determined in a system, for example, which can include a cell free reaction mixture comprising a fragment of the p300 protein comprising the amino acid sequence of SEQ ID NO: 3 and P/CAF. Alternatively, the system can comprise a cell extract produced from cells producing both p300 and P/CAF. Determination of the binding of p300 to P/CAF can be carried out as taught herein.

Additionally provided in the present invention is a bioassay for screening substances for the ability to stimulate the binding of CBP to P/CAF, comprising contacting a system in which the binding of CBP to P/CAF can be determined, with the substance under conditions whereby the binding of CBP to P/CAF can occur; determining the amount of CBP binding to P/CAF in the presence of the substance; and comparing the amount of CBP binding to P/CAF in the presence of the substance with the amount of CBP binding to P/CAF in the absence of the substance, an increased amount of CBP binding to P/CAF in the presence of the substance indicating a substance that can stimulate the binding of CBP to P/CAF. The binding of CBP to P/CAF can be determined in a system, for example, which can include a cell free reaction mixture comprising a fragment of the CBP protein comprising the amino acid sequence of SEQ ID NO:9 and P/CAF. Alternatively, the system can comprise a cell extract produced from cells producing both CBP and P/CAF. Determination of the binding of CBP to P/CAF can be carried out as taught herein.

Transcription modulating activity of P/CAF The present invention contemplates a method for inhibiting the transcription modulating activity of P/CAF in a subject, comprising administering to the subject a transcription modulating activity inhibiting amount of a substance in a pharmaceutically acceptable carrier. For example, the substance can be identified according to the protocols provided herein as one that can inhibit the transcription modulating activity of P/CAF by preventing the binding of P/CAF to p300/CBP or by inhibiting the histone acetyhransferase activity of P/CAF as well as by any other inhibitory mechanism as identified by the protocols provided herein. Inhibition of the transcription modulating activity of P/CAF in a subject is desirable, for example, to inhibit HIV TAT-mediated transcription and therefore, the method of the present invention can be used to treat HIV-infected subjects.

The substance can be in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the substance, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

The transcription modulating activity and/or histone acetyhransferase activity of

P/CAF can be inhibited in a subject by administering to the subject a substance which binds p300/CBP at the P/CAF binding site or a substance which binds the P/CAF protein at the p300/CBP binding site, the ultimate result being that P/CAF and p300/CBP do not bind with one another and P/CAF cannot exert its transcription modulating and/or histone acetyhransferase effect. The substance can be a protein, such as an antibody which binds the P/CAF protein binding site at or near the p300/CBP binding site, thereby preventing its binding or an antibody which binds the p300/CBP protein at or near the P/CAF binding site, thereby preventing its binding. The substance can also bind the histone acetyhransferase site on P/CAF or at the acetylation site on the histone, thereby preventing acetylation by P/CAF.

The substance which binds p300/CBP, the P/CAF protein or the histone and has the net effect of inhibiting the transcription modulating effect and or histone acetyhransferase activity of P/CAF in the cell can be delivered to a cell in the subject by mechanisms well known in the art.

Alternatively, a nucleic acid encoding a protein which binds either to p300/CBP or the P/CAF protein and has the net effect of inhibiting the transcription modulating effect and/or histone acetyhransferase activity of P/CAF in the cell can be delivered to a cell in the subject by gene transduction mechanisms well known in the art. For example, nucleic acid can be introduced by liposomes as well as via retroviral or adeno-associated viral vectors, as described below.

The substance which inhibits the transcription modulating effect and/or histone acetyhransferase activity of P/CAF can be an antisense RNA or an antisense DNA which binds the RNA or DNA of P/CAF, thereby preventing translation or transcription of the RNA or DNA encoding P/CAF and having the net effect of inhibiting the transcription modulating effect and/or histone acetyhransferase activity of P/CAF by inhibiting P/CAF production. The antisense RNA of the present invention can be generated from the nucleic acid of SEQ ID NO: 14 (human) or SEQ ID NO: 15 (mouse). Furthermore, the antisense DNA can be a phosphorothioate oligodeoxyribonucleotide having the nucleotide sequence of SEQ ID NO: 16 (human) or of SEQ ID NO: 17 (mouse). The mouse antisense RNA can be used to inhibit the activity of mouse P/CAF, having the nucleotide sequence of SEQ ID NO: 18 and the amino acid sequence of SEQ ID NO: 8 The present invention also contemplates an antisense nucleic acid sequence which can bind the DNA or RNA of any of the transcription factors or other proteins now known or later identified to bind P/CAF, thereby inhibiting expression of the gene products of these proteins and having the net effect of inhibiting the transcription modulating effect and or histone acetyhransferase activity of P/CAF.

The antisense nucleic acid can comprise a typical nucleic acid, but the antisense nucleic acid can also be a modified nucleic acid or a derivative of a nucleic acid such as a phosphorothioate analogue of a nucleic acid. The composition can comprise, for example, an antisense RNA that specifically binds an RNA encoded by the gene encoding the serum protein. Antisense RNAs can be synthesized and used by standard methods (62).

Antisense RNA can inhibit gene expression by forming an RNA/RNA duplex between the antisense RNA and the RNA transcribed from the target gene. The precise mechanism by which this duplex formation decreases the production of the protein encoded by the endogenous gene probably involves binding of complementary regions of the normal sense mRNA and the antisense RNA strand with duplex formation in a manner that blocks RNA processing and translation. Alternative mechanisms include the formation of a triplex between the antisense RNA and duplex DNA or the formation of an DNA-RNA duplex with subsequent degradation of DNA-RNA hybrids by RNAse H. Furthermore, an antigene effect can result from certain DNA-based oligonucleotides via triple-helix formation between the oligomer and double-stranded DNA which results in the repression of gene transcription. Regardless of the specific molecular mechanism, the present invention results in inhibition of expression of the P/CAF gene by the introduced and replicated DNA resulting in inhibition of the transcription modulating and/or histone acetyhransferase activity of P/CAF, by a reduction in the expression of the nucleic acid to which the antisense nucleic acid is hybridized, and therefore a reduction of the gene product from the targeted gene.

The antisense nucleic acid may be obtained by any number of techniques known to one skilled in the art. One method of constructing an antisense nucleic acid is to synthesize a recombinant antisense DNA molecule. For example, oligonucleotide synthesis procedures are routine in the art and oligonucleotides coding for a particular protein or regulatory region are readily obtainable through automated DNA synthesis A nucleic acid for one strand of a double-stranded molecule can be synthesized and hybridized to its complementary strand. One can design these oligonucleotides such that the resulting double-stranded molecule has either internal restriction sites or appropriate 5' or 3' overhangs at the termini for cloning into an appropriate vector. Double-stranded molecules coding for relatively large proteins or regulatory regions can be synthesized by first constructing several different double-stranded molecules that code for particular regions of the protein or regulatory region, followed by ligating these DNA molecules together. Once the appropriate DNA molecule is synthesized, this DNA can be cloned downstream of a promoter in an antisense orientation. Techniques such as this are routine in the art and are well documented.

An example of another method of obtaining an antisense nucleic acid is to isolate that nucleic acid from the organism in which it is found and clone it in an antisense orientation. For example, a DNA or cDNA library can be constructed and screened for the presence of the nucleic acid of interest. Methods of constructing and screening such libraries are well known in the art and kits for performing the construction and screening steps are commercially available (for example, Stratagene Cloning Systems, La Jolla, CA). Once isolated, the nucleic acid can be directly cloned into an appropriate vector in an antisense orientation, or if necessary, be modified to facilitate the subsequent cloning steps. Such modification steps are routine, an example of which is the addition of oligonucleotide linkers which contain restriction sites to the termini of the nucleic acid General methods are set forth in Sambrook et al. (39).

The DNA that is introduced into the cell is in an expression orientation that is antisense to a corresponding endogenous DNA or RNA of the cells. For example, where an endogenous DNA comprises a gene which encodes for a particular protein, the introduced DNA is in an expression orientation opposite the expression of the endogenous DNA; that is the DNA operatively linked to a promoter is in an antisense expression orientation relative to the corresponding endogenous gene. The introduced DNA may be homologous to the entire transcribed gene or homologous to only part of the transcribed gene. Alternatively, the sequence of the introduced DNA may be divergent to that of the endogenous DNA but only divergent to the extent that hybridization of the nucleic acids occurs, thereby preventing transcription. One skilled in the art can determine the maximum extent of this divergence by routine screening of antisense DNAs corresponding to an endogenous DNA of the cell. In this manner, one skilled in the art can readily determine which fragments, or alternatively the extent of homology of the fragments or the entire gene that is necessary to inhibit gene expression.

The antisense nucleic acids of the present invention can be made according to protocols standard in the art, as well as described in the Examples provided herein. The antisense nucleic acids can be administered to a subject according to the gene transduction protocols standard in the art, as described below.

The present invention also contemplates a method for stimulating the transcription modulating activity and/or histone acetyhransferase activity of P/CAF in a subject comprising administering to the subject a substance, in a pharmaceutically acceptable carrier, determined according to the methods taught herein, to have a stimulatory affect on the transcription modulating and or histone acetyhransferase activity of P/CAF. The substance can be one which has been identified, according to the protocols provided herein, to stimulate histone acetyhransferase activity in P/CAF or promote binding of P/CAF to p300/CBP. The stimulation of the transcription modulation activity and/or histone acetyhransferase activity of P/CAF in a subject is desirable, for example, to activate tumor suppressor p53 (which promotes apoptosis) or to activate the muscle differentiation factor, MyoD. Thus, the method of the present invention can be employed to treat cancer and to promote muscle differentiation in conditions where muscle differentiation is desired. The substance can be delivered to a cell in the subject by mechanisms well known in the art.

Further contemplated in the present invention is a method for promoting binding of P/CAF to p300/CBP in a subject, comprising administering to the subject a substance identified by the methods provided herein to promote binding of P/CAF to either p300 or CBP

Additionally, a nucleic acid encoding a protein which stimulates the transcription modulating activity and/or histone acetyhransferase activity of P/CAF can be delivered to a cell in the subject by gene transduction mechanisms, as described below.

Also provided in the present invention is a method of inhibiting the cell cycle progression inducing effect of an oncoprotein which binds p300/CBP in a subject comprising transducing the cells of the subject with a vector comprising a nucleic acid encoding the P/CAF protein; inducing expression of the nucleic acid in the cell to produce the P/CAF in an amount which will allow the P/CAF gene product to replace the oncoprotein bound to p300/CBP, whereby the replacement of the oncoprotein bound to p300/CBP by the P/CAF gene product inhibits the cell cycle progression inducing effect of the oncoprotein. The oncoprotein which binds p300/CBP in the cell can be the adenovirus ElA oncoprotein.

A method for providing a functional P/CAF protein to a subject in need of the functional P/CAF protein is also provided, comprising transducing the cells of the subject with a vector comprising a nucleic acid encoding the P/CAF protein and inducing expression of the nucleic acid to produce the functional P/CAF protein in the cell, thereby providing the functional P/CAF protein to the subject. The transduction of the vector nucleic acid into the subject's cells can be carried out according to standard gene therapy protocols well known in the art (see, for example, U.S. Patent No. 5,339,346).

Screening assays for p300/CBP

The present invention also provides a bioassay for screening substances for the ability to inhibit the histone acetyhransferase activity of p300/CBP comprising contacting a system, in which histone acetylation by p300/CBP can be determined, with the substance under conditions whereby histone acetylation by p300/CBP can occur; determining the amount of histone acetylation by p300/CBP in the presence of the substance; and comparing the amount of histone acetylation by p300/CBP in the presence of the substance with the amount of histone acetylation by p300/CBP in the absence of the substance, a decreased amount of histone acetylation by p300/CBP in the presence of the substance indicating a substance that can inhibit the histone acetyhransferase activity of p300/CBP. The acetylation of histones by p300/CBP can be determined in a system including, for example, either core histones (histones H2A, H2B, H3 and H4) or the nucleosome core particles (146 base pairs of DNA wrapped around the octamer of core histones) as substrates, the P300/CBP protein and radiolabeled acetyl-CoA (e.g., [l-¹⁴C]acetyl CoA). The presence of acetylated histones can be detected by autoradiography after separation by SDS-PAGE as described herein in the Examples. Thus, the compound to be tested for the ability to inhibit the histone acetyhransferase activity of p300/CBP can be added to this system and assayed for acetyhransferase inhibiting ability.

Also provided in the present invention is a bioassay for screening substances for the ability to inhibit the binding of a transcriptional factor to p300/CBP, comprising contacting a system in which the binding of a transcriptional factor to p300/CBP can be determined, with the substance under conditions whereby the binding of the transcriptional factor and p300/CBP can occur; determining the amount of transcriptional factor binding to p300/CBP in the presence of the substance; and comparing the amount of transcriptional factor binding to p300/CBP in the presence of the substance with the amount of transcriptional factor binding to p300/CBP in the absence of the substance, a decreased amount of transcriptional factor binding to p300/CBP in the presence of the substance indicating a substance that can inhibit the binding of a transcriptional factor to p300/CBP. The binding of a transcriptional factor to p300/CBP can be determined in a system, for example, which can include a cell free reaction mixture comprising a transcriptional factor which binds p300/CBP and p300/CBP. Alternatively, the system can comprise a cell extract produced from cells producing both a transcriptional factor which binds p300/CBP and p300/CBP. The transcriptional factor which binds p300/CBP can be selected from, but is not limited to the group consisting of nuclear hormone receptors, CREB, c-Jun/v-Jun, c-Myb/v-Myb, YYI, Sap- la, c-Fos, MyoD and SRC-1, as well as any other transcriptional factor now known or later identified to bind p300/CBP. The screening assay of the present invention can also be used to identify substances which inhibit the binding of p300/CBP to other components to which it is known to bind, for example, P/CAF, PP90_RSK, TFIIB, ElA, SN40 large T antigen, as well as any other substances now known or later identified to bind p300/CBP. Determination of the binding of a transcriptional factor or other substance to p300/CBP can be carried out as taught in the Examples herein as well as by protocols described in the literature.

The present invention further contemplates a bioassay for screening substances for the ability to stimulate the histone acetyhransferase activity of p300/CBP comprising contacting a system, in which histone acetylation by p300/CBP can be determined, with the substance; determining the amount of histone acetylation by p300/CBP in the presence of the substance; and comparing the amount of histone acetylation by p300/CBP in the presence of the substance with the amount of histone acetylation by p300/CBP in the absence of the substance, an increased amount of histone acetylation by p300/CBP in the presence of the substance indicating a substance that can stimulate the histone acetyhransferase activity of p300/CBP. The acetylation of histones by p300/CBP can be determined in a system including, for example, either core histones (histones H2A, H2B, H3 and H4) or the nucleosome core particles (146 base pairs of DΝA wrapped around the octamer of core histones) as substrates, the p300/CBP protein and radiolabeled acetyl-CoA (e.g., [l-¹⁴C]acetyl CoA). The presence of acetylated histones can be detected by autoradiography after separation by SDS-PAGE as described herein in the Examples. Thus, the compound to be tested for the ability to stimulate the histone acetyhransferase activity of p300/CBP can be added to this system and assayed for stimulating ability.

The present invention further provides a bioassay for screening substances for the ability to stimulate binding of a component, which binds p300/CBP, to p300/CBP, comprising contacting a system in which the binding of the component to p300/CBP can be determined, with the substance under conditions whereby the binding of the component to p300/CBP can occur; determining the amount of component binding to p300/CBP in the presence of the substance; and comparing the amount of component binding to p300/CBP in the presence of the substance with the amount of component binding to p300/CBP in the absence of the substance, an increased amount of component binding to p300/CBP in the presence of the substance indicating a substance that can stimulate the binding of the component to p300/CBP. The binding of the component to p300/CBP can be determined in a system, for example, which can include a cell free reaction mixture comprising the component and p300/CBP. Alternatively, the system can comprise a cell extract produced from cells producing both the component and p300/CBP. The component which binds p300/CBP can be any of the transcriptional factors or other proteins which are known or are identified in the future to bind p300/CBP, as set forth above. Determination of the binding of the component to p300/CBP can be canied out as taught in the Examples provided herein and according to protocols available in the literature.

Histone acetyltransferase activity of p300/CBP

A method for inhibiting the histone acetyltransferase activity of p300/CBP in a subject is provided in the present invention, comprising administering to the subject a histone acetyltransferase activity inhibiting amount of a substance in a pharmaceutically acceptable carrier. The mechanism of the inhibitory action of the substance can be the inhibition of the binding of a DNA-binding transcription factor, such as, for example, a nuclear hormone receptor, CREB, c-Jun/v-Jun, c-Myb/v-Myb, YY1, Sap- la, c-Fos, MyoD or SRC-1, to p300/CBP.

The histone acetyltransferase activity of p300/CBP can be inhibited in a subject by administering to the subject a substance which binds p300/CBP at the transcription factor binding site or a substance which binds the transcription factor protein at the p300/CBP binding site, the ultimate result being that the transcription factor and p300/CBP do not bind with one another and p300/CBP cannot acetylate histones The substance which binds either to the transcription factor or the p300/CBP protein and has the net effect of inhibiting the histone acetyltransferase activity of p300/CBP in the cell can be identified according to the screening methods provided herein and delivered to a cell in the subject by mechanisms well known in the art. The substance can be a protein, such as an antibody which binds the p300/CBP protein binding site at or near the DNA-binding transcription factor binding site, thereby preventing its binding or an antibody which binds the DNA-binding transcription factor at or near the p300/CBP binding site, thereby preventing its binding. The substance can also bind the histone acetyltransferase site on p300/CBP (aa 1195-1673 on p300 or aa 1174-1850 on CBP) or at the acetylation site on the histone, thereby preventing acetylation by p300/CBP.

Additionally, the substance can be a nucleic acid which can be expressed in the cell to produce a protein which inhibits the histone acetyltransferase activity of p300/CBP. For example, a nucleic acid encoding a protein which binds either to a transcription factor or the p300/CBP protein and has the net effect of inhibiting the histone acetyltransferase activity of p300/CBP in the cell can be delivered to a cell in the subject by gene transduction mechanisms well known in the art. For example, nucleic acid can be introduced by liposomes as well as via retroviral or adeno-associated viral vectors, as described below.

The substance which inhibits the histone acetyltransferase activity of p300/CBP can be an antisense RNA or an antisense DNA which binds the RNA or DNA of p300/CBP thereby preventing translation or transcription of the RNA or DNA encoding p300/CBP and having the net effect of inhibiting the histone acetyltransferase activity of P/CAF by inhibiting p300/CBP production. The antisense RNA or DNA of the present invention can be produced and introduced into cells according to the same methods as set forth above for P/CAF antisense nucleic acids.

The present invention also contemplates a method for stimulating the histone acetyltransferase activity of p300/CBP in a subject comprising administering to the subject a histone acetyltransferase activity stimulating amount of a substance, in a pharmaceutically acceptable carrier, determined according to the methods taught herein, to have a stimulatory affect on the histone acetyltransferase activity of p300/CBP. The substance can exert a stimulatory effect by promoting the binding of a DNA-binding transcription factor of the present invention to p300/CBP. The substance can be delivered to a cell in the subject by mechanisms well known in the art. A nucleic acid encoding a protein which stimulates the transcription modulating activity of p300/CBP can be delivered to a cell in the subject by gene transduction mechanisms, as described below.

Gene transduction

In the methods described above which include gene transduction into cells (i.e., addition of exogenous DNA into cells), the nucleic acids of the present invention can be in a vector for delivering the nucleic acids to the site for expression of the P/CAF protein. The vector can be one of the commercially available preparations, such as the pGM plasmid (Promega). Vector delivery can be by liposome, using commercially available liposome preparations or newly developed liposomes having the features of the present liposomes. Additionally, vector delivery can be via a viral system, including, but not limited to, retroviral, adenoviral and adeno-associated viral systems. Other delivery methods can be adopted and routinely tested according to the methods taught herein.

The modes of administration of the liposome will vary predictably according to the disease being treated and the tissue being targeted. For example, for treating cancer in either the lung or the liver, which are both sinks for liposomes, intravenous delivery is reasonable. For other localized cancers, as well as precancerous conditions, catheterization of an artery upstream from the target organ is a preferred mode of delivery, because it avoids significant clearance of the liposome by the lung and liver. For cancerous lesions at a number of other sites (e.g., skin cancer, localized dysplasias), topical delivery is expected to be effective and may be preferred, because of its convenience. Leukemias and other disorders involving dysregulated proliferation of certain isolatable cell populations may be more readily treated by ex vivo administration of the nucleic acid.

The liposomes may be administered topically, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally or the like, although intravenous or topical administration is typically preferred. The exact amount of the liposomes required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the disease being treated, the particular compound used, its mode of administration and the like Thus, it is not possible to specify an exact amount. However, an appropriate amount may be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

Parenteral administration, if used, is generally characterized by injection.

Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant level of dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.

Topical administration can be by creams, gels, suppositories and the like. Ex vivo (extracorporeal) delivery can be as typically used in other contexts.

The present invention is more particularly described in the following examples which are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art EXAMPLES

I. P/CAF studies.

Cloning and characterization of P/CAF protein.

In human cells, CBP binds to c-Jun in a phosphorylation-dependent manner in association with stimulation of transcription (9). In yeast, GCN4 is believed to be a c- Jun counterpart on the basis of similarities in DNA recognition (15) as well as the participation of both proteins in UN signaling pathways (16). Yeast genetic screening has led to the isolation of various cofactors for GCΝ4, including GCN5 (yGCN5), ADA2 (yADA2) and ADA3 (yADA3) (17-19). These factors are considered to function as a complex (or in a common pathway) based on genetic and protein-protein interaction studies (18-22). Finally, p300/CBP and yADA2 exhibit significant sequence similarity within a 50 amino acid region including a Zn²⁺ finger motif (3). Human counterparts to yGCN5, yADA2, or yADA3 that interact with p300/CBP to mediate transcriptional activation by c-Jun were searched for in various nucleotide sequence databases.

Comparison of the yGCN5 protein sequence with various databases (23) revealed significant similarities with the two randomly sequenced human cDNAs,

ETS05039 (24) (P=4.0xl0^'15) and NIB2000-5R (P=6.5xl0^"9). Given that these cDNAs were truncated, human fetal liver and fetal brain cDNA libraries (Clontech) were screened with ETS05039 and NIB2000-5R, respectively and complete clones were isolated from the human fetal liver cDNA library. The complete sequences revealed that the ETS05039- and NTB2000-5R-derived clones are encoded by distinct genes but are highly related within the protein coding regions (68% identity at the DNA level; 75% identity and 86% similarity at the protein level). The former encodes an N-terminal region with no sequence similarity to any proteins in the databases besides the yGCN5- related C-terminal region, whereas the latter encodes only the yGCN5-related region. Given that p300/CBP-binding activity was observed in the former polypeptide as shown below, it was designated p300/CBP-associated factor (P/CAF), having the amino acid sequence of SEQ ID NO.l and the nucleotide sequence of SEQ ID NO 10 and the latter was named human GCN5 (hGCN5), having the amino acid sequence of SEQ ID NO 5 and the nucleotide sequence of SEQ ID NO 11

Additionally, an RNA blot (Clontech) was hybridized with a random-primed probe made from the cDNA encoding P/CAF RNA blotting indicated that transcripts detected by the P/CAF and hGCN5 cDNAs are ubiquitously expressed, but the former is most abundant in heart and skeletal muscle, whereas the latter is most abundant in pancreas and skeletal muscle

P/CAF-p300/CBP interaction in vitro

The P/CAF binding site was presumed to reside in the C terminal one third of CBP (residues 1,678-2,442) because it was observed that this region, when fused to a DNA binding domain, activates transcription (4) in a manner repressed by coexpression of l2S ElA This region was divided into 6 overlapping fragments and each was expressed in E. coli as a glutathione-S-transferase (GST) fusion protein GST-CBP fusions were incubated with recombinant P/CAF protein and, subsequently, purified using glutathione-Sepharose. Co-purified P/CAF was detected by immunoblotting analysis

To construct GST-fusions, various regions of CBP and p300 were amplified by PCR A series of deletions of the CBP segment B was created by site-directed in vitro mutagenesis (30). These fragments were subcloned into pGEX-2T (Pharmacia) GST- fusions were expressed in E. coli and extracted with buffer B [20 mM Tris-HCl (pH 8 0), 5 mM MgCl₂, 10% glycerol, 1 mM AEBSF, 0 1% NP40, 10 μg/ml of aprotinin, 10 μg/ml of leupeptin, 1 μg/ml of pepstatin A, 1 mM DTT] containing 0 1 M KC1 for these experiments GST-CBP-segment B was purified by glutathione-Sepharose and phenyl- Sepharose chromatographic steps, P/CAF, hGCN5, and ElA were expressed as FLAG- fusions in Sf9 cells via baculovirus vectors and affinity-purified with M2-agarose (ref 30, Kodak-IBI). For interaction, a crude E. coli extract containing 20 pmol of GST- fusion was incubated with 40-60 pmol of P/CAF or El A in a total volume of 50 μl of buffer B with 0.1 M KC1 on ice for 10 min. Samples were further incubated with 10 μl (packed volume) of glutathione-Sepharose at 4°C for 30 min, washed four times with 200 μl of buffer B containing 0.1 M KC1, and eluted with 20 μl of buffer E [50 mM Tris-HCl (pH 8.0), 0.2 M KC1, 20 mM glutathione] for 60 min. Interacting proteins were detected by anti-FLAG immunoblotting or silver staining.

For p300 interactions, the segment spanning residues 1763-1966 (segment B') of p300, which is analogous to the CBP segment-B, was used. Twenty percent of the P/CAF and hGCN5 inputs and 100% of the El A input were also analyzed. In the GST precipitation assays, almost identical amounts of the GST fusions were recovered in all samples. Interaction between P/CAF and CBP (segment B) was determined in the absence and in the presence of El A. Control reactions with GST-CBP alone and without GST-CBP were also performed. Input proteins were analyzed.

Two CBP segments, A and B, interacted specifically with P/CAF. The stronger interaction was observed in the latter segment, which does not include the yADA2-like Zn²⁺ finger. Given that the CBP segment-B is well conserved in p300 (66% identity, 75% similarity), the binding of P/CAF to p300 in vitro was also analyzed. For this experiment, the p300 segment spanning residues 1763-1966, termed segment B', which is analogous to the CBP segment-B, was used. Like CBP, p300 interacted specifically with P/CAF. These studies demonstrated that P/CAF binds specifically to both ρ300 and CBP in vitro. In contrast to P/CAF, hGCN5 did not bind to CBP or p300.

These studies also demonstrated that the Zn²⁺ finger region of p300/CBP, which shares sequence similarity with yADA2, is not essential for the interaction with P/CAF Cloning of a human structural homolog of yADA2, termed hADA2 (25) has revealed that, unlike the sequence similarity between p300/CBP and yADA2, which is restricted to a 50 amino acid region, hADA2 shares extensive similarity (30% identity, 52% similarity) to yADA2 over the entire protein sequence. Moreover, a computer search of the complete genomic sequence of Saccharomyces cerevisiae revealed that yeast does not have counterparts of p300/CBP or P/CAF. Thus, the p300/CBP-P/CAF pathway may have been acquired during metazoan evolution.

Action of ElA in vitro

Previous reports indicated that ElA binds to both the p300 segment spanning residues 1767-1816 and the CBP segment spanning residues 1805-1854 (7). These interactions were reconfirmed in the present system; thus, both p300 and CBP segments covering the previously identified regions interacted with ElA.

For further mapping, a series of deletions was introduced within the CBP segment-B and tested for interactions with P/CAF and ElA. Deletions of residues 1801-1825 or 1824-1851 markedly reduced interactions with both P/CAF and ElA, whereas deletion of residues 1850-1878 did not affect these interactions. Furthermore, deletion of residues 1801 - 1851 completely abolished interactions with both P/CAF and ElA. These data indicate that residues 1801-1851 of CBP are critical for interaction with both P/CAF and ElA. Taken together with the evidence that CBP segment A (aa residues 1,678-1,880) also binds to these factors, the above findings demonstrate that P/CAF and ElA bind to the same or very closely spaced sites on CBP.

Evidence that both P/CAF and ElA recognize the same p300/CBP segments raises the possibility of direct competition between P/CAF and El A for binding to p300/CBP. To test this possibility, a competition experiment was performed with the use of affinity purified recombinant proteins. The interaction of P/CAF with the CBP- segment B was progressively inhibited by the addition of increasing amounts of ElA In contrast, no inhibition was caused by an El A mutant which does not bind to p300/CBP (El AΔN). Similar results were obtained with the p300-segment B', leading to the conclusion that P/CAF and ElA compete for the same binding sites in p300/CBP. P/CAF-p300/CBP interaction in vivo

The in vivo interaction between P/CAF and p300/CBP was established by co- immunoprecipitation from a human osteosarcoma cell extract. Proteins in this extract were immunoprecipitated with rabbit anti-P/CAF, rabbit anti-CBP and anti-p300 antibodies. For controls, cell extract was precipitated with rabbit control IgG or mouse anti-HA monoclonal antibody. The precipitates were analyzed by immunoblotting with anti-P/CAF, anti-CBP and anti-p300 antibodies.

Osteosarcoma cells were transfected with either control vector or El A- or ElAΔN-expression vectors. Extract from the transfected subpopulation was immunoprecipitated with anti-P/CAF or control IgG. The precipitates were analyzed by immunoblotting with anti-p300 and anti-P/CAF antibodies.

Rabbit anti-P/CAF antibody was raised to the P/CAF segment spanning residues 125-397 and purified by immunoaffinity chromatography (33). A mixture of monoclonal antibodies raised to the human p300 segment spanning residues 1572-2371 (5) and rabbit polyclonal antibodies raised to the mouse CBP segment spanning residues 2-23 (for immunoprecipitation) and 1736-2179 (immunoblotting) were purchased from Upstate Biotechnology. Approximately 2 x 10⁷ human osteosarcoma U-2 OS cells (ATCC accession number HTB 96) were extracted with 10 ml of lysis buffer [25 mM HEPES-KOH (pH 7.2), 150 mM potassium acetate, 2 mM EDTA, 1 mM DTT, 1 mM AEBSF, 10 μg/ml of aprotinin, 10 μg/ml of leupeptin, 1 μg/ml of pepstatin A, 20 mM sodium fluoride, 0.1% NP40], Two to 10 ml of extract were incubated with 2 μg of the respective antibody for four hours at 4°C. Fifty μl (packed volume) of protein- A Trisacryl (Pierce) were added and incubation was continued for two hours. The matrix was washed four times with 1 ml of the lysis buffer, then boiled in 2x SDS sample buffer. Human osteosarcoma U-2 OS cells were transfected with 20 μg of the indicated plasmid and 1 μg of sorting plasmid (pCMV-IL2R) (31). The transfected subpopulation was purified by magnetic affinity cell sorting (32). Extract from approximately 2 x 10⁵ sorted cells was immunoprecipitated as described. Anti-P/CAF antibody specifically detected a 95 kDa protein, which is very close to the calculated value for the full-length P/CAF, in the immunoprecipitates Anti- P/CAF antibody co-immunoprecipitated both CBP and p300. Similarly, anti-CBP antibody also co-immunoprecipitated P/CAF However, anti-p300 antibody did not co- immunoprecipitate P/CAF. This is most likely due to steric interference since the anti- p300 antibody was raised to the p300 segment spanning residues 1572-2371 which includes the P/CAF binding region These data demonstrate that P/CAF forms complexes with both p300 and CBP in vivo

Action of ElA in vivo

The in vitro experiments described herein indicate that P/CAF and ElA compete for the binding sites in p300/CBP Thus, a study was conducted to determine whether El A targets the endogenous interaction between P/CAF and p300 An El A-expression vector was transiently transfected into human osteosarcoma cells and the transfected subpopulation was purified by cell sorting Then, the interaction between P/CAF and p300 in transfected cells was examined by co-immunoprecipitation with anti-P/CAF antibody The endogenous interaction of P/CAF with p300 was drastically inhibited by expression of El A. On the other hand, no inhibition was observed by the El A mutant lacking the p300 binding domain (E1AΔN), indicating that ElA disrupts the P/CAF- p300 complex in vivo through an interaction with p300

Cell cycle regulation by P/CAF

Given that binding of P/CAF to p300/CBP is inhibited by El A, experiments were performed to evaluate whether P/CAF, by binding to and forming a functional complex with p300, is involved in the regulation of entry into S phase This possibility was addressed by examining whether transient expression of P/CAF would affect the rate of Gl/S transit in HeLa cells P/CAF negatively affected the distribution of cells between Gl and S phases in this assay

HeLa cells were transfected by electroporation with 7 μg of P/CAF-expression plasmid and/or 3 μg of the full-length or the N-terminally deleted (Δ2-36) ElA 12S- expression plasmid as indicated. These plasmids were constructed by subcloning FLAG-P/CAF and ElA cDNAs into pCX (34) and pcDNAI (Invitrogen), respectively. All samples, in addition, contained 1 μg of sorting plasmid (pCMV-EL2R) (31) and carrier plasmid (pCX) to normalize the total amount of DNA to 11 μg. After transfection, cells were incubated in Dulbecco's modified Eagle's medium with 10% fetal bovine calf serum for 12 h, and subsequently labeled in medium containing 10 μM bromo-deoxyuridine (BrdU) for 30 min. Subsequently, the transfected subpopulation was purified by magnetic affinity cell sorting and nuclei were analyzed by dual parameter flow cytometry as described (32).

The fraction of cells accumulating in S phase in control cultures was 23%, compared to 15% in P/CAF-transfected cells. This effect was reproducible in multiple independent experiments. In parallel experiments to verify the utility of this experimental protocol, plasmids encoding E2F-1, simian virus 40 small t, cyclin A or cyclin E increased the accumulation of cells in S phase, whereas plasmids encoding the cyclin-dependent kinase inhibitors p21 or p27 reduced the number of S phase cells.

On the basis of evidence that ElA and P/CAF compete for binding sites on p300, it seemed possible that cotransfection of P/CAF with El A would oppose the mitogenic effect caused by El A. As shown by the data herein, this is indeed the case. ElA alone has mitogenic activity in this experimental setting, while the El A mutant lacking the p300 binding domain (El AΔN) has very weak activity. Comparable expression levels between wild type and mutant El A in the transfected cells were revealed by immunoblotting analysis with anti-El A. Intriguingly, when P/CAF was cotransfected with El A, the mitogenic activity of El A was significantly counteracted by P/CAF. These results show that P/CAF and ElA mediate antagonistic effects on cell cycle progression.

In the course of assessing P/CAF activity, it was also revealed that p300 is able to inhibit cell cycle progression under the same assay conditions. These findings suggest that P/CAF and p300, perhaps by forming a complex, act in concert to suppress cell cycle progression.

Histone acetyltransferase activity in P/CAF Acetylation of the N-terminal histone tails has been considered to play a crucial role in accessibility of transcription factors to nucleosomal templates (26-27). Recently, yGCN5 has been identified as a histone acetyltransferase (28). On the basis of this information, intrinsic histone acetyhransferase activity in P/CAF and hGCN5 was examined. As substrates, the core histones (histones H2A, H2B, H3 and H4) and the nucleosome core particles (146 base pairs of DNA wrapped around the octamer of core histones) were used.

Activity of hGCN5 and P/CAF that acetylates free histones or histones in the nucleosome core particle (35) was measured as described (36). Each reaction contained 0.3 pmol of affinity purified FL AG-hGCN5 or FLAG-P/CAF, 4 pmol of the histone octamer or the nucleosome core particle and 10 pmol of [l-¹⁴C]acetyl-CoA. The histone octamer dissociated into dimers or tetramers under assay conditions. Acetylated histones were detected by autoradiography after separation by SDS-PAGE.

P/CAF and hGCN5 acetylated the core histones with almost the same efficiency

Both factors acetylated histones H3 and H4, but preferentially H3. In contrast, very weak or no acetylation by hGCN5 was detected in the nucleosome core particles. Remarkably, significant acetylation by P/CAF was observed in a nucleosomal context. Although all core histones are acetylated in the nucleus, P/CAF and hGCN5 did not acetylate histones H2A and H2B in vitro.

Direct function of P/CAF is likely to involve its intrinsic histone acetyltransferase activity. Although exact molecular mechanisms by which acetylation of core histones contribute to transcription remains undefined, acetylation of the histones is considered to play an important role in transcriptional regulation (26-27). The positively charged N- terminal tails of core histones are believed to affect nucleosome structure by interacting with DNA at or near the nucleosome-spacer junction. Acetylation of the histone tails presumably destabilizes the nucleosome and facilitates access by regulatory factors. Likewise, there is a general correlation between the level of acetylation and transcriptional activity of nucleosomal domains. The findings of the present invention provide insights into the mechanisms of targeted histone acetylation.

Cellular factor p300/CBP binds to various sequence-specific factors that are involved in cell growth and/or differentiation, including CREB (3,4), c-Jun (9), Fos (1 1), c-Myb (12) and nuclear receptors (13). P/CAF could stimulate the activation function of these factors via promoter-specific histone acetylation. The present invention demonstrates that ElA appears to perturb normal cellular regulation by disrupting the connection between p300/CBP and its associated histone acetyhransferase.

H. D300/CBP studies.

Purification of ElA associated histone acetyltransferase.

FLAG-epitope tagged ElA (or ΔE1 A) was expressed in Sf9 cells (ATCC accession number CRL 1711) by infecting recombinant baculovirus (43). All purification steps were carried out at 4°C. Extract was prepared from infected cells by one cycle of freeze and thaw in buffer B (20 mM Tris-HCl, pH 8.0; 5 mM MgCl₂; 10% glycerol; 1 mM PMSF; 10 mMβ-mercaptoethanol; 0.1% Tween 20) containing 0.1 M KC1 and the complete protease inhibitor cocktail (Boehringer Mannheim). To prepare El A-immobilized beads, the extract was incubated with M2 anti-FLAG antibody agarose (Kodak-IBI) for four hours with rotating and subsequently washed with the same buffer three times. The resulting beads were incubated with HeLa (ATCC accession number CCL 2) nuclear extract for four to eight hours and thereafter washed with the same buffer six times. Finally, FLAG-E1 A was eluted from the beads along with associated polypeptides by incubating with the same buffer containing 0.1 mg/ml FLAG peptide. For further purification, eluted polypeptides were dialyzed in 0.05 M KCl-buffer B and subsequently loaded onto a SMART Mono Q column (Pharmacia) equilibrated with the same 0.05 M KCl-buffer B. After washing, the column was developed with a linear gradient of 0.05-1.0 M KC1 in buffer B. Mono Q fractions were concentrated with a MICROCON spin-filter (Amicon) and consequently loaded onto a SMART Superdex 200 column (Pharmacia) equilibrated with 0.1 M KCl-buffer B.

Histone acetyltransferase assays

Filter binding assays were performed as described (80) with minor modifications. Samples were incubated at 30°C for 10-60 minutes in 30 ml of assay buffer containing 50 mM Tris-HCl, pH 8.0; 10% glycerol; 1 mM DTT; 1 mM PMSF; 10 mM sodium butyrate; 6 pmol of [³H]acetyl CoA (4.3 mCi/mmole, Amersham Life Science Inc.), and 33 mg/ml of calf thymus histones (Sigma Chemical Co.). In experiments where synthetic peptides were substituted for core histones, 50 pmol of each peptide were used. After incubation, the reaction mixture was spotted onto Whatman P-81 phosphocellulose filter paper and washed for 30 minutes with 0.2 M sodium carbonate buffer pH 9.2 at room temperature with 2-3 changes of the buffer. The dried filters were counted in a liquid scintillation counter.

PAGE analysis was done as above except that 90 pmol of [¹⁴C]acetyl CoA (55 mCi/mmole, Amersham Life Science Inc.) and 9 pmol of core histones or mononucleosomes were used. Core histones and mononucleosomes were prepared as described (35). For trypsin digestion, reaction mixtures were further incubated with various amounts of trypsin on ice for 30 minutes. The samples were analyzed on one dimensional SDS-PAGE gels or two dimensional gels, where the first dimension was an acid-urea-PAGE gel (44) and the second dimension was an SDS-PAGE gel.

Protein expression

For baculovirus expression, cDNAs corresponding to p300 portions of aa 1-670, aa 671-1194 and aa 1135-2414 were amplified by PCR (EXPAND High Fidelity PCR System; Boehringer Mannheim) as KpnI-NotI fragments. The resulting fragments were subcloned into a baculovirus transfer vector having the FLAG-tag sequence (43). The recombinant viruses were isolated using the BACULOGOLD system (Pharmingen), according to the manufacturer's protocol and were infected into Sf9 cells (ATCC accession number CRL 171 1) to express FLAG-p300. Recombinant proteins were affinity purified with M2 anti-FLAG antibody-immobilized agarose (Kodak-IBI) according to the manufacturer's protocol.

For bacterial expression, cDNAs encoding the p300 portions and the CBP portion (aa 1174-1850) were first subcloned into the baculovirus transfer vector having the FLAG-tag as described above. Thereafter, the Xhol and NotI fragments encoding FLAG-p300 or FLAG-CBP fusions were resubcloned into the E. coli expression vector pΕT-28c (Novagene) digested with Sail and NotI. Recombinant proteins were expressed in E. coli BL21(DE3) and affinity purified with M2-antibody agarose.

Histone acetyltransferases that associate with ElA

Although the adenovirus ElA 12S protein (ElA) inhibits transcription in a variety of genes via direct binding to p300/CBP (45), ElA also stimulates transcription in some contexts (46). Thus, p300/CBP-bound ElA was tested to determine whether it might recruit histone acetyltransferases or deacetylases to regulate transcription. In addition, experiments were conducted as described below to determine if p300/CBP per se is a histone acetyltransferase.

Initially, recombinant FLAG-epitope tagged ElA was immobilized on anti-FLAG antibody beads. Immobilized ElA was incubated with a HeLa nuclear extract for affinity purification of ElA-associated polypeptides. FLAG-E1A was then eluted from the beads, along with ElA-associated polypeptides, by incubating with FLAG-peptide. Although El A per se has no histone acetyltransferase activity, ElA recruited significant amounts of histone acetyltransferase activity from the nuclear extract. It is very unlikely that this activity is derived from P/CAF given that ElA and P/CAF cannot bind to p300/CBP simultaneously (43). Consistent with this, no P/CAF was detected in these fractions by immunoblotting. The ElA N-terminus, a region that is not highly conserved among the various adenovirus serotypes, is involved in p300/CBP binding in vivo. Mutations in the N-terminal region lead to loss of the ability for p300/CBP binding without affecting RB binding (1,47). Thus, the requirement of the El A N-terminal region for the recruitment of histone acetyltransferase activity was tested. In contrast to the wild type, the N-terminal deleted form of ElA (ΔN-E1 A) recruited only a background level of acetyltransferase activity. In agreement with previous reports (47), the ΔN-E1A showed no ability to interact with p300/CBP, although it still retained the ability to interact with a variety of other polypeptides, including RB.

To define the relationship between p300/CBP and histone acetyltransferase activity, affinity purified El A-binding polypeptides were separated by Mono Q ion-exchange column. Both p300/CBP and the acetyltransferase activity were coeluted at 140 mM KCl, while most of polypeptides were eluted at 260 mM KCl. The active fraction of Mono Q column (-140 mM KCl) was further separated by Superdex-200 gel filtration column. Both ρ300/CBP and the acetyltransferase activity coeluted after the void volume, indicating that p300/CBP is involved in the histone acetyltransferase activity.

p300 is a histone acetyltransferase

The data provided herein indicate that p300 per se, or a polypeptide(s) associated with ρ300, possesses histone acetyltransferase activity. To test the former possibility, the acetyltransferase activity of recombinant p300 was measured. p300 was divided into three fragments, each of which was expressed in and purified from Sf9 cells via a baculovirus expression vector. Histone acetyltransferase activity was readily detected in the C-terminal fragment containing amino acids 1135-2414, whereas no activity was found in the other fragments, demonstrating conclusively that p300 per se is a histone acetyltransferase. p300/CBP-histone acetyltransferase domain

To map the histone acetyltransferase domain of p300, a series of deletions was prepared. Given the poor conservation of the glutamine-rich region (aa 1815-2414) in the C. elegans p300/CBP homolog (6), the p300 fragment encoding aa 1135-1810 was expressed in and purified from E. coli. Importantly, this candidate region of p300 (aa 1135-1810) showed significant histone acetyltransferase activity. For further mapping within this region, a series of N-terminal deletions was constructed. Deletion of 60 residues, resulting in a fragment containing aa 1195-1810, had no effect on the acetyltransferase activity, whereas the deletion of 185 residues, yielding a fragment comprising aa residues 1320-1810, completely eliminated the acetyltransferase activity.

Next, a series of C-terminal deletions was analyzed to determine the requirement of the P/CAF (or ElA) -binding domain. The p300 fragments lacking the ElA binding domain (aa 1195-1760, 1195-1706 and 1195-1673) still retained the acetyltransferase activity, whereas the further truncated mutant (aa 1195-1652) completely lost the acetyltransferase activity. Consistent with these results, the internal deletion of residues 1418-1720 showed no acetyltransferase activity. These data demonstrate that the histone acetyltransferase domain is located between the bromodomain and the El A-binding domain. Given that the histone acetyltransferase domain is highly conserved between p300 and CBP (91% similarity), the corresponding region of CBP, aa residues 1174-1850, was expressed to confirm the acetyltransferase activity. As expected, comparable activity was detected, indicating that both p300 and CBP are histone acetyltransferases.

Among various acetyltransferases including histone acetyltransferases GCN5 and

P/CAF, putative acetyl-CoA binding sites are conserved (48). However, multiple alignment analysis (49) showed that the p300/CBP histone acetyltransferase domain does not belong to this group. Moreover, comparison of the p300/CBP histone acetyltransferase domain with peptide sequence databases (23) showed no sequence similarity to any other proteins. Accordingly, this invention shows that p300/CBP represents a novel class of acetyltransferases in that it does not have the conserved motif found among previously described acetyltransferases (48). p300 acetylates all core histones in mononucleosomes

Substrate specificity for acetylation by p300 was also examined. As substrates, histone octamers and mononucleosomes (146 base pairs of DNA wrapped around the octamer of core histones) were used. Given that the histone octamer dissociates into dimers or tetramers under physiological conditions, the histone octamer is referred to here as core histones. When core histones were used, p300 acetylated all four proteins, but preferentially H3 and H4. More importantly, in a nucleosomal context, p300 acetylated all four core histones nearly stoichiometrically. In contrast, p300 acetylated neither BSA nor lysozyme.

Hyperacetylated histones are believed to be linked with transcriptionally active chromatin (26,27,50,51). Hyperacetylated forms are found in histones H4, H3 and H2B, which have multiple acetylation sites in vivo. Thus, the level of acetylation by p300 was also tested.

Mononucleosomes treated with p300 were analyzed by two-dimensional gel electrophoresis. A Coomassie blue-stained gel and the corresponding autoradiogram showed that a significant amount of histones, especially H4, were hyperacetylated. Importantly, acetylation levels by p300 were very close to those of hyperacetylated histones prepared from HeLa nuclei treated with sodium butyrate, a histone deacetylase inhibitor. In contrast, no acetylated forms were detected in the reaction without p300. These results indicate that p300 acetylates histones in mononucleosomes to the hyperacetylated state by targeting multiple lysine residues.

p300 acetylates the four lysines in the histone H4 N-terminal tail in vitro which are acetylated in vivo

Lysines at positions 5, 8, 12 and 16 of histone H4 are acetylated in vivo (51). Recent studies with yeast histone acetyltransferases demonstrate the position-specific acetylation by distinct acetyltransferases, i.e., while cytoplasmic acetyltransferases for histone deposition and chromatin assembly modify positions 5 and 12, GCN5 modifies positions 8 and 16 (52). Accordingly, the positions of acetylation by ρ300 were also determined. A series of synthetic peptides containing acetylated lysines at various positions was used to determine the acetylation site-specificity of p300. Consistent with the two-dimensional gel electrophoresis analysis, the experiments with peptide substrates showed that p300 acetylates all four lysines in the histone H4 that are acetylated in vivo. These results are consistent with the view that deposition-related diacetylated histones are deacetylated during maturation of chromatin (53).

p300 preferentially acetylates the N-terminal histone tail

Histone acetyltransferases modify specific lysine residues in the N-terminal tail of core histones but not the C-terminal globular domain in vivo (26,27,50,51). Structural models of nucleosomes (54,55,56) suggest that most of the lysine residues in the C-terminal globular domain are buried. Therefore, experiments were conducted to examine whether restricted acetylation of the N-terminal tail resulted from the substrate specificity of the enzyme or inaccessibility of the enzyme to the core domain in nucleosomes. The globular domains of all core histones contain a long helix flanked on either side by a loop segment and short helix, termed the "histone fold" (54,55,56). The histone fold is involved in formation of the stable H2A-H2B and H3-H4 hetero-dimers, consisting of extensive hydrophobic contacts between the paired molecules. Therefore, it is likely that a histone monomer cannot fold properly, thereby increasing access of the histone acetyltransferase to the core domain. Based on these considerations, experiments were conducted to determine whether p300 acetylates free histone H4 in a N-terminal-specific manner.

Histone H4 was acetylated with p300 and subsequently the histone tail was removed by partial digestion with trypsin. The distributions of radioactivity between intact and core histones were compared. While the globular core histone domain was predominant at the higher trypsin concentrations, radioactivity was detected mostly in the intact histone. These data demonstrate that p300 preferentially acetylates the N-terminal tail of histone H4. HI. P/CAF interaction with MyoD

Tissue culture and transfection experiments

C₂C₁₂ mouse cells (ATCC accession number CRL 1772) were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 20% fetal bovine serum (FBS) until they reached confluence. Differentiation was induced by switching medium to differentiation medium (DM), consisting of DMEM containing 2% horse serum. C₃H/10Tl/2 fibroblasts (ATCC accession number CCL 226) were grown in DMEM supplemented with 10% FBS. Cells were transfected by the calcium phosphate precipitation method. Total amounts of transfected DNA were equalized by empty vector DNA. After 12 h incubation in medium containing the precipitated DNA, the cells were washed and incubated in fresh DMEM containing 10% FBS for an additional 24 h. Afterwards, differentiation was induced by incubating in DM for 36 to 72 h. Chloramphenicol acetyltransferase (CAT) assays were performed as previously described (64,69). The quantities of cell extracts used for CAT assays were normalized toβ-galactosidase activity by cotransfection of 1 mg of the β-galactosidase expression vector, pON260.

Expression vectors used for transfection experiments are as follows: pCX-P/CAF for P/CAF (43); pCMV-bp300 for p300 (65), pCMV-p300 (1869-2414) (64) and pCMV-p300 (1514-1922) (60) for p300 wild type and mutants; pElA12S, pEl A12S R2G, pElA12S D2-36 and pEl A12S D121-130 for ElA wild type and mutants (66,67,68); and pEMSV-MyoD for MyoD (64).

The antisense P/CAF RNA expression vector, pcDNA3 P/CAF-AS, was created as follows. The 2.5 Kb EcoRI-Kpnl fragment containing the entire P/CAF open reading frame was isolated from pCX-P/CAF (43). This fragment was subcloned into the EcoRI-Kpnl sites of plasmid pcDNA3 (Invitrogen) so that the antisense P/CAF RNA is driven under the CMV promoter. Reporter genes employed were 4RE-CAT and MCK-CAT (69). 4RE-CAT is driven by a synthetic promoter containing 4 copies of the E-box, whereas MCK-CAT is driven by the native MCK promoter (nucleotides -1256 to +7).

Microinjection and immunofluorescence

Cells were grown on small glass slides, subdivided into numbered squares of 2 mm x 2 mm and microinjected with purified and concentrated antibodies, as previously described (70). For immunofluorescence, cells were fixed in either 2% paraformaldehyde or 1 :2 methanol/acetone solution, preincubated with 5% BS A/PBS and incubated with the primary antibodies for 30 min at 37° C. Subsequently, antibody was visualized by incubating with either rhodamine- or fluorescein-conjugated secondary antibody for 30 min at 37° C. Injected antibodies were stained with a rhodamine-conjugated secondary antibody and nuclei were counter-stained by DAPI as previously described (69).

Antibodies employed are as follows; rabbit polyclonal affinity purified anti-P/CAF antibody (43), rabbit polyclonal anti-p300/CBP antiserum (71), mouse monoclonal anti-MyoD antibody (clone 5.8 A, kindly provided by P. Houghton), goat polyclonal anti-c-Jun affinity purified antibody (Santa Cruz) and rabbit pre-immune serum.

Immunoprecipitation and DNA affinity purification

Cells were resuspended in lysis buffer (20 mM NaPO₄, 150 mM NaCl, 5mM MgCl₂, 0.1% NP40, 1 mM DTT, 10 mM sodium fluoride, 0.1 mM sodium vanadate, 1 mM phenylmethylsulfonyl-fluoride and 10 mg/ml each of leupeptin, aprotinin and pepstatin). After 30 min incubation on ice, samples were centrifuged at 12,000 x g for 30 min and supernatants were used as cell extracts. Extracts were pre-cleared by incubating with rabbit pre-immune serum and protein A/G Plus- Agarose (Santa Cruz) for 2 h at 4 C. For immunoprecipitation, the supernatants were incubated with the respective antibodies for 3 h at 4 C. Protein A/G Plus- Agarose was added, and incubation continued for 3 h. The matrix was washed with lysis buffer, then boiled in 2 X SDS sample buffer. Immunoblotting was performed by using the ECL chemiluminescent detection kit (Amersham) according to the manufacturer's protocol

Affinity purification of E-box-bound complexes was done as previously described (69). Briefly, 100 ng of the biotinylated double stranded DNA containing the E-box were immobilized on streptavidin-conjugated magnetic beads and incubated with 500 mg of cell extracts in the presence of poly dl-dC. After extensive washing, bound proteins were eluted with SDS sample buffer and analyzed by immunoblotting

In vitro protein-protein interaction assays The CBP-B fragment and its deletion derivatives were expressed as

GST-fusions described previously (43). MyoD and ElA (43) were expressed as FLAG-fusion proteins in Sf9 cells via a baculovirus expression system and affinity-purified on M2 anti-FLAG antibody-agarose (Kodak-IBI) Crude E coli extracts containing GST-fusions were incubated with various amounts of MyoD and/or ElA in 50 ml of buffer B (20 mM Tris-HCl, pH 8 0, 0 1 M KCl, 5 mM MgCl₂, 10% glycerol, and 0.1% Nonidet P-40) on ice for 10 min. GST-precipitation was performed as described (43). MyoD and ElA were detected by immunoblotting with anti-FLAG M2 antibody. For the interaction between P/CAF and MyoD, 1.5 pmol of FLAG-P/CAF and 15 pmol of FLAG-MyoD were incubated in 50 ml of buffer B on ice for 10 min. The mixture was further incubated with 2 mg of anti-P/CAF (43) or anti-hADA2 antibody for 60 min. The immunocomplexes were precipitated by incubation with 10 ml of protein A-Trisacryl (Pierce) and rotated for 1-4 hr at 4oC The matrix was washed 4 times with 200 ml of buffer B and boiled in 10 ml of 2 X SDS sample buffer The proteins were resolved on a 4%-20% gradient SDS-PAGE and subjected to immunoblotting with the anti-FLAG M2 antibody The blot was developed with the SUPERSIGNAL chemiluminescent substrates (Pierce). P/CAF coactivates muscle-specific transcription

P/CAF and MyoD were co-transfected into mouse C3H10T1/2 fibroblasts, and MyoD-mediated transcription was determined from reporter activity driven by the artificial (4RE) and the naturally-occurring muscle creatine kinase (MCK) promoters. Overexpression of P/CAF stimulated MyoD-dependent transcription several folds in both promoters. Similar results were obtained for the myoD activated myogenin promoter Transcriptional activation was further stimulated by co-transfecting with MyoD, P/CAF and p300 expression vectors, suggesting that P/CAF may function by forming a complex with p300/CBP. Consistent with the lack of DNA binding capacity in P/CAF, overexpression of P/CAF alone did not increase the basal transcriptional activity of either enhancer. To test whether P/CAF and p300/CBP function in the same pathway, two dominant negative forms of p300 were employed which specifically inhibit p300/CBP-mediated transcription (60,64). The p300 segment spanning residues 1514-1922 inhibits the MyoD-dependent activation via direct interaction with MyoD (60), whereas the p300 segment spanning residues 1869-2414 inhibit it without direct interaction (64). Both dominant negative mutants inhibited MyoD-coactivation by P/CAF), suggesting that P/CAF and p300/CBP function in the same pathway.

For further elucidation of the activation mechanism by P/CAF, the effect of El A, which inhibits MyoD-dependent transcription and differentiation (66,72,73) via direct interaction with p300/CBP (65,78), was tested. Expression of El A in C3H10T1/2 fibroblasts inhibited stimulation of MyoD-directed transcription by P/CAF overexpression. ElA mutants lacking p300/CBP-binding activity, E1A D2-36 and ElA R2G (67,79), had almost no effect. On the other hand, an ElA mutant retaining p300/CBP-binding activity, ElA D121-130, behaved like the wild type. Since ElA associates with p300/CBP, but not with P/CAF, these results suggest that P/CAF functions in MyoD-directed transcription via interaction with p300/CBP.

To address the role of P/CAF as a myogenic coactivator in a more relevant environment, P/CAF was overexpressed in proliferating C2C12 myoblasts which express endogenous myogenic bHLH factors. As observed in fibroblasts, overexpression of P/CAF stimulated muscle specific transcription. Concomitant expression of exogenous p300 increased P/CAF-mediated coactivation. The repression exerted by wild type ElA, but not mutant ElA D2-36, on P/CAF coactivation of MyoD was also observed in muscle cells.

Similar experiments were performed with myogenic cell lines that were stably transformed with wild type or mutant El A-expressing vectors (66). Coactivation by P/CAF was inhibited by wild type ElA or the El A mutant that retains p300/CBP-binding activity (E1AΔ121-130). In contrast, ElA mutants that lack p300/CBP-binding (ElA Δ2-36 and ElA R2G) allowed transcriptional coactivation by P/CAF. Taken together, these experiments show that P/CAF coactivates MyoD-directed transcription via interaction with p300/CBP.

P/CAF stimulates myogenic differentiation

Given that P/CAF potentiates MyoD-directed transcription, the ability of P/CAF to assist MyoD in promoting myogenic differentiation was investigated. To this aim, C3H10T1/2 fibroblasts were transiently transfected with P/CAF and MyoD expression vectors. An expression vector for the green fluorescent protein (GFP) was co-transfected to identify transfected cells. After incubation in differentiation medium, the myogenic conversion of transfected cells was determined by simultaneous expression of the GFP and the differentiation-specific marker myosin heavy chain (MHC). Forced expression of MyoD in fibroblasts caused muscle differentiation in 12% of the transfected fibroblasts. This myogenic conversion was 20% by co-expressing MyoD and P/CAF. As observed in transcription experiments, stimulation of differentiation by P/CAF was counteracted by co-transfection with the p300 dominant negative mutant, p300 (1869-2414). Consistent with a general role for coactivators, overexpression of P/CAF alone was unable to differentiate fibroblasts.

Similar experiments were done using proliferating C2C12 myoblasts in which the differentiation program is already committed. Most of the myoblasts differentiated into myotubes by overexpressing P/CAF, whereas only a modest effect was observed by overexpressing p300. In contrast, differentiation was inhibited slightly by overexpressing c-Jun. This inhibitory effect presumably was caused by titration of p300/CBP, which associates directly with c-Jun (74). A similar inhibition was observed in the p300 dominant negative mutant. Consistent with the transcriptional effect, ElA almost completely inhibited differentiation. The ElA mutant RG2, lacking p300/CBP-binding capability but retaining the retinoblastoma protein (Rb)-binding capability, only partially inhibited differentiation, although this same mutant inhibited transcription as severely as the wild type. Taken together, these data show that P/CAF stimulates muscle differentiation by coactivating MyoD function via p300/CBP association.

P/CAF is essential for myogenic transcription and differentiation

To test the necessity of P/CAF for myogenic transcription, experiments were conducted whereby P/CAF synthesis was inhibited by expressing antisense P/CAF RNA A vector from which the P/CAF mRNA is transcribed in the antisense orientation (P/CAF- AS) was transfected with P/CAF and MyoD expression vectors into fibroblasts and MyoD-dependent transcription was examined. Cotransfection of the antisense expression vector strongly inhibited MyoD-dependent transcription below the level of induction elucidated by MyoD alone, demonstrating that expression of P/CAF antisense RNA inhibits not only the coactivation exerted by exogenous P/CAF but also that of endogenous P/CAF. These results indicate that P/CAF is essential for MyoD-dependent transcription.

Studies were also carried out to determine whether expression of P/CAF antisense RNA inhibits myogenic differentiation. C3H10T1/2 fibroblasts were transiently transfected with various expression vectors with or without the P/CAF antisense RNA expression vector. Expression of P/CAF antisense RNA reduced MyoD-mediated myogenic conversion of fibroblasts. Expression of P/CAF antisense RNA also counteracted the stimulatory effect of both P/CAF and p300 on myogenic differentiation. These data support the view that P/CAF and p300/CBP coactivate MyoD-dependent transcription in the same pathway. More drastic inhibition was observed in C2C12 myoblasts in similar experiments. Therefore, it can be concluded that P/CAF is essential for transcription of muscle specific genes and hence differentiation into myotubes.

To further confirm the essential role of P/CAF for myogenic differentiation, we blockage experiments by antibody microinjection were performed. Antibodies were injected into the cytoplasm of proliferating C2C12 myoblasts to prevent the nuclear transport of newly synthesized target proteins. After incubating in the differentiation medium, the degree of differentiation was determined. Microinjection of an anti-P/CAF antibody almost completely inhibited differentiation. Similar results were obtained by microinjecting anti-p300/CBP antibodies. Although microinjection of either anti-p300/CBP or P/CAF antibody was sufficient to inhibit differentiation, an even greater inhibition was observed by coinjecting both of them. Microinjection of anti-P/CAF or anti-p300/CBP antibody did not interfere with induction of p53 by DNA damaging agents, showing specificity of the inhibition by the antibodies. In contrast to anti-P/CAF or anti-p300/CBP antibodies, the injection of anti-MyoD antibody only partially inhibited differentiation, supporting the view of functional redundancy between MyoD and Myf-5 (75,76). Injection of anti-c-Jun antibody or control antibody did not interfere with muscle differentiation.

Similar experiments were performed with C3H10T1/2 fibroblasts stably expressing MyoD. In these cells, either anti-p300/CBP or anti-P/CAF antibody completely inhibited muscle differentiation. In contrast to myoblasts, anti-MyoD antibody completely blocked differentiation in the fibroblasts expressing MyoD.

Anti-c-Jun and control antibodies did not interfere with differentiation. Taken together, these results demonstrate that P/CAF and p300/CBP are indispensable for activation of the myogenic program. p300/CBP, P/CAF and MyoD form a multimeric complex in vivo

The data described above indicate that P/CAF stimulates MyoD-directed transcription via association with p300/CBP. Thus, experiments were conducted to investigate whether P/CAF, p300/CBP and MyoD could associate in a complex. First, cellular extracts derived from C2C12 myotubes were subjected to immunoprecipitation. Both anti-MyoD and anti-p300/CBP antibodies co-precipitated P/CAF. In a complementary experiment, both anti-p300/CBP and anti-P/CAF antibodies also co-precipitated MyoD, suggesting that these factors form a multimeric protein complex in myotubes.

Next, attempts were made to detect this complex on the E-box, the DNA binding site for MyoD. Immobilized DNA containing an E-box sequence was incubated with myotube extracts. After extensive washing, P/CAF, p300/CBP and MyoD were analyzed by immunoblotting. P/CAF, p300/CBP and MyoD were all affinity purified on the immobilized DNA, whereas they were not purified on the control DNA lacking the E-box. Given that P/CAF and p300/CBP per se cannot bind to DNA, these observations indicate that P/CAF and p300/CBP are recruited through MyoD at the E-box sites to form a multi-protein complex.

Complex formation is inhibited by viral transforming factors

Since the oncoviral proteins ElA and large T antigen inhibit myogenic transcription and differentiation, the effect of these factors on the formation of complexes on the E-box was tested. Importantly, very small amouts of P/CAF and p300/CBP were co-purified on the E-box from myocyte extracts which stably express ElA or large T antigen, although MyoD was detected under these conditions. The lower recovery of MyoD from El A-expressing muscle cells could reflect the low level of MyoD in the extracts (66). These results indicate that ElA and large T antigen dissociate P/CAF and p300/CBP from MyoD without altering MyoD binding to DNA.

Consistent with the previous observations that transiently expressed E 1 A prevents interaction between P/CAF and p300/CBP in vivo (43), the association between p300/CBP and P/CAF was abolished in myoblasts stably transformed by wild type ElA but not in those clones transformed with the El A mutant R2G unable to bind ρ300/CBP. Similarly, the interaction between p300/CBP and P/CAF was abolished by large T antigen but not by the mutant protein that localizes into the cytoplasm (77).

Interaction between MyoD, P/CAF and CBP in vitro

Previous interaction experiments in vitro indicate that the CBP region spanning residues 1801 to 1850 is crucial for interaction with both P/CAF and ElA (43). While most sequence-specific factors bind to CBP sites distinct from the P/CAF/E1 A binding sites, MyoD interacts with an overlapping CBP fragment called the CH3 region

(60,64,65). To understand how P/CAF, p300/CBP and MyoD associate, the CBP sites important for MyoD binding were mapped more precisely. Consistent with previous reports (60,64,65), the CBP fragment spanning residues 1801-2000 (fragment B) bound MyoD. Moreover, deletion of residues 1801 to 1850 within fragment B completely abolished interaction with MyoD, which is similar to the results obtained with P/CAF and ElA. Importantly, an internal deletion of residues 1850-1878 abolished the MyoD interaction with CBP, while it did not affect binding of El A or P/CAF (43). These results suggest that MyoD and P/CAF bind to distinct sites of p300/CBP, albeit the binding sites may overlap. Moreover, a direct interaction was observed between MyoD and P/CAF, which may contribute to stabilization of the multimeric complex.

These data show that ElA prevents not only p300/CBP-interaction with P/CAF but also that with MyoD in vivo. To obtain evidence that this inhibition is due to the direct action by El A, competition experiments were performed in vitro. Importantly, the interaction between CBP and MyoD was strongly inhibited by addition of El A, implicating that ElA inhibits myogenic transcription by disrupting multiple interactions.

Although the present process has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the invention except as and to the extent that they are included in the accompanying claims.

Throughout this application various publications are referenced by numbers within parentheses. Full citations for these publications are as follows. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

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80 BrowneU and Allis PNAS 92:6364-6368 (1995). SEQUENCE LISTING

(1) GENERAL INFORMATION

(1) APPLICANT: The United States of America, as repesented by the Secretary, Department of Health and Human Services, c/o National Institutes of Health, Office of Technology Transfer, 6011 Executive Boulevard, Suite 325, Rockville, Maryland 20842

(li) TITLE OF THE INVENTION: METHODS AND COMPOSITIONS FOR p300/CBP-ASSOCIATED TRANSCRIPTIONAL CO-FACTOR P/CAF

(ill) NUMBER OF SEQUENCES: 18

(IV) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: NEEDLE & ROSENBERG, P.C.

(B) STREET: Suite 1200, 127 Peachtree Street, NE

(C) CITY: Atlanta

(D) STATE: GA

(E) COUNTRY: USA

(F) ZIP: 30303

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette

(B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: DOS

(D) SOFTWARE: FastSEQ for Windows Version 2.0

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: 23-JUL-1997

(C) CLASSIFICATION:

(Vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: Corresponding U.S. Serial No. 60/022,273

(B) FILING DATE: 23-July-1996

(Vlll) ATTORNEY/AGENT INFORMATION:

(A) NAME: Miller, Mary L

(B) REGISTRATION NUMBER: 39,303

(C) REFERENCE/DOCKET NUMBER: 14014.0238/P

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 404/688-0770

(B) TELEFAX: 404/688-9880

(C) TELEX:

(2) INFORMATION FOR SEQ ID NO:l:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 832 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(li) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:

Met Ser Glu Ala Gly Gly Ala Gly Pro Gly Gly Cys Gly Ala Gly Ala

1 5 ^" 10 15

Gly Ala Gly Ala Gly Pro Gly Ala Leu Pro Pro Gin Pro Ala Ala Leu

20 25 30

Pro Pro Ala Pro Pro Gin Gly Ser Pro Cys Ala Ala Ala Ala Gly Gly

35 40 45

Ser Gly Ala Cys Gly Pro Ala Thr Ala Val Ala Ala Ala Gly Thr Ala

50 55 60

Glu Gly Pro Gly Gly Gly Gly Ser Ala Arg lie Ala Val Lys Lys Ala 65 70 75 80

Gin Leu Arg Ser Ala Pro Arg Ala Lys Lys Leu Glu Lys Leu Gly Val

85 90 95

Tyr Ser Ala Cys Lys Ala Glu Glu Ser Cys Lys Cys Asn Gly Trp Lys

100 105 110

Asn Pro Asn Pro Ser Pro Thr Pro Pro Arg Ala Asp Leu Gin Gin lie

115 120 125 lie Val Ser Leu Thr Glu Ser Cys Arg Ser Cys Ser His Ala Leu Ala

130 135 140

Ala His Val Ser His Leu Glu Asn Val Ser Glu Glu Glu Met Asn Arg 145 150 155 160

Leu Leu Gly lie Val Leu Asp Val Glu Tyr Leu Phe Thr Cys Val His

165 170 175

Lys Glu Glu Asp Ala Asp Thr Lys Gin Val Tyr Phe Tyr Leu Phe Lys

180 185 190

Leu Leu Arg Lys Ser lie Leu Gin Arg Gly Lys Pro Val Val Glu Gly

195 200 205

Ser Leu Glu Lys Lys Pro Pro Phe Glu Lys Pro Ser lie Glu Gin Gly

210 215 220

Val Asn Asn Phe Val Gin Tyr Lys Phe Ser His Leu Pro Ala Lys Glu 225 230 235 240

Arg Gin Thr lie Val Glu Leu Ala Lys Met Phe Leu Asn Arg lie Asn

245 250 255

Tyr Trp His Leu Glu Ala Pro Ser Gin Arg Arg Leu Arg Ser Pro Asn

260 265 270

Asp Asp lie Ser Gly Tyr Lys Glu Asn Tyr Thr Arg Trp Leu Cys Tyr

275 280 285

Cys Asn Val Pro Gin Phe Cys Asp Ser Leu Pro Arg Tyr Glu Thr Thr

290 295 300

Gin Val Phe Gly Arg Thr Leu Leu Arg Ser Val Phe Thr Val Met Arg 305 310 315 320

Arg Gin Leu Leu Glu Gin Ala Arg Gin Glu Lys Asp Lys Leu Pro Leu

325 330 335

Glu Lys Arg Thr Leu lie Leu Thr His Phe Pro Lys Phe Leu Ser Met

340 345 350

Leu Glu Glu Glu Val Tyr Ser Gin Asn Ser Pre lie Trp Asp Gin Asp

355 360 365

Phe Leu Ser Ala Ser Ser Arg Thr Ser Gin Leu Gly lie Gin Thr Val

370 375 380 lie Asn Pro Pro Pro Val Ala Gly Thr lie Ser Tyr Asn Ser Thr Ser 385 390 395 400

Ser Ser Leu Glu Gin Pro Asn Ala Gly Ser Ser Ser Pro Ala Cys Lys

405 410 415

Ala Ser Ser Gly Leu Glu Ala Asn Pro Gly Glu Lys Arg Lys Met Thr

420 425 430

Asp Ser His Val Leu Glu Glu Ala Lys Lys Pro Arg Val Met Gly Asp

435 440 445 lie Pro Met Glu Leu lie Asn Glu Val Met Ser Thr lie Thr Asp Pro

450 455 460

Ala Ala Met Leu Gly Pro Glu Thr Asn Phe Leu Ser Ala His Ser Ala 465 470 475 480 Arg Asp Glu Ala Ala Arg Leu Glu Glu Arg Arg Gly Val He Glu Phe

485 490 495

His Val Val Gly Asn Ser Leu Asn Gin Lys Pro Asn Lys Lys He Leu

500 505 510

Met Trp Leu Val Gly Leu Gin Asn Val Phe Ser His Gin Leu Pro Arg

515 520 525

Met Pro Lys Glu Tyr He Thr Arg Leu Val Phe Asp Pro Lys His Lys

530 535 540

Thr Leu Ala Leu He Lys Asp Gly Arg Val He Gly Gly He Cys Phe 545 550 555 560

Arg Met Phe Pro Ser Gin Gly Phe Thr Glu He Val Phe Cys Ala Val

565 570 575

Thr Ser Asn Glu Gin Val Lys Gly Tyr Gly Thr His Leu Met Asn His

580 585 590

Leu Lys Glu Tyr His He Lys His Asp He Leu Asn Phe Leu Thr Tyr

595 600 605

Ala Asp Glu Tyr Ala He Gly Tyr Phe Lys Lys Gin Gly Phe Ser Lys

610 615 620

Glu He Lys He Pro Lys Thr Lys Tyr Val Gly Tyr He Lys Asp Tyr 625 630 635 640

Glu Gly Ala Thr Leu Met Gly Cys Glu Leu Asn Pro Arg He Pro Tyr

645 650 655

Thr Glu Phe Ser Val He He Lys Lys Gin Lys Glu He He Lys Lys

660 665 670

Leu He Glu Arg Lys Gin Ala Gin He Arg Lys Val Tyr Pro Gly Leu

675 680 685

Ser Cys Phe Lys Asp Gly Val Arg Gin He Pro He Glu Ser He Pro

690 695 700

Gly He Arg Glu Thr Gly Trp Lys Pro Ser Gly Lys Glu Lys Ser Lys 705 710 715 720

Glu Pro Arg Asp Pro Asp Gin Leu Tyr Ser Thr Leu Lys Ser He Leu

725 730 735

Gin Gin Val Lys Ser His Gin Ser Ala Trp Pro Phe Met Glu Pro Val

740 745 750

Lys Arg Thr Glu Ala Pro Gly Tyr Tyr Glu Val He Arg Ser Pro Met

755 760 765

Asp Leu Lys Thr Met Ser Glu Arg Leu Lys Asn Arg Tyr Tyr Val Ser

770 775 780

Lys Lys Leu Phe Met Ala Asp Leu Gin Arg Val Phe Thr Asn Cys Lys 785 790 795 800

Glu Tyr Asn Ala Pro Glu Ser Glu Tyr Tyr Lys Cys Ala Asn He Leu

805 810 815

Glu Lys Phe Phe Phe Ser Lys He Lys Glu Ala Gly Leu He Asp Lys 820 825 830

(2) INFORMATION FOR SEQ ID NO: 2:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 481 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: None

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

Met Leu Glu Glu Glu Val Tyr Ser Gin Asn Ser Pro He Trp Asp Gin

1 5 10 15

Asp Phe Leu Ser Ala Ser Ser Arg Thr Ser Gin Leu Gly He Gin Thr

20 25 30

Val He Asn Pro Pro Pro Val Ala Gly Thr He Ser Tyr Asn Ser Thr 35 40 45 Ser Ser Ser Leu Glu Gin Pro Asn Ala Gly Ser Ser Ser Pro Ala Cys

50 55 60

Lys Ala Ser Ser Gly Leu Glu Ala Asn Pro Gly Glu Lys Arg Lys Met 65 70 75 80

Thr Asp Ser His Val Leu Glu Glu Ala Lys Lys Pro Arg Val Met Gly

85 90 95

Asp He Pro Met Glu Leu He Asn Glu Val Met Ser Thr He Thr Asp

100 105 110

Pro Ala Ala Met Leu Gly Pro Glu Thr Asn Phe Leu Ser Ala His Ser

115 120 125

Ala Arg Asp Glu Ala Ala Arg Leu Glu Glu Arg Arg Gly Val He Glu

130 135 140

Phe His Val Val Gly Asn Ser Leu Asn Gin Lys Pro Asn Lys Lys He 145 150 155 160

Leu Met Trp Leu Val Gly Leu Gin Asn Val Phe Ser His Gin Leu Pro

165 170 175

Arg Met Pro Lys Glu Tyr He Thr Arg Leu Val Phe Asp Pro Lys His

180 185 190

Lys Thr Leu Ala Leu He Lys Asp Gly Arg Val He Gly Gly He Cys

195 200 205

Phe Arg Met Phe Pro Ser Gin Gly Phe Thr Glu He Val Phe Cys Ala

210 215 220

Val Thr Ser Asn Glu Gin Val Lys Gly Tyr Gly Thr His Leu Met Asn 225 230 235 240

His Leu Lys Glu Tyr His He Lys His Asp He Leu Asn Phe Leu Thr

245 250 255

Tyr Ala Asp Glu Tyr Ala He Gly Tyr Phe Lys Lys Gin Gly Phe Ser

260 265 270

Lys Glu He Lys He Pro Lys Thr Lys Tyr Val Gly Tyr He Lys Asp

275 280 285

Tyr Glu Gly Ala Thr Leu Met Gly Cys Glu Leu Asn Pro Arg He Pro

290 295 300

Tyr Thr Glu Phe Ser Val He He Lys Lys Gin Lys Glu He He Lys 305 310 315 320

Lys Leu He Glu Arg Lys Gin Ala Gin He Arg Lys Val Tyr Pro Gly

325 330 335

Leu Ser Cys Phe Lys Asp Gly Val Arg Gin He Pro He Glu Ser He

340 345 350

Pro Gly He Arg Glu Thr Gly Trp Lys Pro Ser Gly Lys Glu Lys Ser

355 360 365

Lys Glu Pro Arg Asp Pro Asp Gin Leu Tyr Ser Thr Leu Lys Ser He

370 375 380

Leu Gin Gin Val Lys Ser His Gin Ser Ala Trp Pro Phe Met Glu Pro 385 390 395 400

Val Lys Arg Thr Glu Ala Pro Gly Tyr Tyr Glu Val He Arg Ser Pro

405 410 415

Met Asp Leu Lys Thr Met Ser Glu Arg Leu Lys Asn Arg Tyr Tyr Val

420 425 430

Ser Lys Lys Leu Phe Met Ala Asp Leu Gin Arg Val Phe Thr Asn Cys

435 440 445

Lys Glu Tyr Asn Ala Pro Glu Ser Glu Tyr Tyr Lys Cys Ala Asn He

450 455 460

Leu Glu Lys Phe Phe Phe Ser Lys He Lys Glu Ala Gly Leu He Asp 465 470 475 480

Lys

(2) INFORMATION FOR SEQ ID NO: 3:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 203 amino acids

(B) TYPE: amino acid

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

(11) MOLECULE TYPE: None

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

Arg Val Val Gin His Thr Lys Gly Cys Lys Arg Lys Thr Asn Gly Gly

1 5 10 15

Cys Pro He Cys Lys Gin Leu He Ala Leu Cys Cys Tyr His Ala Lys

20 25 30

His Cys Gin Glu Asn Lys Cys Pro Val Pro Phe Cys Leu Asn He Lys

35 40 45

Gin Lys Leu Arg Gin Gin Gin Leu Gin His Arg Leu Gin Gin Ala Gin

50 55 60

Met Leu Arg Arg Arg Met Ala Ser Met Arg Thr Gly Val Val Gly Gin 65 70 75 80

Gin Gin Gly Leu Pro Ser Pro Thr Pro Ala Thr Pro Thr Thr Pro Thr

85 90 95

Gly Gin Gin Pro Thr Thr Pro Gin Thr Pro Gin Pro Thr Ser Gin Pro

100 105 110

Gin Pro Thr Pro Pro Asn Ser Met Pro Pro Tyr Leu Pro Arg Thr Gin

115 120 125

Ala Ala Gly Pro Val Ser Gin Gly Lys Ala Ala Gly Gin Val Thr Pro

130 135 140

Pro Thr Pro Pro Gin Thr Ala Gin Pro Pro Leu Pro Gly Pro Pro Pro 145 150 155 160

Thr Ala Val Glu Met Ala Met Gin He Gin Arg Ala Ala Glu Thr Gin

165 170 175

Arg Gin Met Ala His Val Gin He Phe Gin Arg Pro He Gin His Gin

180 185 190

Met Pro Pro Met Thr Pro Met Ala Pro Met Gly 195 200

(2) INFORMATION FOR SEQ ID NO: 4:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 351 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: None

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

Met Ser Glu Ala Gly Gly Ala Gly Pro Gly Gly Cys Gly Ala Gly Ala

1 5 10 15

Gly Ala Gly Ala Gly Pro Gly Ala Leu Pro Pro Gin Pro Ala Ala Leu

20 25 30

Pro Pro Ala Pro Pro Gin Gly Sεr Pro Cys Ala Ala Ala Ala Gly Gly

35 40 45

Ser Gly Ala Cys Gly Pro Ala Thr Ala Val Ala Ala Ala Gly Thr Ala

50 55 60

Glu Gly Pro Gly Gly Gly Gly Ser Ala Arg He Ala Val Lys Lys Ala 65 70 75 80

Gin Leu Arg Ser Ala Pro Arg Ala Lys Lys Leu Glu Lys Leu Gly Val

85 90 95

Tyr Ser Ala Cys Lys Ala Glu Glu Ser Cys Lys Cys Asn Gly Trp Lys

100 105 110

Asn Pro Asn Pro Ser Pro Thr Pro Pro Arg Ala Asp Leu Gin Gin He

115 120 125

He Val Ser Leu Thr Glu Ser Cys Arg Ser Cys Ser His Ala Leu Ala 130 135 140 Ala His Val Ser His Leu Glu Asn Val Ser Glu Glu Glu Met Asn Arg 145 150 155 160

Leu Leu Gly He Val Leu Asp Val Glu Tyr Leu Phe Thr Cys Val His

165 170 175

Lys Glu Glu Asp Ala Asp Thr Lys Gin Val Tyr Phe Tyr Leu Phe Lys

180 185 190

Leu Leu Arg Lys Ser He Leu Gin Arg Gly Lys Pro Val Val Glu Gly

195 200 205

Ser Leu Glu Lys Lys Pro Pro Phe Glu Lys Pro Ser He Glu Gin Gly

210 215 220

Val Asn Asn Phe Val Gin Tyr Lys Phe Ser His Leu Pro Ala Lys Glu 225 230 235 240

Arg Gin Thr He Val Glu Leu Ala Lys Met Phe Leu Asn Arg He Asn

245 250 255

Tyr Trp His Leu Glu Ala Pro Ser Gin Arg Arg Leu Arg Ser Pro Asn

260 265 270

Asp Asp He Ser Gly Tyr Lys Glu Asn Tyr Thr Arg Trp Leu Cys Tyr

275 280 285

Cys Asn Val Pro Gin Phe Cys Asp Ser Leu Pro Arg Tyr Glu Thr Thr

290 295 300

Gin Val Phe Gly Arg Thr Leu Leu Arg Ser Val Phe Thr Val Met Arg 305 310 315 320

Arg Gin Leu Leu Glu Gin Ala Arg Gin Glu Lys Asp Lys Leu Pro Leu

325 330 335

Glu Lys Arg Thr Leu He Leu Thr His Phe Pro Lys Phe Leu Ser 340 345 350

(2) INFORMATION FOR SEQ ID NO: 5:

(I) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 476 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(II) MOLECULE TYPE: None

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

Met Leu Glu Glu Glu He Tyr Gly Ala Asn Ser Pro He Trp Glu Ser

1 5 10 15

Gly Phe Thr Met Pro Pro Ser Glu Gly Thr Gin Leu Val Pro Arg Pro

20 25 30

Ala Ser Val Ser Ala Ala Val Val Pro Ser Thr Pro He Phe Ser Pro

35 40 45

Ser Met Gly Gly Gly Ser Asn Ser Ser Leu Ser Leu Asp Ser Ala Gly

50 55 60

Ala Glu Pro Met Pro Gly Glu Lys Arg Thr Leu Pro Glu Asn Leu Thr 65 70 75 80

Leu Glu Asp Ala Lys Arg Leu Arg Val Met Gly Asp He Pro Met Glu

85 90 95

Leu Val Asn Glu Val Met Leu Thr He Thr Asp Pro Ala Ala Met Leu

100 105 110

Gly Pro Glu Thr Ser Leu Leu Ser Ala Asn Ala Ala Arg Asp Glu Thr

115 120 125

Ala Arg Leu Glu Glu Arg Arg Gly He He Glu Phe His Val He Gly

130 135 140

Asn Ser Leu Thr Pro Lys Ala Asn Arg Arg Val Leu Leu Trp Leu Val 145 150 155 160

Gly Leu Gin Asn Val Phe Ser His Gin Leu Pro Arg Met Pro Lys Glu

165 170 175

Tyr He Ala Arg Leu Val Phe Asp Pro Lys His Lys Thr Leu Ala Leu 180 185 190 He Lys Asp Gly Arg Val He Gly Gly He Cys Phe Arg Met Phe Pro

195 200 205

Thr Gin Gly Phe Thr Glu He Val Phe Cys Ala Val Thr Ser Asn Glu

210 215 220

Gin Val Lys Gly Tyr Gly Thr His Leu Met Asn His Leu Lys Glu Tyr 225 230 235 240

His He Lys His Asn He Leu Tyr Phe Leu Thr Tyr Ala Asp Glu Tyr

245 250 255

Ala He Gly Tyr Phe Lys Lys Gin Gly Phe Ser Lys Asp He Lys Val

260 265 270

Pro Lys Ser Arg Tyr Leu Gly Tyr He Lys Asp Tyr Glu Gly Ala Thr

275 280 285

Leu Met Glu Cys Glu Leu Asn Pro Arg He Pro Tyr Thr Glu Leu Ser

290 295 300

His He He Lys Lys Gin Lys Glu He He Lys Lys Leu He Glu Arg 305 310 315 320

Lys Gin Ala Gin He Arg Lys Val Tyr Pro Gly Leu Ser Cys Phe Lys

325 330 335

Glu Gly Val Arg Gin He Pro Val Glu Ser Val Pro Gly He Arg Glu

340 345 350

Thr Gly Trp Lys Pro Leu Gly Lys Glu Lys Gly Lys Glu Leu Lys Asp

355 360 365

Pro Asp Gin Leu Tyr Thr Thr Leu Lys Asn Leu Leu Ala Gin He Lys

370 375 380

Ser His Pro Ser Ala Trp Pro Phe Met Glu Pro Val Lys Lys Ser Glu 385 390 395 400

Ala Pro Asp Tyr Tyr Glu Val He Arg Phe Pro He Asp Leu Lys Thr

405 410 415

Met Thr Glu Arg Leu Arg Ser Arg Tyr Tyr Val Thr Arg Lys Leu Phe

420 425 430

Val Ala Asp Leu Gin Arg Val He Ala Asn Cys Arg Glu Tyr Asn Pro

435 440 445

Pro Asp Ser Glu Tyr Cys Arg Cys Ala Ser Ala Leu Glu Lys Phe Phe

450 455 460

Tyr Phe Lys Leu Lys Glu Gly Gly Leu He Asp Lys 465 470 475

(2) INFORMATION FOR SEQ ID NO: 6:

(I) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2414 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(II) MOLECULE TYPE: None

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

Met Ala Glu Asn Val Val Glu Pro Gly Pro Pro Ser Ala Lys Arg Pro

1 5 10 15

Lys Leu Ser Ser Pro Ala Leu Ser Ala Ser Ala Ser Asp Gly Thr Asp

20 25 30

Phe Gly Ser Leu Phe Asp Leu Glu His Asp Leu Pro Asp Glu Leu He

35 40 45

Asn Ser Thr Glu Leu Gly Leu Thr Asn Gly Gly Asp He Asn Gin Leu

50 55 60

Gin Thr Ser Leu Gly Met Val Gin Asp Ala Ala Ser Lys His Lys Gin 65 70 75 80

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

85 90 95

Val Gly Gly Pro Gly Gin Val Met Ala Ser Gin Ala Gin Gin Ser Ser 100 105 110 Pro Gly Leu Gly Leu He Asn Ser Met Val Lys Ser Pro Met Thr Gin

115 120 125

Ala Gly Leu Thr Ser Pro Asn Met Gly Met Gly Thr Ser Gly Pro Asn

130 135 140

Gin Gly Pro Thr Gin Ser Thr Gly Met Met Asn Ser Pro Val Asn Gin 145 150 155 160

Pro Ala Met Gly Met Asn Thr Gly Thr Asn Ala Gly Met Asn Pro Gly

165 170 175

Met Leu Ala Ala Gly Asn Gly Gin Gly He Met Pro Asn Gin Val Met

180 185 190

Asn Gly Ser He Gly Ala Gly Arg Gly Arg Gin Asp Met Gin Tyr Pro

195 200 205

Asn Pro Gly Met Gly Ser Ala Gly Asn Leu Leu Thr Glu Pro Leu Gin

210 215 220

Gin Gly Ser Pro Gin Met Gly Gly Gin Thr Gly Leu Arg Gly Pro Gin 225 230 235 240

Pro Leu Lys Met Gly Met Met Asn Asn Pro Asn Pro Tyr Gly Ser Pro

245 250 255

Tyr Thr Gin Asn Pro Gly Gin Gin He Gly Ala Ser Gly Leu Gly Leu

260 265 270

Gin He Gin Thr Lys Thr Val Leu Ser Asn Asn Leu Ser Pro Phe Ala

275 280 285

Met Asp Lys Lys Ala Val Pro Gly Gly Gly Met Pro Asn Met Gly Gin

290 295 300

Gin Pro Ala Pro Gin Val Gin Gin Pro Gly Leu Val Thr Pro Val Ala 305 310 315 320

Gin Gly Met Gly Ser Gly Ala His Thr Ala Asp Pro Glu Lys Arg Lys

325 330 335

Leu He Gin Gin Gin Leu Val Leu Leu Leu His Ala His Lys Cys Gin

340 345 350

Arg Arg Glu Gin Ala Asn Gly Glu Val Arg Gin Cys Asn Leu Pro His

355 360 365

Cys Arg Thr Met Lys Asn Val Leu Asn His Met Thr His Cys Gin Ser

370 375 380

Gly Lys Ser Cys Gin Val Ala His Cys Ala Ser Ser Arg Gin He He 385 390 395 400

Ser His Trp Lys Asn Cys Thr Arg His Asp Cys Pro Val Cys Leu Pro

405 410 415

Leu Lys Asn Ala Gly Asp Lys Arg Asn Gin Gin Pro He Leu Thr Gly

420 425 430

Ala Pro Val Gly Leu Gly Asn Pro Ser Ser Leu Gly Val Gly Gin Gin

435 440 445

Ser Ala Pro Asn Leu Ser Thr Val Ser Gin He Asp Pro Ser Ser He

450 455 460

Glu Arg Ala Tyr Ala Ala Leu Gly Leu Pro Tyr Gin Val Asn Gin Met 465 470 475 480

Pro Thr Gin Pro Gin Val Gin Ala Lys Asn Gin Gin Asn Gin Gin Pro

485 490 495

Gly Gin Ser Pro Gin Gly Met Arg Pro Met Ser Asn Met Ser Ala Ser

500 505 510

Pro Met Gly Val Asn Gly Gly Val Gly Val Gin Thr Pro Ser Leu Leu

515 520 525

Ser Asp Ser Met Leu His Ser Ala He Asn Ser Gin Asn Pro Met Met

530 535 540

Ser Glu Asn Ala Ser Val Pro Ser Leu Gly Pro Met Pro Thr Ala Ala 545 550 555 560

Gin Pro Ser Thr Thr Gly He Arg Lys Gin Trp His Glu Asp He Thr

565 570 575

Gin Asp Leu Arg Asn His Leu Val His Lys Leu Val Gin Ala He Phe

580 585 590

Pro Thr Pro Asp Pro Ala Ala Leu Lys Asp Arg Arg Met Glu Asn Leu 595 600 605 Val Ala Tyr Ala Arg Lys Val Glu Gly Asp Met Tyr Glu Ser Ala Asn

610 615 620

Asn Arg Ala Glu Tyr Tyr His Leu Leu Ala Glu Lys He Tyr Lys He 625 630 635 640

Gin Lys Glu Leu Glu Glu Lys Arg Arg Thr Arg Leu Gin Lys Gin Asn

645 650 655

Met Leu Pro Asn Ala Ala Gly Met Val Pro Val Ser Met Asn Pro Gly

660 665 670

Pro Asn Met Gly Gin Pro Gin Pro Gly Met Thr Ser Asn Gly Pro Leu

675 680 685

Pro Asp Pro Ser Met He Arg Gly Ser Val Pro Asn Gin Met Met Pro

690 695 700

Arg He Thr Pro Gin Ser Gly Leu Asn Gin Phe Gly Gin Met Ser Met 705 710 715 720

Ala Gin Pro Pro He Val Pro Arg Gin Thr Pro Pro Leu Gin His His

725 730 735

Gly Gin Leu Ala Gin Pro Gly Ala Leu Asn Pro Pro Met Gly Tyr Gly

740 745 750

Pro Arg Met Gin Gin Pro Ser Asn Gin Gly Gin Phe Leu Pro Gin Thr

755 760 765

Gin Phe Pro Ser Gin Gly Met Asn Val Thr Asn He Pro Leu Ala Pro

770 775 780

Ser Ser Gly Gin Ala Pro Val Ser Gin Ala Gin Met Ser Ser Ser Ser 785 790 795 800

Cys Pro Val Asn Ser Pro He Met Pro Pro Gly Ser Gin Gly Ser His

805 810 815

He His Cys Pro Gin Leu Pro Gin Pro Ala Leu His Gin Asn Ser Pro

820 825 830

Ser Pro Val Pro Ser Arg Thr Pro Thr Pro His His Thr Pro Pro Ser

835 840 845

He Gly Ala Gin Gin Pro Pro Ala Thr Thr He Pro Ala Pro Val Pro

850 855 860

Thr Pro Pro Ala Met Pro Pro Gly Pro Gin Ser Gin Ala Leu His Pro 865 870 875 880

Pro Pro Arg Gin Thr Pro Thr Pro Pro Thr Thr Gin Leu Pro Gin Gin

885 890 895

Val Gin Pro Ser Leu Pro Ala Ala Pro Ser Ala Asp Gin Pro Gin Gin

900 905 910

Gin Pro Arg Ser Gin Gin Ser Thr Ala Ala Ser Val Pro Thr Pro Asn

915 920 925

Ala Pro Leu Leu Pro Pro Gin Pro Ala Thr Pro Leu Ser Gin Pro Ala

930 935 940

Val Ser He Glu Gly Gin Val Ser Asn Pro Pro Ser Thr Ser Ser Thr 945 950 955 960

Glu Val Asn Ser Gin Ala He Ala Glu Lys Gin Pro Ser Gin Glu Val

965 970 975

Lys Met Glu Ala Lys Met Glu Val Asp Gin Pro Glu Pro Ala Asp Thr

980 985 990

Gin Pro Glu Asp He Ser Glu Ser Lys Val Glu Asp Cys Lys Met Glu

995 1000 1005

Ser Thr Glu Thr Glu Glu Arg Ser Thr Glu Leu Lys Thr Glu He Lys

1010 1015 1020

Glu Glu Glu Asp Gin Pro Ser Thr Ser Ala Thr Gin Ser Ser Pro Ala 025 1030 1035 1040

Pro Gly Gin Ser Lys Lys Lys He Phe Lys Pro Glu Glu Leu Arg Gin

1045 1050 1055

Ala Leu Met Pro Thr Leu Glu Ala Leu Tyr Arg Gin Asp Pro Glu Ser

1060 1065 1070

Leu Pro Phe Arg Gin Pro Val Asp Pro Gin Leu Leu Gly He Pro Asp

1075 1080 1085

Tyr Phe Asp He Val Lys Ser Pro Met Asp Leu Ser Thr He Lys Arg 1090 1095 1100 Lys Leu Asp Thr Gly Gin Tyr Gin Glu Pro Trp Gin Tyr Val Asp Asp 105 1110 1115 1120

He Trp Leu Met Phe Asn Asn Ala Trp Leu Tyr Asn Arg Lys Thr Ser

1125 1130 1135

Arg Val Tyr Lys Tyr Cys Ser Lys Leu Ser Glu Val Phe Glu Gin Glu

1140 1145 1150

He Asp Pro Val Met Gin Ser Leu Gly Tyr Cys Cys Gly Arg Lys Leu

1155 1160 1165

Glu Phe Ser Pro Gin Thr Leu Cys Cys Tyr Gly Lys Gin Leu Cys Thr

1170 1175 1180

He Pro Arg Asp Ala Thr Tyr Tyr Ser Tyr Gin Asn Arg Tyr His Phe 185 1190 1195 1200

Cys Glu Lys Cys Phe Asn Glu He Gin Gly Glu Ser Val Ser Leu Gly

1205 1210 1215

Asp Asp Pro Ser Gin Pro Gin Thr Thr He Asn Lys Glu Gin Phe Ser

1220 1225 1230

Lys Arg Lys Asn Asp Thr Leu Asp Pro Glu Leu Phe Val Glu Cys Thr

1235 1240 1245

Glu Cys Gly Arg Lys Met His Gin He Cys Val Leu His His Glu He

1250 1255 1260

He Trp Pro Ala Gly Phe Val Cys Asp Gly Cys Leu Lys Lys Ser Ala 265 1270 1275 1280

Arg Thr Arg Lys Glu Asn Lys Phe Ser Ala Lys Arg Leu Pro Ser Thr

1285 1290 1295

Arg Leu Gly Thr Phe Leu Glu Asn Arg Val Asn Asp Phe Leu Arg Arg

1300 1305 1310

Gin Asn His Pro Glu Ser Gly Glu Val Thr Val Arg Val Val His Ala

1315 1320 1325

Ser Asp Lys Thr Val Glu Val Lys Pro Gly Met Lys Ala Arg Phe Val

1330 1335 1340

Asp Ser Gly Glu Met Ala Glu Ser Phe Pro Tyr Arg Thr Lys Ala Leu 345 1350 1355 1360

Phe Ala Phe Glu Glu He Asp Gly Val Asp Leu Cys Phe Phe Gly Met

1365 1370 1375

His Val Gin Glu Tyr Gly Ser Asp Cys Pro Pro Pro Asn Gin Arg Arg

1380 1385 1390

Val Tyr He Ser Tyr Leu Asp Ser Val His Phe Phe Arg Pro Lys Cys

1395 1400 1405

Leu Arg Thr Ala Val Tyr His Glu He Leu He Gly Tyr Leu Glu Tyr

1410 1415 1420

Val Lys Lys Leu Gly Tyr Thr Thr Gly His He Trp Ala Cys Pro Pro 425 1430 1435 1440

Ser Glu Gly Asp Asp Tyr He Phe His Cys His Pro Pro Asp Gin Lys

1445 1450 1455

He Pro Lys Pro Lys Arg Leu Gin Glu Trp Tyr Lys Lys Met Leu Asp

1460 1465 1470

Lys Ala Val Ser Glu Arg He Val His Asp Tyr Lys Asp He Phe Lys

1475 1480 1485

Gin Ala Thr Glu Asp Arg Leu Thr Ser Ala Lys Glu Leu Pro Tyr Phe

1490 1495 1500

Glu Gly Asp Phe Trp Pro Asn Val Leu Glu Glu Ser He Lys Glu Leu 505 1510 1515 1520

Glu Gin Glu Glu Glu Glu Arg Lys Arg Glu Glu Asn Thr Ser Asn Glu

1525 1530 1535

Ser Thr Asp Val Thr Lys Gly Asp Ser Lys Asn Ala Lys Lys Lys Asn

1540 1545 1550

Asn Lys Lys Thr Ser Lys Asn Lys Ser Ser Leu Ser Arg Gly Asn Lys

1555 1560 1565

Lys Lys Pro Gly Met Pro Asn Val Ser Asn Asp Leu Ser Gin Lys Leu

1570 1575 1580

Tyr Ala Thr Met Glu Lys His Lys Glu Val Phe Phe Val He Arg Leu 585 1590 1595 1600 He Ala Gly Pro Ala Ala Asn Ser Leu Pro Pro He Val Asp Pro Asp

1605 1610 1615

Pro Leu He Pro Cys Asp Leu Met Asp Gly Arg Asp Ala Phe Leu Thr

1620 1625 1630

Leu Ala Arg Asp Lys His Leu Glu Phe ser Ser Leu Arg Arg Ala Gin

1635 1640 1645

Trp Ser Thr Met Cys Met Leu Val Glu Leu His Thr Gin Ser Gin Asp

1650 1655 1660

Arg Phe Val Tyr Thr Cys Asn Glu Cys Lys His His Val Glu Thr Arg 665 1670 1675 1680

Trp His Cys Thr Val Cys Glu Asp Tyr Asp Leu Cys He Thr Cys Tyr

1685 1690 1695

Asn Thr Lys Asn His Asp His Lys Met Glu Lys Leu Gly Leu Gly Leu

1700 1705 1710

Asp Asp Glu Ser Asn Asn Gin Gin Ala Ala Ala Thr Gin Ser Pro Gly

1715 1720 1725

Asp Ser Arg Arg Leu Ser He Gin Arg Cys He Gin Ser Leu Val His

1730 1735 1740

Ala Cys Gin Cys Arg Asn Ala Asn Cys Ser Leu Pro Ser Cys Gin Lys 745 1750 1755 1760

Met Lys Arg Val Val Gin His Thr Lys Gly Cys Lys Arg Lys Thr Asn

1765 1770 1775

Gly Gly Cys Pro He Cys Lys Gin Leu He Ala Leu Cys Cys Tyr His

1780 1785 1790

Ala Lys His Cys Gin Glu Asn Lys Cys Pro Val Pro Phe Cys Leu Asn

1795 1800 1805

He Lys Gin Lys Leu Arg Gin Gin Gin Leu Gin His Arg Leu Gin Gin

1810 1815 1820

Ala Gin Met Leu Arg Arg Arg Met Ala Ser Met Gin Arg Thr Gly Val 825 1830 1835 1840

Val Gly Gin Gin Gin Gly Leu Pro Ser Pro Thr Pro Ala Thr Pro Thr

1845 1850 1855

Thr Pro Thr Gly Gin Gin Pro Thr Thr Pro Gin Thr Pro Gin Pro Thr

1860 1865 1870

Ser Gin Pro Gin Pro Thr Pro Pro Asn Ser Met Pro Pro Tyr Leu Pro

1875 1880 1885

Arg Thr Gin Ala Ala Gly Pro Val Ser Gin Gly Lys Ala Ala Gly Gin

1890 1895 1900

Val Thr Pro Pro Thr Pro Pro Gin Thr Ala Gin Pro Pro Leu Pro Gly 905 1910 1915 1920

Pro Pro Pro Thr Ala Val Glu Met Ala Met Gin He Gin Arg Ala Ala

1925 1930 1935

Glu Thr Gin Arg Gin Met Ala His Val Gin He Phe Gin Arg Pro He

1940 1945 1950

Gin His Gin Met Pro Pro Met Thr Pro Met Ala Pro Met Gly Met Asn

1955 1960 1965

Pro Pro Pro Met Thr Arg Gly Pro Ser Gly His Leu Glu Pro Gly Met

1970 1975 1980

Gly Pro Thr Gly Met Gin Gin Gin Pro Pro Trp Ser Gin Gly Gly Leu 985 1990 1995 2000

Pro Gin Pro Gin Gin Leu Gin Ser Gly Met Pro Arg Pro Ala Met Met

2005 2010 2015

Ser Val Ala Gin His Gly Gin Pro Leu Asn Met Ala Pro Gin Pro Gly

2020 2025 2030

Leu Gly Gin Val Gly He Ser Pro Leu Lys Pro Gly Thr Val Ser Gin

2035 2040 2045

Gin Ala Leu Gin Asn Leu Leu Arg Thr Leu Arg Ser Pro Ser Ser Pro

2050 2055 2060

Leu Gin Gin Gin Gin Val Leu Ser He Leu His Ala Asn Pro Gin Leu 065 2070 2075 2080

Leu Ala Ala Phe He Lys Gin Arg Ala Ala Lys Tyr Ala Asn Ser Asn 2085 2090 2095 Pro Gin Pro He Pro Gly Gin Pro Gly Met Pro Gin Gly Gin Pro Gly

2100 2105 2110

Leu Gin Pro Pro Thr Met Pro Gly Gin Gin Gly Val His Ser Asn Pro

2115 2120 2125

Ala Met Gin Asn Met Asn Pro Met Gin Ala Gly Val Gin Arg Ala Gly

2130 2135 2140

Leu Pro Gin Gin Gin Pro Gin Gin Gin Leu Gin Pro Pro Met Gly Gly 145 2150 2155 2160

Met Ser Pro Gin Ala Gin Gin Met Asn Met Asn His Asn Thr Met Pro

2165 2170 2175

Ser Gin Phe Arg Asp He Leu Arg Arg Gin Gin Met Met Gin Gin Gin

2180 2185 2190

Gin Gin Gin Gly Ala Gly Pro Gly He Gly Pro Gly Met Ala Asn His

2195 2200 2205

Asn Gin Phe Gin Gin Pro Gin Gly Val Gly Tyr Pro Pro Gin Pro Gin

2210 2215 2220

Gin Arg Met Gin His His Met Gin Gin Met Gin Gin Gly Asn Met Gly 225 2230 2235 2240

Gin He Gly Gin Leu Pro Gin Ala Leu Gly Ala Glu Ala Gly Ala Ser

2245 2250 2255

Leu Gin Ala Tyr Gin Gin Arg Leu Leu Gin Gin Gin Met Gly Ser Pro

2260 2265 2270

Val Gin Pro Asn Pro Met Ser Pro Gin Gin His Met Leu Pro Asn Gin

2275 2280 2285

Ala Gin Ser Pro His Leu Gin Gly Gin Gin He Pro Asn Ser Leu Ser

2290 2295 2300

Asn Gin Val Arg Ser Pro Gin Pro Val Pro Ser Pro Arg Pro Gin Ser 305 2310 2315 2320

Gin Pro Pro His Ser Ser Pro Ser Pro Arg Met Gin Pro Gin Pro Ser

2325 2330 2335

Pro His His Val Ser Pro Gin Thr Ser Ser Pro His Pro Gly Leu Val

2340 2345 2350

Ala Ala Gin Ala Asn Pro Met Glu Gin Gly His Phe Ala Ser Pro Asp

2355 2360 2365

Gin Asn Ser Met Leu Ser Gin Leu Ala Ser Asn Pro Gly Met Ala Asn

2370 2375 2380

Leu H s Gly Ala Ser Ala Thr Asp Leu Gly Leu Ser Thr Asp Asn Ser 385 2390 2395 2400

Asp Leu Asn Ser Asn Leu Ser Gin Ser Thr Leu Asp He His 2405 2410 2

(2) INFORMATION FOR SEQ ID NO: 7:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2441 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(li) MOLECULE TYPE: None

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

Met Ala Glu Asn Leu Leu Asp Gly Pro Pro Asn Pro Lys Arg Ala Lys

1 5 10 15

Leu Ser Ser Pro Gly Phe Ser Ala Asn Asp Asn Thr Asp Phe Gly Ser

20 25 30

Leu Phe Asp Leu Glu Asn Asp Leu Pro Asp Glu Leu He Pro Asn Gly

35 40 45

Glu Leu Ser Leu Leu Asn Ser Gly Asn Leu Val Pro Asp Ala Ala Ser

50 55 60

Lys His Lys Gin Leu Ser Glu Leu Leu Arg Gly Gly Ser Gly Ser Ser 65 70 75 80 He Asn Pro Gly He Gly Asn Val Ser Ala Ser Ser Pro Val Gin Gin

85 90 95

Gly Leu Gly Gly Gin Ala Gin Gly Gin Pro Asn Ser Thr Asn Met Ala

100 105 110

Ser Leu Gly Ala Met Gly Lys Ser Pro Leu Asn Gin Gly Asp Ser Ser

115 120 125

Thr Pro Asn Leu Pro Lys Gin Ala Ala Ser Thr Ser Gly Pro Thr Pro

130 135 140

Pro Ala Ser Gin Ala Leu Asn Pro Gin Ala Gin Lys Gin Val Gly Leu 145 150 155 160

Val Thr Ser Ser Pro Ala Thr Ser Gin Thr Gly Pro Gly He Cys Met

165 170 175

Asn Ala Asn Phe Asn Gin Thr His Pro Gly Leu Leu Asn Ser Asn Ser

180 185 190

Gly His Ser Leu Met Asn Gin Ala Gin Gin Gly Gin Ala Gin Val Met

195 200 205

Asn Gly Ser Leu Gly Ala Ala Gly Arg Gly Arg Gly Ala Gly Met Pro

210 215 220

Tyr Pro Ala Pro Ala Met Gin Gly Ala Thr Ser Ser Val Leu Ala Glu 225 230 235 240

Thr Leu Thr Gin Val Ser Pro Gin Met Ala Gly His Ala Gly Leu Asn

245 250 255

Thr Ala Gin Ala Gly Gly Met Thr Lys Met Gly Met Thr Gly Thr Thr

260 265 270

Ser Pro Phe Gly Gin Pro Phe Ser Gin Thr Gly Gly Gin Gin Met Gly

275 280 285

Ala Thr Gly Val Asn Pro Gin Leu Ala Ser Lys Gin Ser Met Val Asn

290 295 300

Ser Leu Pro Ala Phe Pro Thr Asp He Lys Asn Thr Ser Val Thr Thr 305 310 315 320

Val Pro Asn Met Ser Gin Leu Gin Thr Ser Val Gly He Val Pro Thr

325 330 335

Gin Ala He Ala Thr Gly Pro Thr Ala Asp Pro Glu Lys Arg Lys Leu

340 345 350

He Gin Gin Gin Leu Val Leu Leu Leu His Ala His Lys Cys Gin Arg

355 360 365

Arg Glu Gin Ala Asn Gly Glu Val Arg Ala Cys Ser Leu Pro His Cys

370 375 380

Arg Thr Met Lys Asn Val Leu Asn His Met Thr His Cys Gin Ala Pro 385 390 395 400

Lys Ala Cys Gin Val Ala His Cys Ala Ser Ser Arg Gin He He Ser

405 410 415

His Trp Lys Asn Cys Thr Arg His Asp Cys Pro Val Cys Leu Pro Leu

420 425 430

Lys Asn Ala Ser Asp Lys Arg Asn Gin Gin Thr He Leu Gly Ser Pro

435 440 445

Ala Ser Gly He Gin Asn Thr He Gly Ser Val Gly Ala Gly Gin Gin

450 455 460

Asn Ala Thr Ser Leu Ser Asn Pro Asn Pro He Asp Pro Ser Ser Met 465 470 475 480

Gin Arg Ala Tyr Ala Ala Leu Gly Leu Pro Tyr Met Asn Gin Pro Gin

485 490 495

Thr Gin Leu G n Pro Gin Val Pro Gly Gin Gin Pro Ala Gin Pro Pro

500 505 510

Ala His Gin Gin Met Arg Thr Leu Asn Ala Leu Gly Asn Asn Pro Met

515 520 525

Ser Val Pro Ala Gly Gly He Thr Thr Asp Gin Gin Pro Pro Asn Leu

530 535 540

He Ser Glu Ser Ala Leu Pro Thr Ser Leu Gly Ala Thr Asn Pro Leu 545 550 555 560

Met Asn Asp Gly Ser Asn Ser Gly Asn He Gly Ser Leu Ser Thr He 565 570 575 Pro Thr Ala Ala Pro Pro Ser Ser Thr Gly Val Arg Lys Gly Trp His

580 585 590

Glu His Val Thr Gin Asp Leu Arg Ser His Leu Val His Lys Leu Val

595 600 605

Gin Ala He Phe Pro Thr Pro Asp Pro Ala Ala Leu Lys Asp Arg Arg

610 615 620

Met Glu Asn Leu Val Ala Tyr Ala Lys Lys Val Glu Gly Asp Met Tyr 625 630 635 640

Glu Ser Ala Asn Ser Arg Asp Glu Tyr Tyr His Leu Leu Ala Glu Lys

645 650 655

He Tyr Lys He Gin Lys Glu Leu Glu Glu Lys Arg Arg Thr Arg Leu

660 665 670

His Lys Gin Gly He Leu Gly Asn Gin Pro Ala Leu Pro Ala Ser Gly

675 680 685

Ala Gin Pro Pro Val He Pro Pro Ala Gin Ser Val Arg Pro Pro Asn

690 695 700

Gly Pro Leu Pro Leu Pro Val Asn Arg Met Gin Val Ser Gin Gly Met 705 710 715 720

Asn Ser Phe Asn Pro Met Ser Leu Gly Asn Val Gin Leu Pro Gin Ala

725 730 735

Pro Met Gly Pro Arg Ala Ala Ser Pro Met Asn H s Ser Val Gin Met

740 745 750

Asn Ser Met Ala Ser Val Pro Gly Met Ala He Ser Pro Ser Arg Met

755 760 765

Pro Gin Pro Pro Asn Met Met Gly Thr His Ala Asn Asn He Met Ala

770 775 780

Gin Ala Pro Thr Gin Asn Gin Phe Leu Pro Gin Asn Gin Phe Pro Ser 785 790 795 800

Ser Ser Gly Ala Met Ser Val Asn Ser Val Gly Met Gly Gin Pro Ala

805 810 815

Ala Gin Ala Gly Val Ser Gin Gly Gin Glu Pro Gly Ala Ala Leu Pro

820 825 830

Asn Pro Leu Asn Met Leu Ala Pro Gin Ala Ser Gin Leu Pro Cys Pro

835 840 845

Pro Val Thr Gin Ser Pro Leu His Pro Thr Pro Pro Pro Ala Ser Thr

850 855 860

Ala Ala Gly Met Pro Ser Leu Gin His Pro Thr Ala Pro Gly Met Thr 865 870 875 880

Pro Pro Gin Pro Ala Ala Pro Thr Gin Pro Ser Thr Pro Val Ser Ser

885 890 895

Gly Gin Thr Pro Thr Pro Thr Pro Gly Ser Val Pro Ser Ala Ala Gin

900 905 910

Thr Gin Ser Thr Pro Thr Val Gin Ala Ala Ala Gin Ala Gin Val Thr

915 920 925

Pro Gin Pro Gin Thr Pro Val Gin Pro Pro Ser Val Ala Thr Pro Gin

930 935 940

Ser Ser Gin Gin Gin Pro Thr Pro Val His Thr Gin Pro Pro Gly Thr 945 950 955 960

Pro Leu Ser Gin Ala Ala Ala Ser He Asp Asn Arg Val Pro Thr Pro

965 970 975

Ser Thr Val Thr Ser Ala Glu Thr Ser Ser Gin Gin Pro Gly Pro Asp

980 985 990

Val Pro Met Leu Glu Met Lys Thr Glu Val Gin Thr Asp Asp Ala Glu

995 1000 1005

Pro Glu Pro Thr Glu Ser Lys Gly Glu Pro Arg Ser Glu Met Met Glu

1010 1015 1020

Glu Asp Leu Gin Gly Ser Ser Gin Val Lys Glu Glu Thr Asp Thr Thr 025 1030 1035 1040

Glu Gin Lys Ser Glu Pro Met Glu Val Glu Glu Lys Lys Pro Glu Val

1045 1050 1055

Lys Val Glu Ala Lys Glu Glu Glu Glu Asn Ser Ser Asn Asp Thr Ala 1060 1065 1070 Ser Gin Ser Thr Ser Pro Ser Gin Pro Arg Lys Lys He Phe Lys Pro

1075 1080 1085

Glu Glu Leu Arg Gin Ala Leu Met Pro Thr Leu Glu Ala Leu Tyr Arg

1090 1095 1100

Gin Asp Pro Glu Ser Leu Pro Phe Arg Gin Pro Val Asp Pro Gin Leu 105 1110 1115 1120

Leu Gly He Pro Asp Tyr Phe Asp He Val Lys Asn Pro Met Asp Leu

1125 1130 1135

Ser Thr He Lys Arg Lys Leu Asp Thr Gly Gin Tyr Gin Glu Pro Trp

1140 1145 1150

Gin Tyr Val Asp Asp Val Arg Leu Met Phe Asn Asn Ala Trp Leu Tyr

1155 1160 1165

Asn Arg Lys Thr Ser Arg Val Tyr Lys Phe Cys Ser Lys Leu Ala Glu

1170 1175 1180

Val Phe Glu Gin Glu He Asp Pro Val Met Gin Ser Leu Gly Tyr cys 185 1190 1195 1200

Cys Gly Arg Lys Tyr Glu Phe Ser Pro Gin Thr Leu Cys Cys Tyr Gly

1205 1210 1215

Lys Gin Leu Cys Thr He Pro Arg Asp Ala Ala Tyr Tyr Ser Tyr Gin

1220 1225 1230

Asn Arg Tyr His Phe Cys Gly Lys Cys Phe Thr Glu He Gin Gly Glu

1235 1240 1245

Asn Val Thr Leu Gly Asp Asp Pro Ser Gin Pro Gin Thr Thr He Ser

1250 1255 1260

Lys Asp Gin Phe Glu Lys Lys Lys Asn Asp Thr Leu Asp Pro Glu Pro 265 1270 1275 1280

Phe Val Asp Cys Lys Glu Cys Gly Arg Lys Met His Gin He Cys Val

1285 1290 1295

Leu His Tyr Asp He He Trp Pro Ser Gly Phe Val Cys Asp Asn Cys

1300 1305 1310

Leu Lys Lys Thr Gly Arg Pro Arg Lys Glu Asn Lys Phe Ser Ala Lys

1315 1320 1325

Arg Leu Gin Thr Thr Arg Leu Gly Asn His Leu Glu Asp Arg Val Asn

1330 1335 1340

Lys Phe Leu Arg Arg Gin Asn His Pro Glu Ala Gly Glu Val Phe Val 345 1350 1355 1360

Arg Val Val Ala Ser Ser Asp Lys Thr Val Glu Val Lys Pro Gly Met

1365 1370 1375

Lys Ser Arg Phe Val Asp Ser Gly Glu Met Ser Glu Ser Phe Pro Tyr

1380 1385 1390

Arg Thr Lys Ala Leu Phe Ala Phe Glu Glu He Asp Gly Val Asp Val

1395 1400 1405

Cys Phe Phe Gly Met His Val Gin Asp Thr Ala Leu He Ala Pro His

1410 1415 1420

Gin He Gin Gly Cys Val Tyr He Ser Tyr Leu Asp Ser He His Phe 425 1430 1435 1440

Phe Arg Pro Arg Cys Leu Arg Thr Ala Val Tyr His Glu He Leu He

1445 1450 1455

Gly Tyr Leu Glu Tyr Val Lys Lys Leu Val Tyr Val Thr Ala His He

1460 1465 1470

Trp Ala Cys Pro Pro Ser Glu Gly Asp Asp Tyr He Phe His Cys His

1475 1480 1485

Pro Pro Asp Gin Lys He Pro Lys Pro Lys Arg Leu Gin Glu Trp Tyr

1490 1495 1500

Lys Lys Met Leu Asp Lys Ala Phe Ala Glu Arg He He Asn Asp Tyr 505 1510 1515 1520

Lys Asp He Phe Lys Gin Ala Asn Glu Asp Arg Leu Thr Ser Ala Lys

1525 1530 1535

Glu Leu Pro Tyr Phe Glu Gly Asp Phe Trp Pro Asn Val Leu Glu Glu

1540 1545 1550

Ser He Lys Glu Leu Glu Gin Glu Glu Glu Glu Arg Lys Lys Glu Glu 1555 1560 1565 Ser Thr Ala Ala Ser Glu Thr Pro Glu Gly Ser Gin Gly Asp Ser Lys

1570 1575 1580

Asn Ala Lys Lys Lys Asn Asn Lys Lys Thr Asn Lys Asn Lys Ser Ser 585 1590 1595 1600

He Ser Arg Ala Asn Lys Lys Lys Pro Ser Met Pro Asn Val Ser Asn

1605 1610 1615

Asp Leu Ser Gin Lys Leu Tyr Ala Thr Met Glu Lys His Lys Glu Val

1620 1625 1630

Phe Phe Val He His Leu His Ala Gly Pro Val He Ser Thr Gin Pro

1635 1640 1645

Pro He Val Asp Pro Asp Pro Leu Leu Ser Cys Asp Leu Met Asp Gly

1650 1655 1660

Arg Asp Ala Phe Leu Thr Leu Ala Arg Asp Lys His Trp Glu Phe Ser 665 1670 1675 1680

Ser Leu Arg Arg Ser Lys Trp Ser Thr Leu Cys Met Leu Val Glu Leu

1685 1690 1695

His Thr Gin Gly Gin Asp Arg Phe Val Tyr Thr Cys Asn Glu Cys Lys

1700 1705 1710

His His Val Glu Thr Arg Trp His Cys Thr Val Cys Glu Asp Tyr Asp

1715 1720 1725

Leu Cys He Asn Cys Tyr Asn Thr Lys Ser His Thr His Lys Met Val

1730 1735 1740

Lys Trp Gly Leu Gly Leu Asp Asp Glu Gly Ser Ser Gin Gly Glu Pro 745 1750 1755 1760

Gin Ser Lys Ser Pro Gin Glu Ser Arg Arg Leu Ser He Gin Arg Cys

1765 1770 1775

He Gin Ser Leu Val His Ala Cys Gin Cys Arg Asn Ala Asn Cys Ser

1780 1785 1790

Leu Pro Ser Cys Gin Lys Met Lys Arg Val Val Gin His Thr Lys Gly

1795 1800 1805

Cys Lys Arg Lys Thr Asn Gly Gly Cys Pro Val Cys Lys Gin Leu He

1810 1815 1820

Ala Leu Cys Cys Tyr His Ala Lys His Cys Gin Glu Asn Lys Cys Pro 825 1830 1835 1840

Val Pro Phe Cys Leu Asn He Lys His Asn Val Arg Gin Gin Gin He

1845 1850 1855

Gin His Cys Leu Gin Gin Ala Gin Leu Met Arg Arg Arg Met Ala Thr

1860 1865 1870

Met Asn Thr Arg Asn Val Pro Gin Gin Ser Leu Pro Ser Pro Thr Ser

1875 1880 1885

Ala Pro Pro Gly Thr Pro Thr Gin Gin Pro Ser Thr Pro Gin Thr Pro

1890 1895 1900

Gin Pro Pro Ala Gin Pro Gin Pro Ser Pro Val Asn Met Ser Pro Ala 905 1910 1915 1920

Gly Phe Pro Asn Val Ala Arg Thr Gin Pro Pro Thr He Val Ser Ala

1925 1930 1935

Gly Lys Pro Thr Asn Gin Val Pro Ala Pro Pro Pro Pro Ala Gin Pro

1940 1945 1950

Pro Pro Ala Ala Val Glu Ala Ala Arg Gin He Glu Arg Glu Ala Gin

1955 1960 1965

Gin Gin Gin His Leu Tyr Arg Ala Asn He Asn Asn Gly Met Pro Pro

1970 1975 1980

Gly Arg Asp Gly Met Gly Thr Pro Gly Ser Gin Met Thr Pro Val Gly 985 1990 1995 2000

Leu Asn Val Pro Arg Pro Asn Gin Val Ser Gly Pro Val Met Ser Ser

2005 2010 2015

Met Pro Pro Gly Gin Trp Gin Gin Ala Pro He Pro Gin Gin Gin Pro

2020 2025 2030

Met Pro Gly Met Pro Arg Pro Val Met Ser Met Gin Ala Gin Ala Ala

2035 2040 2045

Val Ala Gly Pro Arg Met Pro Asn Val Gin Pro Asn Arg Ser He Ser 2050 2055 2060 Pro Ser Ala Leu Gin Asp Leu Leu Arg Thr Leu Lys Ser Pro Ser Ser 065 2070 2075 2080

Pro Gin Gin Gin Gin Gin Val Leu Asn He Leu Lys Ser Asn Pro Gin

2085 2090 2095

Leu Met Ala Ala Phe He Lys Gin Arg Thr Ala Lys Tyr Val Ala Asn

2100 2105 2110

Gin Pro Gly Met Gin Pro Gin Pro Gly Leu Gin Ser Gin Pro Gly Met

2115 2120 2125

Gin Pro Gin Pro Gly Met His Gin Gin Pro Ser Leu Gin Asn Leu Asn

2130 2135 2140

Ala Met Gin Ala Gly Val Pro Arg Pro Gly Val Pro Pro Pro Gin Pro 145 2150 2155 2160

Ala Met Gly Gly Leu Asn Pro Gin Gly Gin Ala Leu Asn He Met Asn

2165 2170 2175

Pro Gly His Asn Pro Asn Met Thr Asn Met Asn Pro Gin Tyr Arg Glu

2180 2185 2190

Met Val Arg Arg Gin Leu Leu Gin His Gin Gin Gin Gin Gin Gin Gin

2195 2200 2205

Gin Gin Gin Gin Gin Gin Gin Gin Asn Ser Ala Ser Leu Ala Gly Gly

2210 2215 2220

Met Ala Gly His Ser Gin Phe Gin Gin Pro Gin Gly Pro Gly Gly Tyr 225 2230 2235 2240

Ala Pro Ala Met Gin Gin Gin Arg Met Gin Gin His Leu Pro He Gin

2245 2250 2255

Gly Ser Ser Met Gly Gin Met Ala Ala Pro Met Gly Gin Leu Gly Gin

2260 2265 2270

Met Gly Gin Pro Gly Leu Gly Ala Asp Ser Thr Pro Asn He Gin Gin

2275 2280 2285

Ala Leu Gin Gin Arg He Leu Gin Gin Gin Gin Met Lys Gin Gin He

2290 2295 2300

Gly Ser Pro Gly Gin Pro Asn Pro Met Ser Pro Gin Gin His Met Leu 305 2310 2315 2320

Ser Gly Gin Pro Gin Ala Ser His Leu Pro Gly Gin Gin He Ala Thr

2325 2330 2335

Ser Leu Ser Asn Gin Val Arg Ser Pro Ala Pro Val Gin Ser Pro Arg

2340 2345 2350

Pro Gin Ser Gin Pro Pro His Ser Ser Pro Ser Pro Arg He Gin Pro

2355 2360 2365

Gin Pro Ser Pro His His Val Ser Pro Gin Thr Gly Thr Pro His Pro

2370 2375 2380

Gly Leu Ala Val Thr Met Ala Ser Ser Met Asp Gin Gly His Leu Gly 385 2390 2395 2400

Asn Pro Glu Gin Ser Ala Met Leu Pro Gin Leu Asn Thr Pro Asn Arg

2405 2410 2415

Ser Ala Leu Ser Ser Glu Leu Ser Leu Val Gly Asp Thr Thr Gly Asp

2420 2425 2430

Thr Leu Glu Lys Phe Val Glu Gly Leu 2435 2440

(2) INFORMATION FOR SEQ ID NO: 8:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 813 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(li) MOLECULE TYPE: None

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

Met Ala Glu Ala Gly Gly Ala Gly Ser Pro Ala Leu Pro Pro Ala Pro 1 5 10 15 Pro His Gly Ser Pro Arg Thr Leu Ala Thr Ala Ala Gly Ser Ser Ala

20 25 30

Ser Cys Gly Pro Ala Thr Pro Val Ala Ala Ala Gly Thr Ala Glu Gly

35 40 45

Pro Gly Gly Gly Gly Ser Ala Arg He Ala Val Lys Lys Ala Gin Leu

50 55 60

Arg Ser Ala Pro Arg Ala Lys Lys Leu Glu Lys Leu Gly Val Tyr Ser 65 70 75 80

Ala Cys Lys Ala Glu Glu Ser Cys Lys Cys Asn Gly Trp Lys Asn Pro

85 90 95

Asn Pro Ser Pro Thr Pro Pro Arg Gly Asp Leu Gin Gin He He Val

100 105 110

Ser Leu Thr Glu Ser Cys Arg Ser Cys Ser His Ala Leu Ala Ala His

115 120 125

Val Ser His Leu Glu Asn Val Ser Glu Glu Glu Met Asp Arg Leu Leu

130 135 140

Gly He Val Leu Asp Val Glu Tyr Leu Phe Thr Cys Val His Lys Glu 145 150 155 160

Glu Asp Ala Asp Thr Lys Gin Val Tyr Phe Tyr Leu Phe Lys Leu Leu

165 170 175

Arg Lys Ser He Leu Gin Arg Gly Lys Pro Val Val Glu Gly Ser Leu

180 185 190

Glu Lys Lys Pro Pro Phe Glu Lys Pro Ser He Glu Gin Gly Val Asn

195 200 205

Asn Phe Val Gin Tyr Lys Phe Ser His Leu Pro Ser Lys Glu Arg Gin

210 215 220

Thr Thr He Glu Leu Ala Lys Met Phe Leu Asn Arg He Asn Tyr Trp 225 230 235 240

H s Leu Glu Ala Pro Ser Gin Arg Arg Leu Arg Ser Pro Asn Asp Asp

245 250 255

He Ser Gly Tyr Lys Glu Asn Tyr Thr Arg Trp Leu Cys Tyr Cys Asn

260 265 270

Val Pro Gin Phe Cys Asp Ser Leu Pro Arg Tyr Glu Thr Thr Lys Val

275 280 285

Phe Gly Arg Thr Leu Leu Arg Ser Val Phe Thr He Met Arg Arg Gin

290 295 300

Leu Leu Glu Gin Ala Arg Gin Lys Lys Asp Lys Leu Pro Leu Glu Lys 305 310 315 320

Arg Thr Leu He Leu Thr His Phe Pro Lys Phe Leu Ser Met Leu Glu

325 330 335

Glu Glu Val Tyr Ser Gin Asn Ser Pro He Trp Asp Gin Asp Phe Leu

340 345 350

Ser Ala Ser Ser Arg Thr Ser Pro Leu Gly He Gin Thr Val He Ser

355 360 365

Pro Pro Val Thr Gly Thr Ala Leu Phe Ser Ser Asn Ser Thr Ser His

370 375 380

Glu Gin He Asn Gly Gly Arg Thr Ser Pro Gly Cys Arg Gly Ser Ser 385 390 395 400

Gly Leu Glu Ala Asn Pro Gly Glu Lys Arg Lys Met Asn Asn Ser His

405 410 415

Ala Pro Glu Glu Ala Lys Arg Ser Arg Val Met Gly Asp He Pro Val

420 425 430

Glu Leu He Asn Glu Val Met Ser Thr He Thr Asp Pro Ala Gly Met

435 440 445

Leu Gly Pro Glu Thr Asn Phe Leu Ser Ala His Ser Ala Arg Asp Glu

450 455 460

Ala Ala Arg Leu Glu Glu Arg Arg Gly Val He Glu Phe His Val Val 465 470 475 480

Gly Asn Ser Leu Asn Gin Lys Pro Asn Lys Lys He Leu Met Trp Leu

485 490 495

Val Gly Leu Gin Asn Val Phe Ser His Gin Leu Pro Arg Met Pro Lys 500 505 510 Glu Tyr He Thr Arg Leu Val Phe Asp Pro Lys His Lys Thr Leu Ala

515 520 525

Leu He Lys Asp Gly Arg Val He Gly Gly He Cys Phe Arg Met Phe

530 535 540

Pro Ser Gin Gly Phe Thr Glu He Val Phe Cys Ala Val Thr Ser Asn 545 550 555 560

Glu Gin Val Lys Gly Tyr Gly Thr His Leu Met Asn H s Leu Lys Glu

565 570 575

Tyr His He Lys His Glu He Leu Asn Phe Leu Thr Tyr Ala Asp Glu

580 585 590

Tyr Ala He Gly Tyr Phe Lys Lys Gin Gly Phe Ser Lys Glu He Lys

595 600 605

He Pro Lys Thr Lys Tyr Val Gly Tyr He Lys Asp Tyr Glu Gly Ala

610 615 620

Thr Leu Met Gly Cys Glu Leu Asn Pro Gin He Pro Tyr Thr Glu Phe 625 630 635 640

Ser Val He He Lys Lys Gin Lys Glu He He Lys Lys Leu He Glu

645 650 655

Arg Lys Gin Ala Gin He Arg Lys Val Tyr Pro Gly Leu Ser Cys Phe

660 665 670

Lys Asp Gly Val Arg Gin He Pro He Glu Ser He Pro Gly He Arg

675 680 685

Glu Thr Gly Trp Lys Pro Ser Gly Lys Glu Lys Ser Lys Glu Pro Lys

690 695 700

Asp Pro Glu His Val Tyr Ser Thr Leu Lys Asn He Leu Gin Gin Val 705 710 715 720

Lys Asn His Pro Asn Ala Trp Pro Phe Met Glu Pro Val Lys Arg Thr

725 730 735

Glu Ala Pro Gly Tyr Tyr Glu Val He Arg Phe Pro Met Asp Leu Lys

740 745 750

Thr Met Ser Glu Arg Leu Arg Asn Arg Tyr Tyr Val Ser Lys Lys Leu

755 760 765

Phe Met Ala Asp Leu Gin Arg Val Phe Thr Asn Cys Lys Glu Tyr Asn

770 775 780

Pro Pro Glu Ser Glu Tyr Tyr Lys Cys Ala Ser He Leu Glu Lys Phe 785 790 795 800

Phe Phe Ser Lys He Lys Glu Ala Gly Leu He Asp Lys 805 810

(2) INFORMATION FOR SEQ ID NO: 9:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 50 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

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

His Thr Lys Gly Cys Lys Arg Lys Thr Asn Gly Gly Cys Pro Val Cys

1 5 10 15

Lys Gin Leu He Ala Leu Cys Cys Tyr His Ala Lys H s Cys Gin Glu

20 25 30

Asn Lys Cys Pro Val Pro Phe Cys Leu Asn He Lys His Asn Val Arg

35 40 45

Gin Gin 50

(2) INFORMATION FOR SEQ ID NO: 10:

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2204 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

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

ACCCACTCCC CCCAGAGCCG ACCTGCAGCA AATAATTGTC AGTCTAACAG AATCCTGTCG 60

GAGTTGTAGC CATGCCCTAG CTGCTCATGT TTCCCACCTG GAGAATGTGT CAGAGGAAGA 120

AATGAACAGA CTCCTGGGAA TAGTATTGGA TGTGGAATAT CTCTTTACCT GTGTCCACAA 180

GGAAGAAGAT GCAGATACCA AACAAGTTTA TTTCTATCTA TTTAAGCTCT TGAGAAAGTC 240

TATTTTACAA AGAGGAAAAC CTGTGGTTGG AAGGCTCTTT GGAAAAGAAA CCCCCATTTG 300

AAAAACCTAG CATTGAACAG GGTGTGAATA ACTTTGTGCA GTACAAATTT AGTCACCTGC 360

CAGCAAAAAG AAAGGCAAAC CAATAGTTGA GTTGGCAAAA ATGTTCCTAA ACCGCATCAC 420

CTATTGGCAT CTGGAGGCAC CATCTCAACG AGACTGCGAT CTCCAATGAT GATATTCTGG 480

ATACAAAGAG AACTACACAA GGTGGCTGTG TTACTGCAAC GTGCCACAGT TCTGCGACAG 540

TCTACCTCGG TACGAAACCA CACAGGTGTT TGGGAGAACA TCGTTCGCTC GGTCTTCACT 600

GTTATGAGGC GACAACTCCT GGAACAAGCA AGACAGGAAA AAGATAAACT GCCTCTTGAA 660

AAACGAACTC TAATCCTCAC TCATTTCCCA AAATTTCTGT CCATGCTAGA AGAAGAAGTA 720

TATAGTCAAA ACTCTCCCAT CTGGGATCAC CATTTTCTCT CAGCCTCTTC CAGAACCAGC 780

CAGCTAGGCA TCCAAACAGT TATCAATCAC CTCCTGTGGC TGGGACAATT TCATACAATT 840

CAACCTCATC TTCCCTTGAG CAGCCAAACG CAGGGAGCAG CAGTCCTGCC TGCAAAGCCT 900

CTTCTGGACT TGAGGCAAAC CCAGGAGAAA AGAGGAAAAT GACTGATTCT CATGTTCTGG 960

AGGAGGCCAA GAAACCCCGA GTTATGGGGG ATATTCCGAT GGAATTAATC AACGAGGTTA 1020

TGTCTACCAT CACGGACCCT GCAGCAATGC TTGGACCAGA GACCAATTTT CTGTCAGCAC 1080

ACTCGGCCAG GGATGAGGCG GCAAGGTTGG AAGAGCGCAG GGGTGTAATT GAATTTCACG 1140

TGGTTGGCAA TTCCCTCAAC CAGAAACCAA ACAAGAAGAT CCTGATGTGG CTGGTTGGCC 1200

TACAGAACGT TTTCTCCCAC CAGCTGCCCC GAATGCCAAA AGAATACATC ACACGGCTCG 1260

TCTTTGACCC GAAACACAAA ACCCTTGCTT TAATTAAAGA TGGCCGTGTT ATTGGTGGTA 1320

TCTGTTTCCG TATGTTCCCA TCTCAAGGAT TCACAGAGAT TGTCTTCTGT GCTGTAACCT 1380

CAAATGAGCA AGTCAAGGGC TATGGAACAC ACCTGATGAA TCATTTGAAA GAATATCACA 1440

TAAAGCATGA CATCCTGAAC TTCCTCACAT ATGCAGATGA ATATGCAATT GGATACTTTA 1500

AGAAACAGGG TTTCTCCAAA GAAATTAAAA TACCTAAAAC CAAATATGTT GGCTATATCA 1560

AGGATTATGA AGGAGCCACT TTAATGGGAT GTGAGCTAAA TCCACGGATC CCGTACACAG 1620

AATTTTCTGT CATCATTAAA AAGCAGAAGG AGATAATTAA AAAACTGATT GAAAGAAAAC 1680

AGGCACAAAT TCGAAAAGTT TACCCTGGAC TTTCATGTTT TAAAGATGGA GTTCGACAGA 1740

TTCCTATAGA AAGCATTCCT GGAATTAGAG AGACAGGCTG GAAACCGAGT GGAAAAGAGA 1800

AAAGTAAAGA GCCCAGAGAC CCTGACCAGC TTTACAGCAC GCTCAAGAGC ATCCTCCAGC 1860

AGGTGAAGAG CCATCAAAGC GCTTGGCCCT TCATGGAACC TGTGAAGAGA ACAGAAGCTC 1920

CAGGATATTA TGAAGTTATA AGGTCCCCCA TGGATCTCAA AACCATGAGT GAACGCCTCA 1980

AGAATAGGTA CTACGTGTCT AAGAAATTAT TCATGGCAGA CTTACAGCGA GTCTTTACCA 2040

ATTGCAAAGA GTACAACGCC CCTGAGAGTG AATACTACAA ATGTGCCAAT ATCCTGGAGA 2100

AATTCTTCTT CAGTAAAATT AAGGAAGCTG GATTAATTGA CAAGTGATTT TTTTTCCCCC 2160

TCTGCTTCTT AGAAACTCAC CAAGCAGTGT GCCTAAAGCA AGGT 2204

(2) INFORMATION FOR SEQ ID NO: 11:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2093 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

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

GAATTCCGGC GAAACCACTC ATGTCTTTGG GCGAAGCCTT CTCCGGTCCA TTTTCACCGT 60

TACCCGCCGG CAGCTGCTGG AAAAGTTCCG AGTGGAGAAG GACAAATTGG TGCCCGAGAA 120

GAGGACCCTC ATCCTCACTC ACTTCCCCAA GTAAGGCTCC TTCTGGCCTA CCAGGATTTG 180

GCCCCAAGTT CACATCCTCC CTGTTGTCCC CTTTTTTCCA GGAAGGCTTC CTGGATTGGT 240

CCCTCCTCTC CCTCCATGGG CCTTTTGGGA TCTGGGCGTC TACCTGGCAG ACTTGCCCAT 300

GGCCCAGAAG CAACTTGCTA GTACTAGTCT GGGGATGGCA GATTCCTGTC CATGCTGGAG 360

GAGGAGATCT ATGGGGCAAA CTCTCCAATC TGGGAGTCAG GCTTCACCAT GCCACCCTCA 420 GAGGGGACAC AGCTGGTTCC CCGGCCAGCT TCAGTCAGTG CAGCGGTTGT TCCCAGCACC 480

CCCATCTTCA GCCCCAGCAT GGGTGGGGGC AGCAACAGCT CCCTGAGTCT GGATTCTGCA 540

GGGGCCGAGC CTATGCCAGG CGAGAAGAGG ACGCTCCCAG AGAACCTGAC CCTGGAGGAT 600

GCCAAGCGGC TCCGTGTGAT GGGTGACATC CCCATGGAGC TGGTCAATGA GGTCATGCTG 660

ACCATCACTG ACCCTGCTGC CATGCTGGGG CCTGAGACGA GCCTGCTTTC GGCCAATGCG 720

GCCCGGGATG AGACAGCCCG CCTGGAGGAG CGCCGCGGCA TCATCGAGTT CCATGTCATC 780

GGCAACTCAC TGACGCCCAA GGCCAACCGG CGGGTGTTGC TGTGGCTCGT GGGGCTGCAG 840

AATGTCTTTT CCCACCAGCT GCCGCGCATG CCTAAGGAGT ATATCGCCCG CCTCGTCTTT 900

GACCCGAAGC ACAAGACTCT GGCCTTGATC AAGGATGGGC GGGTCATCGG TGGCATCTGC 960

TTCCGCATGT TTCCCACCCA GGGCTTCACG GAGATTGTCT TCTGTGCTGT CACCTCGAAT 1020

GAGCAGGTCA AGGGTTATGG GACCCACCTG ATGAACCACC TGAAGGAGTA TCACATCAAG 1080

CACAACATTC TCTACTTCCT CACCTACGCC GACGAGTACG CCATCGGCTA CTTCAAAAAG 1140

CAGGGTTTCT CCAAGGACAT CAAGGTGCCC AAGAGCCGCT ACCTGGGCTA CATCAAGGAC 1200

TACGAGGGAG CGACGCTGAT GGAGTGTGAG CTGAATCCCC GCATCCCCTA CACGGAGCTG 1260

TCCCACATCA TCAAGAAGCA GAAAGAGATC ATCAAGAAGC TGATTGAGCG CAAACAGGCC 1320

CAGATCCGCA AGGTCTACCC GGGGCTCAGC TGCTTCAAGG AGGGCGTGAG GCAGATCCCT 1380

GTGGAGAGCG TTCCTGGCAT TCGAGAGACA GGCTGGAAGC CATTGGGGAA GGAGAAGGGG 1440

AAGGAGCTGA AGGACCCCGA CCAGCTCTAC ACAACCCTCA AAAACCTGCT GGCCCAAATC 1500

AAGTCTCACC CCAGTGCCTG GCCCTTCATG GAGCCTGTGA AGAAGTCGGA GGCCCCTGAC 1560

TACTACGAGG TCATCCGCTT CCCCATTGAC CTGAAGACCA TGACTGAGCG GCTGCGAAGC 1620

CGCTACTACG TGACCCGGAA GCTCTTTGTG GCCGACCTGC AGCGGGTCAT CGCCAACTGT 1680

CGCGAGTACA ACCCCCCGGA CAGCGAGTAC TGCCGCTGTG CCAGCGCCCT GGAGAAGTTC 1740

TTCTACTTCA AGCTCAAGGA GGGAGGCCTC ATTGACAAGT AGGCCCATCT TTGGGCCGCA 1800

GCCCTGACCT GGAATGTCTC CACCTCGGAT TCTGATCTGA TCCTTAGGGG GTGCCCTGGC 1860

CCCACGGACC CGACTCAGCT TGAGACACTC CAGCCAAGGG TCCTCCGGAC CCGATCCTGC 1920

AGCTCTTTCT GGACCTTCAG GCACCCCCAA GCGTGCAGCT CTGTCCCAGC CTTCACTGTG 1980

TGTGAGAGGT CTCCTGGGTT GGGGCCCAGC CCCTCTAGAG TAGCTGGTGG CCAGGGATGA 2040

ACCTTGCCCA GCCGTGGTGG CCCCCAGGCC TGGTCCCCAA GAGCCCGGAA TTC 2093

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9046 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

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

CCTTGTTTGT GTGCTAGGCT GGGGGGGAGA GAGGGCGAGA GAGAGCGGGC GAGAGTGGGC 60

AAGCAGGACG CCGGGCTGAG TGCTAACTGC GGGACGCAGA GAGTGCGGAG GGGAGTCGGG 120

TCGGAGAGAG GCGGCAGGGG CCAGAACAGT GGCAGGGGGC CCGGGGCGCA CGGGCTGAGG 180

CGACCCCCAG CCCCCTCCCG TCCGCACACA CCCCCACCGC GGTCCAGCAG CCGGGCCGGC 240

GTCGACGCTA GGGGGGACCA TTACATAACC CGCGCCCCGG CCGTCTTCTC CCGCCGCCGC 300

GGCGCCCGAA CTGAGCCCGG GGCGGGCGCT CCAGCACTGG CCGCCGGCGT GGGGCGTAGC 360

AGCGGCCGTA TTATTATTTC GCGGAAAGGA AGGCGAAGGA GGGGAGCGCC GGCGCGAGGA 420

GGGGCCGCCT GCGCCCGCCG CCGGAGCGGG GCCTCCTCGG TGGGCTCCGC GTCGGCGCGG 480

GCGTGCGGGC GGCGCTGCTC GGCCCGGCCC CCTCGGCCCT CTGGTCCGGC CAGCTCCGCT 540

CCCGGCGTCC TTGCCGCGCC TCCGCCGGCC GCCGCGCGAT GTGAGGCGGC GGCGCCAGCC 600

TGGCTCTCGG CTCGGGCGAG TTCTCTGCGG CCATTAGGGG CCGGTGCGGC GGCGGCGCGG 660

AGCGCGGCGG CAGGAGGAGG GTTCGGAGGG TGGGGGCGCA GGCCCGGGAG GGGGCACCGG 720

GAGGAGGTGA GTGTCTCTTG TCGCCTCCTC CTCTCCCCCC TTTTCGCCCC CGCCTCCTTG 780

TGGCGATGAG AAGGAGGAGG ACAGCGCCGA GGAGGAAGAG GTTGATGGCG GCGGCGGAGC 840

TCCGAGAGAC CTCGGCTGGG CAGGGGCCGG CCGTGGCGGG CCGGGGACTG CGCCTCTAGA 900

GCCGCGAGTT CTCGGGAATT CGCCGCAGCG GACCGGCCTC GGCGAATTTG TGCTCTTGTG 960

CCCTCCTCCG GGCTTGGGCC AGGCCGGCCC CTCGCACTTG CCCTTACCTT TTCTATCGAG 1020

•TCCGCATCCC TCTCCAGCCA CTGCGACCCG GCGAAGAGAA AAAGGAACTT CCCCCACCCC 1080

CTCGGGTGCC GTCGGAGCCC CCCAGCCCAC CCCTGGGTGC GGCGCGGGGA CCCCGGGCCG 1140

AAGAAGAGAT TTCCTGAGGA TTCTGGTTTT CCTCGCTTGT ATCTCCGAAA GAATTAAAAA 1200

TGGCCGAGAA TGTGGTGGAA CCGGGGCCGC CTTCAGCCAA GCGGCCTAAA CTCTCATCTC 1260

CGGCCCTCTC GGCGTCCGCC AGCGATGGCA CAGATTTTGG CTCTCTATTT GACTTGGAGC 1320

ACGACTTACC AGATGAATTA ATCAACTCTA CAGAATTGGG ACTAACCAAT GGTGGTGATA 1380 TTAATCAGCT TCAGACAAGT CTTGGCATGG TACAAGATGC AGCTTCTAAA CATAAACAGC 1440

TGTCAGAATT GCTGCGATCT GGTAGTTCCC CTAACCTCAA TATGGGAGTT GGTGGCCCAG 1500

GTCAAGTCAT GGCCAGCCAG GCCCAACAGA GCAGTCCTGG ATTAGGTTTG ATAAATAGCA 1560

TGGTCAAAAG CCCAATGACA CAGGCAGGCT TGACTTCTCC CAACATGGGG ATGGGCACTA 1620

GTGGACCAAA TCAGGGTCCT ACGCAGTCAA CAGGTATGAT GAACAGTCCA GTAAATCAGC 1680

CTGCCATGGG AATGAACACA GGGACGAATG CGGGCATGAA TCCTGGAATG TTGGCTGCAG 1740

GCAATGGACA AGGGATAATG CCTAATCAAG TCATGAACGG TTCAATTGGA GCAGGCCGAG 1800

GGCGACAGGA TATGCAGTAC CCAAACCCAG GCATGGGAAG TGCTGGCAAC TTACTGACTG 1860

AGCCTCTTCA GCAGGGCTCT CCCCAGATGG GAGGACAAAC AGGATTGAGA GGCCCCCAGC 1920

CTCTTAAGAT GGGAATGATG AACAACCCCA ATCCTTATGG TTCACCATAT ACTCAGAATC 1980

CTGGACAGCA GATTGGAGCC AGTGGCCTTG GTCTCCAGAT TCAGACAAAA ACTGTACTAT 2040

CAAATAACTT ATCTCCATTT GCTATGGACA AAAAGGCAGT TCCTGGTGGA GGAATGCCCA 2100

ACATGGGTCA ACAGCCAGCC CCGCAGGTCC AGCAGCCAGG TCTGGTGACT CCAGTTGCCC 2160

AAGGGATGGG TTCTGGAGCA CATACAGCTG ATCCAGAGAA GCGCAAGCTC ATCCAGCAGC 2220

AGCTTGTTCT CCTTTTGCAT GCTCACAAGT GCCAGCGCCG GGAACAGGCC AATGGGGAAG 2280

TGAGGCAGTG CAACCTTCCC CACTGTCGCA CAATGAAGAA TGTCCTAAAC CACATGACAC 2340

ACTGCCAGTC AGGCAAGTCT TGCCAAGTGG CACACTGTGC ATCTTCTCGA CAAATCATTT 2400

CACACTGGAA GAATTGTACA AGACATGATT GTCCTGTGTG TCTCCCCCTC AAAAATGCTG 2460

GTGATAAGAG AAATCAACAG CCAATTTTGA CTGGAGCACC CGTTGGACTT GGAAATCCTA 2520

GCTCTCTAGG GGTGGGTCAA CAGTCTGCCC CCAACCTAAG CACTGTTAGT CAGATTGATC 2580

CCAGCTCCAT AGAAAGAGCC TATGCAGCTC TTGGACTACC CTATCAAGTA AATCAGATGC 2640

CGACACAACC CCAGGTGCAA GCAAAGAACC AGCAGAATCA GCAGCCTGGG CAGTCTCCCC 2700

AAGGCATGCG GCCCATGAGC AACATGAGTG CTAGTCCTAT GGGAGTAAAT GGAGGTGTAG 2760

GAGTTCAAAC GCCGAGTCTT CTTTCTGACT CAATGTTGCA TTCAGCCATA AATTCTCAAA 2820

ACCCAATGAT GAGTGAAAAT GCCAGTGTGC CCTCCCTGGG TCCTATGCCA ACAGCAGCTC 2880

AACCATCCAC TACTGGAATT CGGAAACAGT GGCACGAAGA TATTACTCAG GATCTTCGAA 2940

ATCATCTTGT TCACAAACTC GTCCAAGCCA TATTTCCTAC GCCGGATCCT GCTGCTTTAA 3000

AAGACAGACG GATGGAAAAC CTAGTTGCAT ATGCTCGGAA AGTTGAAGGG GACATGTATG 3060

AATCTGCAAA CAATCGAGCG GAATACTACC ACCTTCTAGC TGAGAAAATC TATAAGATCC 3120

AGAAAGAACT AGAAGAAAAA CGAAGGACCA GACTACAGAA GCAGAACATG CTACCAAATG 3180

CTGCAGGCAT GGTTCCAGTT TCCATGAATC CAGGGCCTAA CATGGGACAG CCGCAACCAG 3240

GAATGACTTC TAATGGCCCT CTACCTGACC CAAGTATGAT CCGTGGCAGT GTGCCAAACC 3300

AGATGATGCC TCGAATAACT CCACAATCTG GTTTGAATCA ATTTGGCCAG ATGAGCATGG 3360

CCCAGCCCCC TATTGTACCC CGGCAAACCC CTCCTCTTCA GCACCATGGA CAGTTGGCTC 3420

AACCTGGAGC TCTCAACCCG CCTATGGGCT ATGGGCCTCG TATGCAACAG CCTTCCAACC 3480

AGGGCCAGTT CCTTCCTCAG ACTCAGTTCC CATCACAGGG AATGAATGTA ACAAATATCC 3540

CTTTGGCTCC GTCCAGCGGT CAAGCTCCAG TGTCTCAAGC ACAAATGTCT AGTTCTTCCT 3600

GCCCGGTGAA CTCTCCTATA ATGCCTCCAG GGTCTCAGGG GAGCCACATT CACTGTCCCC 3660

AGCTTCCTCA ACCAGCTCTT CATCAGAATT CACCCTCGCC TGTACCTAGT CGTACCCCCA 3720

CCCCTCACCA TACTCCCCCA AGCATAGGGG CTCAGCAGCC ACCAGCAACA ACAATTCCAG 3780

CCCCTGTTCC TACACCACCA GCCATGCCAC CTGGGCCACA GTCCCAGGCT CTACATCCCC 3840

CTCCAAGGCA GACACCTACA CCACCAACAA CACAACTTCC CCAACAAGTG CAGCCTTCAC 3900

TTCCTGCTGC ACCTTCTGCT GACCAGCCCC AGCAGCAGCC TCGCTCACAG CAGAGCACAG 3960

CAGCGTCTGT TCCTACCCCA AACGCACCGC TGCTTCCTCC GCAGCCTGCA ACTCCACTTT 4020

CCCAGCCAGC TGTAAGCATT GAAGGACAGG TATCAAATCC TCCATCTACT AGTAGCACAG 4080

AAGTGAATTC TCAGGCCATT GCTGAGAAGC AGCCTTCCCA GGAAGTGAAG ATGGAGGCCA 4140

AAATGGAAGT GGATCAACCA GAACCAGCAG ATACGCAGCC GGAGGATATT TCAGAGTCTA 4200

AAGTGGAAGA CTGTAAAATG GAATCTACCG AAACAGAAGA GAGAAGCACT GAGTTAAAAA 4260

CTGAAATAAA AGAGGAGGAA GACCAGCCAA GTACTTCAGC TACCCAGTCA TCTCCGGCTC 4320

CAGGACAGTC AAAGAAAAAG ATTTTCAAAC CAGAAGAACT ACGACAGGCA CTGATGCCAA 4380

CATTGGAGGC ACTTTACCGT CAGGATCCAG AATCCCTTCC CTTTCGTCAA CCTGTGGACC 4440

CTCAGCTTTT AGGAATCCCT GATTACTTTG ATATTGTGAA GAGCCCCATG GATCTTTCTA 4500

CCATTAAGAG GAAGTTAGAC ACTGGACAGT ATCAGGAGCC CTGGCAGTAT GTCGATGATA 4560

TTTGGCTTAT GTTCAATAAT GCCTGGTTAT ATAACCGGAA AACATCACGG GTATACAAAT 4620

ACTGCTCCAA GCTCTCTGAG GTCTTTGAAC AAGAAATTGA CCCAGTGATG CAAAGCCTTG 4680

GATACTGTTG TGGCAGAAAG TTGGAGTTCT CTCCACAGAC ACTGTGTTGC TACGGCAAAC 4740

AGTTGTGCAC AATACCTCGT GATGCCACTT ATTACAGTTA CCAGAACAGG TATCATTTCT 4800

GTGAGAAGTG TTTCAATGAG ATCCAAGGGG AGAGCGTTTC TTTGGGGGAT GACCCTTCCC 4860

AGCCTCAAAC TACAATAAAT AAAGAACAAT TTTCCAAGAG AAAAAATGAC ACACTGGATC 4920

CTGAACTGTT TGTTGAATGT ACAGAGTGCG GAAGAAAGAT GCATCAGATC TGTGTCCTTC 4980

ACCATGAGAT CATCTGGCCT GCTGGATTCG TCTGTGATGG CTGTTTAAAG AAAAGTGCAC 5040

GAACTAGGAA AGAAAATAAG TTTTCTGCTA AAAGGTTGCC ATCTACCAGA CTTGGCACCT 5100

TTCTAGAGAA TCGTGTGAAT GACTTTCTGA GGCGACAGAA TCACCCTGAG TCAGGAGAGG 5160 TCACTGTTAG AGTAGTTCAT GCTTCTGACA AAACCGTGGA AGTAAAACCA GGCATGAAAG 5220

CAAGGTTTGT GGACAGTGGA GAGATGGCAG AATCCTTTCC ATACCGAACC AAAGCCCTCT 5280

TTGCCTTTGA AGAAATTGAT GGTGTTGACC TGTGCTTCTT TGGCATGCAT GTTCAAGAGT 5340

ATGGCTCTGA CTGCCCTCCA CCCAACCAGA GGAGAGTATA CATATCTTAC CTCGATAGTG 5400

TTCATTTCTT CCGTCCTAAA TGCTTGAGGA CTGCAGTCTA TCATGAAATC CTAATTGGAT 5460

ATTTAGAATA TGTCAAGAAA TTAGGTTACA CAACAGGGCA TATTTGGGCA TGTCCACCAA 5520

GTGAGGGAGA TGATTATATC TTCCATTGCC ATCCTCCTGA CCAGAAGATA CCCAAGCCCA 5580

AGCGACTGCA GGAATGGTAC AAAAAAATGC TTGACAAGGC TGTATCAGAG CGTATTGTCC 5640

ATGACTACAA GGATATTTTT AAACAAGCTA CTGAAGATAG ATTAACAAGT GCAAAGGAAT 5700

TGCCTTATTT CGAGGGTGAT TTCTGGCCCA ATGTTCTGGA AGAAAGCATT AAGGAACTGG 5760

AACAGGAGGA AGAAGAGAGA AAACGAGAGG AAAACACCAG CAATGAAAGC ACAGATGTGA 5820

CCAAGGGAGA CAGCAAAAAT GCTAAAAAGA AGAATAATAA GAAAACCAGC AAAAATAAGA 5880

GCAGCCTGAG TAGGGGCAAC AAGAAGAAAC CCGGGATGCC CAATGTATCT AACGACCTCT 5940

CACAGAAACT ATATGCCACC ATGGAGAAGC ATAAAGAGGT CTTCTTTGTG ATCCGCCTCA 6000

TTGCTGGCCC TGCTGCCAAC TCCCTGCCTC CCATTGTTGA TCCTGATCCT CTCATCCCCT 6060

GCGATCTGAT GGATGGTCGG GATGCGTTTC TCACGCTGGC AAGGGACAAG CACCTGGAGT 6120

TCTCTTCACT CCGAAGAGCC CAGTGGTCCA CCATGTGCAT GCTGGTGGAG CTGCACACGC 6180

AGAGCCAGGA CCGCTTTGTC TACACCTGCA ATGAATGCAA GCACCATGTG GAGACACGCT 6240

GGCACTGTAC TGTCTGTGAG GATTATGACT TGTGTATCAC CTGCTATAAC ACTAAAAACC 6300

ATGACCACAA AATGGAGAAA CTAGGCCTTG GCTTAGATGA TGAGAGCAAC AACCAGCAGG 6360

CTGCAGCCAC CCAGAGCCCA GGCGATTCTC GCCGCCTGAG TATCCAGCGC TGCATCCAGT 6420

CTCTGGTCCA TGCTTGCCAG TGTCGGAATG CCAATTGCTC ACTGCCATCC TGCCAGAAGA 6480

TGAAGCGGGT TGTGCAGCAT ACCAAGGGTT GCAAACGGAA AACCAATGGC GGGTGCCCCA 6540

TCTGCAAGCA GCTCATTGCC CTCTGCTGCT ACCATGCCAA GCACTGCCAG GAGAACAAAT 6600

GCCCGGTGCC GTTCTGCCTA AACATCAAGC AGAAGCTCCG GCAGCAACAG CTGCAGCACC 6660

GACTACAGCA GGCCCAAATG CTTCGCAGGA GGATGGCCAG CATGCAGCGG ACTGGTGTGG 6720

TTGGGCAGCA ACAGGGCCTC CCTTCCCCCA CTCCTGCCAC TCCAACGACA CCAACTGGCC 6780

AACAGCCAAC CACCCCGCAG ACGCCCCAGC CCACTTCTCA GCCTCAGCCT ACCCCTCCCA 6840

ATAGCATGCC ACCCTACTTG CCCAGGACTC AAGCTGCTGG CCCTGTGTCC CAGGGTAAGG 6900

CAGCAGGCCA GGTGACCCCT CCAACCCCTC CTCAGACTGC TCAGCCACCC CTTCCAGGGC 6960

CCCCACCTAC AGCAGTGGAA ATGGCAATGC AGATTCAGAG AGCAGCGGAG ACGCAGCGCC 7020

AGATGGCCCA CGTGCAAATT TTTCAAAGGC CAATCCAACA CCAGATGCCC CCGATGACTC 7080

CCATGGCCCC CATGGGTATG AACCCACCTC CCATGACCAG AGGTCCCAGT GGGCATTTGG 7140

AGCCAGGGAT GGGACCGACA GGGATGCAGC AACAGCCACC CTGGAGCCAA GGAGGATTGC 7200

CTCAGCCCCA GCAACTACAG TCTGGGATGC CAAGGCCAGC CATGATGTCA GTGGCCCAGC 7260

ATGGTCAACC TTTGAACATG GCTCCACAAC CAGGATTGGG CCAGGTAGGT ATCAGCCCAC 7320

TCAAACCAGG CACTGTGTCT CAACAAGCCT TACAAAACCT TTTGCGGACT CTCAGGTCTC 7380

CCAGCTCTCC CCTGCAGCAG CAACAGGTGC TTAGTATCCT TCACGCCAAC CCCCAGCTGT 7440

TGGCTGCATT CATCAAGCAG CGGGCTGCCA AGTATGCCAA CTCTAATCCA CAACCCATCC 7500

CTGGGCAGCC TGGCATGCCC CAGGGGCAGC CAGGGCTACA GCCACCTACC ATGCCAGGTC 7560

AGCAGGGGGT CCACTCCAAT CCAGCCATGC AGAACATGAA TCCAATGCAG GCGGGCGTTC 7620

AGAGGGCTGG CCTGCCCCAG CAGCAACCAC AGCAGCAACT CCAGCCACCC ATGGGAGGGA 7680

TGAGCCCCCA GGCTCAGCAG ATGAACATGA ACCACAACAC CATGCCTTCA CAATTCCGAG 7740

ACATCTTGAG ACGACAGCAA ATGATGCAAC AGCAGCAGCA ACAGGGAGCA GGGCCAGGAA 7800

TAGGCCCTGG AATGGCCAAC CATAACCAGT TCCAGCAACC CCAAGGAGTT GGCTACCCAC 7860

CACAGCCGCA GCAGCGGATG CAGCATCACA TGCAACAGAT GCAACAAGGA AATATGGGAC 7920

AGATAGGCCA GCTTCCCCAG GCCTTGGGAG CAGAGGCAGG TGCCAGTCTA CAGGCCTATC 7980

AGCAGCGACT CCTTCAGCAA CAGATGGGGT CCCCTGTTCA GCCCAACCCC ATGAGCCCCC 8040

AGCAGCATAT GCTCCCAAAT CAGGCCCAGT CCCCACACCT ACAAGGCCAG CAGATCCCTA 8100

ATTCTCTCTC CAATCAAGTG CGCTCTCCCC AGCCTGTCCC TTCTCCACGG CCACAGTCCC 8160

AGCCCCCCCA CTCCAGTCCT TCCCCAAGGA TGCAGCCTCA GCCTTCTCCA CACCACGTTT 8220

CCCCACAGAC AAGTTCCCCA CATCCTGGAC TGGTAGCTGC CCAGGCCAAC CCCATGGAAC 8280

AAGGGCATTT TGCCAGCCCG GACCAGAATT CAATGCTTTC TCAGCTTGCT AGCAATCCAG 8340

GCATGGCAAA CCTCCATGGT GCAAGCGCCA CGGACCTGGG ACTCAGCACC GATAACTCAG 8400

ACTTGAATTC AAACCTCTCA CAGAGTACAC TAGACATACA CTAGAGACAC CTTGTATTTT 8460

GGGAGCAAAA AAATTATTTT CTCTTAACAA GACTTTTTGT ACTGAAAACA ATTTTTTTGA 8520

ATCTTTCGTA GCCTAAAAGA CAATTTTCCT TGGAACACAT AAGAACTGTG CAGTAGCCGT 8580

TTGTGGTTTA AAGCAAACAT GCAAGATGAA CCTGAGGGAT GATAGAATAC AAAGAATATA 8640

TTTTTGTTAT GGGCTGGTTA CCACCAGCCT TTCTTCCCCT TTGTGTGTGT GGTTCAAGTG 8700

TGCACTGGGA GGAGGCTGAG GCCTGTGAAG CCAAACAATA TGCTCCTGCC TTGCACCTCC 8760

AATAGGTTTT ATTATTTTTT TTAAATTAAT GAACATATGT AATATTAATG AACATATGTA 8820

ATATTAATAG TTATTATTTA CTGGTGCAGA TGGTTGACAT TTTTCCCTAT TTTCCTCACT 8880

TTATGGAAGA GTTAAAACAT TTCTAAACCA GAGGACAAAA GGGGTTAATG TTACTTTGAA 8940 ATTACATTCT ATATATATAT AAATATATAT AAATATATAT TAAAATACCA GTTTTTTTTC 9000 TCTGGGTGCA AAGATGTTCA TTCTTTTAAA AAATGTTTAA AAAAAA 9046

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7326 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

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

ATGGCCGAGA ACTTGCTGGA CGGACCGCCC AACCCCAAAC GAGCCAAACT CAGCTCGCCC 60

GGCTTCTCCG CGAATGACAA CACAGATTTT GGATCATTGT TTGACTTGGA AAATGACCTT 120

CCTGATGAGC TGATCCCCAA TGGAGAATTA AGCCTTTTAA ACAGTGGGAA CCTTGTTCCA 180

GATGCTGCGT CCAAACATAA ACAACTGTCA GAGCTTCTTA GAGGAGGCAG CGGCTCTAGC 240

ATCAACCCAG GGATAGGCAA TGTGAGTGCC AGCAGCCCTG TGCAACAGGG CCTTGGTGGC 300

CAGGCTCAGG GGCAGCCGAA CAGTACAAAC ATGGCCAGCT TAGGTGCCAT GGGCAAGAGC 360

CCTCTGAACC AAGGAGACTC ATCAACACCC AACCTGCCCA AACAGGCAGC CAGCACCTCT 420

GGGCCCACTC CCCCTGCCTC CCAAGCACTG AATCCACAAG CACAAAAGCA AGTAGGGCTG 480

GTGACCAGTA GTCCTGCCAC ATCACAGACT GGACCTGGGA TCTGCATGAA TGCTAACTTC 540

AACCAGACCC ACCCAGGCCT TCTCAATAGT AACTCTGGCC ATAGCTTAAT GAATCAGGCT 600

CAACAAGGGC AAGCTCAAGT CATGAATGGA TCTCTTGGGG CTGCTGGAAG AGGAAGGGGA 660

GCTGGAATGC CCTACCCTGC TCCAGCCATG CAGGGGGCCA CAAGCAGTGT GCTGGCGGAG 720

ACCTTGACAC AGGTTTCCCC ACAAATGGCT GGCCATGCTG GACTAAATAC AGCACAGGCA 780

GGAGGCATGA CCAAGATGGG AATGACTGGT ACCACAAGTC CATTTGGACA ACCCTTTAGT 840

CAAACTGGAG GGCAGCAGAT GGGAGCCACT GGAGTGAACC CCCAGTTAGC CAGCAAACAG 900

AGCATGGTCA ATAGTTTACC TGCTTTTCCT ACAGATATCA AGAATACTTC AGTCACCACT 960

GTGCCAAATA TGTCCCAGTT GCAAACATCA GTGGGAATTG TACCCACACA AGCAATTGCA 1020

ACAGGCCCCA CAGCAGACCC TGAAAAACGC AAACTGATAC AGCAGCAGCT GGTTCTACTG 1080

CTTCATGCCC ACAAATGTCA GAGACGAGAG CAAGCAAATG GAGAGGTTCG NGCCTGTTCT 1140

CTCCCACACT GTCGAACCAT GAAAAACGTT TTGAATCACA TGACACATTG TCAGGCTCCC 1200

AAAGCCTGCC AAGTTGCCCA TTGTGCATCT TCACGACAAA TCATCTCTCA TTGGAAGAAC 1260

TGCACACGAC ATGACTGTCC TGTTTGCCTC CCTTTGAAAA ATGCCAGTGA CAAGCGAAAC 1320

CAACAAACCA TCCTGGGATC TCCAGCTAGT GGAATTCAAA ACACAATTGG TTCTGTTGGT 1380

GCAGGGCAAC AGAATGCCAC TTCCTTAAGT AACCCAAATC CCATAGACCC CAGTTCCATG 1440

CAGCGGGCCT ATGCTGCTCT AGGACTCCCC TACATGAACC AGCCTCAGAC GCAGCTGCAG 1500

CCTCAGGTTC CTGGCCAGCA ACCAGCACAG CCTCCAGCCC ACCAGCAGAT GAGGACTCTC 1560

AATGCCCTAG GAAACAACCC CATGAGTGTC CCAGCAGGAG GAATAACAAC AGATCAACAG 1620

CCACCAAACT TGATTTCAGA ATCAGCTCTT CCAACTTCCT TGGGGGCTAC CAATCCACTG 1680

ATGAATGATG GTTCAAACTC TGGTAACATT GGAAGCCTCA GCACGATACC TACAGCAGCG 1740

CCTCCTTCCA GCACTGGTGT TCGAAAAGGC TGGCATGAAC ATGTGACTCA GGACCTACGG 1800

AGTCATCTAG TCCATAAACT CGTTCAAGCC ATCTTCCCAA CTCCAGACCC TGCAGCTCTG 1860

AAAGATCGCC GCATGGAGAA CCTGGTTGCC TATGCTAAGA AAGTGGAGGG AGACATGTAT 1920

GAGTCTGCTA ATAGCAGGGA TGAATACTAT CATTTATTAG CAGAGAAAAT CTATAAAATA 1980

CAAAAAGAAC TAGAAGAAAA GCGGAGGACA CGTTTACATA AGCAAGGCAT CCTGGGTAAC 2040

CAGCCAGCTT TACCAGCTTC TGGGGCTCAG CCCCCTGTGA TTCCACCAGC CCAGTCTGTA 2100

AGACCTCCAA ATGGGCCCCT GCCTTTGCCA GTGAATCGCA TGCAGGTTTC TCAAGGGATG 2160

AATTCATTTA ACCCAATGTC CCTGGGAAAC GTCCAGTTGC CACAGGCACC CATGGGACCT 2220

CGTGCAGCCT CCCCTATGAA CCACTCTGTG CAGATGAACA GCATGGCCTC AGTTCCGGGT 2280

ATGGCCATTT CTCCTTCACG GATGCCTCAG CCTCCAAATA TGATGGGCAC TCATGCCAAC 2340

AACATTATGG CCCAGGCACC TACTCAGAAC CAGTTTCTGC CACAGAACCA GTTTCCATCA 2400

TCCAGTGGGG CAATGAGTGT GAACAGTGTG GGCATGGGGC AACCAGCAGC CCAGGCAGGT 2460

GTTTCACAGG GTCAGGAACC TGGAGCTGCT CTCCCTAACC CTCTGAACAT GCTGGCACCC 2520

CAGGCCAGCC AGCTGCCTTG CCCACCAGTG ACACAGTCAC CATTGCACCC GACTCCACCT 2580

CCTGCTTCCA CAGCTGCTGG CATGCCCTCT CTCCAACATC CAACGGCACC AGGAATGACC 2640

CCTCCTGAGC CAGCAGCTCC CACTCAGCCA TCTACTCCTG TGTCATCTGG GCAGACTCCT 2700

ACCCCAACTC CTGGCTCAGT GCCCAGCGCT GCCCAAACAC AGAGTACCCC TACAGTCCAG 2760

GCAGCAGCAC AGGCTCAGGT GACTCCACAG CCTCAGACCC CAGTGCAGCC ACCATCTGTG 2820

GCTACTCCTC AGTCATCACA GCAGCAACCA ACGCCTGTGC ATACTCAGCC ACCTGGCACA 2880

CCGCTTTCTC AGGCAGCAGC CAGCATTGAT AATAGAGTCC CTACTCCCTC CACTGTGACC 2940 AGTGCTGAAA CCAGTTCCCA GCAGCCAGGA CCCGATGTGC CCATGCTGGA AATGAAGACA 3000

GAGGTGCAGA CAGATGATGC TGAGCCTGAA CCTACTGAAT CCAAGGGGGA ACCTCGGTCT 3060

GAGATGATGG AAGAGGATTT ACAAGGTTCT TCCCAAGTAA AAGAAGAGAC AGATACGACA 3120

GAGCAGAAGT CAGAGCCAAT GGAAGTAGAA GAAAAGAAAC CTGAAGTAAA AGTGGAAGCT 3180

AAAGAGGAAG AAGAGAACAG TTCGAACGAC ACAGCCTCAC AATCAACATC TCCTTCCCAG 3240

CCACGCAAAA AAATCTTTAA ACCCGAGGAG CTACGCCAGG CACTTATGCC AACTCTAGAA 3300

GCACTCTATC GACAGGACCC AGAGTCTTTG CCTTTTCGTC AGCCTGTAGA TCCTCAGCTC 3360

CTAGGAATCC CAGATTATTT TGATATAGTG AAGAATCCTA TGGACCTTTC TACCATCAAA 3420

CGAAAGCTGG ACACAGGGCA ATATCAAGAA CCCTGGCAGT ATGTGGATGA TGTCAGGCTT 3480

ATGTTCAACA ATGCGTGGCT ATATAATCGT AAAACGTCCC GTGTATATAA ATTTTGCAGT 3540

AAACTTGCAG AGGTCTTTGA ACAAGAAATT GACCCTGTCA TGCAGTCTCT TGGATATTGC 3600

TGTGGACGAA AGTATGAGTT CTCCCCACAG ACTTTGTGCT GTTACGGAAA GCAGCTGTGT 3660

ACAATTCCTC GTGATGCAGC CTACTACAGC TATCAGAATA GGTATCATTT CTGTGGGAAG 3720

TGTTTCACAG AGATCCAGGG CGAGAATGTG ACCCTGGGTG ACGACCCTTC CCAACCTCAG 3780

ACGACAATTT CCAAGGATCA ATTTGAAAAG AAGAAAAATG ATACCTTAGA TCCTGAACCT 3840

TTTGTTGACT GCAAAGAGTG TGGCCGGAAG ATGCATCAGA TTTGTGTTCT ACACTATGAC 3900

ATCATTTGGC CTTCAGGTTT TGTGTGTGAC AACTGTTTGA AGAAAACTGG CAGACCTCGG 3960

AAAGAAAACA AATTCAGTGC TAAGAGGCTG CAGACCACAC GATTGGGAAA CCACTTAGAA 4020

GACAGAGTGA ATAAGTTTTT GCGGCGCCAG AATCACCCTG AAGCTGGGGA GGTTTTTGTC 4080

AGAGTGGTGG CCAGCTCAGA CAAGACTGTG GAGGTCAAGC CGGGAATGAA GTCAAGGTTT 4140

GTGGATTCTG GAGAGATGTC GGAATCTTTC CCATATCGTA CCAAAGCACT CTTTGCTTTT 4200

GAGGAGATCG ATGGAGTCGA TGTGTGCTTT TTTGGGATGC ATGTGCAAGA TACGGCTCTG 4260

ATTGCCCCCC ACCAAATACA AGGCTGTGTA TACATATCTT ATCTGGACAG TATTCATTTC 4320

TTCCGGCCCC GCTGCCTCCG GACAGCTGTT TACCATGAGA TCCTCATCGG ATATCTCGAG 4380

TATGTGAAGA AATTGGTGTA TGTGACAGCA CATATTTGGG CCTGTCCCCC AAGTGAAGGA 4440

GATGACTATA TCTTTCATTG CCACCCCCCT GACCAGAAAA TCCCCAAACC AAAACGACTA 4500

CAGGAGTGGT ACAAGAAGAT GCTGGACAAG GCGTTTGCAG AGAGGATCAT TAACGACTAT 4560

AAGGACATCT TCAAACAAGC GAACGAAGAC AGGCTCACGA GTGCCAAGGA GTTGCCCTAT 4620

TTTGAAGGAG ATTTCTGGCC TAATGTGTTG GAAGAAAGCA TTAAGGAACT AGAACAAGAA 4680

GAAGAAGAAA GGAAAAAAGA AGAGAGTACT GCAGCGAGTG AGACTCCTGA GGGCAGTCAG 4740

GGTGACAGCA AAAATGCGAA GAAAAAGAAC AACAAGAAGA CCAACAAAAA CAAAAGCAGC 4800

ATTAGCCGCG CCAACAAGAA GAAGCCCAGC ATGCCCAATG TTTCCAACGA CCTGTCGCAG 4860

AAGCTGTATG CCACCATGGA GAAGCACAAG GAGGTATTCT TTGTGATTCA TCTGCATGCT 4920

GGGCCTGTTA TCAGCACTCA GCCCCCCATC GTGGACCCTG ATCCTCTGCT TAGCTGTGAC 4980

CTCATGGATG GGCGAGATGC CTTCCTCACC CTGGCCAGAG ACAAGCACTG GGAATTCTCT 5040

TCCTTACGCC GCTCCAAATG GTCCACTCTG TGCATGCTGG TGGAGCTGCA CACACAGGGC 5100

CAGGACCGCT TTGTTTATAC CTGCAATGAG TGCAAACACC ATGTGGAAAC ACGCTGGCAC 5160

TGCACTGTGT GTGAGGACTA TGACCTTTGT ATCAATTGCT ACAACACAAA GAGCCACACC 5220

CATAAGATGG TGAAGTGGGG GCTAGGCCTA GATGATGAGG GCAGCAGTCA GGGTGAGCCA 5280

CAGTCCAAGA GCCCCCAGGA ATCCCGGCGT CTCAGCATCC AGCGCTGCAT CCAGTCCCTG 5340

GTGCATGCCT GCCAGTGTCG CAATGCCAAC TGCTCACTGC CGTCTTGCCA GAAGATGAAG 5400

CGAGTCGTGC AGCACACCAA GGGCTGCAAG CGCAAGACTA ATGGAGGATG CCCAGTGTGC 5460

AAGCAGCTCA TTGCTCTTTG CTGCTACCAC GCCAAACACT GCCAAGAAAA TAAATGCCCT 5520

GTGCCCTTCT GCCTCAACAT CAAACATAAC GTCCGCCAGC AGCAGATCCA GCACTGCCTG 5580

CAGCAGGCTC AGCTCATGCG CCGGCGAATG GCAACCATGA ACACCCGCAA TGTGCCTCAG 5640

CAGAGTTTGC CTTCTCCTAC CTCAGCACCA CCCGGGACTC CTACACAGCA GCCCAGCACA 5700

CCCCAAACAC CACAGCCCCC AGCCCAGCCT CAGCCTTCAC CTGTTAACAT GTCACCAGCA 5760

GGCTTCCCTA ATGTAGCCCG GACTCAGCCC CCAACAATAG TGTCTGCTGG GAAGCCTACC 5820

AACCAGGTGC CAGCTCCCCC ACCCCCTGCC CAGCCCCCAC CTGCAGCAGT AGAAGCAGCC 5880

CGGCAAATTG AACGTGAGGC CCAGCAGCAG CAGCACCTAT ACCGAGCAAA CATCAACAAT 5940

GGCATGCCCC CAGGACGTGA CGGTATGGGG ACCCCAGGAA GCCAAATGAC TCCTGTGGGC 6000

CTGAATGTGC CCCGTCCCAA CCAAGTCAGT GGGCCTGTCA TGTCTAGTAT GCCACCTGGG 6060

CAGTGGCAGC AGGCACCCAT CCCTCAGCAG CAGCCGATGC CAGGCATGCC CAGGCCTGTA 6120

ATGTCCATGC AGGCCCAGGC AGCAGTGGCT GGGCCACGGA TGCCCAATGT GCAGCCAAAC 6180

AGGAGCATCT CGCCAAGTGC CCTGCAAGAC CTGCTACGGA CCCTAAAGTC ACCCAGCTCT 6240

CCTCAGCAGC AGCAGCAGGT GCTGAACATC CTTAAATCAA ACCCACAGCT AATGGCAGCT 6300

TTCATCAAAC AGCGCACAGC CAAGTATGTG GCCAATCAGC CTGGCATGCA GCCCCAGCCC 6360 GGACTTCAAT CCCAGCCTGG TATGCAGCCC CAGCCTGGCA TGCACCAGCA GCCTAGTTTG 6420

CAAAACCTGA ACGCAATGCA AGCTGGTGTG CCACGGCCTG GTGTGCCTCC ACCACAACCA 6480

GCAATGGGAG GCCTGAATCC CCAGGGACAA GCTCTGAACA TCATGAACCC AGGACACAAC 6540

CCCAACATGA CAAACATGAA TCCACAGTAC CGAGAAATGG TGAGGAGACA GCTGCTACAG 6600

CACCAGCAGC AGCAGCAGCA ACAGCAGCAG CAGCAGCAGC AACAACAAAA TAGTGCCAGC 6660

TTGGCCGGGG GCATGGCGGG ACACAGCCAG TTCCAGCAGC CACAAGGACC TGGAGGTTAT 6720 GCCCCAGCCA TGCAGCAGCA ACGCATGCAA CAGCACCTCC CCATCCAGGG CAGCTCCATG 6780

GGCCAGATGG CTGCTCCAAT GGGACAACTT GGCCAGATGG GGCAGCCTGG GCTAGGGGCA 6840

GACAGCACCC CTAATATCCA GCAGGCCCTG CAGCAACGGA TTCTGCAGCA GCAGCAGATG 6900

AAGCAACAAA TTGGGTCACC AGGCCAGCCG AACCCCATGA GCCCCCAGCA GCACATGCTC 6960

TCAGGACAGC CACAGGCCTC ACATCTCCCT GGCCAGCAGA TCGCCACATC CCTTAGTAAC 7020

CAGGTGCGAT CTCCAGCCCC TGTGCAGTCT CCACGGCCCC AATCCCAACC TCCACATTCC 7080

AGCCCGTCAC CACGGATACA ACCCCAGCCT TCACCACACC ATGTTTCACC CCAGACTGGA 7140

ACCCCTCACC CTGGACTCGC AGTCACCATG GCCAGCTCCA TGGATCAGGG ACACCTGGGG 7200

AACCCTGAAC AGAGTGCAAT GCTCCCCCAG CTGAATACCC CCAACAGGAG CGCACTGTCC 7260

AGTGAACTGT CCCTGGTTGG TGATACCACG GGAGACACAC TAGAAAAGTT TGTGGAGGGT 7320

TTGTAG 7326

(2) INFORMATION FOR SEQ ID NO: 14:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2499 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

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

TCACTTGTCA ATTAATCCAG CTTCCTTAAT TTTACTGAAG AAGAATTTCT CCAGGATATT 60

GGCACATTTG TAGTATTCAC TCTCAGGGGC GTTGTACTCT TTGCAATTGG TAAAGACTCG 120

CTGTAAGTCT GCCATGAATA ATTTCTTAGA CACGTAGTAC CTATTCTTGA GGCGTTCACT 180

CATGGTTTTG AGATCCATGG GGGACCTTAT AACTTCATAA TATCCTGGAG CTTCTGTTCT 240

CTTCACAGGT TCCATGAAGG GCCAAGCGCT TTGATGGCTC TTCACCTGCT GGAGGATGCT 300

CTTGAGCGTG CTGTAAAGCT GGTCAGGGTC TCTGGGCTCT TTACTTTTCT CTTTTCCACT 360

CGGTTTCCAG CCTGTCTCTC TAATTCCAGG AATGCTTTCT ATAGGAATCT GTCGAACTCC 420

ATCTTTAAAA CATGAAAGTC CAGGGTAAAC TTTTCGAATT TGTGCCTGTT TTCTTTCAAT 480

CAGTTTTTTA ATTATCTCCT TCTGCTTTTT AATGATGACA GAAAATTCTG TGTACGGGAT 540

CCGTGGATTT AGCTCACATC CCATTAAAGT GGCTCCTTCA TAATCCTTGA TATAGCCAAC 600

ATATTTGGTT TTAGGTATTT TAATTTCTTT GGAGAAACCC TGTTTCTTAA AGTATCCAAT 660

TGCATATTCA TCTGCATATG TGAGGAAGTT CAGGATGTCA TGCTTTATGT GATATTCTTT 720

CAAATGATTC ATCAGGTGTG TTCCATAGCC CTTGACTTGC TCATTTGAGG TTACAGCACA 780

GAAGACAATC TCTGTGAATC CTTGAGATGG GAACATACGG AAACAGATAC CACCAATAAC 840

ACGGCCATCT TTAATTAAAG CAAGGGTTTT GTGTTTCGGG TCAAAGACGA GCCGTGTGAT 900

GTATTCTTTT GGCATTCGGG GCAGCTGGTG GGAGAAAACG TTCTGTAGGC CAACCAGCCA 960

CATCAGGATC TTCTTGTTTG GTTTCTGGTT GAGGGAATTG CCAACCACGT GAAATTCAAT 1020

TACACCCCTG CGCTCTTCCA ACCTTGCCGC CTCATCCCTG GCCGAGTGTG CTGACAGAAA 1080

ATTGGTCTCT GGTCCAAGCA TTGCTGCAGG GTCCGTGATG GTAGACATAA CCTCGTTGAT 1140

TAATTCCATC GGAATATCCC CCATAACTCG GGGTTTCTTG GCCTCCTCCA GAACATGAGA 1200

ATCAGTCATT TTCCTCTTTT CTCCTGGGTT TGCCTCAAGT CCAGAAGAGG CTTTGCAGGC 1260

AGGACTGCTG CTCCCTGCGT TTGGCTGCTC AAGGGAAGAT GAGGTTGAAT TGTATGAAAT 1320

TGTCCCAGCC ACAGGAGGTG GATTGATAAC TGTTTGGATG CCTAGCTGGC TGGTTCTGGA 1380

AGAGGCTGAG AGAAAATCCT GATCCCAGAT GGGAGAGTTT TGACTATATA CTTCTTCTTC 1440

TAGCATGGAC AGAAATTTTG GGAAATGAGT GAGGATTAGA GTTCGTTTTT CAAGAGGCAG 1500

TTTATCTTTT TCCTGTCTTG CTTGTTCCAG GAGTTGTCGC CTCATAACAG TGAAGACCGA 1560

GCGAAGCAAT GTTCTCCCAA ACACCTGTGT GGTTTCGTAC CGAGGTAGAC TGTCGCAGAA 1620

CTGTGGCACG TTGCAGTAAC ACAGCCACCT TGTGTAGTTC TCTTTGTATC CAGAAATATC 1680

ATCATTGGGA GATCGCAGTC TTCGTTGAGA TGGTGCCTCC AGATGCCAAT AGTTGATGCG 1740

GTTTAGGAAC ATTTTTGCCA ACTCAACTAT TGTTTGCCTT TCTTTTGCTG GCAGGTGACT 1800

AAATTTGTAC TGCACAAAGT TATTCACACC CTGTTCAATG CTAGGTTTTT CAAATGGGGG 1860

TTTCTTTTCC AAAGAGCCTT CAACCACAGG TTTTCCTCTT TGTAAAATAG ACTTTCTCAA 1920

GAGCTTAAAT AGATAGAAAT AAACTTGTTT GGTATCTGCA TCTTCTTCCT TGTGGACACA 1980

GGTAAAGAGA TATTCCACAT CCAATACTAT TCCCAGGAGT CTGTTCATTT CTTCCTCTGA 2040

CACATTCTCC AGGTGGGAAA CATGAGCAGC TAGGGCATGG CTACAACTCC GACAGGATTC 2100

TGTTAGACTG ACAATTATTT GCTGCAGGTC GGCTCTGGGG GGAGTGGGTG AGGGGTTAGG 2160

GTTTTTCCAG CCATTACATT TACAAGACTC CTCGGCCTTG CAGGCGGAGT ACACTCCGAG 2220

TTTCTCCAGT TTCTTGGCCC GCGGAGCGGA GCGTAGTTGC GCTTTCTTCA CGGCGATTCG 2280

GGCCGAGCCA CCGCCTCCCG GTCCTTCGGC CGTGCCCGCT GCAGCCACTG CCGTCGCCGG 2340

ACCGCAGGCG CCCGAGCCCC CGGCGGCAGC GGCGCAGGGG GAGCCCTGCG GGGGCGCGGG 2400 CGGAAGCGCC GCAGGCTGCG GGGGCAGCGC CCCGGGCCCG GCCCCTGCCC CGGCTCCTGC 2460 CCCGCAGCCG CCCGGCCCGG CCCCGCCAGC CTCGGACAT 2499

(2) INFORMATION FOR SEQ ID NO: 15:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2442 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

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

TCACTTGTCA ATCAACCCTG CTTCCTTAAT TTTACTGAAG AAGAACTTCT CCAGGATGCT 60

GGCGCATTTG TAGTACTCGC TCTCGGGAGG GTTGTACTCC TTGCAGTTGG TGAACACTCG 120

TTGCAAGTCC GCCATGAATA ACTTCTTAGA CACATAGTAC CTGTTCCTGA GGCGTTCACT 180

CATGGTTTTC AGATCCATGG GGAACCTTAT AACTTCATAA TATCCCGGAG CTTCTGTTCT 240

CTTCACTGGT TCCATGAAAG GCCAAGCATT TGGATGGTTC TTCACCTGCT GCAGGATGTT 300

CTTGAGGGTG CTGTAAACGT GCTCAGGGTC TTTGGGCTCT TTACTTTTCT CTTTTCCACT 360

TGGTTTCCAG CCTGTCTCTC TGATTCCAGG AATGCTTTCT ATAGGAATCT GCCGAACTCC 420

ATCTTTGAAA CACGAAAGTC CAGGGTAGAC TTTTCGAATC TGGGCTTGTT TTCTTTCTAT 480

CAGCTTTTTA ATGATCTCCT TCTGCTTTTT AATGATGACA GAGAACTCTG TGTATGGGAT 540

CTGAGGGTTC AGCTCACATC CCATCAAAGT GGCCCCTTCA TAATCCTTGA TGTAGCCAAC 600

ATATTTGGTT TTAGGTATTT TGATTTCTTT GGAGAAACCC TGCTTCTTGA AATAGCCGAT 660

GGCATACTCA TCTGCATATG TGAGGAAGTT GAGGATCTCG TGCTTTATGT GGTATTCTTT 720

GAGATGGTTC ATCAGGTGGG TTCCATAGCC CTTGACTTGT TCATTTGAGG TTACTGCACA 780

GAAAACAATC TCTGTGAATC CCTGGGATGG AAACATCCGG AAACAGATAC CACCAATGAC 840

ACGGCCATCT TTAATTAAAG CAAGGGTTTT GTGTTTCGGG TCAAAGACGA GCCGTGTGAT 900

GTACTCTTTG GGCATTCTGG GCAGCTGGTG GGAAAACACA TTCTGGAGGC CCACGAGCCA 960

CATCAGGATC TTCTTGTTTG GTTTCTGGTT CAGGGAGTTG CCCACCACGT GGAATTCAAT 1020

GACACCCCTG CGTTCTTCCA GCCGTGCCGC CTCATCTCTG GCCGAATGGG CTGACAGAAA 1080

ATTGGTCTCT GGTCCAAGCA TCCCTGCAGG GTCTGTGATG GTAGACATGA CCTCATTGAT 1140

CAATTCCACG GGAATATCCC CCATCACTCG AGATCTCTTG GCCTCCTCGG GAGCATGAGA 1200

GTTGTTCATT TTCCTCTTTT CTCCCGGGTT TGCTTCAAGC CCAGAAGAGC CTCTGCATCC 1260

AGGACTTGTT CTCCCTCCAT TGATCTGCTC ATGGGAAGTT GAATTTGAAC TGAACAATGC 1320

TGTCCCAGTA ACAGGAGGAC TGATTACTGT TTGGATTCCT AGCGGGCTGG TTCTGGAAGA 1380

GGCTGAGAGA AAATCCTGAT CCCAGATAGG AGAATTTTGA CTATACACTT CTTCTTCCAA 1440

CATGGACAGA AACTTTGGGA AATGTGTGAG GATAAGCGTG CGTTTCTCAA GAGGCAGTTT 1500

GTCTTTTTTC TGTCTGGCTT GTTCCAAGAG CTGTCGTCTC ATGATGGTGA AGACCGAGCG 1560

AAGCAATGTT CTCCCAAACA CCTTTGTGGT TTCGTACCGA GGTAAGCTGT CACAGAACTG 1620

CGGTACATTG CAGTAGCACA ACCACCTTGT GTAGTTTTCC TTGTATCCAG AGATGTCATC 1680

ATTGGGAGAC CGTAGTCTCC GCTGAGATGG AGCCTCCAGA TGCCAGTAGT TGATGCGGTT 1740

CAGAAACATC TTGGCCAGCT CGATCGTTGT CTGCCTCTCT TTCGATGGCA AGTGACTAAA 1800

CTTGTACTGC ACGAAGTTGT TCACACCCTG TTCAATACTG GGCTTCTCAA ATGGCGGCTT 1860

CTTCTCCAAG GAGCCTTCAA CCACAGGTTT TCCTCTTTGT AAAATTGACT TTCTCAAGAG 1920

CTTGAATAGG TAGAAGTACA CTTGTTTGGT ATCTGCATCT TCTTCTTTGT GGACGCAGGT 1980

GAAGAGGTAC TCCACATCCA ACACAATTCC CAGGAGTCTG TCCATCTCTT CCTCTGACAC 2040

ATTCTCCAAG TGAGAAACGT GAGCAGCAAG GGCATGGCTA CAGCTTCGAC AGGATTCTGT 2100

CAAACTGACA ATTATCTGCT GGAGGTCTCC TCTTGGTGGA GTAGGAGAGG GGTTAGGGTT 2160

CTTCCAGCCA TTGCATTTAC AGGACTCCTC TGCCTTGCAG GCGGAGTACA CGCCGAGTTT 2220

CTCCAGCTTC TTCGCCCGCG GAGCAGAGCG CAACTGCGCC TTCTTCACGG CGATCCGGGC 2280

CGAGCCGCCT CCTCCCGGTC CCTCGGCGGT GCCCGCCGCG GCCACCGGCG TCGCTGGCCC 2340

GCAGGAAGCA GAGCTCCCGG CAGCGGTGGC CAGGGTCCGG GGGGAACCGT GCGGGGGCGC 2400

GGGAGGCAGT GCTGGGGACC CGGCCCCGCC AGCCTCGGCC AT 2442

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

CCCGCCAGCC TCGGACATGC 20 (2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

CCCGCCAGCC TCGGCCATGC 20 (2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2442 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

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

ATGGCCGAGG CTGGCGGGGC CGGGTCCCCA GCACTGCCTC CCGCGCCCCC GCACGGTTCC 60

CCCCGGACCC TGGCCACCGC TGCCGGGAGC TCTGCTTCCT GCGGGCCAGC GACGCCGGTG 120

GCCGCGGCGG GCACCGCCGA GGGACCGGGA GGAGGCGGCT CGGCCCGGAT CGCCGTGAAG 180

AAGGCGCAGT TGCGCTCTGC TCCGCGGGCG AAGAAGCTGG AGAAACTCGG CGTGTACTCC 240

GCCTGCAAGG CAGAGGAGTC CTGTAAATGC AATGGCTGGA AGAACCCTAA CCCCTCTCCT 300

ACTCCACCAA GAGGAGACCT CCAGCAGATA ATTGTCAGTT TGACAGAATC CTGTCGAAGC 360

TGTAGCCATG CCCTTGCTGC TCACGTTTCT CACTTGGAGA ATGTGTCAGA GGAAGAGATG 420

GACAGACTCC TGGGAATTGT GTTGGATGTG GAGTACCTCT TCACCTGCGT CCACAAAGAA 480

GAAGATGCAG ATACCAAACA AGTGTACTTC TACCTATTCA AGCTCTTGAG AAAGTCAATT 540

TTACAAAGAG GAAAACCTGT GGTTGAAGGC TCCTTGGAGA AGAAGCCGCC ATTTGAGAAG 600

CCCAGTATTG AACAGGGTGT GAACAACTTC GTGCAGTACA AGTTTAGTCA CTTGCCATCG 660

AAAGAGAGGC AGACAACGAT CGAGCTGGCC AAGATGTTTC TGAACCGCAT CAACTACTGG 720

CATCTGGAGG CTCCATCTCA GCGGAGACTA CGGTCTCCCA ATGATGACAT CTCTGGATAC 780

AAGGAAAACT ACACAAGGTG GTTGTGCTAC TGCAATGTAC CGCAGTTCTG TGACAGCTTA 840

CCTCGGTACG AAACCACAAA GGTGTTTGGG AGAACATTGC TTCGCTCGGT CTTCACCATC 900

ATGAGACGAC AGCTCTTGGA ACAAGCCAGA CAGAAAAAAG ACAAACTGCC TCTTGAGAAA 960

CGCACGCTTA TCCTCACACA TTTCCCAAAG TTTCTGTCCA TGTTGGAAGA AGAAGTGTAT 1020

AGTCAAAATT CTCCTATCTG GGATCAGGAT TTTCTCTCAG CCTCTTCCAG AACCAGCCCG 1080

CTAGGAATCC AAACAGTAAT CAGTCCTCCT GTTACTGGGA CAGCATTGTT CAGTTCAAAT 1140

TCAACTTCCC ATGAGCAGAT CAATGGAGGG AGAACAAGTC CTGGATGCAG AGGCTCTTCT 1200

GGGCTTGAAG CAAACCCGGG AGAAAAGAGG AAAATGAACA ACTCTCATGC TCCCGAGGAG 1260

GCCAAGAGAT CTCGAGTGAT GGGGGATATT CCCGTGGAAT TGATCAATGA GGTCATGTCT 1320

ACCATCACAG ACCCTGCAGG GATGCTTGGA CCAGAGACCA ATTTTCTGTC AGCCCATTCG 1380

GCCAGAGATG AGGCGGCACG GCTGGAAGAA CGCAGGGGTG TCATTGAATT CCACGTGGTG 1440

GGCAACTCCC TGAACCAGAA ACCAAACAAG AAGATCCTGA TGTGGCTCGT GGGCCTCCAG 1500

AATGTGTTTT CCCACCAGCT GCCCAGAATG CCCAAAGAGT ACATCACACG GCTCGTCTTT 1560

GACCCGAAAC ACAAAACCCT TGCTTTAATT AAAGATGGCC GTGTCATTGG TGGTATCTGT 1620

TTCCGGATGT TTCCATCCCA GGGATTCACA GAGATTGTTT TCTGTGCAGT AACCTCAAAT 1680

■GAACAAGTCA AGGGCTATGG AACCCACCTG ATGAACCATC TCAAAGAATA CCACATAAAG 1740

CACGAGATCC TCAACTTCCT CACATATGCA GATGAGTATG CCATCGGCTA TTTCAAGAAG 1800

CAGGGTTTCT CCAAAGAAAT CAAAATACCT AAAACCAAAT ATGTTGGCTA CATCAAGGAT 1860

TATGAAGGGG CCACTTTGAT GGGATGTGAG CTGAACCCTC AGATCCCATA CACAGAGTTC 1920

TCTGTCATCA TTAAAAAGCA GAAGGAGATC ATTAAAAAGC TGATAGAAAG AAAACAAGCC 1980

CAGATTCGAA AAGTCTACCC TGGACTTTCG TGTTTCAAAG ATGGAGTTCG GCAGATTCCT 2040 ATAGAAAGCA TTCCTGGAAT CAGAGAGACA GGCTGGAAAC CAAGTGGAAA AGAGAAAAGT 2100

AAAGAGCCCA AAGACCCTGA GCACGTTTAC AGCACCCTCA AGAACATCCT GCAGCAGGTG 2160

AAGAACCATC CAAATGCTTG GCCTTTCATG GAACCAGTGA AGAGAACAGA AGCTCCGGGA 2220

TATTATGAAG TTATAAGGTT CCCCATGGAT CTGAAAACCA TGAGTGAACG CCTCAGGAAC 2280

AGGTACTATG TGTCTAAGAA GTTATTCATG GCGGACTTGC AACGAGTGTT CACCAACTGC 2340

AAGGAGTACA ACCCTCCCGA GAGCGAGTAC TACAAATGCG CCAGCATCCT GGAGAAGTTC 2400

TTCTTCAGTA AAATTAAGGA AGCAGGGTTG ATTGACAAGT GA 2442

Claims

What is claimed is:

1. A purified protein designated P/CAF having a molecular weight of about 93,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions and which acetylates histones.

2. The protein of claim 1 consisting of the amino acid sequence of SEQ ID NO: 1

3. The protein of claim 1 comprising the amino acid sequence of SEQ ID NO:2.

4. The protein of claim 1, which also binds to the amino acid sequence of SEQ ID NO:3 on a p300 cellular protein and to amino acid residues 1805-1854 of a CBP cellular protein (SEQ ID NO:9).

5. A fragment of the protein of claim 1 having histone acetyltransferase activity.

6. A polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.

7. A fragment of the protein of claim 1 which binds to the amino acid sequence of SEQ ED NO: 3 on the p300 cellular protein and the amino acid sequence of SEQ ED NO:9 on the CBP cellular protein.

8. A polypeptide consisting of the amino acid sequence of SEQ ED NO:4.

9. A nucleic acid consisting of the nucleotide sequence of SEQ ID NO: 10.

10. A nucleic acid having a nucleotide sequence which encodes the protein of claim 1.

11 A nucleic acid having a nucleotide sequence which encodes the protein of claim 2

12 A nucleic acid having a nucleotide sequence which encodes the protein of claim 3

13 A nucleic acid consisting of the nucleotide sequence which encodes the protein of claim 4

14 A nucleic acid complementary to and which selectively hybridizes with the nucleic acid of claim 11 under stringent hybridization conditions

15. A fragment of the nucleic acid of claim 9, which encodes a polypeptide that acetylates histones

16 A fragment of the nucleic acid of claim 9, which encodes a polypeptide which binds to the amino acid sequence of SEQ ID NO: 3 on the p300 cellular protein and the amino acid sequence of SEQ ID NO: 9 on the CBP cellular protein

7 A purified antibody which specifically binds the protein of claim 1

18 A purified antibody which specifically binds the protein of claim 2

19 A purified antibody which specifically binds the protein of claim 3.

20 A purified antibody which specifically binds the protein of claim 4

21 An assay for screening substances for the ability to inhibit or stimulate the histone acetyltransferase activity of P/CAF comprising a) contacting the substance with a system in which histone acetylation by P/CAF can be determined, b) determining the amount of histone acetylation by P/CAF in the presence of the substance; and c) comparing the amount of histone acetylation by P/CAF in the presence of the substance with the amount of histone acetylation by P/CAF in the absence of the substance, a decreased or increased amount of histone acetylation by P/CAF in the presence of the substance indicating a substance that can inhibit or stimulate, respectively, the histone acetyltransferase activity of P/CAF.

22. An assay for screening substances for the ability to inhibit binding of P/CAF to p300/CBP comprising: a) contacting the substance with a system in which the P/CAF binding of P300/CBP can be determined; b) determining the amount of P/CAF binding of p300/CBP in the presence of the substance; and c) comparing the amount of binding of P/CAF to p300/CBP in the presence of the substance with the amount of binding of P/CAF to p300/CBP in the absence of the substance, a decreased amount of binding of P/CAF to p300/CBP in the presence of the substance indicating a substance that can inhibit the ability to inhibit binding of P/CAF to p300/CBP.

23. The method of claim 22, wherein the system consists of a cell free reaction mixture comprising a fragment of the p300 protein comprising amino acid residues 1767-1816 (SEQ ID NO:3) and the protein of claim 4.

24. The method of claim 22, wherein the system consists of a cell free reaction mixture comprising a fragment of the CBP protein comprising amino acid residues 1805-1854 (SEQ ID NO:9) and the protein of claim 4.

25. The method of claim 22, wherein the system consists of a cell extract produced from cells producing both p300 and P/CAF.

26. An assay for screening substances for the ability to inhibit or stimulate the histone acetyltransferase activity of p300/CBP comprising: a) contacting the substance with a system in which histone acetylation by p300/CBP can be determined; b) determining the amount of histone acetylation by p300/CBP in the presence of the substance; and c) comparing the amount of histone acetylation by p300/CBP in the presence of the substance with the amount of histone acetylation by p300/CBP in the absence of the substance, a decreased or increased amount of histone acetylation by p300/CBP in the presence of the substance indicating a substance that can inhibit or stimulate, respectively, the histone acetyltransferase activity of p300/CBP.

27. An assay for screening substances for the ability to inhibit binding of a DNA- binding transcription factor to p300/CBP comprising: a) contacting the substance with a system in which the DNA-binding transcription factor binding of P300/CBP can be determined; b) determining the amount of DNA-binding transcription factor binding of p300/CBP in the presence of the substance; and c) comparing the amount of binding of DNA-binding transcription factor to p300/CBP in the presence of the substance with the amount of binding of DNA-binding transcription factor to p300/CBP in the absence of the substance, a decreased amount of binding of DNA-binding transcription factor to p300/CBP in the presence of the substance indicating a substance that can inhibit the ability to inhibit binding of DNA- binding transcription factor to p300/CBP.

28. The method of claim 27, wherein the system consists of a cell free reaction mixture comprising a DNA-binding transcription factor and p300/CBP.

29. The method of claim 27, wherein the system consists of a cell free reaction mixture comprising a fragment of the CBP protein comprising a DNA-binding transcription factor and p300/CBP.

30. The method of claim 27, wherein the system consists of a cell extract produced from cells producing both a DNA-binding transcription factor and p300/CBP.

31. The method of claim 27, wherein the DNA-binding transcription factor is selected from the group consisting of a nuclear hormone receptor, CREB, c-Jun/v-Jun, c-Myb/v-Myb, YY1, Sap-la, c-Fos, MyoD and SRC-1.

32. A method for inhibiting the transcription modulating activity of P/CAF in a subject, comprising administering to the subject a transcription modulating activity inhibiting amount of a substance in a pharmaceutically acceptable carrier.

33. The method of claim 32, wherein the substance can inhibit the transcription modulating activity of P/CAF by preventing the binding of P/CAF to p300/CBP.

34. A method for stimulating the transcription modulating activity of P/CAF in a subject, comprising administering to the subject a transcription modulating activity stimulating amount of a substance in a pharmaceutically acceptable carrier.

35. The method of claim 34, wherein the substance can stimulate the transcription modulating activity of P/CAF by promoting the binding of P/CAF to p300/CBP.

36. The method of claim 34, wherein the substance can stimulate the transcription modulating activity of P/CAF by stimulating the histone acetlytransferase activity of P/CAF.

37. A method for inhibiting the histone acetyltransferase activity of p300/CBP in a subject, comprising administering to the subject a histone acetyltransferase activity inhibiting amount of a substance in a pharmaceutically acceptable carrier.

38. The method of claim 37, wherein the substance can inhibit the transcription modulating activity of p300/CBP by preventing the binding of a DNA-binding transcription factor to p300/CBP.

39. The method of claim 38, wherein the DNA-binding transcription factor is selected from the group consisting of a nuclear hormone receptor, CREB, c-Jun/v-Jun, c-Myb/v-Myb, YY1, Sap- la, c-Fos, MyoD and SRC-1.

40. The method of claim 37, wherein the substance is an antibody which binds p300/CBP.

41. A method for stimulating the histone acetyltransferase activity of p300/CBP in a subject, comprising administering to the subject a histone acetyltransferase activity stimulating amount of a substance in a pharmaceutically acceptable carrier.

42. The method of claim 41, wherein the substance can stimulate the histone acetyltransferase activity of p300/CBP by promoting the binding of a DNA-binding transcription factor to p300/CBP.