JP2003529321A - Isolated nucleic acids of the P-HYDE family, P-HYDE proteins, and methods of inducing susceptibility to inducing cell death in cancer - Google Patents

Abstract

  (57) [Summary] The present invention provides p-Hyde genes of the p-Hyde family, analogs, fragments, variants, and atypical isolated nucleic acids. The present invention provides polypeptides, fusion proteins, chimeras, fusions, antisense molecules, antibodies, and uses thereof. The present invention also provides a method of inducing susceptibility to apoptosis by p-Hyde, a method of inhibiting tumor growth by p-Hyde, and a method of cancer by p-Hide alone or in combination with radiotherapy or a UV mimetic drug. A method of treating a subject having The present invention also relates to therapies for human cancers having a mutation in the p-Hyde gene, including gene therapy, protein replacement therapy, and protein mimetics. The invention further relates to screening drugs for cancer therapy. Finally, the invention relates to the screening of p-Hyde genes for mutations, useful for diagnosing a predisposition to cancer.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention provides isolated nucleic acids of the p-Hyde gene, proteins of the p-Hyde family, analogs, fragments, mimetics, variants, compounds and variants thereof. . The present invention provides a method for inducing susceptibility to apoptosis by p-Hyde, a method for suppressing tumor growth by p-Hyde, and radiation alone or by radiation.
A method of treating a subject with cancer with chemotherapy or p-Hyde in combination with a UV mimetic.

BACKGROUND OF THE INVENTION Prostate cancer is the most common malignancy in men with more than 317,000 new cases in the United States and the second leading cause of cancer death in men (Bori).
ng et al. , 1993; Steiner et al. , 1995).
Much remains unknown about the molecular mechanisms responsible for the development, progression, and metastasis of prostate cancer. As many as 20% of prostate cancers occur in men under the age of 65 (Silberg,
1986), suggesting that the development of prostate cancer is associated with genetic factors as well as aging (Silberg, 1987; McL.
ellan and Norman, 1995; Carter et al. ，
1992). A genetic linkage study of 691 affected families reveals that the age at onset of the proband and the presence of multiple affected family members are important determinants of increased risk of prostate cancer. became. The inherited pattern of the putative prostate cancer gene is autosomal dominant, and the penetrance is considered to be 88% (S
teinberg, 1990). Therefore, genetic factors play an important role in the development of prostate cancer.

[0003] Like many carcinomas, the formation of prostate cancer involves the initiation of tumors.
iation), promotion, conversion (c)
inversion, and progression in a multi-step process (Carter et al., 1990; Sandbe).
rg, 1992). This process is driven by chromosomal instability, spontaneous mutations, and genetic and epigenetic changes induced by carcinogens. Chromosome instability results in complete or partial increases or decreases in chromosomes, translocations, and other abnormalities. Spontaneous mechanisms are age-related and include activation of oncogenes or inactivation of tumor suppressor genes by gene mutations. These mutations lead to misincorporation of nucleotides during DNA replication of the coding region, alterations in intron-exon junction sequences that affect splicing mechanisms, and abnormalities in control sequences that alter regulation of key genes. These mutations lead to a series of DNA repair mechanisms,
And p53 (Effert et al., 1992, Isaacs et.
al, 1991; Mellon et al. , 1992) and p21 (El-
Dairy et al. , 1994) and PCNA (Templetone e)
t al. , 1996) to avoid gene surveillance by its associated gene product. Genetic and epigenetic changes induced by carcinogens initiate tumors as a result of the direct DNA damaging effects of carcinogens, which alter gene expression. Tumor initiation is followed by tumor promotion such that the affected cells selectively and replicate through altered signaling and proliferative responses to growth factors, resistance to cytotoxicity, and deregulation of terminal differentiation. And clonal amplification ability (Yuspa and Poirier,
1998; Weinstein, 1987). Finally, tumor promotion
It progresses through other genetic mutational events that result in loss of hormone sensitivity, increased cell death, invasion, altered programmed cell death, and metastasis. Therefore, the initiation and progression of cancers are well defined in that genetic alterations or mutations of key genes are ultimately different with respect to many important cellular activities including cell proliferation, differentiation, and programmed cell death. It is a multi-step process that directs various cell phenotypes. However, the exact mutational events responsible for the multistep progression of prostate cancer are unknown. A more detailed understanding of the molecular mechanisms responsible for prostate cancer may lead to new therapies to combat and perhaps even prevent prostate cancer.

DISCLOSURE OF THE INVENTION The present invention provides an isolated nucleic acid encoding a p-Hyde gene of the p-Hyde family. The p-Hyde gene as shown herein has (1)
In vivo reduction of tumor growth, (2) induction of susceptibility to UV or chemotherapy-induced DNA damage-induced apoptosis, and (3) UV.
Prevention of DNA repair by up-regulation of apoptosis as a result of damage by and damage to DNA repair.

The present invention provides a novel class of genes that act as inhibitors of DNA repair enzymes and induce susceptibility of cancer cells to cell death. The present invention also provides an isolated nucleic acid (allele, analog, fragment, mimetic, mutant, synthetic, or synthetic nucleic acid encoding a mammalian p-Hyde protein that induces susceptibility of cancer cells to cell death. (Including variants). The present invention includes an isolated nucleic acid encoding an human p-Hyde protein that induces susceptibility of cancer cells to cell death, including alleles, analogs, fragments, mimetics, variants, compounds, or variants thereof. )I will provide a.

In the present invention, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7
, Or a nucleic acid having the nucleotide sequence as set forth in SEQ ID NO: 9.

Further, in the present invention, a fragment of the polypeptide having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 (SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or at least 15 of SEQ ID NO: 10 (25
, 30, 50, 60, or 63) comprising a contiguous amino acid).

Further, in the present invention, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:
7 or at least about 82%, 84% with the nucleic acid sequence as set forth in SEQ ID NO: 9
Nucleic acid molecules having nucleotide sequences that are 85%, 87%, 90%, 92%, 95%, or 98% identical.

The present invention also hybridizes with a nucleic acid molecule containing SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 or a complementary sequence thereof under stringent conditions. A nucleic acid molecule encoding a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.

Further, in the present invention, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:
7 or at least about 82%, 84%, 85%, 8 with the amino acid sequence of SEQ ID NO: 9
Included is an isolated p-Hyde protein having an amino acid sequence that is 7%, 90%, 95%, or 98% identical.

Further, in the present invention, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:
7 or SEQ ID NO: 9 and at least about 65%, preferably 75%, 80%, 85
An isolated p-Hyde protein encoded by a nucleic acid molecule having a nucleotide sequence that is 100% or 95% identical; and SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or An isolated p-Hyde protein encoded by a nucleic acid molecule having a nucleotide sequence that hybridizes under stringent hybridization conditions with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 9 or a complementary sequence thereof. Be done.

The p-Hyde protein of the invention, or a portion thereof having biological activity, is operably linked to a non-p-Hyde polypeptide (eg, a heterologous amino acid sequence), and
-Hyde fusion protein may be formed.

The present invention provides a vector containing an isolated nucleic acid encoding a P-Hyde gene. The present invention provides a replication-defective recombinant E1 / E3 deleted adenovirus (AdRSVpH containing a truncated RSV promoter and p-Hyde cDNA gene).
yde) is provided.

The present invention provides a sequence of nucleotides present in a nucleic acid encoding p-Hyde,
Or, an oligonucleotide of at least 15 nucleotides capable of specifically hybridizing with a sequence complementary to a nucleic acid encoding p-Hyde is provided. The present invention provides antisense molecules, triplex oligonucleotides, or ribozymes that can specifically hybridize with isolated nucleic acids encoding p-Hyde.

The present invention is for making a polypeptide comprising growing a host vector system under suitable conditions which permit the production of the polypeptide, and recovering the polypeptide so produced. To provide a method. In one embodiment,
The method for obtaining the polypeptide in a purified form is as follows: (a) introducing the vector into a suitable host cell; (b) culturing the obtained cell to produce the polypeptide; (c) step (b) A.) Recovering the polypeptide produced in); and (d) purifying the polypeptide so recovered.

The present invention provides a polypeptide comprising the amino acid sequence of p-Hyde. The present invention provides fusion proteins or chimeras that include the polypeptides. The present invention provides an antibody that specifically binds to a polypeptide. The invention provides a pharmaceutical composition comprising an amount of polypeptide and a pharmaceutically effective carrier or diluent.

The present invention comprises (a) obtaining a suitable nucleic acid sample from a subject; and (b) step (a)
To determine whether the subject carries a mutation in the p-Hyde gene by determining whether the nucleic acid sample from is a nucleic acid encoding a mutant p-Hyde or is derived therefrom. What is provided is a method for determining whether a subject carries a mutation in the p-Hyde gene, comprising: In one embodiment,
The nucleic acid sample in step (a) contains mRNA corresponding to a transcript of DNA encoding the mutant p-Hyde, and the determination in step (b) allows (i) binding of the mRNA with the oligonucleotide. Contacting the mRNA with an oligonucleotide to form a complex under conditions; (ii) isolating the complex thus formed; and (iii) identifying the mRNA within the isolated complex By determining whether the mRNA is or is derived from a nucleic acid encoding a mutant p-Hyde.

The present invention is a method for screening a tumor sample from a human subject for somatic modification of the p-Hyde gene in the tumor, the method comprising:
A first selected from the group consisting of: a Hyde gene, a p-Hyde RNA derived from the tumor, and a p-Hyde cDNA prepared from mRNA derived from the tumor sample.
Of p-Hyde gene derived from the non-tumor sample of the subject, p-Hyde RNA derived from the non-tumor sample, and p-H prepared from mRNA derived from the non-tumor sample.
yde cDNA compared to a second sequence selected from the group consisting of the p-Hyde gene, p-Hyde RNA, or p-Hyde from the tumor sample.
cDNA sequence, p-Hyde gene, p-Hyde R from the non-tumor sample
Differences with the sequence of NA or p-Hyde cDNA were detected in p-Hy in the tumor sample.
Provided is a method comprising using as an index of somatic modification of de gene.

The present invention is a method for screening tumor samples from human subjects for the presence of somatic alterations of the p-Hyde gene in the tumor, for analyzing differences between polypeptides, The p-Hyde polypeptide from the tumor sample from the subject is compared to a p-Hyde polypeptide from a non-tumor sample from the subject, wherein the non- P- from tumor sample
A method comprising: using a difference from a Hyde polypeptide as an indicator of the presence of somatic modification of the p-Hyde gene in the tumor sample, wherein the comparison comprises (i) the full length polypeptide in each sample. Or detecting any of the truncated polypeptides, or (ii) an epitope of the modified p-Hyde polypeptide or a wild-type p-
A method performed by contacting an antibody that specifically binds to any of the epitopes of the Hyde polypeptide with the p-Hyde polypeptide from each sample and detecting antibody binding.

The present invention provides (a) under conditions that allow the binding of p-Hyde with a chemical compound,
Contacting p-Hyde with a chemical compound; (b) Chemical compound and p-Hy
identifying a chemical compound capable of inducing susceptibility to cell death by detecting specific binding to de; and (c) determining whether the chemical compound inhibits p-Hyde. What is provided is a method for identifying a chemical compound capable of inducing susceptibility to cell death, comprising:

The present invention comprises the steps of obtaining cells and a deletion in the E1 and E3 regions of the genome,
And contacting a cell of interest with a replication-defective adenovirus type 5 expression vector containing an adenovirus genome having an insertion of a nucleic acid encoding p-Hyde under the control of the Rous sarcoma virus promoter in its region, Provided is a method of inhibiting the growth of cancer cells, comprising the step of inhibiting the growth of prostate cancer cells.

The present invention comprises: 1) obtaining a sample of prostate cells from a subject; 2) inserting a deletion in the E1 and E3 regions of the genome and inserting a p-Hyde cDNA under the control of the Rous sarcoma virus promoter. Contacting a cell with a replication-defective adenovirus type 5 expression vector containing an adenovirus genome having a gene in its region; and 3
Inhibiting the growth of cancer cells by introducing the cells into a subject. The method comprises the steps of: inhibiting the growth of cancer cells.

The present invention provides a quantity of a nucleic acid encoding a p-Hyde protein, p-Hyd.
Introducing a nucleic acid encoding a fragment of e protein or a nucleic acid encoding a mutant p-Hyde protein into cancer cells to suppress the growth of cancer cells in the subject, and Provide a way to suppress.

The present invention provides a therapeutically effective amount of a nucleic acid encoding a p-Hyde protein, p.
In a subject by administering to the subject a pharmaceutical composition comprising a nucleic acid encoding a fragment of the Hyde protein or a nucleic acid encoding the mutant p-Hyde protein and a pharmaceutically acceptable carrier or diluent. Provided is a method of suppressing the growth of cancer cells in a subject, which comprises suppressing the growth of cancer cells.

The present invention provides a quantity of a nucleic acid encoding a p-Hyde protein, p-Hyd.
Inducing susceptibility to apoptosis of a cancer cell in a subject, which comprises inducing susceptibility to apoptosis by introducing a nucleic acid encoding a fragment of e protein or a nucleic acid encoding a mutant p-Hyde protein into the cancer cell Provide a way to do.

The present invention provides a therapeutically effective amount of a nucleic acid encoding a p-Hyde protein, p
Against apoptosis by administering to a subject a pharmaceutical composition comprising a nucleic acid encoding a fragment of Hyde protein or a nucleic acid encoding a mutant p-Hyde protein and a pharmaceutically acceptable carrier or diluent. Provided is a method of inducing susceptibility of cancer cells to apoptosis in a subject, comprising inducing susceptibility.

The present invention provides a therapeutically effective amount of a nucleic acid encoding a p-Hyde protein,
By administering to a subject a pharmaceutical composition comprising a nucleic acid encoding a fragment of p-Hyde protein or a nucleic acid encoding a mutant p-Hyde protein and a pharmaceutically acceptable carrier or diluent, There is provided a method of treating a subject having cancer, comprising treating a subject having.

The present invention relates to 1) a therapeutically effective amount of a nucleic acid encoding a p-Hyde protein, a nucleic acid encoding a fragment of a p-Hyde protein in combination with radiation, chemotherapy or a UV mimetic. Or a nucleic acid encoding a mutant p-Hyde protein, and 2) administering a pharmaceutical composition containing a pharmaceutically acceptable carrier or diluent to the subject, thereby treating the subject with cancer. Methods of treating a subject having cancer are provided.

The present invention has a therapeutically effective amount of 1) a deletion in the E1 and E3 regions of the genome,
And a replication-defective adenovirus type 5 expression vector containing an adenovirus genome having an insertion of the full-length sense p-Hyde cDNA under the control of the Rous sarcoma virus promoter in its region, and 2) a suitable carrier or diluent Provided is a method of treating a subject with cancer, comprising treating the subject with cancer by administering the pharmaceutical composition to the subject. In one embodiment, the cancer is selected from the group consisting of: melanoma; lymphoma; leukemia; and prostate, colorectal, pancreatic, breast, brain, or gastric carcinoma.

Finally, the present invention provides the means necessary to create gene-based therapies directed at cancer cells. These therapeutic agents place all or part of the p-Hyde locus, which is placed in a suitable vector or more directly transported to the target cell, so that the function of the p-Hyde protein is reproduced. It may be in the form of a polynucleotide containing it. The therapeutic agent may also take the form of a polypeptide based on either part or all of the p-Hyde protein sequence. These are the in vivo pH
It functionally replenishes the activity of yde.

BEST MODE FOR CARRYING OUT THE INVENTION The present invention provides a novel gene class that acts as an inhibitor of DNA repair enzyme and induces susceptibility of cancer cells to cell death. Functionally, p-Hyde is
It has been associated with inhibition of tumor growth in vivo and increased susceptibility to apoptosis induced by UV irradiation or FUrD treatment. The up-regulation of apoptosis by UV damage correlates with the presence of intact photoproducts in prostate cancer cell lines stably transfected with p-Hyde. As monotherapy
Or p-Hy in human gene therapy in combination with radiation or chemotherapy
The use of de is useful against cancer or hyperproliferative human diseases. In one embodiment, a protein class such as P-Hyde is a nucleotide excision repair (NER) pathway in prostate cancer, MGMT CN in colon cancer cell lines.
A repair pathway, O ₆ methylguanine methyltransferase enzyme (O ₆ MgMT)
And a DNA repair enzyme inhibitor that downregulates the 6,4 photoproduct (6,4PP). The gene class is characterized by the inclusion of a leucine zipper binding domain and a death domain that causes cell apoptosis. Human p-Hyde (I) and human p-Hyde40 (I
I) is an example described herein of a family of molecules with certain homologous sequences and conserved structural and functional characteristics (“p-Hyde family”).

The present invention provides isolated nucleic acids (alleles, analogs, fragments, mimetics, variants, compounds thereof) encoding a mammalian p-Hyde protein that induces susceptibility of cancer cells to cell death. , Or a variant). The present invention provides isolated nucleic acids (alleles, analogs, fragments, mimetics, variants, compounds, or variants thereof) encoding human p-Hyde proteins that induce susceptibility of cancer cells to cell death. Including).

As used interchangeably herein, “p-Hyde activity”, “p
"Hyde biological activity" or "p-Hyde functional activity" refers to a p-Hyde protein, polypeptide, as determined in vivo or in vitro according to standard techniques for inducing cell apoptosis. , Or the nucleic acid molecule is pH
Activity on yde-responsive cells. p-Hyde means p-Hyde
Refers to a gene family that has activity. An example of such a gene is rat pH
yde, human p-Hyde (I), and human p-Hyde40 (II).
In one embodiment, these sequences are at the nucleotide level SEQ ID NO: 7.
Including SEQ ID NO: 8 at the amino acid level. In addition, the family p-Hyde
Examples of gene members of, include the isolated nucleotide sequence of SEQ ID NO: 1, 3, 5 or 7 at the nucleotide level and SEQ ID NO: 2 at the amino acid level,
Included are 4, 6 or 8 amino acid sequences.

In one embodiment, the p-Hyde gene has a nucleotide sequence that has at least 85% similarity to the nucleic acid encoding the sequence of SEQ ID NO: 1.
In another embodiment, the nucleic acid has a nucleotide sequence that has at least 87% similarity to the nucleic acid encoding the sequence of SEQ ID NO: 1. In another embodiment, the nucleic acid has a nucleotide sequence that has at least 90% similarity to the nucleic acid encoding the sequence of SEQ ID NO: 1. In another embodiment, the nucleic acid has a nucleotide sequence that has at least 95% similarity to the nucleic acid encoding the sequence of SEQ ID NO: 1. In another embodiment, the nucleic acid fragment comprises a fragment starting with the nucleic acid at position 1 of SEQ ID NO: 1 and ending at position 557 of SEQ ID NO: 1. In another embodiment, the nucleic acid comprises a fragment starting at position 1 of SEQ ID NO: 1 and ending at position 158 of SEQ ID NO: 1. In another embodiment, the nucleic acid starts at the nucleic acid at position 50 of SEQ ID NO: 1 and ends at 120 of SEQ ID NO: 1.
Includes fragments that end in position. The nucleic acid is DNA, cDNA, genomic DNA, or RNA.

Human p-Hyde Nucleic Acid Coding Region: (SEQ ID NO: 1) In another embodiment, the nucleic acid encodes an amino acid sequence having a sequence as set forth in SEQ ID NO: 2. In one embodiment, the amino acid fragment is
It includes a fragment starting at amino acid 1 of SEQ ID NO: 2 and ending at position 101. In one embodiment, the amino acid fragment begins at the amino acid at position 1 of SEQ ID NO: 2, 8
It contains a fragment that ends at position 0. In one embodiment, the amino acid fragment comprises a fragment starting at the amino acid at position 1 and ending at position 60 of SEQ ID NO: 2. In one embodiment the amino acid is at least 85 with the nucleic acid encoding the sequence of SEQ ID NO: 2.
It has a% similarity. In another embodiment, the amino acids have at least 90% similarity with the nucleic acid encoding the sequence of SEQ ID NO: 2. In another embodiment, the amino acids have at least 95% similarity to the nucleic acid encoding the sequence of SEQ ID NO: 2.

Amino Acid Sequence of Human p-Hyde: (SEQ ID NO: 2) In one embodiment, the p-Hyde gene is a nucleotide having at least 75% similarity to the nucleic acid encoding the sequence of SEQ ID NO: 3. Has an array.
In another embodiment, the nucleic acid has a nucleotide sequence that has at least 85% similarity to the nucleic acid encoding the sequence of SEQ ID NO: 3. In another embodiment, the nucleic acid has a nucleotide sequence that has at least 90% similarity to the nucleic acid encoding the sequence of SEQ ID NO: 3. In another embodiment, the nucleic acid has a nucleotide sequence that has at least 95% similarity to the nucleic acid encoding the sequence of SEQ ID NO: 3. In another embodiment, the nucleic acid fragment comprises a fragment starting with the nucleic acid at position 1 of SEQ ID NO: 3 and ending at position 557 of SEQ ID NO: 3. In another embodiment, the nucleic acid comprises a fragment that begins at position 1 of SEQ ID NO: 3 and ends at position 158 of SEQ ID NO: 3. In another embodiment, the nucleic acid begins at the nucleic acid at position 50 of SEQ ID NO: 3 and is 120 of SEQ ID NO: 3.
Includes fragments that end in position. The nucleic acid is DNA, cDNA, genomic DNA, or RNA.

Nucleic acid sequence of human p-Hyde40: (SEQ ID NO: 3) In another embodiment, the nucleic acid encodes an amino acid sequence having a sequence as set forth in SEQ ID NO: 4. In one embodiment, the amino acid fragment is
It includes a fragment starting at amino acid 1 of SEQ ID NO: 4 and ending at position 101. In one embodiment, the amino acid fragment begins at the amino acid at position 1 of SEQ ID NO: 4,
It contains a fragment that ends at position 0. In one embodiment, the amino acid fragment comprises a fragment beginning at the amino acid at position 1 and ending at position 60 of SEQ ID NO: 4. In one embodiment the amino acid is at least 85 with the nucleic acid encoding the sequence of SEQ ID NO: 4.
It has a% similarity. In another embodiment, the amino acids have at least 90% similarity to the nucleic acid encoding the sequence of SEQ ID NO: 4. In another embodiment, the amino acids have at least 95% similarity to the nucleic acid encoding the sequence of SEQ ID NO: 4.

Amino Acid Sequence of Human p-Hyde40: (SEQ ID NO: 4) In one embodiment, the p-Hyde gene is a nucleotide having at least 75% similarity to the nucleic acid encoding the sequence of SEQ ID NO: 5. Has an array.
In another embodiment, the nucleic acid has a nucleotide sequence that has at least 85% similarity to the nucleic acid encoding the sequence of SEQ ID NO: 5. In another embodiment, the nucleic acid has a nucleotide sequence that has at least 90% similarity to the nucleic acid encoding the sequence of SEQ ID NO: 5. In another embodiment, the nucleic acid has a nucleotide sequence that has at least 95% similarity to the nucleic acid encoding the sequence of SEQ ID NO: 5. In another embodiment, the nucleic acid fragment comprises a fragment starting with the nucleic acid at position 1 of SEQ ID NO: 5 and ending at position 557 of SEQ ID NO: 5. In another embodiment, the nucleic acid comprises a fragment that begins with the nucleic acid at position 1 of SEQ ID NO: 5 and ends at position 158 of SEQ ID NO: 5. In another embodiment, the nucleic acid begins at the nucleic acid at position 50 of SEQ ID NO: 5 and ends at 120 of SEQ ID NO: 5.
Includes fragments that end in position. The nucleic acid is DNA, cDNA, genomic DNA, or RNA.

Rat p-Hyde Nucleic Acid Sequence: (SEQ ID NO: 5) In another embodiment, the nucleic acid encodes an amino acid sequence having a sequence as set forth in SEQ ID NO: 6. In one embodiment, the amino acid fragment is
It includes a fragment starting at amino acid 1 of SEQ ID NO: 6 and ending at position 101. In one embodiment, the amino acid fragment begins at the amino acid at position 1 of SEQ ID NO: 6,
It contains a fragment that ends at position 0. In one embodiment, the amino acid fragment comprises a fragment starting at the amino acid at position 1 and ending at position 60 of SEQ ID NO: 2. In one embodiment, the amino acid fragment comprises a fragment beginning at amino acid position 61 and ending at position 241 of SEQ ID NO: 6. In one embodiment, the amino acid fragment is SEQ ID NO: 6.
It includes a fragment starting at amino acid position 81 and ending at position 361. In one embodiment, the amino acid fragment comprises a fragment starting at amino acid position 81 and ending at position 421 of SEQ ID NO: 2. In another embodiment, the amino acid is SEQ ID NO: 6.
Of at least 70% similarity to the nucleic acid encoding the sequence of In another embodiment, the amino acids have at least 75% similarity to the nucleic acid encoding the sequence of SEQ ID NO: 6. In another embodiment, the amino acids have at least 80% similarity to the nucleic acid encoding the sequence of SEQ ID NO: 6. In one embodiment the amino acid has at least 85% similarity to the nucleic acid encoding the sequence of SEQ ID NO: 6. In another embodiment, the amino acids have at least 90% similarity to the nucleic acid encoding the sequence of SEQ ID NO: 6. In another embodiment, the amino acids have at least 95% similarity to the nucleic acid encoding the sequence of SEQ ID NO: 6.

Amino acid sequence of rat p-Hyde: (SEQ ID NO: 6) Further, the present invention provides an isolated nucleic acid encoding an amino acid sequence tccctgg.
ccacacgcctgtgtggggctctggctctc (SEQ ID NO: 7) is provided. Furthermore, the present invention relates to the amino acid sequence AAPCVAYVLLSLVYLPG.
VLAAALQLLRGTKYQRFPDWLDHWLQHRKQIGLLSF
Provided is an isolated nucleic acid encoding F (SEQ ID NO: 8). Furthermore, the invention provides an isolated nucleic acid encoding the amino acid sequence NFIRDVLQPYIRKDENK.

Due to the degeneracy of the genetic code, the present invention is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5.
, Or is different from the nucleotide sequence of SEQ ID NO: 7, thus SEQ ID NO: 1,
Further included is a nucleic acid molecule encoding a p-Hyde protein identical to the protein encoded by the nucleotide sequence set forth in SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7. Those skilled in the art will appreciate that DNA sequence polymorphisms that result in changes in the amino acid sequence of p-Hyde may exist within a population (eg, the human population). Such genetic polymorphisms in the p-Hyde gene may exist among individuals within a population due to natural allelic variation. An allele is one of a group of genes that are alternatively present at a particular locus. Such natural allelic variations typically result in 1-5% variance of the nucleotide sequence of the p-Hyde gene. Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals. This can be easily done in a variety of individuals by using hybridization probes to identify the same locus. Any and all such nucleotide variations in p-Hyde that result from natural allelic variation and do not alter the functional activity of p-Hyde, and resulting amino acid polymorphisms are included within the scope of the invention. Shall be provided. Furthermore, a nucleic acid molecule encoding a p-Hyde protein (p-Hyde homolog) derived from another species having a nucleotide sequence different from the nucleotide sequence of human p-Hyde is also included in the scope of the present invention. And P-Hyde cDNA of the present invention
Nucleic acid molecules corresponding to naturally occurring allelic variants and homologues of A can be prepared using human cDNA or a portion thereof as a hybridization probe under stringent hybridization conditions according to standard hybridization techniques. It can be isolated based on its identity to the human p-Hyde nucleic acid disclosed in the specification. For example, splice variants of human and mouse p-Hyde cDNA can be isolated based on their identity with human and mouse p-Hyde.

As used herein, the term “hybridize under stringent conditions” refers to nucleotide sequences that are at least 60% (65%, 70%, preferably 75%) identical to each other. The conditions of hybridization and washing, which typically remain hybridized to each other, shall be stated. Such stringent conditions are known to those of skill in the art and can be found in Current Pr
otocols in Molecular Biology, John Wi
ley & Sons, N.M. Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringent hybridization conditions is hybridization in 6 × sodium chloride / sodium citrate (SSC) at about 45 ° C., followed by 0.2 × SSC, 0 at 50-65 ° C.
． One or more washes in 1% SDS. Preferably under stringent conditions with the coding or non-coding sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 7 (or “sense” or “antisense” sequences). An isolated nucleic acid molecule of the invention that hybridizes corresponds to a naturally occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a naturally-occurring nucleotide sequence (eg, encoding a native protein). P that can exist in the group
-In addition to naturally occurring allelic variants of the Hyde sequence, without altering the biological capacity of the p-Hyde protein, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7 One skilled in the art will further appreciate that introducing mutations into the nucleotide sequence can result in changes in the amino acid sequence of the encoded p-Hyde protein. Mutations can be introduced by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.

A “conservative amino acid substitution” is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. A family of amino acid residues with similar side chains has been defined in the art. These families include amino acids with basic side chains (eg lysine, arginine, histidine), amino acids with acidic basic side chains (eg aspartic acid, glutamic acid), amino acids with uncharged polar side chains. (For example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having a non-polar side chain (for example, alanine, valine, leucine, isoleucine, proline, phenylalanine,
Methionine, tryptophan), amino acids with beta branched side chains (eg threonine, valine, isoleucine) and amino acids with aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). Therefore, a predicted nonessential amino acid residue in p-Hyde is preferably replaced with another amino acid residue of the same side chain family. Alternatively, mutations are randomly introduced into all or part of p-Hyde coding by saturation mutagenesis or the like, and the resulting mutants are screened for P-Hyde biological activity. It can also be identified. After mutagenesis, the encoded protein can be recombinantly expressed and the activity of the protein can be determined.

Amino acid residues that are conserved between p-Hyde proteins of different species are not modified (except for conservative substitutions) in order to avoid greatly diminishing or eliminating biological activity. ). Both mouse and human p-Hyde proteins have a conserved pattern of 6 cysteine residues. Such conserved domains and cysteine residues are less likely to tolerate mutations. However, other amino acid residues, such as those that are not conserved or only semiconserved between p-Hyde of various species, such as between mouse and human p-Hyde, are not active. May not be essential to, and therefore is likely to accept, the modification. Thus, another aspect of the invention relates to nucleic acid molecules encoding p-Hyde proteins that contain changes in amino acid residues that are not essential for activity. Such a p-Hyde protein differs in amino acid sequence from SEQ ID NO: 2 or SEQ ID NO: 4, but retains biological activity. In one embodiment, the isolated nucleic acid molecule has an amino acid sequence that is at least about 84%, 85%, 90%, 95%, or 98% identical to the amino acid sequence of SEQ ID NOs: 2, 4, 6, or 8. It includes a nucleotide sequence that encodes a protein that includes the sequence.

As will be readily appreciated by those skilled in the art, p-Hyde-encoding nucleotides include both sense and antisense strands of RNA, cDNA, genomic DNA, synthetic and mixed polymers. , May be chemically or biochemically modified, or may contain non-natural or derivatized nucleotide bases. Such modifications include, for example, labeling, methylation, substitution of one or more naturally occurring nucleotides with analogs, uncharged linkages (eg, methylphosphonates, phosphotriesters, phosphoamidates, Carbamate, etc.), internucleotide modifications such as charged bonds (eg, phosphorothioate, phosphorodithioate, etc.), pendant moieties (eg, polypeptides), intercalators (eg, acridine, psoralen, etc.),
Included are chelating agents, alkylating agents, and modified linkages such as alpha anomeric nucleic acids. Also included are synthetic molecules that exhibit a similar ability to nucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art, eg, are substantially homologous to the primary structural sequence but include, for example, in vivo or in vitro chemical and biochemical modifications, or incorporate uncommon amino acids. The peptide bonds that are included include those that replace the phosphate bonds of the molecular backbone. The nucleic acid may be modified. As will be readily appreciated by those skilled in the art, such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, eg labeling with radionuclides, and various enzymatic modifications. . A variety of polypeptide labeling methods and substituents or labels useful for such purposes are well known in the art and include radioisotopes such as ³² P, labeled anti-ligands (eg, (Antibodies), fluorophores, chemiluminescent agents, enzymes, and antiligands that can act as members of the specific binding pair for the labeled ligand. The choice of label will depend on the sensitivity required, the ease with which the primer will bind, the stability requirements, and the equipment available. Polypeptide labeling methods are well known in the art. For example, Sambrook et
al. , 1989 or Ausubel et al. , 1992.
In addition to substantially full-length p-Hyde, the present invention provides biologically active p-Hyde fragments known to those of skill in the art.

“Isolated” or “substantially pure” as defined herein
Nucleic acid (eg, RNA, DNA, or mixed polymers) means substantially any other cellular component naturally associated with native human sequences or proteins, such as ribosomes, polymerases, and many other human genomic sequences and proteins. Are separated into. The term includes nucleic acid sequences or proteins that have been removed from their naturally occurring environment, including recombinant or cloned DNA isolates, and chemically synthesized analogs or heterologs. Included are analogs biologically synthesized by the system.

“P-Hyde allele” refers to the normal allele of the p-Hyde locus,
And alleles that carry variability that predisposes an individual to developing cancers at many sites, including, for example, breast, ovarian, colorectal, and prostate cancers. Such a predisposition allele is also called a "p-Hyde susceptibility allele".

“P-Hyde locus”, “p-Hyde gene”, “p-Hyde nucleic acid”
, Or “p-Hyde polynucleotide” means a polynucleotide having p-Hyde activity, which is highly likely to be expressed in normal tissues, and is all present in the p-Hyde region (a certain allele). But breast, ovary, colorectal,
And predispose the individual to developing prostate cancer). Mutations in the p-Hyde locus may also be involved in the initiation and / or progression of other types of tumors. The locus is partly indicated by a mutation that predisposes the individual to developing cancer. These mutations are described in p-Hyde below.
Included in the area. The p-Hyde locus shall include coding sequences, intervening sequences, and regulatory elements that regulate transcription and / or translation. The p-Hyde locus shall include all allelic variations of the DNA sequence.

“Nucleic acid” refers to a ribonucleoside (adenosine, guanosine, uridine, or cytidine; “RNA molecule”) or deoxyribonucleoside (deoxyadenosine, deoxyguanosine, single-stranded form or double-stranded helix, Deoxythymidine, or deoxycytidine; "DNA molecule") refers to the phosphoric acid ester polymerized form. Double-stranded helices of DNA-DNA, DNA-RNA, and RNA-RNA are possible. The term nucleic acid molecule, in particular DNA molecule or RNA molecule, refers only to the primary and secondary structure of the molecule and is not limited to a particular tertiary form. Thus, the term specifically includes double-stranded DNA found in linear or circular DNA molecules (eg, restriction fragments), plasmids, and chromosomes. When discussing the structure of a particular double-stranded DNA molecule, the sequence is such that only the sequence on the non-transcribed strand of DNA (ie, the strand having a sequence homologous to mRNA) is given in the 5'to 3'direction. It may be described herein according to convention. "Recombinant DNA" is DNA that has undergone molecular biological manipulation.

The phrase “nucleic acid encoding” refers to a nucleic acid molecule that directs the expression of a particular protein or peptide. Nucleic acid sequences include both DNA strand sequences that are transcribed into RNA and RNA sequences that are translated into proteins. Nucleic acid molecules include both full length nucleic acid sequences and non-full length sequences derived from full length proteins. It is further understood that the sequences include degenerate codons of the native sequence (s) that can be introduced to provide codon preference in a particular host cell.

A “recombinant nucleic acid” is a non-naturally occurring nucleic acid or a nucleic acid created by artificial recombination of two originally isolated sequence segments. This artificial recombination is often accomplished either by chemical synthetic means or by artificial manipulation of isolated nucleic acid segments, such as genetic engineering techniques. They replace certain codons with redundant codons that encode the same or conservative amino acids and are typically performed to introduce or remove sequence recognition sites. Alternatively, it is performed to join nucleic acid segments with the desired function, to produce the desired combination of functions.

As used herein, the term “cancer cell” means a tissue that proliferates through cell proliferation more rapidly than normal, eg, faster than adjacent cells or other cells within the tissue. To do. Neoplastic cells continue to grow even after the growth stimulus has stopped. In general, tumors exhibit or form different tissue weights. The present invention relates to both types of tumors, but is particularly valuable in the treatment of cancer.

In one embodiment, the cancer cells are selected from the group consisting of melanoma; lymphoma; leukemia; and prostate, colorectal, pancreatic, breast, brain, or gastric cancer. Examples of tumors include, but are not limited to, sarcomas and carcinomas, such as, but not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
Endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, periosteoma, mesothelioma, Ewing sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer , Squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma,
Choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, germ tum
or), non-small cell lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma,
Includes medulloblastoma, craniopharyngioma, ependymoma, pineocytoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. In a preferred embodiment, the tumor is melanoma or prostate cells.

Mutations can be made in the nucleic acid encoding p-Hyde such that specific codons are changed to codons encoding different amino acids, but the induction of susceptibility to cell death is maintained. Such mutations are generally made by making minimal nucleotide changes. This kind of substitution mutation changes the codons of the amino acids in the resulting protein in a non-conservative manner (ie, from amino acids belonging to a group of amino acids having a particular size or characteristic to amino acids belonging to another group). ) Or conservatively (ie, by changing codons from amino acids belonging to a group of amino acids having a particular size or characteristic to amino acids belonging to the same group). Such conservative changes generally result in relatively few changes in structure and function of the resulting protein. Non-conservative changes are likely to alter the structure, activity, or function of the resulting protein. The present invention should be considered to include sequences containing conservative changes that do not significantly alter the activity or binding characteristics of the resulting protein. Substitutions for amino acids in the sequence may be selected from other members of the class to which the amino acid belongs. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Amino acids containing aromatic ring structures are phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine.
Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such modifications are not expected to affect the apparent molecular weight as determined by polyacrylamide gel electrophoresis or isoelectric point. This isolated nucleic acid also encodes a mutant p-Hyde or wild-type protein.

The present invention relates to D of the present invention cloned into an expression vector in the antisense orientation.
Further provided is a recombinant expression vector containing the NA molecule. That is, the DNA molecule is
It is operably linked to regulatory sequences in a manner that allows expression (by transcription of a DNA molecule) of an RNA molecule that is antisense to p-Hyde mRNA. A control sequence, such as a viral promoter and / or enhancer, may be chosen which directs the continuous expression of the antisense RNA molecule in a variety of cell types and is operably linked to the nucleic acid cloned in the antisense orientation, or Regulatory sequences which direct the expression of constitutive, tissue-specific, or cell-type specific antisense RNA may be selected. Antisense expression vectors are forms of recombinant plasmids, phagemids, or attenuated viruses in which the antisense nucleic acid is produced under the control of high efficiency control regions, with activity that can be determined by the cell type into which the vector has been introduced. obtain. For a discussion on the regulation of gene expression using antisense genes, see
Weintraub et al. (Reviews-Trends in G
enetics, Vol. 1 (1) 1986).

The present invention provides a replicable vector containing an isolated nucleic acid molecule of a DNA virus. Vectors include, but are not limited to, plasmids, cosmids, phages, or yeast artificial chromosomes (Y containing at least a portion of an isolated nucleic acid molecule.
AC) is included. As an example for obtaining these vectors, both the insert sequence and the vector DNA are exposed to restriction enzymes to create complementary ends that base pair with each other on both molecules, which are then digested with DNA ligase. Can be ligated. Or, insert a linker corresponding to a restriction site in the vector DNA.
It is also possible to ligate to A and then digest it with a restriction enzyme that cuts at that site. Other means are available and known to those skilled in the art. In one embodiment, the adenovirus vector is a replication defective adenovirus type 5 expression vector. In another embodiment, the adenovirus vector has an adenovirus genome having deletions in the E1 and E3 regions of the genome and having an insertion under the control of the promoter of the nucleic acid encoding p-Hyde within that region. Including. The promoter can be the Rous Sarcoma Virus promoter.

Knowledge of the genetic makeup of adenovirus, a 36 kB linear double-stranded DNA virus, allows the replacement of large pieces of adenovirus DNA with foreign sequences up to 7 kB (Grunhaus and Horwitz, 1992). . In contrast to retroviruses, infection of adenovirus DNA into host cells does not result in chromosomal integration, since adenovirus DNA can replicate episomally without potential genotoxicity. Also, adenovirus is structurally stable and no genomic rearrangements have been detected after many amplifications. Adenovirus can infect virtually all epithelial cells regardless of cell cycle stage. So far, adenovirus infections have been thought to be associated with only mild disease in humans, such as acute respiratory disease.

Adenovirus is a gene transfer vector because of its medium-sized genome, easy manipulation, high titer, wide target cell range, and high infectivity. Especially suitable for use. Both ends of the viral genome are 100-2, which are cis factors required for viral DNA replication and packaging.
It contains an inverted base repeat sequence (ITR) of 00 base pairs. The early region (E) and the late region (L) of the genome contain different transcription units that are split at the start of viral DNA replication. The E1 region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and several cellular genes. E2 (E2
Expression of A and E2B) leads to the synthesis of proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell shut off (Renan, 1990).
The products of late genes, which contain most of the viral capsid protein, are expressed only after significant processing of a single primary transcript produced by the major late promoter (MLP). MLPs (located at 16.8 mu) become particularly efficient late in infection, and all mRNAs generated from this promoter have a three-component leader (TL) sequence that makes them a preferred mRNA for translation. I have 5 '.

In the current system, recombinant adenovirus is produced by homologous recombination between shuttle vector and provirus vector. Wild type adenovirus can be generated from this process because of the recombination between the two proviral vectors. Therefore, it is important to isolate a single viral clone from each plaque and investigate its genomic structure. The use of the YAC system is an alternative method for producing recombinant adenovirus.

The production and propagation of current adenovirus vectors that are replication-defective has been limited to Ad5.
It depends on a special helper cell line called 293 which is transformed from human embryonic kidney cells with a DNA fragment and constitutively expresses E1 protein (Grah).
am, et al. , 1977). Since the E3 region can be deleted from the adenovirus genome (Jones and Shenk, 1978), the current adenovirus vectors assisted by 293 cells carry foreign DNA in either or both of the E1, E3 regions. (Graham and Prevec, 19
91). Naturally, adenovirus packages approximately 105% of the wild-type genome (Ghosh-Choudhury, et al., 1987), approximately 2k.
B can provide additional DNA capacity. Combined with approximately 5.5 kB of exchangeable DNA in the E1 and E3 regions, the current maximum capacity of adenovirus vectors is less than 7.5 kB, or about 15% of the total length of the vector.
Is. Over 80% of the adenovirus genome remains in the vector backbone, which is the source of vector-derived cytotoxicity. Also, the replication deficiency of the E1 deleted virus is incomplete. For example, leakage of viral gene expression has been observed with currently available adenoviral vectors at high multiplicity of infection (Mulliga).
n, 1993).

Helper cell lines can be derived from human cells, such as human fetal kidney cells, muscle cells, hematopoietic cells, or other human fetal mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from cells of other mammalian species that are permissive for human adenovirus. Such cells include, for example, Vero cells, or other monkey fetal mesenchymal or epithelial cells. As mentioned above, the preferred helper cell line is 293.

Except for the requirement that the adenovirus vector is replication defective, or at least conditionally replication defective, the nature of the adenovirus virus is not considered critical to the successful practice of the invention. The adenovirus may be of any of the 42 different known serotypes or AF subgroups. Adenovirus 5 of subgroup C
The mold is the preferred starting material for obtaining a conditional replication-defective adenovirus vector for use in the methods of the invention. Adenovirus type 5,
This is because the human adenovirus, whose biochemical and genetic information is known in detail, has been historically used for most of the construction using adenovirus as a vector.

As mentioned above, typical vectors of the invention will be replication defective and will not have the adenovirus E1 region. Therefore, it would be most convenient to introduce the nucleic acid encoding p-Hyde at the position where the E1 coding sequence was removed. However, the position of insertion of the p-Hyde coding region within the adenovirus sequence is not critical to the invention. Nucleic acids encoding p-Hyde transcription units are described by Karlsson et al. Instead of the deleted E3 region in the E3 exchange vector, as described previously by (1986), or in the E4 region where a helper cell line or helper virus complements the E4 deficiency.

Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo. This group of viruses is highly infectious, obtained with high titers, eg 10 ⁹ -10 ¹¹ plaque forming units (PFU) / ml. The life cycle of adenovirus does not require integration into the host cell genome. Foreign genes delivered by adenovirus vectors are episomal and therefore have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus (Cough et al., 1963; Top et a.
l. , 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.

Adenovirus vectors are described in eukaryotic gene expression (Levrero et al.
, 1991; Gomez-Fox et al. , 1992) and vaccine development (Grunhaus and Horwitz, 1992; Grahama.
nd Prevec, 1992). Recently, animal studies have suggested that recombinant adenovirus could be used for gene therapy (St.
ratford-Perricaudet and Perricaudet,
1991; Stratford-Pricaudaud et al. , 1990; R
ich et al. , 1993). For administration experiments of recombinant adenovirus to different tissues, tracheal instillation (Rosenfeld et al., 1991; Ros
enfield et al. , 1992), intramuscular injection (Ragot et al.
． , 1993), peripheral intravenous injection (Herz and Gerard, 1993).
), And stereotaxic inoculation to the brain (Le Gal La Sale et al., 1).
993) are included.

Preferred promoters and other necessary vector sequences may be chosen to be functional in the host, and if preferred include those essentially associated with the p-Hyde gene. Examples of working combinations of cell lines and expression vectors are found in Sambrook et al. , 1989 or Ausubel et a
l. , 1992, and see, for example, Metzger et al.
, 1988. Many useful vectors are known in the art, including Stratagene, New England Biolabs, Promega Biotech (Pr).
commercially available from manufacturers such as Omega Biotech). tr
Promoters such as p, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters may be used in prokaryotic hosts. Useful yeast promoters include promoter regions for metal binding proteins, 3-phosphoglycerate kinase or enolase or glyceraldehyde-3-phosphate dehydrogenase, enzymes responsive to maltose and galactose utilization, and others. Suitable vectors and promoters for use in yeast expression are further described in Hitzeman et al. , EP 74,675A. A preferred non-native mammalian promoter is SV40 (Fi
ers et al. , 1978) or a promoter derived from rodent Moloney leukemia virus, avian sarcoma virus, adenovirus II, bovine papilloma virus or polyoma. In addition, the structure may be linked to an amplifiable gene (eg, DHFR) so that multiple copies of the gene can be made. For preferred enhancers and other expression control sequences, see "Enhancers and Prokaryotic Gene Expression (Ehancers and
Eukaryotic Gene Expression) ", Cold Spr
ing Harbor Press, Cold Spring Harbor.
N. Y. See also (1983).

While such expression vectors are capable of spontaneous replication, they are also replicable by methods well known in the art by being introduced into the genome of the host cell. Expression and cloning vectors may contain a selectable marker, a gene encoding a protein necessary for the survival or growth of the host cell transduced with the vector. The presence of this gene ensures growth of only those host cells expressing the insert. Typical selection genes are: a) conferring resistance to antibiotics or other toxic substrates such as ampicillin, neomycin, methotrexate, b) supplementing auxotrophy, or c) not available in complex media. It encodes a protein that supplies a gene encoding a key nutrient, such as D-alanine racemase for Bacillus. Selection of the appropriate selectable marker may depend on the host cell, and preferred markers for different hosts are well known in the art.

The resulting RNA or vector, into which the vector containing the nucleic acid of interest can be translated in vitro and introduced into host cells by well known methods, eg injection (see Kubo et al., 1988). Changes depending on the type of cell host, electroporation, calcium chloride, rubidium chloride,
By methods well known in the art, including transfection using calcium phosphate, DEAE-dextran or other substrates, micro-fire bombardment, lipofection, (where the vector is an infectious agent such as a retroviral genome) infection. , Can be directly introduced into host cells. Generally Sambrook e
t al. , 1989 and Ausubel et al. , 1992. Introduction of a polynucleotide into a host cell by any method known in the art, including among others those described above, is referred to herein as "trans".
information) ”. A cell into which the above-described nucleic acid has been introduced is also meant to include progeny of such a cell.

Common elements required for expression include a promoter or enhancer sequence for binding to the RNA polymerase and transcription initiation sequences for ribosome binding. For example, microbial expression vectors include a promoter such as the lac promoter, a Shine-Dalgarno sequence for transcription initiation, and the initiation codon AUG. Similarly, eukaryotic expression vectors include RNA polymerase I
Included are heterologous and homologous promoters for I, a downstream polyadenylation signal, a start codon AUG, and a stop codon for ribosome dissociation. Such vectors may be obtained commercially or assembled by methods well known in the art, such as those described by the methods generally described above for constructing vectors.

Viral promoters, cellular promoters / enhancers and inducible promoters / enhancers that can be used in combination with the nucleic acid encoding p-Hyde within the expression construct include: immunoglobulin heavy chains, immunoglobulin light chains Chain, T
-Cell receptors, HLA DQ alpha and DQ beta, beta-interferon, interleukin-2, interleukin-2 receptor, MHC class II5 alpha, MHC class II HLA-DR alpha, beta-actin, muscle creatinine kinase, prealbumin ( Transthyretin), elastase I, metal binding protein, collagenase, albumin gene, alpha-fetoprotein, tau-globulin, beta-globulin, c-fos.
, C-HA-ras, neural cell adhesion molecule (NCAM), alpha1-antitrypsin, H2B (TH2B) histone, mouse or type I collagen, glucose regulatory proteins (GRP94 and GRP78), rat growth hormone, human serum amyloid A. (SAA), troponin I (TN I), platelet-derived growth factor, Duchenne muscle, SV40, polyoma, retrovirus, papillomavirus,
Hepatitis B virus, human immunodeficiency virus, cytomegalovirus, gibbon Ape leukemia virus, MT II, MMTV (mouse mammary glucocorticoid, adenovirus 5E2 include, but are not limited to.

Furthermore, any promoter / enhancer combination (Eukaryotic Promoter Data Base; E
(According to PDB) can also be used to drive the expression of p-Hyde. T
The use of the 3, T7 or SP6 transcription expression system is another possible form. Eukaryotic cells can support cytoplasmic transcription from specific microbial promoters when the preferred microbial polymerase is provided, either as part of the transduction complex or as an additional gene expression construct.

The present invention provides a host cell containing the above vector. The host cell may comprise an isolated DNA molecule that has been artificially introduced into the host cell. Host cells can be eukaryotic cells, microbial cells (such as E. coli), yeast cells, fungal cells, insect cells and animal cells. Suitable animal cells include Vero cells,
HeLa cells, Cos cells, CV1 cells and various primary mammalian cells include but are not limited to.

Another aspect of the present invention relates to a host cell into which the expression vector of the present invention has been introduced. The phrases "host cell" and "recombinant host cell (r
"ecobinant host cell)" are used interchangeably herein. It is understood that such phrases refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain alterations can be carried on to generations, either by mutagenesis or environmental influences,
Such progeny are not, in fact, identical to the parental cell, but are included within the term as used herein. The host cell can be any prokaryotic or eukaryotic cell. For example, the p-Hyde protein is E. coli.
, Insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells), and other suitable host cells known to those of skill in the art. Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the phrase "transformation".
on) ”and“ transfection ”refers to the introduction of foreign nucleic acid (eg, DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. It is intended to mean the various art-recognized techniques for doing so. Suitable methods for transforming or transfecting host cells are described in Sambrook, et al.
(See above) and other laboratory manuals. For stable transfection of mammalian cells, it is known that only a small fraction of cells can integrate foreign DNA into their genome, depending on the expression vector and transfection technique used. To identify and select for these integrations, generally a selectable marker (eg, for resistance to antibiotics) is introduced into the host cell along with the gene of interest.

Preferred selectable markers include those that provide resistance to agents such as G418, hygromycin and methotrexate. The nucleic acid encoding the selectable marker can be introduced into the host cell on a vector similar to that encoding p-Hyde or can be introduced on a separate vector. Cells that are stably transfected with the introduced nucleic acid can be identified by drug selection (eg, cells that have inserted the appropriate marker gene survive while other cells die). A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, is maintained at pH
It can be used to produce (ie, express) the yde protein.

Accordingly, the present invention further provides methods for producing p-Hyde protein using the host cells of the present invention. In one embodiment, the method comprises (
culturing a host cell of the invention (introducing a recombinant expression vector encoding p-Hyde) in a suitable medium so as to produce the p-Hyde protein. In another embodiment, the method further comprises p- from the medium or host cells.
Isolating Hyde. The host cells of the invention can also be used to produce nonhuman transgenic animals.

For example, in one embodiment, the host cell of the invention is a fertilized oocyte or fetal stem cell into which the p-Hyde coding sequence has been introduced. Such host cells can then be used to produce non-human transgenic animals in which the foreign p-Hyde sequences have been introduced into their genome or homologous recombinant animals from which the foreign p-Hyde sequences have been removed. Such animals are useful for studying p-Hyde function and / or activity, and for identifying and / or evaluating modulators of p-Hyde activity. As used herein, a “transgenic animal” is a non-human animal, preferably a mammal, more preferably a rat, or one or more cells of which contain a transgene. It is a rodent such as a mouse. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians and the like.

The transgenic animal of the present invention comprises introducing a p-Hyde-encoding nucleic acid into the male pronucleus of a fertilized oocyte by, for example, microinjection, retroviral infection, and introducing the oocyte into a pseudopregnant female. It can be made by developing in foster animals. For example (SEQ ID NO. 1, 3, 5 or 7)
A p-Hyde cDNA sequence such as those described above can be introduced as a transgene into the genome of non-human animals. Alternatively, a non-human homologue of the human p-Hyde gene can be isolated based on hybridization to the human p-Hyde cDNA and used as a transgene. Transducing sequences and polyadenylation signals may also be included within the transgene to increase the efficiency of expression of the transgene. Tissue-specific regulatory sequence (s) can be operably linked to the p-Hyde transgene to direct the direct expression of the p-Hyde protein in particular cells. Methods for producing transgenic animals via fetal manipulation and microinjection, especially animals such as mice, have become routine in the art, eg, US Pat. No. 4,736,866 and US Pat. 4,870,009,
U.S. Pat. No. 4,873,191, and Hogan, Mouse Fetal Manipulation (Ma.
nipulsing the Mouse Embry), (Cold S
pring Harbor Laboratory Press, Cold S
pring Harbor, N.M. Y. , 1986). Similar methods are used for the production of other transgenic animals. Transgenic founders can be identified based on the presence of the p-Hyde transgene in their genome and / or the expression of p-Hyde mRNA in the tissues or cells of the animal. The transgenic founder animal can then be used to breed additional animals carrying the transgene. Furthermore, transgenic animals carrying a transgene encoding p-Hyde can be bred to other transgenic animals carrying other transgenes.

To create a homologous recombinant animal, a vector is introduced into which an insertion, addition or substitution is introduced, whereby a p-Hyde gene (eg a human or non-human homologue of the p-Hyde gene, eg a rat. The dental p-Hyde gene) is altered and prepared to contain at least a portion of the p-Hyde gene, eg, functionally disrupted. In a preferred embodiment, the endogenous p-Hyde gene is
It is functionally disrupted (ie it no longer encodes a functional protein,
Called a "knock out" vector). Alternatively, the vector may be modified in homologous recombination such that the endogenous p-Hyde gene is mutagenized, otherwise altered, but still encodes a functional protein (eg, the upstream regulatory region may be altered, Can be designed to alter the expression of the endogenous p-Hyde protein).

The term “vector” refers to a viral expression system, a spontaneous self-replicating circular DNA (plasmid), including both expression and non-expression plasmids. Recombinant microorganisms or cell cultures have been described as "expression vector".
r) ”when described as being a host, this includes both extrachromosomal circular DNA and DNA integrated into the host chromosomes. If the vector is carried by the host cell, the vector may either be stably replicated by the cell during mitosis as a spontaneous structure or integrated into the host genome.

The phrase “plasmid” is a spontaneous circular DNA molecule capable of replicating inside a cell, including both expressed and non-expressed forms. When a recombinant microorganism or cell culture is described as hosting an "expression plasmid", it includes potential viral DNA integrated within the host chromosome (s). If the plasmid is carried by the host cell, the plasmid may either be stably replicated by the cell during mitosis as a spontaneous structure or integrated into the host genome.

The following terms are used to describe the sequence relationships between two or more nucleic acid molecules or polynucleotides. "Reference sequence (reference sequence)
ence) "," comparison window ",
"Sequence identity", "percentage of sequence identity", and "substantial identity". A "reference sequence" is a defined sequence used as the basis for sequence comparisons, where a comparison sequence is a subset of a larger sequence, such as a segment of full-length cDNA or gene sequence given in the sequence listing. Or may include the complete cDNA or gene sequence.

The optimal arrangement of the sequences for arranging the comparison windows is Smith and W.
aterman (1981) Adv. Appl. Math. 2: 482 by the local homology algorithm Needleman and Wunsch (19
70) J. Mol. Biol. 48: 443 by the homology sequencing algorithm of Pearson and Lipman (1988) Proc. Natl. A
cad. Sci. (USA) 85: 2444 similarity search methods, or the computational implementation of these algorithms (Wisconsin Ge).
NETICS SOFTWARE PACKAGE RELEASE 7.0,
Genetics Computer Group, 575 Science
Dr. , Madison, Wis., BESTFIT, FASTA and TFASTA).

“Substantial identity” or “substantial sequence identity”
y) ”is the program GAP or BESTFIT with default gap
At least 9 of the two peptide sequences when optionally sequenced, such as
It means to share 0 percent, preferably at least 95 percent sequence identity, more preferably at least 99 percent sequence identity or higher. "Percentage amino acid identity (percentage amino
acid identity) ”or“ percentage amino acid sequence identity (
percentage amino acid sequence identity
"ity)" indicates a comparison of the amino acids of two polypeptides that have an approximately designed percentage of the same amino acids when arranged arbitrarily. For example, "95% amino acid identity (95% amino acid identity
) "Refers to a comparison of the amino acids of two polypeptides that have 95% amino acid identity when arranged arbitrarily. Preferably, non-identical residue positions differ by conservative amino acid substitutions. For example, the substitution of amino acids with similar chemical properties such as charge or polarity may not affect the properties of the protein. Examples include glutamine in place of asparagine and glutamic acid in place of aspartic acid.

The percent identity between two sequences is a function of the number of identical positions shared by the sequences (ie,% identity / position of identical positions (eg overlap) # × 100). Preferably the two sequences are the same length. The determination of percent homology between two sequences can be performed using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm used for comparison of two sequences is
Karlin and Altschul (1993) Proc. Natl. A
cad. Sci. USA 90: 5873-5877, modified as in Karlin and Altschul (1990) Proc. Natl.
Acad. Sci. USA 87: 2264-2268.
Such an algorithm is described in Altshul et al. (1990) J. M
ol. Biol. 215: 403-410, incorporated into the NBLAST and XBLAST programs. BLAST nucleotide searches are performed using p- of the invention.
To obtain nucleotide sequence homology to the Hyde nucleic acid molecule, NBLAST
It can be implemented with a program, score = 100, and character length = 12. BLAST protein searches can be performed with the X13LAST program, score = 50, character length = 3 to obtain amino acid sequence homology to p-Hyde protein molecules of the invention. To obtain a gap adjusted alignment for comparison purposes, gap adjusted BLAST was prepared according to Altschul et al. , (1997) Nucle
ic Acid Res. : 3389-3402. B
When using the LAST and Gap Adjust BLAST programs, the default parameters of the respective programs (eg X13LAST and NBLAST) can be used. http: // www. ncbi. nlm. nih. gov
checking ... Another preferred, non-limiting example of a mathematical algorithm used for sequence comparisons is Myers and Miller, CABIOS 4: 1.
1-17 (1988). Such an algorithm is
It is incorporated into the ALIGN program (version 2.0) which is part of the G sequence alignment software package. ALI for amino acid sequence comparison
When using the GN program, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without gaps. Only exact matches are counted when calculating percent identity.

The present invention provides a nucleic acid having a sequence complementary to that of the isolated nucleic acid of the human p-Hyde gene. In particular, the invention provides an oligonucleotide of at least 15 nucleotides capable of specifically hybridizing to a sequence of nucleotides present in a nucleic acid encoding human p-Hyde. 1
In one embodiment, the nucleic acid is DNA or RNA. In another embodiment, the oligonucleotide is labeled with a detectable marker. In other embodiments, the oligonucleotide is a radioactive isotope, fluorophore or enzyme.

Oligonucleotides that are complementary may be obtained as follows. The polymerase chain reaction is performed with two primers. PCR Protocols: Guides to Methods and Applications (PCR Protocols: A Guide to Me
See methods and Applications) [74]. PC
After R amplification, the PCR-amplified region of viral DNA can be tested for its ability to hybridize to the three specific nucleic acid probes listed above. Alternatively, hybridization of viral DNA to the nucleic acid probe described above may be performed by
It can be performed without amplification and by the Southern blot procedure under stringent hybridization conditions as described herein.

Oligonucleotides for use as probes or PCR primers are solid-phase phosphos initially described by Beaucage and Carruthers [19] using an automated synthesizer, as described in Needham-Van Devanter [69]. It is chemically synthesized according to the Lamidite Triester method. Purification of oligonucleotides is by native acrylamide gel electrophoresis or by Pearson, J. et al. D. and Regni
er, F.I. E. Either by cation exchange HPLC, as described in [75A]. The sequences of synthetic oligonucleotides are described in Maxam, A .; M.
and Gilbert, W.A. It can be confirmed using the chemical decomposition method of [63].

High stringency hybridization conditions are selected at about 5 ° C. below the temperature melting point (Tm) for the specific sequence at the defined ionic strength and pH. The Tm is the temperature (at defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, stringent conditions are those in which the salt concentration is at least about 0.02 molar at pH 7 and the temperature is at least about 60 ° C. Among other things, base content and complementary strand size, the presence of organic solvents,
That is, the combination of parameters is more important than any one absolute measurement, since other factors, such as Enma et al., Include formamide concentration and degree of base mismatch, clearly influence the stringency of hybridization. is there. For example, high stringency can be achieved, for example, by overnight hybridization at about 68 ° C. in 6 × SSC solution, 6 × SS.
Washing with C solution at room temperature, followed by about 6 in 6 × SSC solution in 0.66 × SSX.
Achieved by washing at 8 ° C.

Hybridization with moderate stringency was achieved by eg 1) 3 × sodium chloride, sodium citrate (SSC), 50% formamide, 0.1 at pH 7.5.
Pre-hybridization and hybridization with M Tris buffer, 5 × Denhardt's solution, 2) 4 hours, pre-hybridization at 37 ° C., 3) amount equal to 3,000,000 cpm in 16 hours Hybridization with labeled probe at 37 ° C., 4) Washing in 2 × SSC and 0.1% SDS solution, 5) Washing 4 times for 1 minute each at room temperature, 60 ° C. for 30 minutes each
It may be carried out by washing 4 times with, and 6) drying and exposing the film.

The phrase “selectively hybridize.
“Dicing to)” refers to nucleic acid probes that hybridize, double-stranded, or bind only to a particular target DNA or RNA sequence when the target sequence is present in the preparation of total intracellular DNA or RNA. By selective hybridization is meant a probe that, under different high stringency hybridization conditions, binds to a given target in a manner that is detectable in a manner different than the non-target sequence.
"Complementary" or "target" nucleic acid sequences refer to those nucleic acid sequences that selectively hybridize to a nucleic acid probe. Appropriate annealing conditions depend on, for example, the length of the probe, the base composition, and the number of mismatches and their position on the probe, and must often be determined empirically. For a discussion of nucleic acid probe design and annealing conditions, see, eg, Sa.
mbrook et al. , [81] or Ausubel, F .; , Et a
l. , [8].

When a reference is made to a particular sequencing method, such reference essentially includes the sequence associated with its complementary sequence, and such changes in relevant sequences. Included are the sequences described, including allowance for small amounts of sequence errors, single base changes, deletions, relaxations, etc., such that the listing corresponds to the nucleic acid sequence of the pathogenic organism or disease marker involved.

The primer sets of the present invention are useful in determining the nucleotide sequence of specific p-Hyde alleles using PCR. The set of single-stranded DNA primers is pH
The yde gene itself can be annealed to sequences within or around the p-Hyde gene to initiate amplified DNA synthesis. The complete set of these primers allows the synthesis of sequences, ie all nucleotides of the p-Hyde gene encoding the exons. The primer set preferably allows the synthesis of both introns and exons. Allele-specific primers can also be used. Such primers may only anneal to a particular p-Hyde mutant allele and thus only amplify the product in the presence of the mutant allele as a template.

Nucleic acid probe technology is such that the probe may vary considerably in length and may be labeled with a detectable label such as a radioisotope or a fluorescent dye to facilitate detection of the probe. It is well known to those skilled in the art who can easily understand
The DNA probe molecule, or the full length or fragment of an isolated nucleic acid molecule of a DNA virus, is inserted into a suitable vector, such as a plasmid or bacteriophage, and subsequently transformed into a suitable host cell to It may be produced by amplification and recovery of the DNA probe using methods well known in the art. Alternatively, the probe may be chemically produced by a DNA synthesizer.

An RNA probe is a full-length or fragment of an isolated nucleic acid molecule of a DNA virus,
It may be made by inserting downstream of a bacteriophage promoter such as T3, T7 and SP6. Large amounts of RNA probe may be produced by incubating labeled nucleotides with a linearized isolated nucleic acid molecule of DNA virus or a fragment thereof containing an upstream promoter in the presence of a preferred RNA polymerase.

The nucleic acid probe may be a DNA or RNA fragment, as defined herein. DNA fragments can be obtained, for example, by digesting plasmid DNA or P
It can be prepared by using CR, or Beaucage and Carru
by the phosphoramidite method described by Thers [19] or by Matteucci et al. , Either by the triester method according to [62] (both incorporated herein by reference).
. Double-stranded fragments, if desired, by chemically annealing single-stranded chemically synthesized single strands together under preferred conditions or using DNA polymerase to synthesize complementary strands with appropriate primer sequences. You can get it at. It is understood that the complementary strand is also identified and included, given the specific sequence of the nucleic acid probe. Complementary strands can work equally well in conditions where the target is double-stranded nucleic acid. It is also understood that when a particular sequence is identified for use in a nucleic acid probe, a subsequence of the listed sequences that is 25 base pairs or more in length is also included for use as a probe. To be done.

Nucleic acids of the invention differ from naturally-occurring forms with respect to the identification or localization of one or more amino acid residues (deletion analogs containing less than all protein-specific residues). , Substituted analogs in which one or more specific residues have been replaced by other residues and added analogs in which one or more amino acid residues have been added to the terminal or intermediate portion of the polypeptide) and natural DNA molecules encoding polypeptide analogs, fragments or derivatives of antigenic polypeptides that share some or all of the properties of the forms present in. These molecules include insertion of "preferred" codons for expression by the selected non-mammalian host, provision of sites for cleavage by restriction endonuclease enzymes, and further initiation, termination to facilitate construction of simple expression vectors. Alternatively, the supply of intermediate DNA sequences is included.

The present invention also provides antisense molecules capable of specifically hybridizing to the isolated nucleic acid of the human p-Hyde gene. The present invention provides an antagonist capable of inhibiting the expression of the peptide or polypeptide encoded by the isolated DNA molecule. In one embodiment, the antagonist is capable of hybridizing to the double stranded DNA molecule. In another embodiment, the antagonist is a triple stranded oligonucleotide capable of hybridizing to a DNA molecule. In other embodiments, triple-stranded oligonucleotides can bind to at least a portion of an isolated DNA molecule having a nucleotide sequence.

The antisense molecule may be DNA or RNA or variants thereof (ie DNA or RNA with a protein backbone). The present invention is directed to specific mRNA
In translation of E. coli, development of antisense nucleotides and ribozymes that may be used to interfere with expression of receptor recognition proteins, either by covering the MRNA with an antisense nucleic acid or by cleaving it with a ribozyme To do.

Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a particular MRNA molecule. In the cell, it hybridizes to its MRNA and forms a double-stranded molecule. Cells do not translate MRNA in double-stranded form. Therefore, the antisense nucleic acid interferes with the expression of MRNA within the protein.

Antisense nucleotide or polynucleotide sequences are useful in preventing or reducing expression of the p-Hyde gene, as will be appreciated by those in the art. For example, a polynucleotide vector containing all or part of the p-Hyde gene, or other sequences from the p-Hyde region (particularly those flanking the p-Hyde gene), may be added in the antisense direction under the control of a promoter. It may be placed and introduced intracellularly. Intracellular expression of such antisense constructs may interfere with p-Hyde transcription and / or translation and / or replication. Molecules that hybridize to about 15 nucleotide oligomers and the AUG start codon are particularly effective because they are simple to synthesize and are less problematic upon introduction into cells than larger molecules.

The present invention provides a transgenic non-human mammal comprising at least a portion of an isolated DNA molecule introduced into the mammal at fetal stage. Methods of producing transgenic non-human mammals are known to those of skill in the art.

The present invention provides a polypeptide comprising the amino acid sequence of human p-Hyde.
In one embodiment, the amino acid sequence is SEQ ID NOs. Listed as 2, 4, 6 or 8. The present invention provides chimeras that include fusion proteins or polypeptides. The present invention provides an antibody that specifically binds to a polypeptide. In one embodiment, the antibody is a monoclonal or polyclonal antibody.

The present invention also provides p-Hyde chimeric or fusion proteins. As used herein, "chimeric protein"
) ”Or a fusion protein” comprises a p-Hyde polypeptide operably linked to a non-p-Hyde polypeptide. "P
-Hyde polypeptide (p-Hyde polypeptide) "is p-
Refers to a polypeptide having an amino acid sequence corresponding to Hyde, while "non-p-Hyde polypeptide" (non-p-Hyde polypeptide)
Is a protein that is not essentially identical to a p-Hyde protein, such as p-
It refers to a polypeptide having an amino acid sequence different from Hyde and corresponding to a protein derived from the same or different organism. Within the p-Hyde fusion protein, the p-Hyde polypeptide may correspond to all or a portion of the p-Hyde protein, preferably to at least one biologically active portion of the p-Hyde protein.

Within the fusion protein, the phrase “operably linked.
ed) ”is intended to indicate that the p-Hyde polypeptide and the non-p-Hyde polypeptide are fused in-frame to each other. Non-p-Hyde
The polypeptide can be fused to the N-terminus or C-terminus of the p-Hyde polypeptide. In yet another embodiment, the fusion protein is p-Hyd.
A p-Hyde immunoglobulin fusion protein in which all or part of e is fused to sequences from members of the immunoglobulin protein family. The p-Hyde immunoglobulin fusion proteins of the invention can be incorporated into pharmacological compositions and administered to patients.

The invention further provides a method for preparing a polynucleotide comprising polymerized nucleotides to yield a sequence consisting of at least 8 consecutive nucleotides of the p-Hyde locus, and within the p-Hyde locus. A method of preparing a polypeptide comprising polymerized amino acids to yield a sequence comprising at least 5 encoded amino acids is provided.

The present invention provides an isolated polynucleotide comprising all or part of the p-Hyde locus or a mutated p-Hyde locus. Such a polynucleotide may be an antisense polynucleotide. The present invention also provides recombinant constructs containing such isolated polynucleotides, eg, recombinant constructs suitable for expression in transformed host cells.

The invention also provides a method of detecting a polypeptide comprising a portion of the p-Hyde locus or its expression product in an analyte. Such a method is further described in p
The step of amplifying a portion of the -Hyde locus may be included, and further the step of providing a set of polynucleotides that are primers for amplification of said portion of the p-Hyde locus. This method is effective in either the diagnosis of predisposition to cancer or the diagnosis or prognosis of cancer.

The present invention also provides a method of producing a polypeptide encoded by an isolated molecule, which allows the above host vector systems to produce the polypeptide and thus be produced. Growing the polypeptide under conditions suitable to recover the polypeptide.

In addition, the isolated polypeptide encoded by the isolated DNA molecule may be
It may be linked to a second polypeptide encoded by a nucleic acid molecule to form a fusion protein by expression in a suitable host cell. In one embodiment, the second nucleic acid molecule encodes beta-galactosidase. Other nucleic acid molecules used to form fusion proteins are known to those of skill in the art.

The present invention provides an antibody that specifically binds to the polypeptide encoded by the isolated DNA molecule. In one embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is a polyclonal antibody. The antibody or DNA molecule may be labeled with a radioactive label or a colorimetric, luminescent or fluorescent marker, or a detectable marker including but not limited to gold. The radioactive ^{label, 3 H, 1 4 C,} 32 P, 33 P, 35 S, 36 Cl, 51 Cr, 57 Co, 59 Co, 59 Fe, 90 Y, 125 I, 131 I and ¹⁸⁶ Re are limited Not included, but included. Luminescent markers include, but are not limited to, fluorescein, rhodamine and auramine. Colorimetric markers include but are not limited to biotin and digoxigenin. Methods for producing polyclonal or monoclonal antibodies are known to those of skill in the art.

In addition, the antibody or nucleic acid molecule complex may be detected by a second antibody which may be linked to an enzyme such as alkaline phosphatase or horseradish peroxidase. Other enzymes that may be used are known to those of skill in the art.

When referring to a protein or peptide, “specifically binds to antibody (Spe
"Cially bounds to an antibody" or "
Specific immunological activity (specifically immunoreactive)
“With)” refers to a binding reaction that determines the presence of p-Hyde of the invention in the presence of a heterogeneous population of proteins, including viruses, and other microorganisms other than p-Hyde. Therefore, under the designed immunoassay conditions, the specialized antibody is p-
It binds to the Hyde antigen and does not bind to other antigens present in the sample in significant amounts.
Specific binding to an antibody under such conditions may require an antibody selected for its specificity for a particular protein. For example, to obtain an antibody raised against the human p-Hyde immunogen described herein, to obtain an antibody that is specifically immunoreactive with the p-Hyde protein and not with other proteins. Can be sorted into These antibodies recognize proteins homologous to the human p-Hyde protein. Various immunoassay formats can be used to screen for antibodies that are specifically immunoreactive with a particular protein. For example, solid phase ELISA immunoassays are routinely used to screen proteins for specifically immunoreactive monoclonal antibodies. For a description of immunoassay formats and conditions that can be used to determine specific immune activity, see Harlow
See and Lane [32].

The present invention provides a method for producing an antibody by selecting a specific region on a polypeptide encoded by an isolated DNA molecule of a DNA virus. The protein sequence may be determined from the cDNA sequence. Amino acid sequences may be analyzed by methods well known to those of skill in the art to determine whether they yield hydrophilic or hydrophobic regions within the constructed protein. In the case of cell membrane proteins, the hydrophobic region is well known to form the part of the protein that is inserted within the lipid bilayer of the cell membrane, while the hydrophilic region is in the aqueous environment, on the cell surface. Localize.
Generally, hydrophobic regions may be more immunogenic than hydrophilic regions. Accordingly, hydrophobic amino acid sequences may be selected and used to raise antibodies specific for the polypeptide encoded by the isolated nucleic acid molecule encoded by the DNA virus. Selected peptides may be prepared using commercially available machines. Alternatively, such a cDNA or fragment thereof may be cloned, expressed and the resulting polypeptide recovered and used as an immunogen.

Polyclonal antibodies against these peptides may be produced by immunizing animals with the selected peptides. Monoclonal antibodies may be prepared using hybridoma technology by fusing antibody-producing B cells from the immunized animal with myeloma cells and selecting the resulting hybridoma strains producing the desired antibody. . Alternatively, the monoclonal antibody may be an in
It may be produced by in vitro technology. These antibodies detect the expression of a polypeptide encoded by an isolated DNA molecule of a DNA virus in a living animal, human, or biological tissue or fluid isolated from the animal or human. Useful for.

Antibodies may be directly labeled using conventional labeling techniques well known in the art. Thus, antibodies may be radiolabeled using radioactive isotopes such as ³ H, ¹²⁵ I, ¹³¹ I and ³⁵ S. The antibody may also be labeled with a fluorescent label, an enzyme label, a free radical label, or a bacteriophage label using techniques known in the art. Typical fluorescent labels include fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin and Texas Red.

It is also possible that such specific enzymes may be covalently coupled to other molecules and used as labels for the production of tracer substances. Suitable enzymes include alkaline phosphatase, beta-galactosidase, glucose-6-phosphate dehydrogenase, maleate dehydrogenase and peroxidase. The two principle types of enzyme immunoassays are the enzyme-linked immunosorbent assay (ELISA) and the enzyme-multiplexed immunoassay (EMIT, Shiva (Sy).
va Corporation, Palo Alto, CA), an alloenzyme immunoassay. In an ELISA system, the separation may be carried out, for example, by using an antibody conjugated to a solid phase. The EMIT system relies on the inactivation of the enzyme in the tracer-antibody complex and activity can be measured without the need for a separation step.

In addition, chemiluminescent compounds may be used as labels. Typical chemiluminescent compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters. Similarly, chemiluminescent compounds may be used to label biochemiluminescence, including luciferin, luciferase, and aequorin. Once labeled, the antibodies may be used to identify and quantify immunological counterparts (antibodies or antigenic polypeptides) using techniques well known in the art.

A description of radioimmunoassay (RIA) can be found in Laboratory Technology.
ques in Biochemistry and Molecular B
iology [52], Chard, T .; Entitled "Introduction to Radioimmunoassay and Related Techniques (An Introduction to Radioim
Mune Assay and Related Tequencies) chapters, which are incorporated herein by reference. A description of various types of general immunological assays is described in US Pat. No. 4,376,11 below.
No. 0 (David et al.) Or No. 4,098,876 (Piasio
) Can be seen at.

Immunoassays can be used to detect for the p-Hyde gene, specific peptides, or for antibodies to the virus or peptide. A general overview of applicable techniques is provided in Harlow a, incorporated herein by reference.
nd Lane [32].

In one embodiment, antibodies to human p-Hyde can be used to detect a drug in a sample. Briefly, the targeted sequence is expressed and purified in transfected cells, preferably microbial cells, to produce antibodies to the drug or peptide. The product is injected into a mammal capable of producing the antibody. Either monoclonal or polyclonal antibodies (specific to the gene product (
And any recombinant antibody) can be used in various immunoassays. Such assays include competitive immunoassays, radioimmunoassays, Western blots, ELISAs, indirect immunofluorescence assays and the like. For competitive immunoassays, see pages 567-573 and 584-589 of Harlow and Lane [32].

In a further embodiment of the invention, a commercial test kit suitable for use by a medical professional has or does not have a previously determined binding activity or a previously determined binding activity stress on a suspicious target cell. May be prepared to determine With respect to the testing techniques discussed above, one type of such kit would, of course, be the method of choice, "competitive", "sandwich".
dwitch) "," DASP ", etc., and at least the labeled polypeptide or its binding partner, eg, an antibody specific thereto, and instructions. The kit may also include peripheral agents such as buffers, stabilizers and the like.

Monoclonal or recombinant antibodies can be obtained by various techniques familiar to those skilled in the art. Briefly, immunization of spleens or other lymphocytes from animals immunized with the desired antigen, generally by fusion with myeloma cells (Kohler and Milstein [incorporated herein by reference).
50]). Other methods of immortalization include transformation with the Epstein-Barr virus oncogene or retrovirus, or methods well known in the art. Colonies taken from a single immortalized cell are screened for production of antibodies of the desired specificity and affinity for the antigen, and the yield of monoclonal antibody produced by such cells is transferred into the peritoneal cavity of the spinal host. It may be augmented by various techniques, including injection. New techniques using recombinant phage antibody expression systems can also be used to generate monoclonal antibodies. For example, McCaf
ferty, J et al. , [64], Hoogenboom, H .; R. e
t al. [39], and Marks, J. et al. D. et al. See [60].

Such peptides have specific sequences in recombinantly engineered cells such as microorganisms, yeasts, filamentous fungi, insects (especially with baculovirus vectors), and mammalian cells. May be produced by expressing The person skilled in the art is familiar with the many expression systems available for the expression of herpesvirus proteins.

Briefly, expression of a natural or synthetic nucleic acid encoding a viral protein generally drives the desired sequence for the promoter (either constitutive or inducible) or a portion thereof. It is carried out by ligation so that it can be incorporated into an expression vector. Vectors are suitable for replication or integration in either prokaryotic or eukaryotic cells. A typical cloning vector contains an antibiotic resistance marker, a gene for transformant selection, an inducible or regulatable promoter region, and translation ends useful for the expression of viral genes.

Methods for the expression of cloned genes in microorganisms are also well known. In general, in order to obtain high levels of expression of cloned genes in prokaryotic systems, it is desirable to construct an expression vector containing a strong promoter that directs mRNA transcription. Inclusion of a selectable marker in a DNA vector transformed into E. coli is also useful. Examples of such markers include genes that specify resistance to antibiotics. See [81] above for details on selectable markers and promoters for use in E. coli. Suitable prokaryotic hosts may include plant cells, insect cells, mammalian cells, yeast, and filamentus fungi.

Peptides, peptide fragments derived from nucleic acids are produced by recombinant technology,
It may be purified by standard techniques well known to those skilled in the art. The recombinantly produced sequence can be expressed directly or as a fusion protein. The protein is then purified by a combination of cell lysis (eg sonication) and affinity chromatography. For the fusion product, subsequent digestion of the fusion protein with the preferred proteolytic enzyme releases the desired peptide.

Proteins may be purified to substantial precision by standard techniques well known in the art, including selective precipitation with substances such as ammonium sulfate, column chromatography, immunopurification methods and the like. See, for example, Scopes, R. et al., Incorporated herein by reference. See [84].

The present invention is directed to analogs of isolated nucleic acids and polypeptides that include the amino acid sequences listed above. An analog is an N of a polypeptide containing an amino acid sequence.
Or it may have an N-terminal methionine or N-terminal polyhistidine optionally attached to the COOH terminus.

In another embodiment, the invention contemplates peptide fragments of the polypeptide that are the result of proteolytic digestion of the polypeptide. In another embodiment, the derivative of the polypeptide has one or more chemical moieties attached to it.
In another embodiment, the chemical moiety is a water soluble polymer. In another embodiment, the chemical moiety is polyethylene glycol. In other embodiments, the chemical moieties are mono-, di-, tri- or tetrapegylated. In another embodiment, the chemical moieties are N-terminally monopegylated.

A polypeptide "fragment,""portion," or "segment" is typically at least 5-7 contiguous amino acids, often at least about 7-9 contiguous amino acids, typically Is at least about 9 to 1
It ranges from amino acid residues of 3 contiguous amino acids, most preferably at least about 20-30 or more contiguous amino acids.

Polypeptides of the present invention, if soluble, may be solid supports such as nitrocellulose, nylon, column packed material (eg Sepharose).
rose) beads), magnetic beads, glass wool, plastics, metals, polymer gels, cells or other substrates. Such supports may take the form of, for example, beads, wells, dipsticks, or membranes.

“Target region” refers to a region of nucleic acid that has been amplified and / or detected. The phrase "target sequence" refers to a sequence that will form a stable hybrid under the conditions under which the probe or primer is desired.

Polyethylene glycol (PEG) has very low toxicity in mammals (
Carpenter et al. , 1971), adhesion of PEG to compounds is preferred and useful. For example, PEG adducts of adenosine deaminase have been approved in the United States for use in humans for the treatment of severe combined immunodeficiency syndrome. PE
The second advantage conferred by the coupling of G is that it effectively reduces the immunogenicity and antigenicity of the heterologous compound. The compounds of this invention may be delivered in a microencapsulated device to reduce or prevent a host immune response against the compound or against cells that may produce the compound.

Many activated forms of PEG suitable for direct reaction with proteins have been described. PEG agents useful for reacting with protein amino acids include active esters of carboxylic acids, or carboxylate salt derivatives, especially where the leaving group is N-hydroxysuccinide, p-nitrophenol, imidazole or 1-hydroxyl. -2-
Includes such as being nitrobenzene-4-sulfonic acid. Maleimide or PEG derivatives containing haloacetyl groups are useful agents for modification of the free mercapto group of proteins. Similarly, PEG agents containing aminohydrazine or hydrazine groups are useful for reaction with aldehydes generated by peroxidase oxidation of carbohydrate groups in proteins.

In one embodiment, the amino acid residues of the polypeptides described herein are preferably in the “L” isomeric form. In other embodiments, residues in the "D" isomeric form may be substituted for L-amino acid residues, as long as the desired functional property of lectin activity is preserved by the polypeptide. NH ₂ refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. The abbreviations used herein are
Standard Polypeptide Nomenclature, J. Biol. Chem. , 243: 3552-5.
9 (1969).

It should be noted that all amino acid residue sequences are represented by such formulas that the left and right orientations are in the conventional direction of amino-terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bound to a further sequence of one or more amino acid residues.

Synthetic polypeptides prepared using the well-known techniques of solid phase, solution phase or peptide enrichment techniques or combinations thereof can include natural and unnatural amino acids.
The amino acids used for peptide synthesis were originally Merrifield (
1963, J.M. Am. Chem. Soc. 85: 2149-2154) standard Boc (N-amino protected Nt-butyloxycarbonyl) amino acid resin in standard deprotection, neutralization, conjugation and washing protocols or Carpin.
o and Han (1972, J. Org. Chem. 37: 3403-34.
09), which may be a base-labile N-amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acid first described by G.09. Thus, the polypeptides of the invention include D-amino acids, a mixture of D- and L-amino acids, and various "designer" amino acids (in order to induce special properties.
(Eg, methyl amino acids, C-methyl amino acids, N-methyl amino acids, etc.). Synthetic amino acids include ornithine for lysine, fluorophenylalanine for phenylalanine, norleucine for leucine or isoleucine. Furthermore, alpha-helix, alpha-turn, beta-sheet, beta-turn and cyclic properties can be created by assigning specific amino acids at specific coupling steps.

In one aspect of the invention, the peptide has a special amino acid at the C-terminus that incorporates either a CO ₂ H or CONH ₂ side chain to stimulate a free glycine or glycine-amide group. May be included. Another way to consider this particular residue is to consist of a linker or D or L with side chains attached to the beads.
As an amino acid analog. In one embodiment, pseudo-free C-
The terminal residues may be in the D or L optical configuration, and in other examples racemic D and L isomers may be used.

In a further embodiment, pyroglutamate may be included as the N-terminal residue of the peptide. Although pyroglutamate is not susceptible to sequence by Edman degradation, limiting the substitution to only 50% of the peptides on the beads provided with N-terminal pyroglutamate allows for sequencing. There may be enough non-pyroglutamate peptide left on the beads. Those skilled in the art will readily recognize that this technique can be used to sequence any peptide that incorporates residues resistant to Edman degradation at the N-terminus. Other methods for characterizing individual peptides exhibiting the desired activity are described in detail below. A particular activity of a peptide containing a blocked N-terminal group, eg pyroglutamate, is that the specific N-terminal group is present in 50% of the peptides,
It may be briefly shown by comparing the activity of the intact (100%) blocked peptide with the unblocked (0%) peptide.

Further, the present invention provides for the preparation of peptides with better defined structural properties, and for the preparation of peptides with novel properties, peptidomimetics and peptidomimetics such as ester bonds and peptidomimetics. Envisioning the use of joins. In another embodiment, the peptide is made with reduced peptide bonds, ie, R ₁ —CH ₂ —NH—R ₂ , where R ₁ and R ₂ are amino acid residues or sequences. You may The reduced peptide bond is introduced as a dipeptide subunit. Such molecules may be resistant to peptide bond hydrolysis, eg protease activity. Such peptides may provide ligands with unique functions and activities, such as extended half-life in vivo due to resistance to metabolic degradation or protease activity. Furthermore, it is well known that unnatural peptides show enhanced functional activity in certain systems (Hruby, 1982, Life Sciences.
31: 189-199, Hruby et al. , 1990, Bioche
m J. 268: 249-262), the invention provides a method for producing a compulsory peptide that incorporates random sequences at all other positions.

Unnatural, cyclic or stiffened peptides are characterized by at least two positions in the peptide's sequence where an amino acid or amino acid analogue forces the peptide to undergo cyclization after treatment to form a crosslink, Alternatively, it may be prepared to insert synthetically to provide a chemical functional group tail that can be crosslinked to be rigid. Cyclization may be preferred when incorporating turn-derived amino acids.
Examples of amino acids that can crosslink the peptide are cysteine that forms a disulfide bond, aspartic acid that forms a lactone or lactase, and carboxyl-glutamic acid (Gla) (Bla) (Bla) (Bla) that chelates a transition metal and forms a crosslink.
It is a chelator like a chem). The protected carboxyglutamic acid was added to Zee-Cheng and Olson (1980, Biophys. Bi.
ochem. Res. Commun. 94: 1128-1132) and may be prepared by modifying the synthesis described by A peptide whose peptide sequence comprises at least two crosslinkable amino acids so as to crosslink the peptide to form a forced cyclic or stiffened peptide, for example oxidation of cysteine residues to form a disulfide, or It may be treated by the addition of metal ions to form chelates.

The present invention provides strategies for synthetically preparing crosslinks. For example, different protecting groups may be used when four cysteine residues are incorporated into the peptide sequence (Hiskey, 1981, in The Peptides: Analysi).
s, Synthesis, Biology, Vol. 3, Gross and
Meienhofer, eds. , Academic Press: New Y
ork, pp. 137-167, Ponsanti et al. , 1990,
Tetrahedron 46: 8255- 8266). The first set of cysteines may be deprotected and oxidized, then the second set may be deprotected and oxidized. In this way, a defined set of disulfide bridges can be formed. Alternatively, one set of cysteines and one set of control amino acid analogs may be incorporated such that the crosslinks are of different chemical properties.

The following non-traditional amino acids are modified by a specific structural motif, 1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Kazmierski et al.
． , 1991, J .; Am. Chem. Soc. 113: 2275-2283),
(2S, 3S) -Methyl-phenylalanine, (2S, 3R) -methyl-phenylalanine, (2R, 3S) -methyl-phenylalanine and (2R, 3R).
-Methyl-phenylalanine (Kazmierski and Hruby, 1
991, Tetrahedron Lett. ), 2-aminotetrahydronaphthalene-2-carboxylic acid (Landis. 1989, Ph.D. Thesis).
(University of Arizona), hydroxy-1,2,3,4
-Tetrahydroisoquinoline-3-carboxylate (Miake et a
l. , 1989, J. Takeda Res. Labs. 43: 53-76),
? -Carbolines (D and L) (Kazmierski, 1988, Ph.D.
Thesis, University of Arizona), HIC (histidine isoquinoline carboxylic acid) (Zechel et al., 1991, I.
nt. J. Pep. Protein Res. 43), and HIC (histidine cyclic urea) (Dharaniprada) may be incorporated into the peptide.

The following amino acid analogs and peptidomimetics may be incorporated within the peptide to induce or assist in specific secondary structure. LL-Acp (LL-3-amino-2-propenidone-6-carboxylic acid), dipeptide analogue (Kemp et.
al. , 1985, J. Am. Org. Chem. 50: 5834-5838) and the following references, Nagai and Sato, 1985, Tetrahed.
ron Lett. 26: 647-650, DiMio et al. , 19
89, J.M. Chem. Soc. Perkin Trans. p. 1687, as well as the Gly-Ala turn analog (Kahn et al.
． , 1989, Tetrahedron Lett. 30: 2317), amide bond isostere (Jones et al., 1988, Tetrahedro.
n Lett. 29: 3853-5856), tetrazole (Zabrocki).
et al. , 1988, J. Am. Chem. Soc. 110: 5875-
5880), DTC (Samanen et al., 1990, Int.
Protein Pep. Res. 35: 501: 509), and Olso.
et al. , 1990, J .; Am. Chem. Sci. 112: 323-33
3 and Garvey et al. , 1990, J .; Org. Chem. 56
: Analogue taught in 436. Structurally restricted mimetics of beta-turn and beta-bargio and peptides containing them are described in US Pat. No. 5,440,013 issued to Kahn on August 8, 1995.

The invention further provides methods for modifying or inducing a polypeptide or peptide of the invention. Modifications of peptides are well known to those of skill in the art and include phosphorylation,
Includes carboxymethylation and acylation. The modification may be performed by chemical or enzymatic methods. In another aspect, glycosylated or fatty acylated peptide derivatives may be prepared. The preparation of glycosylated or fatty acylated peptides is well known in the art. Fatty acyl peptide derivatives may also be prepared.
For example, and in a non-limiting manner, free amino groups (N-terminal or lysyl) may be acylated, eg myristoylated. In another embodiment aspect, the structure - (CH ₂₎ an amino acid containing an aliphatic side chain of _n CH _3, may be incorporated in the peptide. This and other peptide-fatty acid conjugates suitable for use in the present invention are described in British Patent GB-8809162.4, International Patent Specification No. PCT / AU89 / 00.
No. 166 and reference 5 above.

In accordance with the present invention, conventional molecular biology, microbiology and recombinant DNA techniques within the skill of the art may be practiced. Such techniques are explained fully in the literature. For example, Sambrook et al. , "Molecular Cl
oning: A Laboratory Manual "(1989)," Cu
current Protocols in Molecular Biology
Volumes I-III [Ausubel, R .; M. , Ed. (199
4)], "Cell Biology: A Laboratory Handb.
Look "Volumes I-III [J. E. Celis, ed. (1994
)], “Current Protocols in Immunology”
Volumes I-III [Coligan, J. et al. E. , Ed, (1994)
], “Oligonucleotide Synthesis” (M. J. Gai
ed. 1984), "Nucleic Acid Hybridizati
on ”[B. D. Hames & S. J. Higgins, eds. (198
5)], “Transscription And Translation” [
B. D. Hames & S. J. Higgis eds. (1984) ","
Animal Cell Culture "[R. I. Freshney, ed
． (1986)], "Immobilized Cells And Enzy.
mes "[IRL Press, (1986)], B.M. Perbal, "AP
"ractional Guide To Molecular Cloning"
(1984). Chemical Motif for Induction A chemical motif suitable for induction may be selected among water-soluble polymers. The polymer selected should be water soluble so that the binding compound does not precipitate in an aqueous environment, such as a physiological environment. Preferably, the polymer may be pharmacologically acceptable for therapeutic use of the final product preparation. Those skilled in the art will select the desired polymer based on considerations such as whether the polymer / component conjugate can be used therapeutically, and if so, desired administration, circulation time, resistance to proteolysis and other considerations. Could be For this component or components, these may be ascertained using the assays provided herein.

Water-soluble polymers are, for example, polyethylene glycol, ethylene glycol /
Copolymer of propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer,
Polyamino acids (either homopolymers or random copolymers) and dextran or poly (n-vinylpyrrolidone) polyethylene glycols, propropylene glycol homopolymers, prolipropylene oxide / ethylene oxide co-polymers, polyoxyethylenated polyols and polyvinyl alcohols May be selected from the group consisting of Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.

The number of polymer molecules so attached may vary and one of ordinary skill in the art can ascertain the effect on function. One may be mono-derivatized, or a di-, tri-, tetra- or some combination of derivatives with similar or different chemical moieties (eg, different weights of polymer such as polyethylene glycol) may be provided. Good. The ratio of polymer molecules to a component or components may change, such as its concentration in the reaction mixture. In general, the optimum ratio (with respect to the efficiency of the reaction such that excess unreacted component or components and polymer are absent) is selected for the desired degree of induction (eg, mono-, di-, tri-, etc.). It may be determined by such factors as the molecular weight of the polymer, whether the polymer is branched or unbranched, and reaction conditions.

The present invention comprises: a) obtaining a nucleic acid sample that is preferable to the subject; (b) step (a)
Whether the subject is a nucleic acid encoding a mutant p-Hyde or is derived therefrom, so as to determine whether the subject has a mutation in the p-Hyde gene. The subject is p-Hyde, including determining
Provide a method for determining whether to have a mutation in a gene. In one embodiment, the nucleic acid sample in step (a) comprises mRNA corresponding to a transcript of DNA encoding the mutant p-Hyde, where the determination of step (b) includes (i) B.) Contacting the mRNA with the oligonucleotide under conditions that allow binding of the mRNA to the oligonucleotide so as to form a complex; (ii)
Isolating the complex so formed, and (iii) thereby determining whether the mRNA is or is derived from a nucleic acid encoding a mutant p-Hyde Includes identifying mRNA in the isolated complex. In another embodiment, determining in step (b) comprises i) differentiating and distinguishing fragments of the nucleic acid sample of step (a) and the isolated nucleic acid of the nucleic acid sample and the isolated nucleic acid. Contacting with a restriction enzyme under conditions permitting digestion into, (ii) isolating a fragment of the nucleic acid, and (iii) whereby the nucleic acid sample encodes the nucleic acid encoding the mutant p-Hyde. And comparing the fragment of the nucleic acid derived from the nucleic acid sample with the fragment of the nucleic acid derived from the isolated nucleic acid to determine whether or not it is derived from.

The invention further provides a method for preparing a polynucleotide containing polymerized nucleotides to yield a sequence consisting of at least 8 contiguous nucleotides of the p-Hyde gene, and a code in the p-Hyde gene. At least 5
Methods are provided for preparing a polypeptide containing a polymerizing amino acid to yield a sequence containing one amino acid.

The present invention provides a pharmacological composition comprising a number of polypeptides and a pharmacologically effective carrier or diluent.

The present invention comprises: a) obtaining a nucleic acid sample that is preferable to the subject; (b) step (a)
Whether the subject is a nucleic acid encoding a mutant p-Hyde or is derived therefrom, so as to determine whether the subject has a mutation in the p-Hyde gene. The subject is p-Hyde, including determining
Provide a method for determining whether to have a mutation in a gene. In one embodiment, the nucleic acid sample in step (a) comprises mRNA corresponding to a transcript of DNA encoding the mutant p-Hyde, where the determination of step (b) includes (i) B.) Contacting the mRNA with the oligonucleotide under conditions that allow binding of the mRNA to the oligonucleotide so as to form a complex; (ii)
Isolating the complex so formed, and (iii) thereby determining whether the mRNA is or is derived from a nucleic acid encoding a mutant p-Hyde Identifying the mRNA in the isolated complex. In another embodiment, determining step (b) includes:
And) contacting the nucleic acid sample of step (a) and the isolated nucleic acid of claim 1 with a restriction enzyme under conditions permitting digestion of the nucleic acid sample and the isolated nucleic acid into different, distinguishable fragments of the nucleic acid. Causing, (iii) isolating a fragment of the nucleic acid, and (iii)
Whether the nucleic acid sample is a nucleic acid encoding a mutant p-Hyde,
Or comparing it with a fragment of the nucleic acid derived from the nucleic acid sample to determine whether it is derived or not.

Detection of point mutations or variants can be performed using techniques well known in the art, p-
It may be carried out by molecular cloning of the Hyde allele and sequencing of the allele (s). Alternatively, the gene sequences can be amplified directly from genomic DNA preparations from tumor tissue using known techniques. D of the amplified sequence
The NA sequence can be determined. There are six well-known methods for a more complete, but still indirect test to confirm the presence of susceptible alleles. 1) Single-stranded structure analysis (SSCA) (Orita et al., 1989), 2) Reduction gradient gel electrophoresis (DGGE) (Wartell et al., 1990, Sh)
effect et al. , 1989), 3) RNase protection assay (F
inkelstein et al. , 1990, Kinszler et a
l. , 1991), 4) allele-specific oligonucleotides (ASOs) (C
oner et al. , 1983), 5) Use of proteins that recognize nucleotide mismatches, such as the E. coli mutS protein (Modrich, 1991).
), And 6) allele-specific PCR (Rano & Kidd, 1989).
. For allele-specific PCR, the primer is labeled with a specific p- at its 3'end.
Those that hybridize with the Hyde mutation are used. No amplification product is observed in the absence of the particular p-Hyde mutation. Amplification-insensitive mutation system (Ampli
fiction Refraction Mutation System:
ARMS) can also be used, European Patent Specification No. 0332435 and New.
ton et al. , 1989. Gene insertions and deletions can also be detected by cloning, sequencing and amplification. In addition, digestive fragment length genetic polymorphisms (RFLPs) to the gene or surrounding marker genes
3.) The probe can be used to score insertion changes in alleles or polymorphic fragments. Such a method is particularly useful for screening individual relationships that influence the presence of p-Hyde mutations found in the individual. Other techniques known in the art for detecting insertions and deletions can also be used.

In a similar fashion, DNA probes can be used to detect mismatches via enzymatic or chemical cleavage. For example, Cotton et al. , 1988,
Shenk et al. , 1975, Novack et al. , 1986
checking ... Alternatively, the mismatch can be detected by a change in electrophoretic mobility of the mismatched duplex compared to the matched duplex. For example, Cariello, 1988.
checking ... With either riboprobes or DNA probes, cellular mRNA or DNA that may contain mutations can be amplified using PCR (see below) prior to hybridization. Changes in the DNA of the p-Hyde gene can also be detected using Southern hybridization, especially if the changes are significant rearrangements such as deletions or insertions.

As noted above, non-PCR based screening assays are also contemplated by the present invention. This procedure hybridizes a nucleic acid probe (or analog, such as a methylphosphate backbone substituted with a regular phosphodiester) to a low concentration of DNA target. The probe may have the enzyme covalently attached to the probe, which covalent attachment does not interfere with the specificity of hybridization. The enzyme-probe-conjugate-target nucleic acid complex is then isolated from the free probe enzyme conjugate and the enzyme is added to the substrate for detection. Enzyme activity, sensitivity is 10sup3 ~
Observe as a change in color development or luminescence output, with an increase of 10 sup6. Examples of oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes are described in Jablonski et a.
l. , 1986.

Two-step label amplification methods are known in the art. These assays work on the principle that small ligands (such as digoxigenin, biotin) attach to nucleic acid probes that can specifically bind p-Hyde. Allele-specific probes are also contemplated within the scope of this example. In one example, the small ligand attached to the nucleic acid probe is specifically recognized by the antibody-enzyme conjugate. In one embodiment of this example, digoxigenin is attached to the nucleic acid probe. Hybridization is detected by an antibody-alkaline phosphatase conjugate, which is a chemiluminescent substrate. For methods for labeling nucleic acid probes according to this embodiment, see Martin et al. , 1990.

In the second example, the small ligand is recognized by a second ligand-enzyme conjugate capable of specifically complexing with the first ligand. A well-known embodiment of this example is the biotin-avidin type interaction. For methods for labeling nucleic acid probes and their use in biotin-avidin-based assays, see Rigby et al. , 1977 and Nguyen et a.
l. , 1992.

It is also contemplated within the scope of the present invention to use a reaction mixture of nucleic acid probes capable of detecting p-Hyde in the nucleic acid probe assay of the present invention. Therefore, in one example for detecting the presence of p-Hyde in a cell sample,
One or more probes complementary to p-Hyde are used, in particular the number of different probes is alternatively 2, 3 or 5 different nucleic acid probe sequences. In another example, a reaction mixture was identified in a population of patients with changes in p-Hyde to detect the presence of a mutation in the p-Hyde gene sequence in the patients, with specific mutations. Where one or more probes complementary to p-Hyde are used. In this embodiment, any number of probes can be used, and may preferably include probes that are compatible with the major gene mutations identified when the individual was previously treated for breast cancer.

The present invention provides a tumor sample from a human patient, the p-Hyde gene from the tumor sample, the p-Hyde RNA from the tumor sample and the mRN from the tumor sample.
The first sequence selected from the group consisting of p-Hyde cDNA prepared from A was used for the p-Hyde gene from the non-tumor sample of the patient, p-Hy from the non-tumor sample.
p-Hyde made from de RNA and mRNA from said non-tumor sample
There is provided a method for screening for somatic alterations of the p-Hyde gene in said tumor comprising comparing with a gene of a second sequence selected from the group consisting of cDNA, wherein p-Hyde from said tumor sample is provided. Gene, p-Hyde RN
P from the non-tumor sample in the sequence of Am or p-Hyde cDNA
The difference from the sequence of -Hyde gene, p-Hyde RNAm, or p-Hyde cDNA suggests somatic alteration of p-Hyde gene in the tumor sample.

The present invention uses p-Hyde polypeptides from said use sample from said patient to analyze tumor samples from human patients for differences between the polypeptides, from non-tumor samples of said patient. Provided is a method for screening for the presence of somatic alterations of the p-Hyde gene in said tumor comprising comparing with a p-Hyde polypeptide, wherein the comparison comprises (i) full length in each sample. Detecting a polypeptide or a cleaved polypeptide, or (ii) altered p
An antibody that specifically binds to either the epitope of the Hyde polypeptide or the epitope of the wild-type p-Hyde polypeptide, p- from each sample.
P-Hyde polypeptide from said tumor sample and p-Hy from said non-tumor sample, performed by contacting with Hyde polypeptide and detecting antibody binding.
Differences with the de polypeptide suggest the presence of somatic alterations of the p-Hyde gene in the tumor samples.

The present invention is particularly useful for screening compounds by using p-Hyde polypeptides or binding fragments thereof in any of a variety of drug screening techniques. The p-Hyde polypeptide or fragment thereof used in such assays may either be free in solution, immobilized on a solid support, or present on the cell surface. . One method of drug screening uses prokaryotic or eukaryotic host cells, which are stably transformed with recombinant polynucleotides expressing the polypeptide or fragment, preferably in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays. For example,
The formation of a complex between the p-Hyde polypeptide or fragment and the agent being tested may be measured, or the formation of a complex between the p-Hyde polypeptide or fragment and a known ligand is tested. The degree of interference with the existing drug may be tested.

Accordingly, the invention provides for contacting such agents with a p-Hyde polypeptide or fragment thereof by methods well known in the art, (i) between the agent and the p-Hyde polypeptide or fragment. The presence of a complex of, or (ii
2.) A method for screening a drug, comprising assaying for the presence of a complex between a p-Hyde polypeptide or fragment and a ligand. In such competitive binding assays, the p-Hyde polypeptide or fragment is typically labeled. The free p-Hyde polypeptide or fragment is separated from the presence of the protein: protein complex and the amount of free (ie uncomplexed) label is determined by the binding or p-Hyde of the agent under test, respectively. pH
yde: measurement of its interference by ligand binding.

Another technique of drug screening provides high power screening for compounds that have a suitable binding affinity for p-Hyde polypeptide, 198
Geysen, PCT Specification No. WO 84/03, issued September 13, 4
No. 564 for more details. Briefly, a number of different small peptide test compounds are synthesized on a solid phase substrate such as plastic pins or some other surface. Peptide test compounds are reacted with p-Hyde polypeptide and washed. Bound p-Hyde polypeptide is then detected by methods well known in the art.

Purified p-Hyde can be coated directly onto plates for use in the drug screening techniques described above. However, non-neutralizing antibodies to the polypeptide can be used to capture the antibody to immobilize the p-Hyde polypeptide on the solid phase. The present invention also relates to the use of a competitive drug screening assay in which a neutralizing antibody capable of specifically binding to p-Hyde polypeptide competes with a test compound for binding to p-Hyde polypeptide or a fragment thereof. I am planning. In this manner, the antibody can be used to detect the presence of any peptide that shares one or more antigenic determinants of the p-Hyde polypeptide.

Additional techniques for drug screening include the use of eukaryotic cell lines or groups of cells (as described above) that carry a non-functional p-Hyde gene. These host cell lines or groups of cells are defective at the p-Hyde polypeptide level. The host cell line or cells are grown in the presence of the drug compound. The host cell growth rate is measured to determine if the compound can regulate the growth of p-Hyde deficient cells.

The goal of ideal drug design is a biologically active polypeptide of interest, or, for example, a more active or stable form of the polypeptide, or, for example, the function of the polypeptide in vivo. Is to produce structural analogs of small molecules (eg, agonists, antagonists, inhibitors) with which it interacts to form drugs that enhance or interfere with. For example Hodgson, 1
See 991. In one approach, the three-dimensional structure of the protein of interest, or for example the p-Hyde receptor or ligand complex, is first determined by X
Determined by line crystallography, by computer modeling, or most commonly by a combination of approaches. Less often, useful information regarding the structure of polypeptides may be obtained by modeling based on the structure of homologous proteins. An example of an ideal drug design is the development of HIV protease inhibitors (Erickson et al., 1990). In addition, peptides (e.g., p-Hyde polypeptide) were labeled with Alanic Scan (Wells).
, 1991). In this technique, an amino acid residue is replaced by Ala and its effect on the activity of the peptide is measured. Each amino acid residue of the peptide is analyzed in this fashion to determine the key regions of the peptide.

It is also possible to isolate target-specific antibodies, select by functional assays and then interpret their crystal structure. In principle, this approach yields a drug core upon which subsequent drug design can be based. By generating anti-idiotypic antibodies (anti-ids) to functional, pharmacologically active antibodies, protein crystallography can be completely ignored. As a sub-image of the sub-image, the binding site for anti-ids is expected to be an analog of the original receptor. The anti-id can then be used to identify and isolate peptides from chemically or biologically produced banks of peptides. The selected peptide now serves as the drug core.

Thus, for example, drugs may be designed that have improved activity or stability of p-Hyde polypeptide or that act as inhibitors, agonists, antagonists of p-Hyde polypeptide activity. Due to the utility of the cloned p-Hyde sequence, a sufficient amount of p-H to carry out analytical studies such as x-ray crystallography.
The yde polypeptide may be available. Moreover, knowledge of the p-Hyde protein sequences provided herein may guide the use of computer modeling techniques in place of or in addition to x-ray structure.

In the present invention, (a) contacting p-Hyde with a chemical compound under conditions that allow a bond between p-Hyde and the chemical compound, (b) p-Hy of the chemical compound
Sensitivity to cell death comprising detecting binding to de, and (c) determining whether the chemical compound inhibits p-Hyde so as to identify the chemical compound capable of inducing susceptibility to cell death. Methods are provided for identifying chemicals that are capable of inducing. Useful diagnostic techniques include in situ hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern blot analysis, single strand structure analysis (SSCA), Rna, as discussed in further detail below.
Se protection assays, allele-specific oligonucleotides (ASO), dot blot analysis and PCR-SSCP include but are not limited.

A rapid preliminary analysis for detecting polymorphisms in DNA sequences can be performed by looking at successive Southern blots of DNA cleavage by one or more restriction enzymes, preferably multiple restriction enzymes. Each blot contains a series of normal individuals and a series of tumors. Southern blots presenting hybridizing fragments, which differ in length from the control DNA when probed with sequences near or containing the p-Hyde gene, suggest possible mutations. Pulsed area gel electrophoresis (PFGE) when using restriction enzymes that produce very large numbers of restriction fragments
To use.

Detection of point mutations may be accomplished by molecular cloning of the p-Hyde allele (s) and sequencing of the allele (s) using techniques well known in the art. Alternatively, the gene sequence can be amplified directly from a genomic DNA preparation from tumor tissue using known techniques. The DNA sequence of the amplified sequence can then be determined. There are six well-known methods for more complete, but indirect, tests to confirm the presence of susceptible alleles. 1) Single-stranded structure analysis (SSCA) (Orita et al., 1989), 2) Reduction gradient gel electrophoresis (DGGE) (Wartell et al., 1990, S).
heffield et al. , 1989), 3) RNase protection assay (
Finkelstein et al. , 1990, Kinszler et
al. , 1991), 4) allele-specific oligonucleotides (ASOs) (
Conner et al. , 1983), 5) E. coli mut.
Use of proteins that recognize nucleotide maladaptation, such as the S protein (Mo
drich, 1991), and 6) allele-specific PCR (Rano &
Kidd, 1989). For allele-specific PCR, plaimer is used to hybridize with a specific p-Hyde mutation at its 3'end. No amplification product is observed in the absence of the particular p-Hyde mutation. Amplification Refractory Mutati
on System: ARMS) can also be used, see European Patent Specification No. 033.
2435 and Newton et al. , 1989. Gene insertions and deletions can also be detected by cloning, sequencing and amplification. In addition, restriction fragment length genetic polymorphism (RFLP) probes to the gene or surrounding marker genes can be used to score insertion changes in alleles or polymorphic fragments. Such a method can be found in p
-Especially useful for screening individual relationships that influence the presence of the Hyde mutation. Other techniques known in the art for detecting insertions and deletions can also be used.

In a similar fashion, DNA probes can be used to detect mismatches via enzymatic or chemical cleavage. For example, Cotton et al. , 1988,
Shenk et al. , 1975, Novack et al. , 1986
checking ... Alternatively, the mismatch can be detected by a change in electrophoretic mobility of the mismatched duplex compared to the matched duplex. For example, Cariello, 1988.
checking ... With either riboprobes or DNA probes, cellular mRNA or DNA that may contain mutations can be amplified using PCR (see below) prior to hybridization. Changes in the DNA of the p-Hyde gene can also be detected using Southern hybridization, especially if the changes are significant rearrangements such as deletions or insertions.

The DNA sequence of the p-Hyde gene amplified by use of PCR may also be screened with allele-specific probes. These probes are nucleic acid oligomers, each containing a region of the p-Hyde gene sequence containing known mutations. For example, one oligomer can be about 30 nucleotides in length, corresponding to a portion of the p-Hyde gene sequence. By using a series of such allelic probes, PCR amplification products can be screened to identify the presence of previously identified mutations in the p-Hyde gene. Hybridization of the allele-specific probe with the amplified p-Hyde sequence can be performed, for example, on a nylon filter. Hybridization to a particular probe under stringent hybridization conditions suggests the presence of similar mutations in the tumor tissue as in the allele-specific probe.

Altered p-Hyde mRNA expression can be detected by any technique known in the art. These include Northern blot analysis, PCR amplification and RNase.
Includes protection. A decrease in mRNA expression suggests an alteration in the wild type p-Hyde gene. Changes in the wild-type p-Hyde gene can also be detected by screening for changes in the wild-type p-Hyde protein. For example, pH
Monoclonal antibody immunoreactivity with yde can be used to sort tissues. Deletion of the cognate antibody may indicate a p-Hyde mutation. Antibodies specific for the product of the mutant allele can also be used to detect the mutant p-Hyde gene product. Such immunological assays can be performed in any conventional format known in the art. These include Western blots, immunohistological assays and ELISA assays. Any method for detecting altered p-Hyde protein can be used to detect alterations in the wild-type p-Hyde gene. Functional assays such as protein binding measurements can be used.
In addition, the assay can be used to detect p-Hyde biochemical function. The discovery of a mutated p-Hyde gene product suggests a change in the wild-type p-Hyde gene. Mutant p-Hyde genes or gene products can also be detected in other human body samples such as serum, stool, urine, and saliva.

The present invention also provides fusion polypeptides, including p-Hyde polypeptides and fragments. The cognate polypeptide may be fused between two or more p-Hyde polypeptide sequences or between proteins associated with the sequence of p-Hyde. Similarly, heterologous fusions may be constructed to display combinations of properties or activities of derived proteins. For example, ligand binding or other regions may be "swapped" between different novel fusion polypeptides or fragments. Such homologous or heterologous fusion polypeptides may exhibit altered binding strength or specificity, for example. Fusion partners include immunoglobulins, microbial beta-galactosidase, trpE, protein A, beta-lactamase, alpha amylase, alcohol dehydrogenase and yeast alpha mating factor. For example, G
odwsli et al. , 1988. Fusion proteins are typically made by the recombinant nucleic acid methods described below, or may be made by chemical synthesis. Techniques for the synthesis of polypeptides are described, for example, in Merrif
field, 1963.

The probe for the p-Hyde allele may be derived from the sequence of the p-Hyde region or its cDNA. The probe may be of any suitable length,
It is all or part of the p-Hyde region and allows specific hybridization to the p-Hyde region. If the target sequence contains a sequence identical to that of the probe, the probe may be shorter, for example in the range of about 8-30 base pairs, as the hybrid is stable under stringent conditions. If some degree of mismatch is expected in the probe, i.e., the probe is likely to hybridize to different regions, use longer probes that hybridize to the target sequence with the required specificity. You may

Probes may include isolated polynucleotides attached to a label or reporter molecule and may be used to isolate other polynucleotide sequences and may be used to isolate sequence similarity by standard methods. have. For techniques for preparing and labeling probes, see, eg, Sambrook et al. , 19
89 or Ausubel et al. , 1992.

Probes containing synthetic oligonucleotides or other polynucleotides of the invention may be derived from naturally occurring or recombinant single-stranded or double-stranded polynucleotides, or chemically synthesized. Good. The probe may also be labeled by nick translation, Klenowfilin reaction, or other methods known in the art. The portion of the polynucleotide sequence that has at least about 8 nucleotides, usually at least about 15 nucleotides and is about 6 kb or less, usually about 1.0 kb or less, is preferred as a probe than the polynucleotide sequence encoding p-Hyde. The probe may also be used to determine whether mRNA encoding p-Hyde is present in cells or tissues.

The present invention obtains cells, the cells of which are under control of the Rous sarcoma virus promoter, deletions in the E1 and E3 regions of the genome, and a region of nucleic acid encoding p-Hyde. Provided is a method for suppressing the growth of cancer cells, which comprises the step of contacting with a replication-defective adenovirus type 5 expression vector containing an adenovirus genome having an insertion therein, thereby suppressing the growth of prostate cancer cells.

The present invention provides for 1) obtaining a sample of prostate cells from a patient, 2) deleting these cells in the E1 and E3 regions of the genome under the control of the Rous sarcoma virus promoter,
And contacting with a replication-defective adenovirus type 5 expression vector containing an adenovirus genome having an insertion in the region of the nucleic acid encoding p-Hyde, and 3) introducing the cell into a patient, thereby Provided is a method for suppressing the growth of prostate cancer cells, which comprises suppressing the growth of cells.

The present invention provides a nucleic acid encoding a p-Hyde protein, p-, in a cancer cell.
Proliferation of cancer cells in a patient, comprising introducing a nucleic acid encoding a fragment of the Hyde protein or a nucleic acid encoding a mutant p-Hyde protein, thereby suppressing the growth of cancer cells in the patient Provide a method of suppressing.

The present invention provides a patient with a therapeutically effective amount of a nucleic acid encoding a p-Hyde protein, a nucleic acid encoding a fragment of a p-Hyde protein, or a mutated p-.
Within a patient, comprising administering a pharmacological composition comprising a nucleic acid encoding a Hyde protein and a pharmaceutically acceptable carrier or diluent, thereby inhibiting the growth of cancer cells in the patient. A method for suppressing the growth of cancer cells is provided.

As demonstrated herein, the apoptotic response is assessed using the DNA laddering assay. DNA was extracted after each treatment (24 h 1 mM hydrourea, followed by 24 h 0.1 mM 5′-dFUrd) and 1.6
Analysis was performed on% agarose gel electrophoresis. According to cell cycle analysis, the apoptotic response of stable transfectants, AT1-H1 and AT3-H1 (AT-1 and AT-3 transfected with pcHYDE), was determined by the parent (AT-1 and AT-3) and pcDNA. Transfected parental cell line (AT1-pc1
And AT3-pc1) consistently and significantly higher. Above all,
The highest apoptotic response was 0.1 mM 5'-dF, as shown in FIG.
It occurred in a synchronous culture under incubation with Urd. Enhancement of the apoptotic response of AT1-H1 and AT3-H1 transfectants after hydroxyurea treatment is due to intracellular thymidine-5-triphosphate (TTP) via direct inhibition of thymidylate synthase by fluorodeoxyuridine. ) "Thymine deficiency death" leading to pool depletion (Kyprianou, 1994, Kyprianou).
et al. , 1994). However, the actual mechanism of apoptosis itself associated with TTP depletion is unknown. Taken together, these data suggest that the apoptotic response in pcHYDE stable transfectants may be due to the downstream effects of the pcHYDE gene product.

The present invention provides a nucleic acid encoding p-Hyde protein, p-, in cancer cells.
A method of inducing susceptibility to apoptosis of cancer cells, comprising introducing a nucleic acid encoding a fragment of Hyde protein or a nucleic acid encoding a mutant p-Hyde protein, thereby inducing susceptibility to apoptosis. provide.

The invention provides a patient with a therapeutically effective amount of a nucleic acid encoding a p-Hyde protein, a nucleic acid encoding a fragment of a p-Hyde protein, or a mutated p-.
Sensitization of cancer cells to apoptosis, including administering a pharmacological composition comprising a nucleic acid encoding a Hyde protein and a pharmaceutically acceptable carrier or diluent, thereby inducing susceptibility to apoptosis. Provide a way to induce.

As demonstrated herein, p-Hyde suppresses cancer growth in vivo. Low levels of p-Hyde expression were observed in the AT3 cell line as shown in Figure 9. For this reason, the AT3 cell line was selected under G418 selection, pcHYDE, p
The construct of p-Hyde in a mammalian expression vector of cDNA 3.1 (-) was transfected. As a negative control, the AT-3 cell line was transfected with only the vector, and the obtained stable transfectant was designated as AT3-pc. AT-
Growth of 3 Parental Cell Lines and Stable Transfectants AT3-H1 and A
Those at T3-H2 were evaluated in vivo. One million cells of each cell line in 0.3 ml Hank's solution were subcutaneously transplanted into each inbred Oscopenhagen rat. In an initial experiment, 3 groups of 5 rats each were injected with each cell line. Tumor size was scored after the steps shown in FIG. These preliminary results indicate that both AT3-H1 and AT3-H2 stable transfectants grow significantly slower than the AT-3 parental cell line, with tumor growth regression in both stable transfectants. suggesting that it was regulated by the pcHYDE gene product.

Interestingly, p-Hyde has the dual ability of acting like a tumor suppressor gene and thus inducing susceptibility to apoptosis, which may be a pathway independent of p53. Prostate cancer growth in rats is significantly inhibited by p-Hyde. Moreover, prostate cancer cells expressing p-Hyde are more susceptible to UV DNA damage that induces the cells to programmed death. As a result of analysis of DNA repair enzyme activity, it was found that the original (6-4) PP
And a defect that resulted in reduced cell viability by the colony formation assay. However, the ability of p-Hyde to induce susceptibility to apoptosis is not limited to UV DNA damage. Fluorodeoxyuridine, a pyrimidine antimetabolite and chemotherapeutic agent related to fluorouracil (5-FU) and used in the treatment of a wide range of human epithelial malignancies, is also p-Hyde.
Induces apoptosis in prostate cancer cells expressing Furthermore, p-Hyd
Cancer cells expressing e are also more sensitive to gamma radiation. Therefore,
The mechanism of cellular DNA damage is different for UV, gamma irradiation, and fluorodeoxyuridine, indicating that the ability to make cells more susceptible to apoptosis is more global in activity. This unique function of p-Hyde may represent a new type of gene that induces susceptibility to apoptosis. This is distinct from the function responsible for tumor suppressor genes such as p53 that induce apoptosis directly and not susceptibility to apoptosis (Y
onish-Rouach et al. , 1991). Furthermore, p-Hyde
Activity, in contrast to bcl-2, which does not occur in the absence of bcl-2, makes cancer cells more susceptible to programmed death of cells (McDonelle).
t al. , 1992).

The present invention provides a nucleic acid encoding a p-Hyde protein, p-, in a cancer cell.
Provided is a method for suppressing cancer cells, which comprises introducing a nucleic acid encoding a fragment of Hyde protein or a nucleic acid encoding a mutant p-Hyde protein, thereby inducing susceptibility to apoptosis.

The present invention provides a patient with a therapeutically effective amount of a nucleic acid encoding a p-Hyde protein, a nucleic acid encoding a fragment of a p-Hyde protein, or a mutated p-.
Provided is a method for suppressing cancer cells, comprising administering a pharmacological composition comprising a nucleic acid encoding a Hyde protein and a pharmacologically acceptable carrier or diluent, thereby suppressing cancer cells. To do.

The present invention provides a therapeutically effective amount of a nucleic acid encoding a p-Hyde protein,
Nucleic acid encoding a fragment of p-Hyde protein, or mutated p-Hyde
Provided is a method of treating a cancer patient, comprising administering a pharmacological composition comprising a nucleic acid encoding a protein and a pharmaceutically acceptable carrier or diluent, thereby treating the cancer patient. .

The invention relates to 1) a nucleic acid encoding a p-Hyde protein, a nucleic acid encoding a fragment of the p-Hyde protein, in combination with radiation therapy, chemotherapy or a UV mimetic agent in a patient. , Or a nucleic acid encoding a mutated p-Hyde protein and 2) administering a pharmacological composition comprising a pharmaceutically acceptable carrier or diluent, thereby treating a cancer patient, Methods of treating cancer patients are provided.

The unique functional properties of the p-Hyde gene may be exploited in the treatment of hyperproliferative damage and cancer. One effective therapeutic strategy may be, for example, treatment of carcinoma cells expressing p-Hyde with a chemotherapeutic agent (such as acetylaminofluorine) or a UV mimetic drug. However, cancer cells are unlikely to produce significant levels of growth inhibition of p-Hyde. As a result, the p-Hyde gene may be introduced into cancer cells by gene therapy. Cancers transformed with vectors containing p-Hyde are not only directly suppressed by p-Hyde as shown in this study, but also DNAs such as chemotherapeutic agents, UV mimetics or radiotherapy.
It may even have a better anti-cancer effect when treated in combination with injury treatment. Since gene therapy may target cancer cells, here enhanced apoptosis may occur more selectively in cancer cells that have undergone DNA damage (UV, radiation therapy or chemotherapy).

Three DNA enzyme repair systems were elevated in the parent compared to p-Hyde transfected cells. Uridine phosphorylase, uridine kinase, and UV damage repair. UV damage repair did not function properly in p-Hyde-transfected cells. FIG. 16 shows that a decrease in DNA repair activity results in a higher concentration of the original photochemical reaction product (64PP). Consistent with these data, p-Hyde-transfected cells also had significantly reduced survival after UV exposure compared to parental AT3 cells as measured by colony formation assay. Thus, DNA repair enzyme deficiency was associated with shorter survival and induction of apoptosis in p-Hyde-transfected prostate cancer.

The present invention provides: 1) an adenovirus genome under the control of the Rous sarcoma virus promoter, having a deletion in the E1 and E3 regions of the genome and an insertion within the region of the nucleic acid encoding p-Hyde. Adenovirus type 5 expression vector containing,
And 2) providing a method of treating a cancer patient, comprising administering to the patient a therapeutically effective amount of a pharmaceutically acceptable composition comprising a suitable carrier or diluent. In one embodiment, the cancer may be selected from the group consisting of melanoma, lymphoma, leukemia and prostate, rectum, pancreas, breast, brain or gastric cancer.

The present invention provides the methods required for the production of therapeutics based on genes directed against cancer cells. These therapeutically suitable agents are located in a preferred vector, p-Hyd
It may take the form of a polynucleotide containing all or part of the e locus, or may be delivered to the target cell in a more direct manner such that the function of the p-Hyde protein is restored. Therapeutic agents may also take the form of polypeptides based on either part of p-Hyde or the entire protein sequence. These may functionally replace the activity of p-Hyde in vivo.

Suitable body fluids include, but are not limited to, serum, plasma, cerebrospinal fluid, lymphocytes, urine, exudate, or exudate. In a preferred embodiment, the suitable body fluid sample is serum or plasma. Further, the body fluid sample may be cells from bone marrow or supernatant from cell culture. Methods of obtaining a suitable body fluid sample from a patient are known to those of skill in the art. Methods for determining antibody or antigen concentration include, but are not limited to, ELISA, IFA and Western blotting.

Diagnostic assays of the invention include nucleic acid hybridization assays, and nucleic acid assays such as those that detect amplification of a particular nucleic acid for detection with respect to the human p-Hyde nucleic acid sequences described herein. possible.

Target-specific probes may be used in nucleic acid hybridization diagnostics. The probe is specific or complementary to the target of interest. For correct allelic differentiation, the probe should be approximately 14 nucleotides long, preferably 20-30 nucleotides. For the more general detection of human p-Hyde of the present invention, the nucleic acid probe is about 50 to about 1000 nucleotides, more preferably about 200 to about 400 nucleotides.

Specific nucleic acid probes can be RNA or DNA polynucleotides or oligonucleotides, or analogs thereof. The probe may be single-stranded or double-stranded nucleotide. The probes of the present invention may be enzymatically or chemically (eg, both incorporated by reference) using methods well known in the art (eg, nick translation, primer extension, reverse transcription, polymerase chain reaction, etc.). As described herein, such as by the phosphoramidite method described by Beaucage and Carruthers [19], or by Matteuc.
ci et al. (By the triester method according to [62]).

Another method for determining the presence of human p-Hyde is in situ hybridization, or more recently, in situ polymerase chain reaction.
In situ PCR was performed according to Neuvo et al. [71] Subcellular localization of polymerase chain reaction (PCR) amplified hepatitis C cDNA (Intracellula
r localization of polymerase chain r
action (PCR) -amplified Hepatitis C c
DNA), Bagasra et al. [10], Detection of type I human immunodeficiency virus provirus in mononuclear cells by in situ polymerase chain reaction (D
inspection of Human immunity science v
irus type I provirus in monocular c
ells by in situ polymerase chain rea
action), and Heniford et al. [35], in situ
Intracellular EGF receptor mRN demonstrated by u reverse transcription polymerase chain reaction
Change in A expression (Variation in cellular EGF rec
eptor mRNA expression demonstrated b
y in situ reverse polymerease chain r
action). In situ hybridization assays are well known and are generally described by Meth, incorporated herein by reference.
ods Enzymol. It is described in [67]. For in situ hybridization, cells are fixed to a solid support, typically a glass slide.
The cells are then contacted with the hybridization solution at a temperature suitable to allow the annealing of the labeled target-specific probe. The probe is preferably labeled with a radioisotope or a fluorescent reporter.

The probes described above may also be used for in situ hybridization, or for locating tissue expressing this gene, or for the presence of this gene or its MRNA in various biological tissues. It is useful for other hybridization assays for. In-situ hybridization is a sensitive localization method that is independent of antigen expression or natural versus denaturing conditions.

Briefly, inhibitory nucleic acid therapeutic approaches target DNA sequences (prem
It can be classified into those that target RNA sequences (including RNA and mRNA), those that target proteins (sense strand approach) and those that cause cleavage or chemical modification of target nucleic acids.

DNA targeting approaches can be divided into several types. Nucleic acids can be designed to bind in the larger groove of double-stranded DNA so as to form a triple helix or "triad" structure. Alternatively, inhibitory nucleic acids can be designed to bind to regions of single-stranded DNA as a result of cleavage of double-stranded DNA during replication or transcription.

More generally, inhibitory nucleic acids are designed to bind to mRNA or mRNA precursors. The inhibitory nucleic acid is used to prevent pre-mRNA maturation.
Inhibitory nucleic acids may be designed to interfere with RNA processing, splicing or translation.

The inhibitory nucleic acid can target the mRNA. In this approach, inhibitory nucleic acids are designed to specifically inhibit translation of the encoded protein. Using this approach, inhibitory nucleic acids can be used to selectively suppress certain cellular functions by inhibiting translation of mRNA encoding key proteins. For example,
An inhibitory nucleic acid complementary to a region of c-myc mRNA inhibits c-mys protein expression in the human promyelocytic cell line HL60, which overexpresses the c-mys protooncogene. Wichstrom E. L. , Et al. [93] and Harel-Bellan, A .; , Et al. See [31A]. He
As described in len and Toulme, inhibitory nucleic acids targeting mRNA have been shown to work by several different mechanisms to inhibit translation of the encoded protein (s).

In conclusion, inhibitory nucleic acids can be used to induce chemical inactivation or cleavage of target genes or mRNA. Chemical cleavage can occur in cells by the induction of crosslinks between inhibitory and target nucleic acids. Other chemical modifications of the target nucleic acid induced by the preferably induced inhibitory nucleic acid may also be used.

Cleavage, and thus inactivation, of target nucleic acids can be affected by the attachment of substituents to inhibitory nucleic acids that can be activated to induce a cleavage reaction. Substituents can affect either chemical or enzymatic cleavage. Alternatively, cleavage can be induced by the use of ribosomes or catalytic RNA. In this approach, the inhibitory nucleic acid may comprise either naturally occurring RNA (ribozyme) or synthetic nucleic acid with catalytic activity.

As used herein, a “pharmaceutical composition”
"al composition" is a therapeutically effective amount of a polypeptide of the invention, together with suitable diluents, preservatives, stabilizers, emulsifiers, adjuvants and / or carriers, useful in SCF (Stem Cell Factor) therapy. Means product. As used herein, a "therapeutically effective amount.
“Tive amount” refers to an amount that provides a therapeutic effect for a given condition and dosing regimen. Such compositions are liquid or lyophilized, or otherwise in dry formulation, to prevent absorption of various buffer components (eg Tris-HCl, acetic acid, phosphate), pH and ionic strength, surface. Additives such as albumin or gelatin, surfactants (eg Tween 20, Tw
een80, Pluronic F68, bile salts, solubilizers (eg glycerol, polyethylene glycol), antioxidants (eg ascorbic acid, sodium metabisulfite), preservatives (eg Thimerosal).
), Benzyl alcohol, parabens), grinding substrates or tension modifiers (eg lactose, mannitol), covalent attachment of polymers such as polyethylene glycol to proteins, complexation with metal ions, or polylactic acid, polyglycolic acid, Incorporation of substances into or on microparticulate preparations of polymerized compounds such as hydrogels or on liposomes, microemulsions, micelles, unilamellar particles, red blood cells or spheroplasts. Such compositions can affect the physical state, solubility, stability, release rate in vivo and clearance rate of SFC in vivo. The choice of composition may depend on the physical and chemical activity of the protein having SFC activity. For example, products derived from the membrane-bound form of SCF may require a formulation containing a detergent. Controlled or sustained release compositions include formulation in lipophilic sustained release formulations (eg fatty acids, waxes, oils). Also contemplated by the present invention are microparticle compositions coated with a polymer (eg, polyoxamer or polyoxamine) and SCF conjugated to antibodies to tissue-specific receptors, ligands or antigens, or to ligands of tissue-specific receptors. Another embodiment of the composition of the present invention is a particulate morphological protective coating, parenteral,
Incorporate protease inhibitors or penetration enhancers for various routes of administration including lung, nose and mouth. In one embodiment, the pharmacological composition is administered parenterally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally, intraventricularly, intracranially.

Further, as used herein, "pharmacologically acceptable carrier (pha).
"rmaceutically acceptable carriers" are well known to those of skill in the art and include, but are not limited to, 0.01-0.1 M and preferably 0.05 M phosphate buffer, or 0.8% saline. Further, such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are polypyrene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, including saline and interference solvents,
Includes alcohol / aqueous solutions, emulsions or suspensions. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, emulsified Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives such as anti-microbial agents, antioxidants, auxiliaries, inert gases and the like may also be present.

The phrase "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. Adjuvants can serve as tissue sustained release formulations that slowly release antigens and as lymphoid activators that nonspecifically enhance immune responses (Hood et al., Immunology. Second).
Ed. 1984, Benjamin / Cummings, Menlo Par
k, California p. 384) Primary challenge with antigen alone, without adjuvant, often does not induce a somatic or cellular immune response. Adjuvants include complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronine polyols, polyanions, peptides, oils or hydrocarbons. Emulsions, keyhole limpet hemocyanins, dinitrophenol, and BCG (bacilli Calmette)
-Guerin) and Corynebacterium parvum are potentially useful human adjuvants, including but not limited to. Preferably, the adjuvant is pharmacologically acceptable.

Controlled or sustained release compositions include lipophilic sustained release formulations (eg fatty acids, waxes,
Oil). Further contemplated by the invention are particulate compositions coated with a polymer (eg, poloxamer or poloxamine) and compounds bound to antibodies to tissue-specific receptors, ligands or antigens bound to ligands of tissue-specific receptors. . Other embodiments of the compositions of the invention incorporate protective coated particles, protease inhibitors or penetration enhancers for various routes of administration including parenteral, pulmonary, nasal, oral.

Upon administration, the compounds often are rapidly cleared from the mucosal surface or circulation, inducing relatively short-term pharmacological activity. Therefore, frequent administration of relatively large amounts of bio-affecting compounds is required to maintain therapeutic efficacy. Compounds modified by covalent attachment of water-soluble polymers such as polyethylene glycol, polyethylene glycol and copolymers of polyethylene glycol and polypropylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline are compared to the corresponding unmodified compounds after intravenous injection. It is known to exhibit a considerably long half-life in the blood (Abuchowski et al., 19).
81, Newmark et al. , 1982, and Katre et
al. , 1987). Such modifications may also increase the solubility of the compound in aqueous solution, eliminate aggregation, increase the physical and chemical stability of the compound, and greatly reduce the immunogenicity and reactivity of the compound. . As a result, the desired in vivo
o Biological activity is obtained by the administration of such polymeric compounds which abduct less frequently or at lower doses than with unmodified compounds.

Dosage: A sufficient amount includes, but is not limited to, about 1 μg / kg to about 1000 mg / kg. The amount may be 10 mg / kg. The pharmaceutically acceptable form of the composition comprises a pharmaceutically acceptable carrier.

The preparation of therapeutic compositions that contain active ingredients is well known to those of ordinary skill in the art. Typically,
Such compositions are prepared either as aerosols of the polypeptide for nasopharyngeal administration or as injectables, as liquid solutions or suspensions, but stable in solution or suspension in liquid prior to injection. It may also be prepared as a solid form. The preparation may be an emulsion type. The therapeutically active ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof. In addition, if desired, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents in tumor tissue which enhance the effectiveness of the active ingredient.

The active ingredient may be formulated into the therapeutic composition in the form of neutralized pharmaceutically acceptable salts. Pharmacologically acceptable salts include acid addition salts (formed with free amino groups of the polypeptide or antibody molecule) and inorganic acids such as hydrochloric acid or phosphoric acid, or acetic acid, oxaphosphoric acid, tartaric acid. , Salts formed with organic acids such as mandelic acid. Salts formed from free carbonyl groups are also inorganic bases such as sodium, potassium, ammonium, calcium, or iron hydroxide, and organic bases such as isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like. You may derive from

If at least about 75% by weight of the protein, DNA, vector of the composition (depending on the category of species to which A and B belong) is “A” (here “A
Is a single protein, DNA molecule, vector, etc.) is substantially
(“B” is composed of one or more proteins, DNA molecules, vectors, etc.). Preferably, "A" comprises at least about 90% by weight of the A + B species in the composition, and most preferably at least about 99% by weight.

“Therapeutically effective amount
The term “amount)” is used herein to reduce a clinically significant impairment of host activity, function, or response by at least 15%, preferably by at least 50%, more preferably by at least 90%, most preferably Preferably it refers to an amount sufficient to prevent.

According to the invention, the components of the invention or components of therapeutic compositions may be parenterally, transmucosally, eg orally, nasally, pulmonary or rectal, or transdermally, parenterally, It may be introduced transmucosally. Preferably, administration also includes, but is not limited to, parenteral, eg, by intravenous injection, or intraarterial, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. Oral or pulmonary introduction activates mucosal immunity. Mucoimmunity is a particularly effective prophylactic treatment because pneumococci generally colonize the nasopharynx and lung mucosa. The term "unit dose" as used in reference to the therapeutic compositions of the present invention is suitable as a unit dose for humans,
Refers to physically discrete units, each unit containing a predetermined amount of the active agent calculated to produce the desired therapeutic effect in association with the required excipient, ie carrier or vehicle.

The present invention also provides a method for supplying wild-type p-Hyde function to cells having a mutant p-Hyde allele. Supply of such a function can be expected to suppress the growth of neoplasms of recipient cells. The wild-type p-Hyde gene or part of this gene may be introduced intracellularly by a vector such that the gene remains extrachromosomal. In such cases, the gene is expressed by the cell at the extrachromosomal site. If the gene fragment is introduced and expressed in cells carrying the mutated p-Hyde allele, the gene fragment should encode part of the p-Hyde protein required for non-neoplastic growth of the cell. is there. More preferably, the wild-type p-Hyde gene or a part thereof is used for endogenous p-Hy existing in the cell.
This is the case of introducing into a mutant cell by a method such as recombination using the de gene. Such recombination requires double recombination to correct for the p-Hyde gene mutation. Vectors for gene transfer in recombination and extrachromosomal maintenance include
Any suitable vector known to those of skill in the art may be used. Techniques for introducing DNA into cells, such as electroporation, calcium phosphate precipitation and virus-mediated transduction, are well known to those of skill in the art, and the choice of the technique is up to the person performing the procedure. Cells transformed with the wild-type p-Hyde gene can be used as a model system in the study of cancer sedation and drug treatments that promote such sedation.

As discussed generally in Abnormalities, p-Hyde genes or fragments, where applicable, can be used in gene therapy to increase the amount of expression products of such genes in cancer cells. Good. Such gene therapy is particularly suitable for use with both cancer and pre-cancerous cells, where p-Hyde polypeptide levels are absent or reduced compared to normal cells. For a given p- in Rumor cells such that the mutated gene is expressed at "normal" levels, but the gene product does not function well.
Increasing the expression level of the Hyde-conferring gene is also useful.

Gene therapy is a generally accepted procedure, eg Friedman, 199.
1 may be performed according to the method described in 1. Cells from a patient's tumor are first analyzed by the diagnostic procedure described above to confirm the production of p-Hyde polypeptide in Rumor cells. A viral or plasmid vector (see details below) containing a copy of the p-Hyde gene that is linked to expression control components and is capable of replicating in Rumor cells is prepared. Suitable vectors are, for example, US Pat. No. 5,252,479 and PCT Application No. WO 93/0.
It is known as described in No. 7282. The vector is then injected into the patient either locally at the Rumor site or systemically (to reach all Rumor cells that may metastasize to other sites). If the transfected gene is not permanently integrated into the genome of each target small cell, its treatment will need to be repeated periodically.

Gene delivery systems known to those of skill in the art may be useful in practicing the gene therapy methods of the invention. These include viral and non-viral transmission methods. Some viruses used as gene transfer vectors include papovaviruses such as SV40 (Madzak et al., 1992), adenovirus (B
erkner, 1992, Berkner et al. , 1988, Gorz
iglia and Kapikian, 1992, Quantin et a
l. , 1992, Rosenfeld et al. , 1992, Wikins
on et al. , 1992, Stratford-Perricaudet.
et al. , 1990), vaccinia virus (Moss, 1992), adeno-associated virus (Muzyczka, 1992, Ohi et al., 19).
90), HSV and EBV herpesviruses (Margolskee)
, 1992, Johnson et al. , 1990, Fink et al.
． , 1992, Breakfield and Geller, 1987, Fre.
ese et al. , 1990), and avian (Brandyopadh)
yay and Temin, 1984, Petropoulos et al.
． , 1992), in rodents (Miller, 1992, Miller et a.
l. , 1985, Serge et al. , 1984, Mann and B
altimore, 1985, Miller et al. , 1988), and of human origin (Shimada et al., 1991, Helseth e.
t al. , 1990, Page et al. , 1990, Buchscha
cher and Panganiban, 1992) retrovirus. Most human gene therapy protocols are based on detoxified rodent retroviruses.

Peptides with p-Hyde activity can be delivered to cells with mutated or deleted p-Hyde alleles. The sequence of the P-Hyde protein has been disclosed (
SEQ ID NO: 2). The protein is produced by expression of the cDNA sequence in a microorganism, for example using known expression vectors. Alternatively, p-Hyde polypeptide can be obtained from p-Hyde producing mammalian cells. In addition, synthetic chemistry techniques can be used to synthesize p-Hyde proteins. Any such technique is capable of delivering the preparations of the present invention, which include the p-Hyde protein. The preparation is substantially free of other human human proteins. This can easily be achieved by synthesis in microorganisms or in vitro.

The p-Hyde molecule may be introduced intracellularly, for example by microinjection or by the use of liposomes. Alternatively, some active molecules may be incorporated into cells either actively or by diffusion. Extracellular application of the P-Hyde gene product may be sufficient to affect rumor growth. Molecules with P-Hyde activity can lead to partial reversal of the neoplastic state. Delivery of other molecules with P-Hyde activity (eg peptides, drugs or organic compounds) may also be used to effect such reversal.

In another embodiment, the active compound may be introduced by vesicles, especially liposomes (Langer, Science 249: 1527-1533 (199).
0), Treat et al. , Liposome in the treatment of infectious diseases and cancer (
Lipossomes in the Therapy of Infectio
us Disease and Cancer), Lopez-Bereste
in and Fidler (eds.), Liss, New York, pp.
． 353-365 (1989), Lopez-Berestein, ibid.
, Pp. 317-327, and generally, ibid.).

In yet another embodiment, the therapeutic compound may be introduced by a controlled release system. For example, the polypeptide may be administered using an intravenous drip, an implantable osmotic pump, a transdermal patch, liposomes, or other dosage forms. In one embodiment, a pump may be used (Langer, supra, Sefton, CRC Crit. Re.
f. Biomed. Eng. 14: 201 (1987), Buchwald e
t al. , Surgery 88: 507 (1980), Saudek et.
al. , N .; Engl. J. Med. 321: 574 (1989)). In another embodiment, polymeric materials can be used (controlled release medical applications (M
medical Application of Controlled Rel
ease), Langer and Wise (eds.), CRC Pres
s. , Boca Raton, Florida (1974), Controlling Bioavailability of Controlled Drugs, Designing and Implementing Drug Production (Controlled Drug).
Bioavailability, Drug Product Design
and Performance), Smolen and Ball (eds
． ), Wiley, New York (1984), Ranger and P.
eppas, J .; Macromol. Sci. Rev. Macromol. Ch
em. 23:61 (1983), see also Levy et al. ，
Science 228: 190 (1985), During et al. , A
nn. Neurol. 25: 351 (1989), Howard et al.
J. Neurosurg. 71: 105 (1989)). In yet another embodiment, the controlled release system may be in the vicinity of the therapeutic target, ie, the brain, thus requiring only a small portion of the systemic dose (eg, Goodson, Medical Applications of Controlled Release of C).
controlled release), supra, vol. 2, pp. 115-1
38 (1984)). Preferably, the controlled release device is introduced into the patient in the vicinity of an inappropriate immunologically active site or tumor site. Another controlled release system is Lange
r (Science 249: 1527-1533 (1990)).

Humans are preferred, but any animal, where administration of the active compound as described above is an effective therapeutic regimen against bacterial infections. Therefore,
As will be readily appreciated by those of skill in the art, the techniques and pharmacological compositions of this invention may be any animal, preferably a mammal, and domesticated animals such as, but not limited to, felines or canines. , Domestic animals such as, but not limited to, bovine, equine, goat, sheep, pig, wildlife (whether in the wild or in the zoo), mouse, rat, rabbit, goat, sheep, pig, dog, It is particularly suitable for application to cats, etc., ie research animals for use in veterinary medicine.

In the therapeutic procedures and compositions of the present invention, a therapeutically effective dose of the active composition is obtained. A therapeutically effective dose can be determined by patient characteristics (as is known to those of skill in the art).
It may be determined by a medical professional based on age, weight, sex, health status, complications, and other diseases. In addition, further conventional studies will provide more clear information on the appropriate doses for the treatment of different patients with different conditions, and experts will be able to determine the treatment situation, age of the recipient and roughly The appropriate dose can be ascertained by taking into consideration the health condition of the patient. In general, the dose for intravenous injection or infusion may be lower than for intraperitoneal, intramuscular, or other routes of administration. Dosage regimens may vary with the circulating half-life and the mode of use. The composition is administered in a manner compatible with the therapeutically effective amount of the dosage regimen. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, a suitable dosage depends on the route of administration and is about 0.1 active ingredient per kg body weight of an individual per day.
~ 20 mg, preferably about 0.5 to about 10 mg, more preferably 1 to several mg
The range is good. Suitable initial dosing regimens and booster doses may also vary, but can be typified by initial administration followed by repeated doses at one or more time intervals by injection or other administration. Alternatively, a blood concentration of 10 nM
Sufficient continuous intravenous infusion to maintain at -10 μM is contemplated.

The present invention contemplates the use of alternating p-Hyde treatment in combination with chemotherapeutic or radiotherapeutic interventions. Expression vectors for "target" cells are used to induce susceptibility of cells such as malignant or metastatic cells to cell death, or to inhibit cell proliferation or kill cells using the techniques and compositions of the invention. And contacting at least one DNA damaging agent. In one embodiment, the cells are contacted with a single composition or a pharmacological formulation containing both agents, or at the same time 2
One separate composition or formulation, where one composition contains the vector and the other D
Contacting the pharmacological formulation by simultaneously contacting the cells, including the NA damaging agent. In another embodiment, treatment with the vector precedes or follows treatment with the DNA damaging agent at intervals of minutes to weeks. Protocols and techniques are known to those of skill in the art.

[0230] DNA damaging agents or agents are known to those of skill in the art and refer to therapeutic methods involving chemical damage or DNA damage when applied to cells. Such agents and agents include radiation and waves such as gamma irradiation, X-rays, UV irradiation, microwaves, electron emission. A variety of chemical compounds described as "chemotherapeutic agents" act to induce DNA damage, all of which are contemplated for use in the combination therapies disclosed herein. ing. Chemotherapeutic agents that may be of benefit include, for example, adriamycin, 5-fluorouracil (5FU), etoposide (VP-6), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP) and hydrogen peroxide. . The invention also includes the use of radiation-based, such as the use of X-rays with cisplatin or the use of etoposide with cisplatin, or the actual compound, in combination with one or more DNA damaging agents.

In another embodiment, the localized tumor site may be radiation treated with DNA damaging radiation such as X-rays, UV light, gamma rays or microwaves. Alternatively,
Administering to a patient a pharmacological compound containing a DNA damaging compound such as adriamycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D, mitomycin C, or more preferably cisplatin in a therapeutically effective amount of tumor cells May be contacted with a DNA damaging agent. DNA damaging agents are
By combining with the p-Hyde expression construct as described above, it may be prepared and utilized as a compound therapeutic composition or kit. Agents that directly crosslink nucleic acids, especially DNA, are foreseen and shown herein to cause DNA damage leading to a synergistic antineoplastic combination. Cisplatin, and other DNA alkylating agents may be used. Cisplatin is clinically widely used in the treatment of cancer with an effective dose of 20 mg / m ² every 3 weeks for a total of 3 courses for 5 days. Cisplatin is not absorbed orally and must be administered by intravenous, subcutaneous, intratumoral or intraperitoneal injection. Agents that damage DNA also include compounds that interfere with DNA replication, mitosis, and chromosome segregation. Such chemotherapeutic compounds include adriamycin, also known as doxorubicin, etoposide, verapamil, podophyllotoxin and the like. Widely clinically used in the treatment of tumors, these compounds are administered at 27-75 mg / adriamycin at 21-day intervals.
m ^2, administered by imaginary pills intravenous 35~50mg / m ^2, intravenous injection or twice the vein orally against etoposide.

Agents that disrupt the synthesis and fidelity of nucleic acid precursors and subunits also lead to DNA damage. In such cases, the number of nucleic acid precursors will increase. Especially useful is
It is a drug that has been extensively tested and is readily available. As such, agents such as 5-fluorouracil (5-FU), which are preferably used by neoplastic tissues, make them useful for targeting to neoplastic cells. Although quite toxic, 5-FU is applicable with a wide range of carriers, including topical, however, intravenous administration at doses in the range of 3-15 mg / kg / day is commonly used.

Other factors that cause DNA damage and are widely used include gamma rays,
Includes what is commonly known as X-rays and / or the direct delivery of radioisotopes to tumor cells. Other forms of DNA damaging factor are contemplated such as microwave and UV irradiation. All of these factors are damaged DNA, DNA precursors, DNA
Most likely to affect a wide range of replication and repair, and chromosome association and maintenance. X-ray doses range from 50 to 200 roentgens per day for long periods (3 to 4 weeks) to 2000 to 6000 roentgens per dose. Radioisotope abundances vary widely and depend on isotope half-life, emission intensity and type, and uptake by neoplastic cells.

Several non-viral methods for introducing expression constructs into mammalian cell culture are also contemplated in the present invention. These include calcium phosphate precipitation (Graham and Van Der Eb, 1973, Chen an.
d Okayama, 1987, Rippe et al. , 1990), DE
AE-Dextran (Gopal, 1985), electroporation method (
Tur-Kaspa et al. , 1986, Potter et al. ，
1984), direct microinjection (Harland and Weintraub,
1985), DNA-containing liposomes (Nicolau and Sene, 19).
82, Fraley et al. , 1979), and Lipofectamine-D.
NA complex, ultrasonic cell lysis (Fechheimer et al., 1987).
), Gene bombardment with fast microprojectiles (Yang et al.,
1990), and receptor-mediated transfection (Wu and Wu
, 1987, Wu and Wu, 1988). Some of these techniques may be well adapted for in vivo or ex vivo. Alternatively, helper cell lines may be isolated from human cells such as human fetal liver cells, myocytes, hematopoietic cells or other human fetal mesenchymal or epithelial cells. Alternatively, helper cells may be excised from cells of other mammalian species that tolerate human adenovirus. Such cells include, for example, Vero cells or other monkey embryonic mesenchymal or epithelial cells. As mentioned above, the preferred helper cell line is 293.

In another embodiment, the active compound is a vesicle, especially a liposome (Langer,
Science 249: 1527-1533 (1990), Treat et.
al. , May be transmitted by liposomes during treatment of infectious diseases and cancer (Lip
osomes in the Therapy of Infectious
Disease and Cancer), Lopez-Berstein
and Fidler (eds.), Liss, New York, pp. 35
3-365 (1989), Lopez-Berestein, ibid. , Pp. Three
17-327, generally see ibid.).

In yet another embodiment, the therapeutic compound may be incorporated into a controlled release system. For example, for example, the polypeptide may be administered using an intravenous drip, an implantable osmotic pump, a transdermal patch, liposomes, or other dosage forms. In one embodiment, a pump may be used (Langer, supra, Sefton, CRC Crit. Re.
f. Biomed. Eng. 14: 201 (1987), Buchwald e
t al. , Surgery 88: 507 (1980), Saudek et.
al. , N .; Engl. J. Med. 321: 574 (1989)). In another embodiment, polymeric materials can be used (controlled release medical applications (M
medical Application of Controlled Rel
ease), Langer and Wise (eds.), CRC Pres
s. , Boca Raton, Florida (1974), Controlling Bioavailability of Controlled Drugs, Designing and Implementing Drug Production (Controlled Drug).
Bioavailability, Drug Product Design
and Performance), Smolen and Ball (eds
． ), Wiley, New York (1984), Ranger and P.
eppas, J .; Macromol. Sci. Rev. Macromol. Ch
em. 23:61 (1983), see also Levy et al. ，
Science 228: 190 (1985), During et al. , A
nn. Neurol. 25: 351 (1989), Howard et al.
J. Neurosurg. 71: 105 (1989)). In yet another embodiment, the controlled release system may be in the vicinity of the therapeutic target, ie, the brain, thus requiring only a small portion of the systemic dose (eg, Goodson, Medical Applications of Controlled Release of C).
controlled release), supra, vol. 2, pp. 115-1
38 (1984)). Preferably, the controlled release device is introduced into the patient in the vicinity of an inappropriate immunologically active site or tumor site. Another controlled release system is Lange
r (Science 249: 1527-1533 (1990)).

As will be readily appreciated by those of skill in the art, the procedures and pharmacological compositions of this invention are suitable for administration to a mammalian, preferably human, subject.

In the therapeutic methods and compositions of the invention, therapeutically effective dosages of active compositions are provided. A therapeutically effective dose may be determined by a medical practitioner based on the characteristics of the patient (age, weight, sex, health status, complications, other diseases, etc.) as is known to those skilled in the art. . In addition, further conventional studies will provide more clear information on the appropriate doses for the treatment of different patients with different conditions, and experts will be able to determine the treatment situation, age of the recipient and roughly The appropriate dose can be ascertained by taking into consideration the health condition of the patient. In general, the dose for intravenous injection or infusion may be lower than for intraperitoneal, intramuscular, or other routes of administration. Dosage regimens may vary with the circulating half-life and the mode of use. The composition is administered in a manner compatible with the therapeutically effective amount of the dosage regimen. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosages will depend on the route of administration and are about 0. 0 active ingredient per kg body weight of an individual per day.
1 to 20 mg, preferably about 0.5 to about 10 mg, more preferably 1 to several m
It may be in the range of g. Suitable initial dosing regimens and booster doses may also vary, but can be typified by initial administration followed by repeated doses at one or more time intervals by injection or other administration. Alternatively, the blood concentration should be 10n
Continuous intravenous infusion sufficient to maintain M-10 μM is contemplated.

The present invention provides a kit containing all of the essential substances, including reagents necessary for suppressing prostate tumor cell growth, transforming prostate cells, or detecting prostate cancer cells in the kit. Good. It generally contains a selective expression construct. It may also include various solvents for the replication of expression constructs and host cells for such replication. Such kits include separate containers for individual reagents. When the composition of the kit is supplied in one or more liquid solutions, the liquid solutions are preferably aqueous solutions, particularly preferred sterile aqueous solutions. in viv
For use in o, the expression construct may be formulated in a pharmaceutically acceptable, injectable composition. In this case, the container itself is an inhalent, syringe, pipette, eyedropper, or eyedropper, because it is a formulation that is applied to the site of infection of the body, such as the lungs, injected into animals, or utilized and mixed with other compositions of the kit. Other such appliances. The composition of the kit may also be supplied in dried or lyophilized form. When the reagents or compositions are supplied in dry form, reconstitution is done with a suitable solvent.
It is envisioned that the solvent will also be supplied in a separate container.

The kits of the invention will also generally include means for containing the vial in a precise containment for commercial sale such as injection or insufflation into a plastic container holding the desired vial. . Regardless of the number of containers, the kit of the invention may also include or package a device to assist in the injection / administration or placement of the underlying compound in the animal's body. Such devices may be inhalents, syringes, pipettes, hoses, measuring spoons, eyedroppers or such approved medical transmission media.

The following examples are present in order to more fully illustrate the preferred embodiments of the invention. However, these should not be construed as limiting the broad scope of the invention.

EXAMPLES Example: p-Hyde Induces Susceptibility of Prostate Cancer Cells to Induce Programmed Cell Death.

Materials and Methods Cell lines: Two rat prostate cancer cell lines (undifferentiated tumor, low metastatic AT-1 and high metastatic MAT-LyLu) were used for cDNA competitive hybridization strategies (Rinaldy). and Steiner, 1997). Other Dunning rat prostate cancer cell lines used for Northern analysis were AT-3, MAT-L.
yLu, MAT-Lu and G. These cell lines are established in-
derived from the Vitro Dunning R3327 rat prostate tumor subline and further from Johns Hopkins Oncology.
Center), Baltimore Maryland (Issacs et
al. 1986) in Isaacs et al. By in-vitro
Developed as a cell line. Human prostate cancer cell line PPC used for Northern analysis
-1, LNCaP, TSU and DU145 are American Tiss
e Culture Collection, Rockville, Mary
Obtained from land. All cells were previously described (Issacs et a
1, 1986), in the presence of 10% fetal bovine serum per ml, 50 units penicillin G and 50 μg streptomycin sulfate, and 250 μM dexamethasone, RPMI 1640 medium (Mediatech (Mediate).
ch), Herndon, Virginia).

Cloning Strategy: Radiolabeled MAT-LyLu cDNA population in the presence of a large excess of competitor non-radiolabeled AT-1 cDNA population.
u cDNA library (Rinaldy and Steiner, 199)
It was used to identify the cDNA clone in 7). One of these cDNAs was new and was named p-Hyde. The prostate cancer associated with p-Hyde cDNA was further characterized.

Characterization of cDNA: Sequencing Is p-Hyde cDNA originally?
Obtained as a ZAP Uni XR clone and as described (Stratagene (
Stratagene), La Jolla, California) in v
It was further subcloned into the pBluescript SK-vector via the ivo excision protocol. The double-stranded cDNA was further subjected to dye terminator cycle sequencing (Dye Terminator Cycle S) using ABI 377 automated DNA sequencer version 3.0.
equencing: Perkin Elmer Fast
er City, California). The reading frame of p-Hyde was changed to the DNA Strider program (Pasteur Institute (Past).
eur Institute).

Northern blot analysis: Supplier (Tel-Test, Inc.
． ), Friendwood, Texas).
Total RNA was isolated from cell lines during exponential growth (70% confluent) using Zol B and subjected to Northern blot analysis. Reverse and forward P
A CR primer was designed and 1.35 showing 1467 bases of its reading frame.
It was used to amplify the kb p-Hyde sequence. Random oligo labeling technology (multiprime DNA labeling system (Multiprime DNA L
abeling System, Amersham) and used as a probe for Northern analysis. Comparative expression of p-Hyde gene was determined by PDI Dis with Quantity One software.
Comparison with internal control (cyclophilin) by scanning autoradiograms in covery Series Scanner.

In Vitro Transcription and Translation: The reading frame of the cDNA was determined by in-vitro transcription (cap mRNA) as described, followed by 75 Ci of L- [ ³⁵ S] methionine (Amersham, Arlington He).
rabbits reticulocyte lysate in the presence of light, Illinois (Stratagene, La Jolla, California).
) Was used to confirm using in-vitro translation. In vitro translation products were further identified by autoradiogram on 10% SDS polyacrylamide gel electrophoresis.

Subcloning of p-Hyde pcDNA3.1 (-): The cDNA insert was pBluescript by double digestion with KpnI and SacI.
Release from SK + vector (SK fragment). This fragment is the original p-Hyde
The sequence is shown, followed by the mammalian shuttle vector pcDNA3.1 (-)
(Invitrogen) dephosphorylated KpnI-S
Ligated within the acI double digest. Use the ligation mixture as Competent DH5? Were used to transfect and were screened for resistance to ampicillin, followed by plasmid preparation using standard cesium chloride density gradient centrifugation. Then, the novel structure of p-Hyde was added to lipofectamine (Gibco / BRL (Gib
co / BRL) to transfect AT3 rat prostate cancer sublines, followed by G418 selection (Rinaldy et al.
, 1988). Eight clones were obtained, two of them, AT3-H1 and AT
3-H2 was used to assess the function of p-Hyde in relation to its apoptosis. In addition, the AT3 cell line was also transfected with pcDNA3.1 (-) vector only and the stable transfected cell line designated AT3-pc was used for AT-H1 and AT-H2 for functional assessment of p-Hyde. Used as a negative control compared to the stable transfectants of.

Apoptosis assay: Apoptosis strength in AT3 parental cell line was determined by AT3-H.
1, assessed relative to AT3-H2 and AT3-pc as a negative control. Two
Two apoptotic agents were used in this assay. (1) 200 J / m2 UV
UV damage and apoptotic intensity of DNA using the administration were assayed at 36 hours after UV irradiation. And (2) 36 h 100 μM fluorodeoxyuridine (FUrD) treatment followed by an apoptosis assay. Following these inductions of apoptosis, cells were harvested in two fractions, floating cells and adherent cells. Both cell fractions were counted using a Neubauer chamber and trypan blue removal. DNA was extracted separately from both fractions and analyzed by visualizing the DNA ladder on 16% agarose gel electrophoresis.

UV-damage repair assay: UV-induced damage in DNA was assayed using a mouse monoclonal antibody that specifically cross-reacts with cyclobutane pyrimidine dimer (TMD-2) and a photochemical reaction product (64M-2). The presence of cyclobutane dimer and 64 photochemical reaction products in the DNA was determined by standard ELISA techniques using both antibodies separately in microtiter plates (100 and 200 ng).
/ Well). In addition, UV resistance was also published (Rinal
dy et al. , 1988) and using a UV gradient assay.

Uridine phosphorylase assay: Cell extracts were treated with 1 mM 5-d for 24 hours.
Prepared from cell pellets before and after induction with FUrd, the corresponding cell extracts were prepared via sonication in 50 mM potassium phosphate buffer, pH 7.4,
Subsequently, it is dialyzed against a reaction buffer solution (50 mM potassium phosphate, pH 7.4).
The amount of protein is determined using a standard Lowry and Biorad assay. Equal amount of protein from all cell lines, 10m as substrate
50 mM potassium phosphate buffer (pH 7.4) containing M uridine or thymidine
In assay for UP activity. After incubation for 30 minutes at 37 ° C., the reaction is terminated by adding methanol, followed by centrifugation. A portion of the supernatant was loaded onto an ERC-ODS-1171 (ERMA CR. Inc) HPLC column (6 x 200 mm). The amount of reaction products uracil or thymidine can be measured with a UV detector at 265 nm compared to a standard. As a negative control, a similar reaction mixture is boiled before incubation.

Construction of cDNA library: In a first step, AT-1 and MAT-L
A cDNA library derived from the yLu cell line was prepared from Stratagene (Stra).
It was prepared using Uni ZAP XR based on the protocol of Tagene). The independent clones obtained were 1.9 million and 3.4 million clones for MAT-LyLu and AT-1, respectively. These unamplified libraries are
The DNA insert population was subjected to PCR amplification. Reverse and forward primers (RP) and forward primers (RP), downstream and upstream of the XhoI and EcoRI cloning sites, were used for amplification of the cDNA insert population. ? Uni
The distance between both primers in ZAP or pBluescript is 22
It was 8 bases.

Competitive primer design: two PCR probes, radiolabeled MAT-LyL
The u cDNA population probe and the non-radiolabeled AT-1 cDNA population probe (cold competitor) were amplified. Radiolabeled MAT-LyLu PCR
The product was concentrated using a S400 Sephacryl spin column. Without inserts?
The major unincorporated ³² P-dCTP, primer dimer, and 228 bp PCR product resulting from DNA amplification were separated from the cDNA. The purified radiolabeled cDNA was added with a 30-fold excess of non-radiolabeled cold competitor AT-1 P.
It was mixed with the CR product and used as a competition-probe to screen the MAT-LyLu cDNA library.

Essentially radiolabeled cDNA of competitive probe and selected cDNA of library
Two unexpected radiolabeled PCR products that could interfere with hybridization between A are: 1) non-exponential growth of the cDNA, 2) lack of cDNA insertion?
It was a 228 bp PCR product derived from DNA. To reduce potential cross-hybridization between these two unexpected PCR products with the selected library vector, excess HindIII digested DNA, Pvu
II digested pBluescript DNA, and the 228 bp PCR product of pBluescript based on both primers were mixed with competitor probe.
Preliminary assessment of this fully mixed competition probe was performed using MAT-LyL with this probe.
Hybridization of the u cDNA library was extremely weak, while the double filter hybridized with a similar probe without the non-radiolabeled AT-1 PCR product was highly positive. This is MAT-
The positive hybridization of the LyLu product clearly suggests that it was due to the radiolabeled MAT-LyLu cDNA of the PCR product that did not compete with the AT-1 cDNA.

Screening of MAT-LyLu cDNA library: unamplified MAT-
Independent cDNA clones of the LyLu phage cDNA library (250,000 clones) were screened with competitive probes as described above. Nineteen amplified signals were observed and then these putative prostate cancer- or metastasis-related plaques were rescreened. As a result of this re-sorting, 12 independent plaques were purified. The cDNA is cleaved and a plasmid-based vector (pBlues
It was subcloned in (script). This means that the helper phage ExAs
This is possible due to the ability of sist (Stratagene) to cleave the cDNA at the pBluescript sequence in a circular form such as filamenta phage, which is secreted from the cell (FIG. 2). Recombinant pBluescri
The pt plasmid was recovered by infecting F'species of E. coli (SOLR strain, Stratagene), ampicillin resistant colonies were selected, and plasmid DNA was extracted. The resulting plasmid was digested with EcoRI and XhoI and the cDNA insert was identified by agarose gel electrophoresis. The results suggested that only 4 plasmids contained the cDNA insert.

Sequencing of candidate genes: All four cDNAs were 3'and 5 '
It was partially sequenced at both ends. These initial sequences were used to search NCBI's Genbank nucleic acid database for homology or similarity. Three c
DNA has a known sequence, 1) 16 specific for valine and phenylalanine
Rat mitochondrial gene encoding s and 12s rRNA and tRNA (accession number # emb / V00680 / MIRNR2), 2) rat nucleolar protein B23.1 mRNA (accession number # gb / J0396)
9 / RATB23NP) and 3) rat nucleolar proteins B23.1 and B
23.2 (accession number # gb / M37041 / RATNICBA7). The results of this competitive hybridization were consistent with the recognition phenotypic differences between both cell lines. MAT-LyLu shows twice the number of nucleoli and mitochondria (Isaacs et al, 1986). This also applies to MAT-LyL
It has been suggested that the u cell line is metabolically more active due to higher gene dosage or gene amplification of nucleolar and mitochondrial tRNAs and their related genes.

Complete sequence of the putative gene, p-Hyde: Fourth c, designated p-Hyde
The DNA (lane 1) had the first sequence that did not match any of the known full-length sequences in NCBI's BLASTN nucleic acid database. Therefore, p-
The Hyde cDNA was fully sequenced using a walk-through sequencing strategy in both directions for the three redundancy. Nine flanks were obtained from each sequencing direction and were synthesized as a 2694 base full length synthetic. GAG localized at the poly (A) end on the 3'end and upstream 27 of poly (A)
A polyadenylation signal sequence for AAA (a slight modification of the conserved AATAAA sequence) was also identified. FIG. 6 shows a sequencing map and a restriction map illustrating the open reading frame of p-Hyde. SEQ ID No. 3 is rat p
The resulting nucleic acid sequence of -Hyde and the resulting amino acid sequence of rat p-Hyde are listed.

Yield of a PCR probe showing the open reading frame p-Hyde gene: FIG.
4 shows PCR amplification of cDNA inserts with three sets of sequencing primers. The PCR product based on primers 11 and 5 was p-Hyde.
The reading frame of cDNA is shown. Random-Oligo-
It was radiolabeled by the Labeling technique and used as a representative probe for the cDNA insert in Northern analysis.

Northern blot analysis of p-Hyde: Several rat prostate cancer sublines (AT-
1, MALT-LyLu, MAT-Lu, AT-3 and G) and human prostate cancer cell lines (PPC, LNCaP, DU145 and TSU) were assessed by Northern blot analysis with PCR radiolabeled probes. The results suggested that the transcription of the human counterpart was slightly smaller compared to rat p-Hyde mRNA. The same blot was further hybridized with cyclophilin cDNA as an internal control. In this autoradiograph, 28S and 1
8S ribosomal RNA was used as a marker. Transcript length was calculated based on the spirin formula, M = 1550 × S ^2.1 . Where M = molecular weight and S = Svedbury constant (McConkey, 1967). The signals of p-Hyde and cyclophilin transcripts were analyzed using a computer densitometer (Quality-On).
e software, PDI) was used to quantify and standardize. The levels of p-Hyde mRNA expression from both rat and human prostate cancer cell lines were compared (Table I). The data suggest that a functional relationship between p-Hyde expression and prostate cancer progression may exist, differential expression of p-Hyde gene in both Dunning rat and human prostate cancer cell lines. Suggest that there is. MAT-
Lu showed the highest level of transcription, while AT-3 had the lowest. These data also indicate that the level of transcription in MAT-LyLu is relatively higher than that of AT-1, and that the novel cDNA is c between MAT-LyLu and AT-1 cDNA.
It was suggested that this was a result of DNA competition. More importantly, a human homolog of p-Hyde exists as shown in human prostate cancer cell lines. The highest level of p-Hyde transcription occurred in PPC-1, while it was lowest in the LNCaP cell line (Table I).

Analysis of predicted amino acid sequence of p-Hyde derived from ORF: cDNA
The open reading frame consists of 1467 bases encoding 489 amino acid residues,
The calculated molecular weight of this protein is 54.8 kD. Further molecular analysis of this predicted amino acid sequence can be found in the Kyte-Doolittle hydrophilicity plot, Ja.
Based on Emini surface probability plot using mes-Wolf antigenicity index and Laser Gene DNAstar software. In addition, the hydrophilic character of the predicted amino acid sequence was used to determine the idea of its antigenic determinants by Anitigen.
Predicted based on Hopp and Woods using the program (Table II
). The results of the first three analyzes were consistent with the fourth. Two peptide regions (residue 113 to residue 131 and residue 223 to residue 249) showed the highest points of hydrophilicity and antigenicity index. Both peptide sequences can be used as immunogenic peptides to raise antibodies. Its application for the detection of p-Hyde translation products in Western analysis was consistent with the highest number of its surface probabilities.

Table II Average Hydrophilicity Amino Acid Sequences 2.53 241-246 1.93 117-122 1.73 119-124 Confirmation of ORFs by in vitro transcription and translation: Confirm reading frame based on sequencing data. In order to do so, the p-Hyde cDNA constructed in pBluescript was digested with KpnI, making the linearized structure accessible to T3-RNA polymerase. This enzyme directs the synthesis of riboprobes with a 5'cap structure (5'me7Gppp5'G analog) similar to that present in eukaryotic mRNAs (Stratagene). 5
'The cap increases the yield and stability of synthesized mRNA, and the in-v using rabbit reticulocyte lysate (Stratagene) in the presence of ³⁵ S-methionine.
It is important in enhancing in vitro translation. Translation products were identified under reducing conditions using polyacrylamide gel electrophoresis with 8M urea. The autoradiograph shown in Figure 13 showed that two translation products (55 and 55.5 kDa protein) were observed.

The molecular weights of these two proteins are in agreement with the calculated molecular weights of the predicted open reading amino acids. In addition, two initiation codons were also identified in this open reading frame associated with the two transcripts obtained via in vitro translation of the riboprobe. The difference in molecular weight between both translation products is consistent with the difference at the four amino acid residues (MSGE) on the C-terminus between both start codons. The difference in molecular weight between both translation products is consistent with the 0.5 kDa molecular weight of MSGE.

Subcloning of p-Hyde Insert into pcDNA3.1 (-) and Transfection into Rat and Human Prostate Cancer Cell Lines: Differential p-Hyde in Rat and Human Prostate Cancer Cell Lines In connection with the expression data, this gene is inserted into cell lines expressing the lowest levels of p-Hyde, leading to subsequent assessment of the function of this gene within its stable transfection. For this purpose, the p-Hyde cDNA was subcloned into the pcDNA3.1 (-) mammalian expression vector (Invitrogen). p
The following features of cDNA 3.1 facilitate subcloning and gene expression. (1) a wide range of multiple cloning sites to facilitate unidirectional cloning, (2) a CMV promoter to drive constitutive transcription and translation of the inserted p-Hyde cDNA, and (3) to facilitate expression. SV40 splice acceptor linked to the SV40 polyadenylation signal of E. coli. The KpnI-XbaI fragment of p-Hyde cDNA containing the original open reading frame was successfully transferred to pcDNA3.
． It was subcloned into the 1 (-) vector. Then Cs containing HYDE insert
Cl-conjugated-purified-pcDNA (designated psHYDE) was labeled with G418 selection using lipofectamine (Gibco / BRL) in rats (AT-1 and AT-3), and human (DU145 and DU145). PPC-1) was transfected into a prostate cancer cell line. In addition to this pcHYDE, all cell lines were also successfully transfected with the pcDNA3.1 (-) vector as a negative control. The results obtained for stable transfections suggested that the pcHYDE gene product was not toxic to the host cells.

In vitro assessment of stable p-Hyde transfectants: pcHYDE function in the listed stable transfections (AT1 and AT3 groups) was assessed in vitro in relation to cell cycle and apoptosis. The purpose of the apoptosis assessment was to hypothesize that p-Hyde is the rat homologue of the putative TSAP-6 in rodents, which is associated with upregulation of the apoptotic response upon induction of the tumor suppressor gene p53 (Amson et al. , 1996). On the other hand, cell cycle assays point to the fact that the MAT-LyLu cell line is highly metastatic and the level of p-Hyde expression in this cell line is relatively higher than in the AT-1 cell line.

Cell cycle analysis: The strategy for cell cycle analysis is 2 of treatment with 1 mM hydroxyurea.
After 4 hours, it is based on stopping the cell population at the G1 and S boundaries (Iwas
aki et al. , 1995). As a result, the G2 period should be zero. Cells were harvested after hydroxyurea release at 0, 10 and 24 hours and subjected to flow cytometer analysis using standard propidium iodide staining.
The results are shown in Table III, and AT1-H1 was obtained 10 hours after the release of hydroxyurea.
Both AT3 and AT3-H1 were relatively slow to enter S phase, while the parental cell lines (AT1 and AT3) were faster. At 24 hours after release, all G2 cells were elevated, suggesting that the cell cycle was in progress. Overall, it is not directly established that slow entry into S phase with both pcHYDE stable transfectants is associated with slower tumor growth in vivo. 0, 10 and 2 after the release of hydroxyurea
The progress of cell cycle reentry at 4 hours was followed with a flow cytometer. Consistently, t. No G2 phase was detected 24 hours after treatment with -hydroxyurea, suggesting that the cell cycle is arrested at the G1 and S phase boundaries.

Overall, arrest of all cell cultures at the G1 and S boundaries by treatment with 1 mM hydroxyurea for 24 hours was confirmed in relation to the 0% population of G2. These results indicate that the slower response entering S phase with AT1-H1 and AT3-H1 may reflect the proliferative properties of these stable transfectants in vivo, pcHYDE. It suggests that this may be due to the effect of the gene product.

Induction of Susceptibility to Apoptosis: The apoptotic response was assessed using the DNA laddering assay. DNA was extracted after each treatment (1 mM hydroxyurea for 24 hours, followed by 0.1 mM 5'-dFUrd for 24 hours),
Analysis was performed on 1.6% agarose gel electrophoresis. Consistent with cell cycle analysis, the apoptotic response of stable transfectants AT1-H1 and AT1-H3 (pcHYDE-transfected AT-1 and AT-3) was shown to be higher in the parent (AT-1 and AT-3) and pcDNA-. It was persistently and significantly higher compared to both transfected parental cell lines (AT1-pc1 and AT3-pc1). Notably, the highest apoptotic response occurred in synchronous culture under incubation with 0.1 mM 5'-dFUrd.

The enhanced apoptotic response with AT1-H1 and AT3-H1 transfectants following hydroxyurea treatment is associated with intracellular thymidine-2-triphosphate via direct inhibition of thymidine synthase by fluorodeoxyuridine. Acid (TTP)
"Thymidine death", which leads to the depletion of
Kypianou, 1994, Kyprianou et al. , 1994)
Is the result of. Taken together, these data suggest that the apoptotic response in pcHYDE stable transfectants is due to the downstream effects of the pcHYDE gene product.

UV-damaged DNA cannot be repaired in p-Hyde-transfected prostate cancer: three DNA enzyme repair systems, uridine phosphorylase, uridine kinase and UV-damage repair compared to p-Hyde-transfected cells. And then rose in the parent stock. UV lesion repair did not function in p-Hyde-transfected cells. The reduced DNA repair function results in a higher concentration of the original photoreaction product (64PP). Consistent with these data, p-Hyde-transfected cells also showed a significant reduction in survival after UV exposure compared to parental AT3 cells, as measured in a colony formation assay. Thus, defects in DNA repair enzymes correlated with shorter survival and induction of apoptosis in p-Hyde-transfected prostate cancer cells.

Northern RNA analysis of p-Hyde confirms p-Hyde overexpression: To confirm the relationship between apoptotic response and p-Hyde at the transcriptional level, A
Total R derived from AT3-H1, AT3-H2 compared to T3 parental cell line
Northern analysis of NA was performed. The results clearly suggest that pcHYDE transcription levels in AT3-H1 and AT3-H2 stable transfectants were significantly higher compared to the AT3 parental cell line. This Northern analysis was performed using AT3, AT3-H1 with PCR products as probes and the internal control cyclophilin.
And AT3-H2 only.

P-Hyde suppresses prostate cancer growth in vivo: The lowest level of p-Hyde expression was observed in the AT3 cell line. Therefore, the AT3 cell line
PH in a mammalian expression vector of pcDNA3.1 (-) with G418 selection
It was transfected with pcHYDE, a construct of yde. As a negative control, the AT-3 cell line was also transfected with the vector alone and the resulting stable transfectant is designated AT3-pc.

Tumor growth and stable transfectants AT3-H1 of the parental AT-3 cell line
And AT3-H2 were evaluated in vivo. One million cells of each cell line in 0.3 ml Hank's solution were subcutaneously transplanted into each flank of Inbreed os Copenhagen rats. 5 in each of these initial experiments
Three groups of single rats were injected with each cell line. Tumor size was scored after the process. These results show that both AT3-H1 and AT3-H2 stable transfectants grew significantly slower than the AT-3 parental cell line, and tumor growth inhibition with both stable transfectants was pcHYDE. It was suggested that it is regulated by the gene product.

Tumor progression represents an accumulation of genetic alterations that affect the expression of oncogenes and tumor suppressor genes, which alters the responsiveness of cells to autocrine and paracline positive and negative growth regulators. The Dunning tumor rat model of prostate cancer tumor progression consists of a range of prostate cancer phenotypes from well-differentiated to poorly-differentiated showing different responses to androgens. In addition, a subline originated and evolved from the same original spontaneous rat prostate tumors, and thus the present study was performed only for tumor progression itself. The development of these cell lines is as a result of tumor progression within single and multiple serial passages.

The screening strategy for competitive hybridization of a cDNA library synthesized from the highly metastatic rat MAT-LyLu cell line resulted in a cDNA clone designated p-Hyde (Rinaldy and Steiner,
1997). The full length of this cDNA, shown as its restriction map, consists of 2713 nucleic acid residues, containing two open reading frames of 1467 and 1452 residues, respectively. 46% of the sequence is untranslated region, most of which is 3'of the gene
At the end. The rat p-Hyde nucleic acid sequence is listed in SEQ ID NO 3, and the human p-Hyde nucleic acid sequence is listed in SEQ ID NO 1.

Expression levels of p-Hyde were determined by Northern blot analysis in both rat and human prostate cancer cell lines and compared to cyclophilin expression as an internal control.
Dunning rat prostate cancer cell lines AT-1, MAT-LyLu, MAT-Lu, A
T-3 and G, and the human prostate cancer cell lines PPC1, LNCaP, TSU and DU145 are all positive at different expression levels (Table I). The transcript size of the human counterpart is slightly smaller compared to rat p-Hyde mRNA. The data also suggest that there is differential expression of the p-Hyde gene in rat and human prostate cancer cell lines, and that there may be a functional correlation between p-Hyde expression and prostate cancer progression. It shows that it is doing. MAT-Lu showed the highest transcription levels, while AT-3 was the lowest. The striking difference between these two cell lines is important in its association with the growth characteristics of the highly metastatic MAT-Lu cell line. These data also indicate that the level of transcription in MAT-LyLu is AT-
1, which is relatively (10%) higher than 1, which indicates that the novel cDNA
It is shown that this is the result of cDNA competition between T-LyLu and AT-1 cDNA (Rinaldy and Steiner, 1997). The important thing is
A human homolog of p-Hyde is present in the human prostate cancer cell line. The highest p-Hyde transcription levels occurred in PPC1, the primary androgen-sensitive human prostate cancer cell line, while lowest in the LNCaP cell line, androgen-insensitive prostate cancer cell line. Thus, p-Hyde does not appear to be specific to mammalian prostate tissue, but is found in other tissues, including placenta and milk, and in other species such as mice and humans, which It suggests that the role is important in basic cell biology.

Tumor Suppression and Induction of Susceptibility to Apoptosis: Interestingly, p-Hyd
e has the dual ability of acting like a tumor suppressor gene and of inducing susceptibility to apoptosis, which may be by a p53-independent pathway. Prostate tumor growth in rats was significantly suppressed by p-Hyde. In addition, p-Hyde-expressing prostate cancer was more susceptible to the UV DNA damage driving these cells to the programmed death of those cells. Analysis of DNA repair enzyme activity suggests a defect that results in the presence of the original (6-4) PP, which results in reduced cell survival in the colony formation assay. However, the ability of p-Hyde to induce susceptibility to apoptosis is not limited to UV DNA damage. Chemotherapeutic agents, fluorodeoxyuridine, pyrimidine antibiotics, which are associated with fluorouracil (5-FU) and have been used for the treatment of a wide range of human epithelial malignancies, also express p-Hyde prostate. Induces apoptosis more easily in cancer cells. Moreover, cancer cells expressing p-Hyde are also sensitive to gamma radiation. Thus, the mechanism of cellular DNA damage is different for UV, gamma irradiation and fluorodeoxyuridine, suggesting that the ability to sensitize cells to apoptosis is more comprehensive in function. This unique function of p-Hyde represents a new class of genes that induce susceptibility to apoptosis. This is a function attributable to tumor suppressor genes such as p53 that directly induces apoptosis (Yonish-Rouch).
et al. , 1991). Furthermore, the p-Hyde activity is bcl-
In contrast to bcl-2 in the absence of 2, but not in the presence of 2, cancer cells become more sensitive to programmed death of cells (McDonnel).
l et al. , 1992).

Use in the Treatment of Human Diseases: These unique functional properties of the p-Hyde gene may be exploited for the treatment of hyperplastic diseases and cancer. For example, one effective treatment strategy is treatment of carcinoma cells expressing p-Hyde with a chemotherapeutic agent (such as acetylaminofluorine) or a UV mimetic drug. However, cancer cells do not appear to produce significant levels of growth inhibitory p-Hyde. As a result, the p-Hyde gene was not only directly suppressed by p-Hyde as shown in this study, but was also treated in combination with DNA damaging therapies such as chemotherapy, UV mimetics or radiation. Sometimes it may have a better anti-cancer effect. Since gene therapy targets cancer cells, increased apoptosis is associated with DNA damage (UV
, Radiation, or chemotherapy) can occur more selectively in cancer cells.

Example 2 Prostate Cancer Gene Therapy Using Adenovirus Expressing the Novel Tumor Suppressor Gene pHyde Materials and Methods Cell line and tissue culture conditions: (ATCC, Rockville, Maryla.
Human prostate cancer cell lines PPC-1, DU145, PC-3, L)
NCaP and TSU-Pr1 were added to 10% fetal bovine serum (Hyclone Laboratories at 37 ° C. and 5% CO ₂₎ .
es), Logan, UT) containing RPMI-1640 medium (Celgro (Ce
llgro), Herndon, VA). Human embryonic kidney cell line 2
93 (ATCC) was grown in D-MEM medium (Cellgro) containing 10% heat-inactivated fetal bovine serum at 37 ° C. and 5% CO ₂ .

Construction of AdRSVpHyde: The rat pHyde cDNA gene was isolated as described in US Pat. No. 09 / 302,457. EcoRI
1. contains the 1467 bp full length coding sequence of the pHyde cDNA after digestion with 2.
The 6 kb fragment was E1 / E under the control of the truncated RSV promoter (395 bp).
It was subcloned into a 3-deleted adenovirus shuttle vector. The obtained adenovirus shuttle vector was co-transfected into 239 cells together with pJM17 and adenovirus type 5 genomic plasmid by the calcium phosphate method. Individual plaques were screened for recombinant AdRSVpHyde by PCR using primers specific for both RSV promoter and pHyde cDNA sequences. A single viral clone was propagated in 293 cells. The culture medium of 293 cells showing complete cytopathic effect (CPE) was collected and adenovirus was added to 2 cells.
It was purified by twice CsCl ₂ gradient ultracentrifugation and concentrated. Viral titration and transfer was performed as previously described. A schematic diagram of AdRSVpHyde is shown in FIG.
The sequence of AdRSVpHyde is listed in FIG.

Northern blot: Cells were extracted and total RNA was isolated with the RNeasy Kit (Qiagen, Santa Clarita, CA).
Total RNA was loaded on a 1.2% polyacrylamide gel and electrophoresed. Nylon membrane (Nybond-N ⁺ , Amersham Life Science (Am
ersham Life Science), Buckinghamshire
, Northern) was performed as described previously. The cDNA probe (pHyde or p53) was used for the random primer method (
Prime-It II Kit, Stratagene,
La Jolla, CA) and labeled with a- ³² P-dCTP. Membranes were hybridized with this probe in Rapid-hyb interference (Amersham Life Science) according to the handling protocol. Membrane for autoradiography
It was exposed to Kodak X-ray film under a single intensifying screen at 80 ° C. The GAPDH cDNA probe was labeled as above and used as an internal standard for normalization.

Western Blot: Cells were treated as previously described (Lu Y, Whit.
ker L, Li X, et al. Rodent PCC4. Co-expression of galectin-1 and its complementary glycoconjugate laminin and lysosomal associated membrane proteins in aza1R fetal carcinoma cells induces butyrate-induced differentiation (Coexpre).
session of galaxine-1 and its complete
entry glycoconjugates laminin and l
ysosome-associated membrane proteins
in Murine PCC4. aza1R embryonal carc
inoma cells induced to differentiati
on by butyrate. ) Mol Cell Differ. 1995
: 3: 175-191), extracted and processed by gel electrophoresis. Cell extract lysate (100 mg) was loaded on a 12% polyacrylamide gel, subjected to sodium dodecyl sulfate (SDS) gel electrophoresis, and then nitrocellulose membrane (Bio-Rad Laboratories),
Hercules, CA). The membrane was treated with blocking buffer (15% non-fat milk, 0.02% sodium azide in phosphate buffered saline) at 4 ° C overnight. This membrane was incubated at room temperature for 1 hour (based on Research Genetics, Inc., based on computer-generated antigenic peptides derived from the pHyde coding sequence.
search Genetics, Inc. Incubated with a rabbit anti-rat pHyde polyclonal antibody (specially synthesized from). Then, the membrane was allowed to stand at room temperature for 1 hour with ¹²⁵ I-labeled secondary antibody (Amersham Life Science (
Amersham Life Science), Arlington Hei
ghts, IL). The membrane was exposed to Kodak X-ray film between two intensifying screens at -80 ° C for autoradiography.

AdRSVpHyde in vitro study: 100 human prostate cancer cells
Or with multiple infection (MOI) of 200, AdRSVpHy in vitro
infected with de. After virus infection, cells were incubated at 37 ° C and cell number was measured 5 days after virus infection.

In vitro studies with DU145 xenograft tumors: DU145 cells (
Male nude mice (Harlan Sprague Dawley) were injected subcutaneously in the flank with 0.2 ml of 1.4 × 10 ⁷ cells in PBS. If the tumor reached an average dose of 80 mm ³ , then 5 × 10 ⁹ pfu
Adenovirus (AdRSVpHyde or control adenovirus A
dRSVlacZ) or PBS alone as an untreated control was directly injected intratumorally. Tumor volume was measured every 3-4 days until the animals were euthanized. All animals were sacrificed at day 52 post viral infection, some of which were in distress and had a tumor burden of> 15% of total body weight. Tumor samples were collected and H & E stained.

Gel Electrophoretic Analysis of DNA Extraction and DNA Fragmentation: Soluble DNA was already analyzed (Oridate N, Lotan D, Xu X-C, Hong WK, an.
d Lotan R. Differential induction of apoptosis by all-trans-retinoic acid and N- (4-hydroxyphenyl) retinamide in human head and neck squamous cell carcinoma cell lines
sis by all-trans-retinoic acid and
N- (4-hydroxyphenyl) retinamide in hum
an head and neck squeamous cell carci
noma cell lines. ) Clin. Cancer Res. 199
6; 2: 855-863). Extracted as described. Briefly, cells floating in the medium were collected 48 hours after transduction by centrifugation. Tris- the pellet
Resuspended in EDTA dry solution (pH 8.0). Cells on ice for 15 minutes, 1
0 mM Tris-HCl (pH 8.0), 10 mM EDTA and 0.5%
Dissolved in Triton X-100. Lysate 1 at 12,000 xg
After centrifugation for 5 minutes, soluble (fragmented) DNA was separated from pellet (original genomic) DNA. Soluble DNA was treated with Rnase A (50 ug / ml) for 1 hour at 37 ° C, followed by proteinase K (1 in 0.5% SDS for 2 hours at 50 ° C.
00 ug / ml). The residual material was extracted with phenol / chloroform, ethanol precipitated and electrophoresed on a 2% agarose gel.

Results Exogenous pHyde expression in DU145 cells: AdRSVpHyde shows mRN
To determine whether rat pHyde can be successfully transduced and expressed at A and protein levels, DU145 cells were treated with AdESVpH at MOI = 200.
Transduced with yde. Cell extracts were collected 48 hours after virus infection and
The yde expression was measured. In DU145 cells, at mRNA level, there was a slight expression of exogenous pHyde (FIG. 2A), but not at protein level, while the apparently high exogenous pHyd induced by AdRSVpHyde.
e expression was present at both mRNA (Figure 2A) and protein (Figure 2B) levels.

Prostate Cancer Cell Proliferation Suppressed by AdRSVHyde: To determine the effect of pHyde on cell proliferation of human prostate cancer cell lines, DU145 and L
NCaP was added to AdRSVpHyde and AdRSVlac in vitro.
Treated with Z (control vector) or without virus. AdRSVp
Hyde significantly suppressed the growth of DU145 and LNCaP cells by 76.9% (Figure 3A) and 83.1% (Figure 3B), respectively, compared to untreated control cells.

AdRSVpHyde Suppressed Prostate Cancer Growth In Vivo: in
To evaluate the effect of AdRSVpHyde treatment on prostate cancer cell growth in vivo, DU145 human prostate tumor was placed on the flank of nude mice at 1.4 × 10 ⁷ PP.
Established in nude mice by subcutaneous injection of C-1 cells. 80 mice on average
When developing a mm ³ volume of cancer, mice were divided into three groups, AdRSVpHyde treated (n = 7), AdRSVlacZ control vector treated (n = 7), and untreated group (n = 7). Treated tumors were injected with a single dose of 5 × 10 ⁹ pfu of either control virus or AdRSVpHyde. Figure 4
Untreated and control virus-treated DU145 tumors were treated with A
Proliferated faster compared to dRSVP Hyde treated tumors. By 53 days after virus injection, the tumor addition in nude mice with untreated and control virus treated DU145 tumors reached 5953mm ³ and 4777mm ^3, respectively. In contrast, DU145 tumors transduced with AsRSVpHyde showed a significant reduction in tumor volume (1515 mm ³ ) compared to untreated and control virus treated-Du145 tumors, ie 25.4% untreated and control virus treated. It showed 31.7% of the DU145 tumor volume (Fig. 4).

Growth inhibition by AdRSVpHyde was associated with p53 expression in prostate cancer cells: One observation in the in vitro study was that the phenotype of DU145 and LNCaP cells transduced by AdRSVpHyde was also altered.
Unlike control and control virus-treated cells, AdRSVpHyde-transduced cells showed obvious morphology of round, detached, and floating dead cells, characteristics of cells undergoing apoptosis, and a rapid decrease in their number. (Fig. 5
). Consistently, AdRSVpHyde showed strong suppression in the growth of these two cell lines. However, there were no apparent morphological differences between untreated control, control virus treated and AdRSVp Hyde treated cells with these strains (Figure 6). Different genes, including p53 and Rb, to determine whether the differential expression of different genes, especially those involved in the apoptotic pathway, accounts for the differential inhibitory effect of AdRSVp Hyde on different prostate cancer cell lines. Were screened at the mRNA level by Northern hybridization. Interesting, but less surprising, p53 is DU145 and LN
Expression was seen only in CaP but not in PC-3, TSU-Pr and PPC cells, whereas the Rb gene was fully expressed at the mRNA level in these cells (Figure 7). Therefore, it is clear that there was a relationship between p53 expression and AdRSVp Hyde-mediated suppression. To determine if pHyde regulates p53 expression, a similar Northern blot in Figure 2A was isolated and labeled with p53.
Rehybridized with probe. In fact, the induction of p53 mRNA was
It was observed in RSVP Hyde transduced DU145 cells (FIG. 8). Furthermore, Ad
Transduction of LNCaP cells with RSVp Hyde shows a DNA laddering tendency, a marker for cells undergoing apoptosis (FIG. 9), pH.
It is suggested that yde expression induced apoptosis in LNCaP cells. Taken together, these results indicate that pHyde may function via the p53 pathway to induce apoptosis, whose growth suppression is associated with p5 in target cells.
3 may be dependent on expression.

Previous studies have shown that pHyde has a dual ability to act like a tumor suppressor gene and induce susceptibility to apoptosis. Prostate tumor growth in rats was significantly suppressed by pHyde. Moreover, prostate cancer cells expressing pHyde were more susceptible to UV DNA damage driving these cells to programmed death of the cells. In this study, AdRSVpH
It was shown that yde effectively suppressed the proliferation of p53-expressing prostate cancer cells both in vitro and in vivo, but not in prostate cancer cells lacking p53 expression. It was One possibility is that pHyde requires p53-dependent pathways (such as DU145 and LNCaP cells) and external cell death triggers such as UV or chemicals (such as methylnitrosourea) that act as co-derivatives of apoptosis. It is possible to induce apoptosis by a p53-independent pathway. D, not PPC-1, PC-3 and TSU-Pr
Consistent with our results (FIG. 10) that only U145 and LNCaP express p53 at the mRNA level, another group was DU145 and L.
Only NCaP cells express p53 at the protein level, PC-3 and TSU
-Pr was found not to be expressed. Interestingly, p in DU145
The 53 protein was claimed to be the mutant p53. ²¹ Therefore, regardless of its wild-type or mutant status, the presence of the p53 protein is considered necessary for pHyde to act alone as a tumor suppressor gene. DU145
The possibility that mutations in the p53 protein in E. coli may not affect its ability for pHyde-mediated growth suppression needs further study. Moreover, the potential suppressive effect of the combination of AdRSVpHyde and UV (or methylnitrosourea) in cells lacking p53 protein expression (such as PC-3 and TSU-Pr) would be further characterized.

One interesting and important finding in this study was that AdRSVpHyde was DU145.
It was the induction of p53 expression in the cells. This not only explains that pHyde acts in a more general manner to induce susceptibility to apoptosis, but also pHyde is a rat homolog of the TSAP-6 gene, or p.
It is possible that pHyde has partial sequence homology with TSAP-6, a human protein allegedly involved in the p52-related pathway, as it may be a member of the TSAP-6-like family involved in the 53-related pathway. I could explain in part. further,
If so, there are really small amounts of the identified cellular proteins that act as regulators of the p53 gene, which is a transcription factor that drives many downstream genes. p
The importance of the finding that Hyde upregulates p53 expression, and subsequent exploration of new cellular regulatory mechanisms, is occurring. However, pHyde
Whether it directly regulates the p53 gene at the transcriptional level was determined in the presence and absence of pHyde protein in DU145 cells.
It will be determined in our current study by using the AT reporter chimeric gene.

In summary, pHyde is a novel tumor suppressor gene, AdRSVpHyde
Effectively suppresses prostate cancer both in vitro and in vivo. AdRSVp Hyde monotherapy or AdRSVp Hyde radiation-
Or combination therapy with chemotherapy-therapy must have effective therapeutic potential for the treatment of locally developed prostate cancer.

Example 3 Another traditional method for detecting apoptosis, the TUNEL assay, was also used to show that AdRSVp Hyde actually causes apoptosis in DU145 cells. 72 hours after viral transduction, TUNEL assay was performed on DU145 cells growing on culture dishes. Untreated control (
15A and 15D) or control virus transformed cells (FIGS. 15B and 15E), there were more fluorescent stained cells in AdRSVp Hyde transformed cells (FIGS. 15C and 15F), which was more than in the latter two cases. Also Ad
It is suggested that more apoptotic cells were present in the RSVp Hyde treated cells. Furthermore, tumor secretion from DU145 xenograft tumors growing in nude mice was significant in AdRSVpHyde treated tumors (FIG. 17B) compared to those of untreated (FIG. 17A) and control virus treated tumors. It indicates that apoptosis had occurred.

TUNEL assay of AdRSVpHyde-transduced DU145 cells showed no comparable apoptotic cell mass (about 10% stained cells, FIG. 15) to a degree compatible with growth suppression (76.9% suppression, FIG. 17A). It was One explanation is that cell growth inhibition was recorded 5 days after AdRSVpHyde transformation (FIG. 17A), whereas in vitro TUNEL staining was 3 of AdRSVpHyde transduction.
That is, it was carried out with cells after a day (FIG. 15). In vivo AdRSVpHyde treated DU1 derived 21 days after AdRSVpHyde transduction
TUNEL staining of 45 xenograft tumor compartments showed significantly higher amounts of apoptotic cells (FIG. 16) compared to in vitro TUBEL staining (FIG. 15).
Therefore, p-Hyde-mediated apoptosis may take time to show a peak of apoptosis not yet available intracellularly 3 days after viral transduction. Other explanations indicate that apoptosis may explain only part of pHyde-mediated growth inhibition, in other words, pHyde-mediated growth inhibition may consist of apoptosis and other known mechanisms. That is. As described below, AdRSVpHyde can inhibit cell growth of other human prostate cancer cell lines TSU, however, AdRSVpHyde transformed TS
No apoptosis was observed in U cells.

To determine if there was a relationship between p53 status and susceptibility to apoptosis by pHyde, four different human prostate cancer cell lines PC3, TSU,
LNCaP and DU145 were screened for exogenous expression of p53 v and their sensitivity to pHyde-mediated growth inhibition and compared for apoptosis. Northern blot analysis showed that PC-3 and TSU cells did not express p53 at the mRNA level, while LNCaP and CU145 cells both expressed p53 mRNA. In contrast, all four cell lines expressed comparable Rb at the mRNA level (Figure 18). Consistently, other groups showed that DU145 and LNCaP cells, but not PC-3 cells, expressed p53 protein by Western blot analysis.

To determine the sensitivity of these four cell lines to pHyde-mediated growth suppression and apoptosis, cells were transduced with AdRSVpHyde and growth monitored. AdRSVpHyde had potent growth inhibition in two cell lines expressing p53, DU145 and LNCaP cells (76.9% and 83.1 respectively compared to untreated controls). % Suppression, Figure 17). Interestingly, for the cell lines PC-3 and TSU that do not express the two p53s, AdRSVpHyde has no suppressive effect on the proliferation of PC-3 cells,
However, it showed a small suppression in TUS cell proliferation (24.5% compared to untreated control) (FIG. 19). However, AdRSVpHyde transduced PC
Both -3 and TSU cells showed no DNA fragments and apoptosis as detected by the TUNEL assay. These results suggest that there may be a relationship between the presence of p53 and the susceptibility of cells to pHyde-mediated apoptosis. p53 may be required for pHyde-mediated apoptosis induction. Furthermore, preliminary results showed that the key apoptotic protease, caspase-3, was elevated following AdRSVpHyde transduction in DU145 cells.

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[0342]

[Sequence list] [Brief description of drawings]

FIG. 1 Schematic of the structure of AdRSVpHyde. The 2664 bp inserted fragment contains the 1467 bp full length pHyde cDNA gene (SEQ ID NO: 1) and the 1166 bp 3'untranslated downstream region. The complete sequence of AdRSVpHyde is set forth in FIG. In particular, the nucleic acid sequence of region A of FIG.
The region A of FIG. 1 and the nucleic acid sequence of the region B of FIG. 1 are described in the region B of FIG.

FIG. 2A: Expression of pHyde by AdRSVpHyde. MOI = 20
DU145 cells transduced with AdRSVpHyde at 0 were harvested within 48 h post infection for extraction of either mRNA or protein. (A) DU1
Expression of pHyde at the mRNA level in 45 cells. In each sample well,
10 mg of total RNA was loaded, electrophoresed on a 12.5% agarose gel, transferred to a nylon membrane, and hybridized with ³² P-labeled pHyde cDNA. Northern blots were excised and rehybridized with GAPDH to examine gel loading.

FIG. 2B. Expression of pHyde by AdRSVpHyde. MOI = 20
DU145 cells transduced with AdRSVpHyde at 0 were harvested within 48 h post infection for extraction of either mRNA or protein. (B) DU1
Expression of pHyde at the protein level in 45 cells. The protein extract (50 mg) was loaded on a 12% SDS-PAGE gel. Rabbit anti-rat p
The Hyde antibody was used as the primary antibody.

FIG. 3: Inhibitory effect of pHyde on prostate cancer cell proliferation. DU145 cells (A) and LNCaP cells (B) were transduced with adenovirus vector at MOI = 100 or left untreated. The number of cells was counted 5 days after virus transduction. Data represent the results from two independent experiments, each performed in duplicate.

FIG. 4. AdRSVpHyde inhibits prostate tumor growth in vivo. DU145 cells (1.4 × 10 ⁷ cells) were subcutaneously injected into the flank of nude mice. When the mean volume of tumor reached 80 mm ³ (about 1 month after tumor cell inoculation), the tumor was left untreated (control) or 5 × 10 ⁹ pfu of control virus A.
dRSVlacZ (control virus) or 5 × 10 ⁹ pfu of AdRSVpH
Yde (AdRSVpHyde) was injected intratumoral (day 0). Tumor size was measured periodically until the 52nd day after virus injection at the time points indicated in the figure.

FIG. 5. Morphological changes of DU145 cells and LNCaP cells transduced with AdRSVpHyde. Cells were transduced with control adenovirus AdRSVlacZ or AdRSVpHyde at MOI = 100. Morphological characteristics of untreated control cells and virus-transduced cells were recorded 5 days after virus transduction. All photographs are at the same magnification (66x). (A) and (D): untreated control cells; (B) and (E): virus control AdRSVlacZ treated cells; (C).
) And (F): AdRSVpHyde treated cells.

FIG. 6: PC-3 cells transduced with AdRSVpHyde, TS
Morphological changes of U cells and PPC-1 cells. Cells were AdR at MOI = 100
Transduced with SVlacZ or AdRSVpHyde. Morphological characteristics of untreated control cells and virus-transduced cells were recorded 5 days after virus transduction. All photographs are at the same magnification (66x). (A, D, G): untreated control cells; (B, E, H): virus control AdRSVlacZ treated cells; (C, F, I).
: AdRSVpHyde treated cells.

FIG. 7. Expression of p53 and Rb mRNA in various prostate cancer cell lines. Each sample well was loaded with 10 mg of total RNA, electrophoresed on a 12.5% agarose gel, transferred to a nylon membrane, and hybridized with a ³² P-labeled p53 or Rb cDNA probe. Northern blots were excised and rehybridized with GAPDH to normalize gel loading.

FIG. 8. AdRSVpHyde induced p53 expression in DU145 cells. Cut the same Northern blot and Figure 2A, ³² P-labeled p53 cDNA
Rehybridized with A.

FIG. 9 shows that AdRSVpHyde induced apoptosis in LNCaP cells. Leave cells untreated or MOI = 1
Transduction with AdRSVpHyde at 00, and the supernatant was collected 48 h after transduction. Soluble DNA was extracted from the floating cells and electrophoresed on a 2% agarose gel.

FIG. 10. Sequence of AdRSVpHyde.

FIG. 11: Comparison of nucleotide sequences of pHyde family genes between human pHyde (I) and human pHyde40 (II).

FIG. 12: Comparison of the amino acid sequences of pHyde family genes between human pHyde (I) and human pHyde40 (II).

FIG. 13A: Comparison of the nucleotide sequences of pHyde family genes between rat pHyde and human pHyde.

FIG. 13B: Comparison of amino acid sequences of pHyde family genes between rat pHyde and human pHyde.

FIG. 14. TUNEL assay of DU145 cells in vitro. DU14
5 cells were left untreated (A, D) or transduced with control virus (B, E) or AdRSVpHyde (C, F) at MOI = 200 and fixed 3 days after transduction And processed for TUNEL assay. The cells were then visualized by fluorescence microscopy. Arrows indicate some apoptotic cells. Magnification: A, B, C: x 20; D, E, F: 40 x.

FIG. 15. TUNEL assay of DU145 cells in vivo. DU145 xenograft tumors (˜80 mm ³ ) growing on nude mice were left untreated (A) or injected with 5 × 10 ⁹ pfu of AdRSVpHyde (B).
Tumors were collected within 21 days. Tumor sections were fixed and processed for TUNEL assay. The sections were counterstained with propidium iodide (red) to show cell nuclei. Cells stained bright yellow showed apoptotic cells. Control virus treatment D
Tumor sections from the U145 tumor showed similar results to (A). Magnification: A, B:
× 20.

FIG. 16: pH for proliferation of prostate cancer cell lines DU145 and LNCaP
Inhibitory effect of yde. DU145 cells (A) and LNCaP cells (B) were
= 100 with adenovirus vector (control virus or AdRSVpHyde)
Transduced in or left untreated. The number of cells was counted 5 days after virus transduction. Data represent the results from two independent experiments, each performed in duplicate. ^* Some error bars were too small to be seen in the figure.

FIG. 17. Expression of Rb and p53 in various prostate cancer cell lines. Various prostate cancer cells were screened for expression of endogenous Rb and p53 at the mRNA level. Each well was loaded with 10 μg of total RNA. 12.5% of sample
Electrophoresis on agarose gel, transfer to nylon membrane, and ³² P-labeled Rb (about 4.
CD of 4 kb transcript) or p53 (approx. 2.5 kb transcript)
Hybridized with NA. Northern blots were then excised and rehybridized with GAPDH cDNA (which showed a transcript of approximately 1.2 kb) to check for RNA integrity and gel loading.

FIG. 18. pHyde on proliferation of prostate cancer cell lines PC-3 and TSU
Inhibitory effect. PC-3 cells (A) and TSU cells (B) were transduced with adenovirus vector (control virus or AdRSVpHyde) at MOI = 100 or left untreated. The number of cells was counted 5 days after virus transduction. Data represent the results from two independent experiments, each performed in duplicate. ^* Some error bars were too small to be seen in the figure.

[Procedure amendment]

[Submission date] February 6, 2002 (2002.2.6)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0040

[Correction method] Change

[Contents of correction]

The amino acid sequence of rat p-Hyde is shown in SEQ ID NO: 6. Furthermore, the present invention provides an isolated nucleic acid encoding an amino acid sequence tccctgg.
ccacacgcctgtgtggggctctggctctc (SEQ ID NO: 7) is provided. Furthermore, the present invention relates to the amino acid sequence AAPCVAYVLLSLVYLPG.
VLAAALQLLRGTKYQRFPDWLDHWLQHRKQIGLLSF
Provided is an isolated nucleic acid encoding F (SEQ ID NO: 8). Further, the invention provides an isolated nucleic acid encoding the amino acid sequence NFIRDVLQPYIRKDENK (SEQ ID NO: 9) .

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0279

[Correction method] Change

[Contents of correction]

Construction of AdRSVpHyde: The rat pHyde cDNA gene was isolated as described in US Pat. No. 09 / 302,457. EcoRI
1. contains the 1467 bp full length coding sequence of the pHyde cDNA after digestion with 2.
The 6 kb fragment was E1 / E under the control of the truncated RSV promoter (395 bp).
It was subcloned into a 3-deleted adenovirus shuttle vector. The obtained adenovirus shuttle vector was co-transfected into 239 cells together with pJM17 and adenovirus type 5 genomic plasmid by the calcium phosphate method. Individual plaques were screened for recombinant AdRSVpHyde by PCR using primers specific for both RSV promoter and pHyde cDNA sequences. A single viral clone was propagated in 293 cells. The culture medium of 293 cells showing complete cytopathic effect (CPE) was collected and adenovirus was added to
It was purified by one round of CsCl ₂ gradient ultracentrifugation and concentrated. Viral titration and transfer was performed as previously described. A schematic diagram of AdRSVpHyde is shown in FIG.
The sequence of AdRSVpHyde (SEQ ID NO: 10 and SEQ ID NO: 11) is shown in FIG.
Listed in.

[Procedure 3]

[Document name to be amended] Statement

[Name of item to be corrected] 0342

[Correction method] Change

[Contents of correction]

[0342]

[Sequence listing] SEQUENCE LISTING <110> The University of Tennessee Research Corporation <120> Isolated nucleic acids of the p-hyde family, p-hyde proteins, and
methods of inducing susceptibility to induction of cell death in cancer <130> 1015318 <140> PCT / US00 / 11456 <141> 2000-05-01 <150> US 60 / 131,607 <151> 1999-04-29 <150> US 09 / 302,457 <151> 1999-04-29 <150> US 09 / 499,817 <151> 1999-11-26 <160> 11 <170> PatentIn version 3.0 <210> 1 <211> 1886 <212> DNA <213> Human <400> 1 ggggagctgc cgcggtcgct ccgagcggcg ggccgcagag ccaccaaaat gccagaagag 60 atggacaagc cactgatcag cctccacctg gtggacagcg atagtagcct tgccaaggtc 120 cccgatgagg cccccaaagt gagcatcctg ggtagcgggg actttgcccg ctccctggcc 180 acacgcctgg tgggctctgg cttcaaagtg gtggtgggga gccgcaaccc caaacgcaca 240 gccaggctgt ttccctcagc ggcccaagtg actttccaag aggaggcagt gagctccccg 300 gaggtcatct ttgtggctgt gttccgggag cactactctt cactgtgcag tctcagtgac 360 cagctggcgg gcaagatcct ggtggatgtg agcaacccta cagagcaaga gcaccttcag 420 catcgtgagt ccaatgctga gtacctggcc tccctcttcc ccacttgcac agtggtcaag 480 gccttcaatg tcatctctgc ctggaccctg caggctggcc caagggatgg taacgggcag 540 gtgcccatct gcggtgacca gccagaagcc aagcgtgctg tctcggagat ggcgctcgcc 600 atgggcttca tgcccgtgga catgggatcc ctggcgtcag cctgggaggt ggaggccatg 660 cccctgcgcc tcctcccggc ctggaaggtg cccaccctgc tggccctggg gctcttcgtc 720 tgcttctatg cctacaactt cgtccgggac gttctgcagc cctatgtgca ggaaagccag 780 aacaagttct tcaagctgcc cgtgtccgtg gtcaacacca cactgccgtg cgtggcctac 840 gtgctgctgt ca ctcgtgta cttgcccggc gtgctggcgg ctgccctgca gctgcggcgc 900 ggcaccaagt accagcgctt ccccgactgg ctggaccact ggctacagca ccgcaagcag 960 atcgggctgc tcagcttctt ctgcgccgcc ctgcacgccc tctacagctt ctgcttgccg 1020 ctgcgccgcg cccaccgcta cgacctggtc aacctggcag tcaagcaggt cttggccaac 1080 aagagccacc tctgggtgga ggaggtctgg cggatggaga tctacctctc cctgggagtg 1140 ctggccctcg gcacgttgtc cctgctggcc gtgacctcac tgccgtccat tgcaaactcg 1200 ctcaactgga gggagttcag cttcgttcag tcctcactgg gctttgtggc cctcgtgctg 1260 agcacactgc acacgctcac ctacggctgg acccgcgcct tcgaggagag ccgctacaag 1320 ttctacctgc ctcccacctt cacgctcacg ctgctggtgc cctgcgtcgt catcctggcc 1380 aaagccctgt ttctcctgcc ctgcatcagc cgcagactcg ccaggatccg gagaggctgg 1440 gagagggaga gcaccatcaa gttcacgctg cccacagacc acgccctggc cgagaagacg 1500 agccacgtat gaggtgcctg ccctgggctc tggaccccgg gcacacgagg gacggtgccc 1560 tgagcccgtt aggttttctt ttcttggtgg tgcaaagtgg tataactgtg tgcaaatagg 1620 aggtttgagg tccaaattcc tgggactcaa atgtatgcag tactattcag aatgatatac 1680 acacatatgt gtatatgtat ttacatatat tccacatata taacaggatt tgcaattata 1740 catagctagc taaaaagttg ggtctctgag atttcaactt gtagatttaa aaacaagtgc 1800 cgtacgttaa gagaagagca gatcatgcta ttgtgacatt tgcagagata tacacacact a860 aaaaaatgtacag <210> 2 <211> 487 <212> PRT <213> Human <400> 2 Met Pro Glu Glu Met Asp Lys Pro Leu Ile Ser Leu His Leu Val Asp 1 5 10 15 Ser Asp Ser Ser Leu Ala Lys Val Pro Asp Glu Ala Pro Lys Val Ser 20 25 30 Ile Leu Gly Ser Gly Asp Phe Ala Arg Ser Leu Ala Thr Arg Leu Val 35 40 45 Gly Ser Gly Phe Lys Val Val Val Gly Ser Arg Asn Pro Lys Arg Thr 50 55 60 Ala Arg Leu Phe Pro Ser Ala Ala Gln Val Thr Phe Gln Glu Glu Ala 65 70 75 80 Val Ser Ser Pro Glu Val Ile Phe Val Ala Val Phe Arg Glu His Tyr 85 90 95 Ser Ser Leu Cys Ser Leu Ser Asp Gln Leu Ala Gly Lys Ile Leu Val 100 105 110 Asp Val Ser Asn Pro Thr Glu Gln Glu His Leu Gln His Arg Glu Ser 115 120 125 Asn Ala Glu Tyr Leu Ala Ser Leu Phe Pro Thr Cys Thr Val Val Lys 130 135 140 Ala Phe Asn Val Ile Ser Ala Trp Thr Leu Gln Ala Gly Pro Arg Asp 145 150 155 160 Gly Asn Gly Gln Val Pro Ile Cys Gly Asp Gln Pro Glu Ala Lys Arg 165 170 175 Ala Val Ser Glu Met Ala Leu Ala Met Gly Phe Met Pro Val Asp Met 180 185 190 Gly Ser Leu Ala Ser Ala Trp Glu Val Glu Ala Met Pro Leu Arg Leu 195 200 205 Leu Pro Ala Tr p Lys Val Pro Thr Leu Leu Ala Leu Gly Leu Phe Val 210 215 220 Cys Phe Tyr Ala Tyr Asn Phe Val Arg Asp Val Leu Gln Pro Tyr Val 225 230 235 240 Gln Glu Ser Gln Asn Lys Phe Phe Lys Leu Pro Val Ser Val Val Asn 245 250 255 Thr Thr Leu Pro Cys Val Ala Tyr Val Leu Leu Ser Leu Val Tyr Leu 260 265 270 Pro Gly Val Leu Ala Ala Ala Leu Gln Leu Arg Arg Gly Thr Lys Tyr 275 280 285 Gln Arg Phe Pro Asp Trp Leu Asp His Trp Leu Gln His Arg Lys Gln 290 295 300 Ile Gly Leu Leu Ser Phe Phe Cys Ala Ala Leu His Ala Leu Tyr Ser 305 310 315 320 Phe Cys Leu Pro Leu Arg Arg Ala His Arg Tyr Asp Leu Val Asn Leu 325 330 335 Ala Val Lys Gln Val Leu Ala Asn Lys Ser His Leu Trp Val Glu Glu 340 345 350 Val Trp Arg Met Glu Ile Tyr Leu Ser Leu Gly Val Leu Ala Leu Gly 355 360 365 Thr Leu Ser Leu Leu Ala Val Thr Ser Leu Pro Ser Ile Ala Asn Ser 370 375 380 Leu Asn Trp Arg Glu Phe Ser Phe Val Gln Ser Ser Leu Gly Phe Val 385 390 395 400 Ala Leu Val Leu Ser Thr Leu His Thr Leu Thr Tyr Gly Trp Thr Arg 405 410 415 Ala Phe Glu Gl u Ser Arg Tyr Lys Phe Tyr Leu Pro Pro Thr Phe Thr 420 425 430 Leu Thr Leu Leu Val Pro Cys Val Val Ile Leu Ala Lys Ala Leu Phe 435 440 445 Leu Leu Pro Cys Ile Ser Arg Arg Leu Ala Arg Ile Arg Arg Gly Trp 450 455 460 Glu Arg Glu Ser Thr Ile Lys Phe Thr Leu Pro Thr Asp His Ala Leu 465 470 475 480 Ala Glu Lys Thr Ser His Val 485 <210> 3 <211> 2118 <212> DNA <213> Human <400> 3 ggggagctgc cgcggtcgct ccgagcggcg ggccgcagag ccaccaaaat gccagaagag 60 atggacaagc cactgatcag cctccacctg gtggacagcg atagtagcct tgccaaggtc 120 cccgatgagg cccccaaagt gagcatcctg ggtagcgggg actttgcccg ctccctggcc 180 acacgcctgg tgggctctgg cttcaaagtg gtggtgggga gccgcaaccc caaacgcaca 240 gccaggctgt ttccctcagc ggcccaagtg actttccaag aggaggcagt gagctccccg 300 gaggtcatct ttgtggctgt gttccgggag cactactctt cactgtgcag tctcagtgac 360 cagctggcgg gcaagatcct ggtggatgtg agcaacccta cagagcaaga gcaccttcag 420 catcgtgagt ccaatgctga gtacctggcc tccctcttcc ccacttgcac agtggtcaag 480 gccttcaatg tcatctctgc ctggaccctg caggctggcc caagggatgg taacgggcag 540 gtgcccatct gcggtgacca gccagaagcc aagcgtgctg tctcggagat ggcgctcgcc 600 atgggcttca tgcccgtgga catgggatcc ctggcgtcag cctgggaggt ggaggccatg 660 cccctgcgcc tcctcccggc ctggaaggtg cccaccctgc tggccctggg gctcttcgtc 720 tgcttctatg cctacaactt cgtccgggac gttctgcagc cctatgtgca ggaaagccag 780 aacaagttct tcaagctgcc cgtgtccgtg gtcaacacca cactgccgtg cgtggcctac 840 gtgctgctgt ca ctcgtgta cttgcccggc gtgctggcgg ctgccctgca gctgcggcgc 900 ggcaccaagt accagcgctt ccccgactgg ctggaccact ggctacagca ccgcaagcag 960 atcgggctgc tcagcttctt ctgcgccgcc ctgcacgccc tctacagctt ctgcttgccg 1020 ctgcgccgcg cccaccgcta cgacctggtc aacctggcag tcaagcaggt cttggccaac 1080 aagagccacc tctgggtgga ggaggtctgg cggatggaga tctacctctc cctgggagtg 1140 ctggccctcg gcacgttgtc cctgctggcc gtgacctcac tgccgtccat tgcaaactcg 1200 ctcaactgga gggagttcag cttcgttcag tgtgtggcaa cttccagtgc aggaaacaca 1260 ggcagtggaa cccgaagacc tgaatctcag tcccaagacc cccacttacc tgccccgcat 1320 catcagacaa gtttcctagg ccctcggagc ttctgctgct cacttgtgcc tgtgtccacc 1380 ccatatggtc atcaagagga tttgagctgg acacgttaaa tgcaggatgc gtgcagccaa 1440 cagtggcatg ctggcttttg agtcctcact gggctttgtg gccctcgtgc tgagcacact 1500 gcacacgctc acctacggct ggacccgcgc cttcgaggag agccgctaca agttctacct 1560 gcctcccacc ttcacgctca cgctgctggt gccctgcgtc gtcatcctgg ccaaagccct 1620 gtttctcctg ccctgcatca gccgcagact cgccaggatc cggagaggct gggagaggga 1680 gagcaccatc aagttcacgc tgcccacaga ccacgccctg gccgagaaga cgagccacgt 1740 atgaggtgcc tgccctgggc tctggacccc gggcacacga gggacggtgc cctgagcccg 1800 ttaggttttc ttttcttggt ggtgcaaagt ggtataactg tgtgcaaata ggaggtttga 1860 ggtccaaatt cctgggactc aaatgtatgc agtactattc agaatgatat acacacatat 1920 gtgtatatgt atttacatat attccacata tataacagga tttgcaatta tacatagcta 1980 gctaaaaagt tgggtctctg agatttcaac ttgtagattt aaaaacaagt gccgtacgtt 2040 aagagaagag cagatcatgc tattgtgaca tttgcagaga tatacacaca ctttttgtac 2100 agaaaaaaaa aaaaaaaa 2118 <210> 4 <211> 456 <212> PRT <213> Human <400> 4 Met Pro Glu Glu Met Asp Lys Pro Leu Ile Ser Leu His Leu Val Asp 1 5 10 15 Ser Asp Ser Ser Leu Ala Lys Val Pro Asp Glu Ala Pro Lys Val Ser 20 25 30 Ile Leu Gly Ser Gly Asp Phe Ala Arg Ser Leu Ala Thr Arg Leu Val 35 40 45 Gly Ser Gly Phe Lys Val Val Val Gly Ser Arg Asn Pro Lys Arg Thr 50 55 60 Ala Arg Leu Phe Pro Ser Ala Ala Gln Val Thr Phe Gln Glu Glu Ala 65 70 75 80 Val Ser Ser Pro Glu Val Ile Phe Val Ala Val Phe Arg Glu His Tyr 85 90 95 Ser Ser Leu Cys Ser Leu Ser Asp Gln Leu Ala Gly Lys Ile Leu Val 100 105 110 Asp Val Ser Asn Pro Thr Glu Gln Glu His Leu Gln His Arg Glu Ser 115 120 125 Asn Ala Glu Tyr Leu Ala Ser Leu Phe Pro Thr Cys Thr Val Val Lys 130 135 140 Ala Phe Asn Val Ile Ser Ala Trp Thr Leu Gln Ala Gly Pro Arg Asp 145 150 155 160 Gly Asn Gly Gln Val Pro Ile Cys Gly Asp Gln Pro Glu Ala Lys Arg 165 170 175 Ala Val Ser Glu Met Ala Leu Ala Met Gly Phe Met Pro Val Asp Met 180 185 190 Gly Ser Leu Ala Ser Ala Trp Glu Val Glu Ala Met Pro Leu Arg Leu 195 200 205 Leu Pro Ala Tr p Lys Val Pro Thr Leu Leu Ala Leu Gly Leu Phe Val 210 215 220 Cys Phe Tyr Ala Tyr Asn Phe Val Arg Asp Val Leu Gln Pro Tyr Val 225 230 235 240 Gln Glu Ser Gln Asn Lys Phe Phe Lys Leu Pro Val Ser Val Val Asn 245 250 255 Thr Thr Leu Pro Cys Val Ala Tyr Val Leu Leu Ser Leu Val Tyr Leu 260 265 270 Pro Gly Val Leu Ala Ala Ala Leu Gln Leu Arg Arg Gly Thr Lys Tyr 275 280 285 Gln Arg Phe Pro Asp Trp Leu Asp His Trp Leu Gln His Arg Lys Gln 290 295 300 Ile Gly Leu Leu Ser Phe Phe Cys Ala Ala Leu His Ala Leu Tyr Ser 305 310 315 320 Phe Cys Leu Pro Leu Arg Arg Ala His Arg Tyr Asp Leu Val Asn Leu 325 330 335 Ala Val Lys Gln Val Leu Ala Asn Lys Ser His Leu Trp Val Glu Glu 340 345 350 Val Trp Arg Met Glu Ile Tyr Leu Ser Leu Gly Val Leu Ala Leu Gly 355 360 365 Thr Leu Ser Leu Leu Ala Val Thr Ser Leu Pro Ser Ile Ala Asn Ser 370 375 380 Leu Asn Trp Arg Glu Phe Ser Phe Val Gln Cys Val Ala Thr Ser Ser 385 390 395 400 Ala Gly Asn Thr Gly Ser Gly Thr Arg Arg Pro Glu Ser Gln Ser Gln 405 410 415 Asp Pro His Le u Pro Ala Pro His His Gln Thr Ser Phe Leu Gly Pro 420 425 430 Arg Ser Phe Cys Cys Ser Leu Val Pro Val Ser Thr Pro Tyr Gly His 435 440 445 Gln Glu Asp Leu Ser Trp Thr Arg 450 455 <210> 5 <211> 2714 <212> DNA <213> Rat <400> 5 gaattcggca cgaggctgcc gaggcactgt gatgtccggg gagatggaca aaccgctcat 60 cagtcgccgc ttggtggaca gtgatggcag tctggctgag gtccccaagg aggctcccaa 120 agtgggcatc ctgggcagcg gggattttgc ccggtccctg gccacacgcc tggtgggctc 180 tggcttcttt gtggtggtgg gaagccgtaa ccccaaacgc actgccggcc tcttcccctc 240 cttagcccaa gtgactttcc aggaggaggc cgtgagctct ccagaggtca tctttgtggc 300 cgtgttccgg gagcactact cctcactgtg cagtcttgct gaccagttgg ctggcaagat 360 cctagtggat gtaagcaacc ccacggagaa ggagcgtctt cagcaccgcc agtcgaacgc 420 cgagtacctg gcctccctct tccctgcctg cactgtggtc aaggccttca acgtcatctc 480 tgcatgggcc ctacaggctg gcccaaggga tgggaacagg caggtgctca tctgcggtga 540 ccagctggaa gccaagcaca ccgtctcaga gatggcgcgc gccatgggtt tcaccccact 600 ggacatggga tccctggcct cagcgaggga ggtagaggcc atacccctgc gcctccttcc 660 atcctggaag gtgcccaccc tcctggccct ggggctaagc acacaaagct atgcctacaa 720 cttcatccgg gacgttctac agccgtacat ccggaaagat gagaacaagt tctacaagat 780 gcccctgtct gtggtcaaca ccacgatacc ctgtgtggct tacgtgctgc tgtccctggt 840 ttacctgcct gg tgtgctgg cagctgccct tcagctgagg agggggacca agtaccagcg 900 cttcccagac tggctggacc attggctgca gcaccgcaag cagatcgggc tactcagctt 960 ttttttcgcc atgctgcacg ctctctacag cttctgcctg ccgctgcgcc gctcccaccg 1020 ctatgatctg gtcaacctgg ctgtgaagca ggtcctggcc aacaagagcc gcctctgggt 1080 tgaggaagaa gtctggcgga tggagatata cctgtccctg ggtgtgctgg ctctgggcat 1140 gctgtcactg ctggcggtta cctcgatccc ttccattgca aactcactca actggaagga 1200 gttcagcttt gtgcagtcca cgctgggctt cgtggccctg atgctgagca caatgcacac 1260 cctcacctac ggctggaccc gtgcttttga ggaaaaccac tacaagttct acctgccacc 1320 cacattcacg ctcacgctgc tcctgccctg tgtcatcatc ctggccaagg gcctcttcct 1380 cctgccctgc ctcagccaca gactcaccaa gatccgcagg ggctgggaga gggatggtgc 1440 cgtcaagttc atgctgcccg ctggccacac acagggggag aaaacaagcc acgtgtgagg 1500 ccctggaaat ggagacaggc acagcttgtg ggggccctgg gctgggttcg ggtctctttt 1560 ctgggatggt atatgcgtgg gtggccgagg tctgaatttc tgggatgcag gtgtatgccg 1620 agatactcag aatggcgtac cacacatgcg ataagtactc acatatattt catatataat 1680 aggatttact attattctta gttaaaaaaa aatagtgggt ccttatattt caacttatgc 1740 agggtcccta tatttcaact tgagcatttc agagcaaatg ccacacatta aacagcagat 1800 cccacccttg tggtagctgc agagacagac agaaacttct ggttatgaga gagactgtat 1860 tttgttggat tctaccttta atccccgttc tctacgttcc cctgttagcc acatcttaac 1920 gttggtgcag agctgggaca agagctggct ctggtgcagc ctcccccatc ccagggctag 1980 gaaacaagcc tctgatgaac agagggacca ggtctggacc ctcctgctcc cgcttccctg 2040 ggctcgagtg gggaggctca gcgggatccc ccgcaatctg tgcaggagtt ttcacaggtc 2100 tgtcctttct tccgggagcg gtctgaagcg gccccatctg atcctagctg agccgagatt 2160 gttccccact ccctgaaagt ccagagtcac cgtggagcct gcaaattgct ccttctgcga 2220 aggtgtgaag tcaccgtctc accagagcca ttaacgaacc tgatcttcag aagaagcata 2280 attgtttccc ctccattaag ttggtggtga ccctctttaa accactgtgc cttctcgcct 2340 ttcccatcac taatttgggc atctccatgg agtggactct tgtcggggca gttcaggggg 2400 gagggaagca ttagagattg cggagaataa ccatcgaagc ctcccttgga tgttcccagg 2460 cgtgccttca ttaaattggt ccctaatgag aatgacaggg gacccctgtt gcctgtatgc 2520 agagaaccag ccttctgagc accca ggaaa cacagtggcc ccacgccctt caggggggtc 2580 ccacgtcccc tttcccatgc ttttgcctcc ctccctcccg gttacaatca accataaaag 2640 tctgcaaata ttgttttttg aattcttaaa gagaccacat cctttgttat taccaaaaaaa700 aaaa a700 aaaa 2700 aaaa <210> 6 <211> 488 <212> PRT <213> Rat <400> 6 Met Ser Gly Glu Met Asp Lys Pro Leu Ile Ser Arg Arg Leu Val Asp 1 5 10 15 Ser Asp Gly Ser Leu Ala Glu Val Pro Lys Glu Ala Pro Lys Val Gly 20 25 30 Ile Leu Gly Ser Gly Asp Phe Ala Arg Ser Leu Ala Thr Arg Leu Val 35 40 45 Gly Ser Gly Phe Phe Val Val Val Gly Ser Arg Asn Pro Lys Arg Thr 50 55 60 Ala Gly Leu Phe Pro Ser Leu Ala Gln Val Thr Phe Gln Glu Glu Ala 65 70 75 80 Val Ser Ser Pro Glu Val Ile Phe Val Ala Val Phe Arg Glu His Tyr 85 90 95 Ser Ser Leu Cys Ser Leu Ala Asp Gln Leu Ala Gly Lys Ile Leu Val 100 105 110 Asp Val Ser Asn Pro Thr Glu Lys Glu Arg Leu Gln His Arg Gln Ser 115 120 125 Asn Ala Glu Tyr Leu Ala Ser Leu Phe Pro Ala Cys Thr Val Val Lys 130 135 140 Ala Phe Asn Val Ile Ser Ala Trp Ala Leu Gln Ala Gly Pro Arg Asp 145 150 155 160 Gly Asn Arg Gln Val Leu Ile Cys Gly Asp Gln Leu Glu Ala Lys His 165 170 175 Thr Val Ser Glu Met Ala Arg Ala Met Gly Phe Thr Pro Leu Asp Met 180 185 190 Gly Ser Leu Ala Ser Ala Arg Glu Val Glu Ala Ile Pro Leu Arg Leu 195 200 205 Leu Pro Ser Tr p Lys Val Pro Thr Leu Leu Ala Leu Gly Leu Ser Thr 210 215 220 Gln Ser Tyr Ala Tyr Asn Phe Ile Arg Asp Val Leu Gln Pro Tyr Ile 225 230 235 240 Arg Lys Asp Glu Asn Lys Phe Tyr Lys Met Pro Leu Ser Val Val Asn 245 250 255 Thr Thr Ile Pro Cys Val Ala Tyr Val Leu Leu Ser Leu Val Tyr Leu 260 265 270 Pro Gly Val Leu Ala Ala Ala Leu Gln Leu Arg Arg Gly Thr Lys Tyr 275 280 285 Gln Arg Phe Pro Asp Trp Leu Asp His Trp Leu Gln His Arg Lys Gln 290 295 300 Ile Gly Leu Leu Ser Phe Phe Phe Ala Met Leu His Ala Leu Tyr Ser 305 310 315 320 Phe Cys Leu Pro Leu Arg Arg Ser His Arg Tyr Asp Leu Val Asn Leu 325 330 335 Ala Val Lys Gln Val Leu Ala Asn Lys Ser Arg Leu Trp Val Glu Glu 340 345 350 Glu Val Trp Arg Met Glu Ile Tyr Leu Ser Leu Gly Val Leu Ala Leu 355 360 365 Gly Met Leu Ser Leu Leu Ala Val Thr Ser Ile Pro Ser Ile Ala Asn 370 375 380 Ser Leu Asn Trp Lys Glu Phe Ser Phe Val Gln Ser Thr Leu Gly Phe 385 390 395 400 Val Ala Leu Met Leu Ser Thr Met His Thr Leu Thr Tyr Gly Trp Thr 405 410 415 Arg Ala Phe Gl u Glu Asn His Tyr Lys Phe Tyr Leu Pro Pro Thr Phe 420 425 430 Thr Leu Thr Leu Leu Leu Pro Cys Val Ile Ile Leu Ala Lys Gly Leu 435 440 445 Phe Leu Leu Pro Cys Leu Ser His Arg Leu Thr Lys Ile Arg Arg Arg Arg Gly 450 455 460 Trp Glu Arg Asp Gly Ala Val Lys Phe Met Leu Pro Ala Gly His Thr 465 470 475 480 Gln Gly Glu Lys Thr Ser His Val 485 <210> 7 <211> 33 <212> DNA <213> Human <400> 7 tccctggcca cacgcctggt gggctctggc ttc 33 <210> 8 <211> 54 <212> PRT <213> Rat <400> 8 Ala Ala Pro Cys Val Ala Tyr Val Leu Leu Ser Leu Val Tyr Leu Pro 1 5 10 15 Gly Val Leu Ala Ala Ala Leu Gln Leu Arg Arg Gly Thr Lys Tyr Gln 20 25 30 Arg Phe Pro Asp Trp Leu Asp His Trp Leu Gln His Arg Lys Gln Ile 35 40 45 Gly Leu Leu Ser Phe Phe 50 <210> 9 <211> 17 <212> PRT <213> Rat <400> 9 Asn Phe Ile Arg Asp Val Leu Gln Pro Tyr Ile Arg Lys Asp Glu Asn 1 5 10 15 Lys <210> 10 <211> 3884 <212> DNA <213> Rat <400> 10 gcggccgcca tcatcaataa tataccttat tttggattga agccaatatg ataatgaggg 60 ggtggagttt gtgacgtggc gcggggcgtg ggaacggggc gggtgacgta gtagtgtggc 120 ggaagtgtga tgttgcaagt gtggcggaac acatgtaagc gacggatgtg gcaaaagtga 180 cgtttttggt gtgcgccggt gtacacagga agtgacaatt ttcgcgcggt tttaggcgga 240 tgttgtagta aatttgggcg taaccgagta agatttggcc attttcgcgg gaaaactgaa 300 taagaggaag tgaaatctga ataattttgt gttactcata gcgcgtaata tttgtctagg 360 gccgcgggga ctttgaccgt ttacgtggag actcgcccag ggcgcgcccc gatgtacggg 420 ccagatatac gcgtatctga ggggactagg gtgtgtttag gcgaaaagcg gggcttcggt 480 tgtacgcggt taggagtccc ctcaggatat agtagtttcg cttttgcata gggaggggga 540 aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca 600 tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga 660 tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt 720 ccgcattgca gagatattgt atttaagtgc ctagctcgat acaataaacg ccatttgacc 780 attcaccaca ttggtgtgca cctccggccc tggccactct cttccgcatc gctgtctgcg 840 ggggccagct g ttgggctcg cggttgagga caaactcttc gcggtctttc cagtactctt 900 ggatcggaaa cccgtcggcc tccgaacggt actccgccgc cgagggacct gagcgagtcc 960 gcatcgaccg gatcggaaaa cctctcgaga aaggcgtgta accagtcaca gtcgctctag 1020 aactagtgga tcccccgggc tgcaggaatt cgataattcg gcacgaggct gccgaggcac 1080 tgtgatgtcc ggggagatgg acaaaccgct catcagtcgc cgcttggtgg acagtgatgg 1140 cagtctggct gaggtcccca aggaggctcc caaagtgggc atcctgggca gcggggattt 1200 tgcccggtcc ctggccacac gcctggtggg ctctggcttc tttgtggtgg tgggaagccg 1260 taaccccaaa cgcactgccg gcctcttccc ctccttagcc caagtgactt tccaggagga 1320 ggccgtgagc tctccagagg tcatctttgt ggccgtgttc cgggagcact actcctcact 1380 gtgcagtctt gctgaccagt tggctggcaa gatcctagtg gatgtaagca accccacgga 1440 gaaggagcgt cttcagcacc gccagtcgaa cgccgagtac ctggcctccc tcttccctgc 1500 ctgcactgtg gtcaaggcct tcaacgtcat ctctgcatgg gccctacagg ctggcccaag 1560 ggatgggaac aggcaggtgc tcatctgcgg tgaccagctg gaagccaagc acaccgtctc 1620 agagatggcg cgcgccatgg gtttcacccc actggacatg ggatccctgg cctcagcgag 1680 ggaggtagag gccataccc c tgcgcctcct tccatcctgg aaggtgccca ccctcctggc 1740 cctggggcta agcacacaaa gctatgccta caacttcatc cgggacgttc tacagccgta 1800 catccggaaa gatgagaaca agttctacaa gatgcccctg tctgtggtca acaccacgat 1860 accctgtgtg gcttacgtgc tgctgtccct ggtttacctg cctggtgtgc tggcagctgc 1920 ccttcagctg aggaggggga ccaagtacca gcgcttccca gactggctgg accattggct 1980 gcagcaccgc aagcagatcg ggctactcag cttttttttc gccatgctgc acgctctcta 2040 cagcttctgc ctgccgctgc gccgctccca ccgctatgat ctggtcaacc tggctgtgaa 2100 gcaggtcctg gccaacaaga gccgcctctg ggttgaggaa gaagtctggc ggatggagat 2160 atacctgtcc ctgggtgtgc tggctctggg catgctgtca ctgctggcgg ttacctcgat 2220 cccttccatt gcaaactcac tcaactggaa ggagttcagc tttgtgcagt ccacgctggg 2280 cttcgtggcc ctgatgctga gcacaatgca caccctcacc tacggctgga cccgtgcttt 2340 tgaggaaaac cactacaagt tctacctgcc acccacattc acgctcacgc tgctcctgcc 2400 ctgtgtcatc atcctggcca agggcctctt cctcctgccc tgcctcagcc acagactcac 2460 caagatccgc aggggctggg agagggatgg tgccgtcaag ttcatgctgc ccgctggcca 2520 cacacagggg gagaaaacaa gcca cgtgtg aggccctgga aatggagaca ggcacagctt 2580 gtgggggccc tgggctgggt tcgggtctct tttctgggat ggtatatgcg tgggtggccg 2640 aggtctgaat ttctgggatg caggtgtatg ccgagatact cagaatggcg taccacacat 2700 gcgataagta ctcacatata tttcatatat aataggattt actattattc ttagttaaaa 2760 aaaaatagtg ggtccttata tttcaactta tgcagggtcc ctatatttca acttgagcat 2820 ttcagagcaa atgccacaca ttaaacagca gatcccaccc ttgtggtagc tgcagagaca 2880 gacagaaact tctggttatg agagagactg tattttgttg gattctacct ttaatccccg 2940 ttctctacgt tcccctgtta gccacatctt aacgttggtg cagagctggg acaagagctg 3000 gctctggtgc agcctccccc atcccagggc taggaaacaa gcctctgatg aacagaggga 3060 ccaggtctgg accctcctgc tcccgcttcc ctgggctcga gtggggaggc tcagcgggat 3120 cccccgcaat ctgtgcagga gttttcacag gtctgtcctt tcttccggga gcggtctgaa 3180 gcggccccat ctgatcctag ctgagccgag attgttcccc actccctgaa agtccagagt 3240 caccgtggag cctgcaaatt gctccttctg cgaaggtgtg aagtcaccgt ctcaccagag 3300 ccattaacga acctgatctt cagaagaagc ataattgttt cccctccatt aagttggtgg 3360 tgaccctctt taaaccactg tgccttctcg cctttcccat cactaatttg ggcatctcca 3420 tggagtggac tcttgtcggg gcagttcagg ggggagggaa gcattagaga ttgcggagaa 3480 taaccatcga agcctccctt ggatgttccc aggcgtgcct tcattaaatt ggtccctaat 3540 gagaatgaca ggggacccct gttgcctgta tgcagagaac cagccttctg agcacccagg 3600 aaacacagtg gccccacgcc cttcaggggg gtcccacgtc ccctttccca tgcttttgcc 3660 tccctccctc ccggttacaa tcaaccataa aagtctgcaa atattgtttt ttgaattatc 3720 aagcttatcg ataccgtcga aacttgttta ttgcagctta taatggttac aaataaagca 3780 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt 3840 ccaaactcat caatgtatct tatcatgtct ggatccgacc tcgg 3884 <210> 11 <211> 32166 <212> DNA <213> Rat <400> 11 atctggaagg tgctgaggta cgatgagacc cgcaccaggt gcagaccctg cgagtgtggc 60 ggtaaacata ttaggaacca gcctgtgatg ctggatgtga ccgaggagct gaggcccgat 120 cacttggtgc tggcctgcac ccgcgctgag tttggctcta gcgatgaaga tacagattga 180 ggtactgaaa tgtgtgggcg tggcttaagg gtgggaaaga atatataagg tgggggtctt 240 atgtagtttt gtatctgttt tgcagcagcc gccgccgcca tgagcaccaa ctcgtttgat 300 ggaagcattg tgagctcata tttgacaacg cgcatgcccc catgggccgg ggtgcgtcag 360 aatgtgatgg gctccagcat tgatggtcgc cccgtcctgc ccgcaaactc tactaccttg 420 acctacgaga ccgtgtctgg aacgccgttg gagactgcag cctccgccgc cgcttcagcc 480 gctgcagcca ccgcccgcgg gattgtgact gactttgctt tcctgagccc gcttgcaagc 540 agtgcagctt cccgttcatc cgcccgcgat gacaagttga cggctctttt ggcacaattg 600 gattctttga cccgggaact taatgtcgtt tctcagcagc tgttggatct gcgccagcag 660 gtttctgccc tgaaggcttc ctcccctccc aatgcggttt aaaacataaa taaaaaacca 720 gactctgttt ggatttggat caagcaagtg tcttgctgtc tttatttagg ggttttgcgc 780 gcgcggtagg cccgggacca gcggtctcgg tcgttgaggg tcctgtgtat tttttccagg 840 acgtggtaaa ggtgactctg gatgttcaga tacatgggca taagcccgtc tctggggtgg 900 aggtagcacc actgcagagc ttcatgctgc ggggtggtgt tgtagatgat ccagtcgtag 960 caggagcgct gggcgtggtg cctaaaaatg tctttcagta gcaagctgat tgccaggggc 1020 aggcccttgg tgtaagtgtt tacaaagcgg ttaagctggg atgggtgcat acgtggggat 1080 atgagatgca tcttggactg tatttttagg ttggctatgt tcccagccat atccctccgg 1140 ggattcatgt tgtgcagaac caccagcaca gtgtatccgg tgcacttggg aaatttgtca 1200 tgtagcttag aaggaaatgc gtggaagaac ttggagacgc ccttgtgacc tccaagattt 1260 tccatgcatt cgtccataat gatggcaatg ggcccacggg cggcggcctg ggcgaagata 1320 tttctgggat cactaacgtc atagttgtgt tccaggatga gatcgtcata ggccattttt 1380 acaaagcgcg ggcggagggt gccagactgc ggtataatgg ttccatccgg cccaggggcg 1440 tagttaccct cacagatttg catttcccac gctttgagtt cagatggggg gatcatgtct 1500 acctgcgggg cgatgaagaa aacggtttcc ggggtagggg agatcagctg ggaagaaagc 1560 aggttcctga gcagctgcga cttaccgcag ccggtgggcc cgtaaatcac acctattacc 1620 gggtgcaact ggtagttaag agagctgcag ctgccgtcat ccctgagcag gggggccact 1680 tcgttaagca tgtccctgac tcgcatgttt tccctgacca aatccgccag aaggcgctcg 1740 ccgcccagcg atagcagttc ttgcaaggaa gcaaagtttt tcaacggttt gagaccgtcc 1800 gccgtaggca tgcttttgag cgtttgacca agcagttcca ggcggtccca cagctcggtc 1860 acctgctcta cggcatctcg atccagcata tctcctcgtt tcgcgggttg gggcggcttt 1920 cgctgtacgg cagtagtcgg tgctcgtcca gacgggccag ggtcatgtct ttccacgggc 1980 gcagggtcct cgtcagcgta gtctgggtca cggtgaaggg gtgcgctccg ggctgcgcgc 2040 tggccagggt gcgcttgagg ctggtcctgc tggtgctgaa gcgctgccgg tcttcgccct 2100 gcgcgtcggc caggtagcat ttgaccatgg tgtcatagtc cagcccctcc gcggcgtggc 2160 ccttggcgcg cagcttgccc ttggaggagg cgccgcacga ggggcagtgc agacttttga 2220 gggcgtagag cttgggcgcg agaaataccg attccgggga gtaggcatcc gcgccgcagg 2280 ccccgcagac ggtctcgcat tccacgagcc aggtgagctc tggccgttcg gggtcaaaaa 2340 ccaggtttcc cccatgcttt ttgatgcgtt tcttacctct ggtttccatg agccggtgtc 2400 cacgctcggt gacgaaaagg ctgtccgtgt ccccgtatac agacttgaga ggcctgtcct 2460 cgagcggtgt tccgcggtcc tcctcgtata gaaactcgga ccactctgag acaaaggctc 2520 gcgtccaggc cagcacgaag gaggctaagt gggaggggta gcggtcgttg tccactaggg 2580 ggtccactcg ctccagggtg tgaagacaca tgtcgccctc ttcggcatca aggaaggtga 2640 ttggtttgta ggtgtaggcc acgtgaccgg gtgttcctga aggggggcta taaaaggggg 2700 tgggggcgcg ttcgtcctca ctctcttccg catcgctgtc tgcgagggcc agctgttggg 2760 gtgagtactc cctctgaaaa gcgggcatga cttctgcgct aagattgtca gtttccaaaa 2820 acgaggagga tttgatattc acctggcccg cggtgatgcc tttgagggtg gccgcatcca 2880 tctggtcaga aaagacaatc tttttgttgt caagcttggt ggcaaacgac ccgtagaggg 2940 cgttggacag caacttggcg atggagcgca gggtttggtt tttgtcgcga tcggcgcgct 3000 ccttggccgc gatgtttagc tgcacgtatt cgcgcgcaac gcaccgccat tcgggaaaga 3060 cggtggtgcg ctcgtcgggc accaggtgca cgcgccaacc gcggttgtgc agggtgacaa 3120 ggtcaacgct ggtggctacc tctccgcgta ggcgctcgtt ggtccagcag aggcggccgc 3180 ccttgcgcga gcagaatggc ggtagggggt ctagctgcgt ctcgtccggg gggtctgcgt 3240 ccacggtaaa gaccccgggc agcaggcgcg cgtcgaagta gtctatcttg catccttgca 3300 agtctagcgc ctgctgccat gcgcgggcgg caagcgcgcg ctcgtatggg ttgagtgggg 3360 gaccccatgg catggggtgg gtgagcgcgg aggcgtacat gccgcaaatg tcgtaaacgt 3420 agaggggctc tctgagtatt ccaagatatg tagggtagca tcttccaccg cggatgctgg 3480 cgcgcacgta atcgtatagt tcgtgcgagg gagcgaggag gtcgggaccg aggttgctac 3540 gggcgggctg ctctgctcgg aagactatct gcctgaagat ggcatgtgag ttggatgata 3600 tggttggacg ctggaagacg ttgaagctgg cgtctgtgag acctaccgcg tcacgcacga 3660 aggaggcgta ggagtcgcgc agcttgttga ccagctcggc ggtgacctgc acgtctaggg 3720 cgcagtagtc cagggtttcc ttgatgatgt catacttatc ctgtcccttt tttttccaca 3780 gctcgcggtt gaggacaaac tcttcgcggt ctttccagta ctcttggatc ggaaacccgt 3840 cggcctccga acggtaagag cctagcatgt agaactggtt gacggcctgg taggcgcagc 3900 atcccttttc tacgggtagc gcgtatgcct gcgcggcctt ccggagcgag gtgtgggtga 3960 gcgcaaaggt gtccctgacc atgactttga ggtactggta tttgaagtca gtgtcgtcgc 4020 atccgccctg ctcccagagc aaaaagtccg tgcgcttttt ggaacgcgga tttggcaggg 4080 cgaaggtgac atcgttgaag agtatctttc ccgcgcgagg cataaagttg cgtgtgatgc 4140 ggaagggtcc cggcacctcg gaacggttgt taattacctg ggcggcgagc acgatctcgt 4200 caaagccgtt gatgttgtgg cccacaatgt aaagttccaa gaagcgcggg atgcccttga 4260 tggaaggcaa ttttttaagt tcctcgtagg tgagctcttc aggggagctg agcccgtgct 4320 ctgaaagggc ccagtctgca agatgagggt tggaagcgac gaatgagctc cacaggtcac 4380 gggccattag catttgcagg tggtcgcgaa aggtcctaaa ctggcgacct atggccattt 4440 tttctggggt gatgcagtag aaggtaagcg ggtcttgttc ccagcggtcc catccaaggt 4500 tcgcggctag gtctcgcgcg gcagtcacta gaggctcatc tccgccgaac ttcatgacca 4560 gcatgaaggg cacgagctgc ttcccaaagg cccccatcca agtataggtc tctacatcgt 4620 aggtgacaaa gagacgctcg gtgcgaggat gcgagccgat cgggaagaac tggatctccc 4680 gccaccaatt ggaggagtgg ctattgatgt ggtgaaagta gaagtccctg cgacgggccg 4740 aacactcgtg ctggcttttg taaaaacgtg cgcagtactg gcagcggtgc acgggctgta 4800 catcctgcac gaggttgacc tgacgaccgc gcacaaggaa gcagagtggg aatttgagcc 4860 cctcgcctgg cgggtttggc tggtggtctt ctacttcggc tgcttgtcct tgaccgtctg 4920 gctgctcgag gggagttacg gtggatcgga ccaccacgcc gcgcgagccc aaagtccaga 4980 tgtccgcgcg cggcggtcgg agcttgatga caacatcgcg cagatgggag ctgtccatgg 5040 tctggagctc ccgcggcgtc aggtcaggcg ggagctcctg caggtttacc tcgcatagac 5100 gggtcagggc gcgggctaga tccaggtgat acctaatttc caggggctgg ttggtggcgg 5160 cgtcgatggc ttgcaagagg ccgcatcccc gcggcgcgac tacggtaccg cgcggcgggc 5220 ggtgggccgc gggggtgtcc ttggatgatg catctaaaag cggtgacgcg ggcgagcccc 5280 cggaggtagg gggggctccg gacccgccgg gagagggggc aggggcacgt cggcgccgcg 5340 cgcgggcagg agctggtgct gcgcgcgtag gttgctggcg aacgcgacga cgcggcggtt 5400 gatctcctga atctggcgcc tctgcgtgaa gacgacgggc ccggtgagct tgagcctgaa 5460 agagagttcg acagaatcaa tttcggtgtc gttgacggcg gcctggcgca aaatctcctg 5520 cacgtctcct gagttgtctt gataggcgat ctcggccatg aactgctcga tctcttcctc 5580 ctggagatct ccgcgtccgg ctcgctccac ggtggcggcg aggtcgttgg aaatgcgggc 5640 catgagctgc gagaaggcgt tgaggcctcc ctcgttccag acgcggctgt agaccacgcc 5700 cccttcggca tcgcgggcgc gcatgaccac ctgcgcgaga ttgagctcca cgtgccgggc 5760 gaagacggcg tagtttcgca ggcgctgaaa gaggtagttg agggtggtgg cggtgtgttc 5820 tgccacgaag aagtacataa cccagcgtcg caacgtggat tcgttgatat cccccaaggc 5880 ctcaaggcgc tccatggcct cgtagaagtc cacggcgaag ttgaaaaact gggagttgcg 5940 cgccgacacg gttaactcct cctccagaag acggatgagc tcggcgacag tgtcgcgcac 6000 ctcgcgctca aaggctacag gggcctcttc ttcttcttca atctcctctt ccataagggc 6060 ctccccttct tcttcttctg gcggcggtgg gggagggggg acacggcggc gacgacggcg 6120 caccgggagg cggtcgacaa agcgctcgat catctccccg cggcgacggc gcatggtctc 6180 ggtgacggcg cggccgttct cgcgggggcg cagttggaag acgccgcccg tcatgtcccg 6240 gttatgggtt ggcggggggc tgccatgcgg cagggatacg gcgctaacga tgcatctcaa 6300 caattgttgt gtaggtactc cgccgccgag ggacctgagc gagtccgcat cgaccggatc 6360 ggaaaacctc tcgagaaagg cgtctaacca gtcacagtcg caaggtaggc tgagcaccgt 6420 ggcgggcggc agcgggcggc ggtcggggtt gtttctggcg gaggtgctgc tgatgatgta 6480 attaaagtag gcggtcttga gacggcggat ggtcgacaga agcaccatgt ccttgggtcc 6540 ggcctgctga atgcgcaggc ggtcggccat gccccaggct tcgttttgac atcggcgcag 6600 gtctttgtag tagtcttgca tgagcctttc taccggcact tcttcttctc cttcctcttg 6660 tcctgcatct cttgcatcta tcgctgcggc ggcggcggag tttggccgta ggtggcgccc 6720 tcttcctccc atgcgtgtga ccccgaagcc cctcatcggc tgaagcaggg ctaggtcggc 6780 gacaacgcgc tcggctaata tggcctgctg cacctgcgtg agggtagact ggaagtcatc 6840 catgtccaca aagcggtggt atgcgcccgt gttgatggtg taagtgcagt tggccataac 6900 ggaccagtta acggtctggt gacccggctg cgagagctcg gtgtacctga gacgcgagta 6960 agccctcgag tcaaatacgt agtcgttgca agtccgcacc aggtactggt atcccaccaa 7020 aaagtgcggc ggcggctggc ggtagagggg ccagcgtagg gtggccgggg ctccgggggc 7080 gagatcttcc aacataaggc gatgatatcc gtagatgtac ctggacatcc aggtgatgcc 7140 ggcggcggtg gtggaggcgc gcggaaagtc gcggacgcgg ttccagatgt tgcgcagcgg 7200 caaaaagtgc tccatggtcg ggacgctctg gccggtcagg cgcgcgcaat cgttgacgct 7260 ctaccgtgca aaaggagagc ctgtaagcgg gcactcttcc gtggtctggt ggataaattc 7320 gcaagggtat catggcggac gaccggggtt cgagccccgt atccggccgt ccgccgtgat 7380 ccatgcggtt accgcccgcg tgtcgaaccc aggtgtgcga cgtcagacaa cgggggagtg 7440 ctccttttgg cttccttcca ggcgcggcgg ctgctgcgct agcttttttg gccactggcc 7500 gcgcgcagcg taagcggtta ggctggaaag cgaaagcatt aagtggctcg ctccctgtag 7560 ccggagggtt attttccaag ggttgagtcg cgggaccccc ggttcgagtc tcggaccggc 7620 cggactgcgg cgaacggggg tttgcctccc cgtcatgcaa gaccccgctt gcaaattcct 7680 ccggaaacag ggacgagccc cttttttgct tttcccagat gcatccggtg ctgcggcaga 7740 tgcgcccccc tcctcagcag cggcaagagc aagagcagcg gcagacatgc agggcaccct 7800 cccctcctcc taccgcgtca ggaggggcga catccgcggt tgacgcggca gcagatggtg 7860 attacgaacc cccgcggcgc cgggcccggc actacctgga cttggaggag ggcgagggcc 7920 tggcgcggct aggagcgccc tctcctgagc ggtacccaag ggtgcagctg aagcgtgata 7980 cgcgtgaggc gtacgtgccg cggcagaacc tgtttcgcga ccgcgaggga gaggagcccg 8040 aggagatgcg ggatcgaaag ttccacgcag ggcgcgagct gcggcatggc ctgaatcgcg 8100 agcggttgct gcgcgaggag gactttgagc ccgacgcgcg aaccgggatt agtcccgcgc 8160 gcgcacacgt ggcggccgcc gacctggtaa ccgcatacga gcagacggtg aaccaggaga 8220 ttaactttca aaaaagcttt aacaaccacg tgcgtacgct tgtggcgcgc gaggaggtgg 8280 ctataggact gatgcatctg tgggactttg taagcgcgct ggagcaaaac ccaaatagca 8340 agccgctcat ggcgcagctg ttccttatag tgcagcacag cagggacaac gaggcattca 8400 gggatgcgct gctaaacata gtagagcccg agggccgctg gctgctcgat ttgataaaca 8460 tcctgcagag catagtggtg caggagcgca gcttgagcct ggctgacaag gtggccgcca 8520 tcaactattc catgcttagc ctgggcaagt tttacgcccg caagatatac catacccctt 8580 acgttcccat agacaaggag gtaaagatcg aggggttcta catgcgcatg gcgctgaagg 8640 tgcttacctt gagcgacgac ctgggcgttt atcgcaacga gcgcatccac aaggccgtga 8700 gcgtgagccg gcggcgcgag ctcagcgacc gcgagctgat gcacagcctg caaagggccc 8760 tggctggcac gggcagcggc gatagagagg ccgagtccta ctttgacgcg ggcgctgacc 8820 tgcgctgggc cccaagccga cgcgccctgg aggcagctgg ggccggacct gggctggcgg 8880 tggcacccgc gcgcgctggc aacgtcggcg gcgtggagga atatgacgag gacgatgagt 8940 acgagccaga ggacggcgag tactaagcgg tgatgtttct gatcagatga tgcaagacgc 9000 aacggacccg gcggtgcggg cggcgctgca gagccagccg tccggcctta actccacgga 9060 cgactggcgc caggtcatgg accgcatcat gtcgctgact gcgcgcaatc ctgacgcgtt 9120 ccggcagcag ccgcaggcca accggctctc cgcaattctg gaagcggtgg tcccggcgcg 9180 cgcaaacccc acgcacgaga aggtgctggc gatcgtaaac gcgctggccg aaaacagggc 9240 catccggccc gacgaggccg gcctggtcta cgacgcgctg cttcagcgcg tggctcgtta 9300 caacagcggc aacgtgcaga ccaacctgga ccggctggtg ggggatgtgc gcgaggccgt 9360 ggcgcagcgt gagcgcgcgc agcagcaggg caacctgggc tccatggttg cactaaacgc 9420 cttcctgagt acacagcccg ccaacgtgcc gcggggacag gaggactaca ccaactttgt 9480 gagcgcactg cggctaatgg tgactgagac accgcaaagt gaggtgtacc agtctgggcc 9540 agactatttt ttccagacca gtagacaagg cctgcagacc gtaaacctga gccaggcttt 9600 caaaaacttg caggggctgt ggggggtgcg ggctcccaca ggcgaccgcg cgaccgtgtc 9660 tagcttgctg acgcccaact cgcgcctgtt gctgctgcta atagcgccct tcacggacag 9720 tggcagcgtg tcccgggaca catacctagg tcacttgctg acactgtacc gcgaggccat 9780 aggtcaggcg catgtggacg agcatacttt ccaggagatt acaagtgtca gccgcgcgct 9840 ggggcaggag gacacgggca gcctggaggc aaccctaaac tacctgctga ccaaccggcg 9900 gcagaagatc ccctcgttgc acagtttaaa cagcgaggag gagcgcattt tgcgctacgt 9960 gcagcagagc gtgagcctta acctgatgcg cgacggggta acgcccagcg tggcgctgga 10020 catgaccgcg cgcaacatgg aaccgggcat gtatgcctca aaccggccgt ttatcaaccg 10080 cctaatggac tacttgcatc gcgcggccgc cgtgaacccc gagtatttca ccaatgccat 10140 cttgaacccg cactggctac cgccccctgg tttctacacc gggggattcg aggtgcccga 10200 gggtaacgat ggattcctct gggacgacat agacgacagc gtgttttccc cgcaaccgca 10260 gaccctgcta gagttgcaac agcgcgagca ggcagaggcg gcgctgcgaa aggaaagctt 10320 ccgcaggcca agcagcttgt ccgatctagg cgctgcggcc ccgcggtcag atgctagtag 10380 cccatttcca agcttgatag ggtctcttac cagcactcgc accacccgcc cgcgcctgct 10440 gggcgaggag gagtacctaa acaactcgct gctgcagccg cagcgcgaaa aaaacctgcc 10500 tccggcattt cccaacaacg ggatagagag cctagtggac aagatgagta gatggaagac 10560 gtacgcgcag gagcacaggg acgtgccagg cccgcgcccg cccacccgtc gtcaaaggca 10620 cgaccgtcag cggggtctgg tgtgggagga cgatgactcg gcagacgaca gcagcgtcct 10680 ggatttggga gggagtggca acccgtttgc gcaccttcgc cccaggctgg ggagaatgtt 10740 ttaaaaaaaa aaaagcatga tgcaaaataa aaaactcacc aaggccatgg caccgagcgt 10800 tggttttctt gtattcccct tagtatgcgg cgcgcggcga tgtatgagga aggtcctcct 10860 ccctcctacg agagtgtggt gagcgcggcg ccagtggcgg cggcgctggg ttctcccttc 10920 gatgctcccc tggacccgcc gtttgtgcct ccgcggtacc tgcggcctac cggggggaga 10980 aacagcatcc gttactctga gttggcaccc ctattcgaca ccacccgtgt gtacctggtg 11040 gacaacaagt caacggatgt ggcatccctg aactaccaga acgaccacag caactttctg 11100 accacggtca ttcaaaacaa tgactacagc ccgggggagg caagcacaca gaccatcaat 11160 cttgacgacc ggtcgcactg gggcggcgac ctgaaaacca tcctgcatac caacatgcca 11220 aatgtgaacg agttcatgtt taccaataag tttaaggcgc gggtgatggt gtcgcgcttg 11280 cctactaagg acaatcaggt ggagctgaaa tacgagtggg tggagttcac gctgcccgag 11340 ggcaactact ccgagaccat gaccatagac cttatgaaca acgcgatcgt ggagcactac 11400 ttgaaagtgg gcagacagaa cggggttctg gaaagcgaca tcggggtaaa gtttgacacc 11460 cgcaacttca gactggggtt tgaccccgtc actggtcttg tcatgcctgg ggtatataca 11520 aacgaagcct tccatccaga catcattttg ctgccaggat gcggggtgga cttcacccac 11580 agccgcctga gcaacttgtt gggcatccgc aagcggcaac ccttccagga gggctttagg 11640 atcacctacg atgatctgga gggtggtaac attcccgcac tgttggatgt ggacgcctac 11700 caggcgagct tgaaagatga caccgaacag ggcgggggtg gcgcaggcgg cagcaacagc 11760 agtggcagcg gcgcggaaga gaactccaac gcggcagccg cggcaatgca gccggtggag 11820 gacatgaacg atcatgccat tcgcggcgac acctttgcca cacgggctga ggagaagcgc 11880 gctgaggccg aagcagcggc cgaagctgcc gcccccgctg cgcaacccga ggtcgagaag 11940 cctcagaaga aaccggtgat caaacccctg acagaggaca gcaagaaacg cagttacaac 12000 ctaataagca atgacagcac cttcacccag taccgcagct ggtaccttgc atacaactac 12060 ggcgaccctc agaccggaat ccgctcatgg accctgcttt gcactcctga cgtaacctgc 12120 ggctcggagc aggtctactg gtcgttgcca gacatgatgc aagaccccgt gaccttccgc 12180 tccacgcgcc agatcagcaa ctttccggtg gtgggcgccg agctgttgcc cgtgcactcc 12240 aagagcttct acaacgacca ggccgtctac tcccaactca tccgccagtt tacctctctg 12300 acccacgtgt tcaatcgctt tcccgagaac cagattttgg cgcgcccgcc agcccccacc 12360 atcaccaccg tcagtgaaaa cgttcctgct ctcacagatc acgggacgct accgctgcgc 12420 aacagcatcg gaggagtcca gcgagtgacc attactgacg ccagacgccg cacctgcccc 12480 tacgtttaca aggccctggg catagtctcg ccgcgcgtcc tatcgagccg cactttttga 12540 gcaagcatgt ccatccttat atcgcccagc aataacacag gctggggcct gcgcttccca 12600 agcaagatgt ttggcggggc caagaagcgc tccgaccaac acccagtgcg cgtgcgcggg 12660 cactaccgcg cgccctgggg cgcgcacaaa cgcggccgca ctgggcgcac caccgtcgat 12720 gacgccatcg acgcggtggt ggaggaggcg cgcaactaca cgcccacgcc gccaccagtg 12780 tccacagtgg acgcggccat tcagaccgtg gtgcgcggag cccggcgcta tgctaaaatg 12840 aagagacggc ggaggcgcgt agcacgtcgc caccgccgcc gacccggcac tgccgcccaa 12900 cgcgcggcgg cggccctgct taaccgcgca cgtcgcaccg gccgacgggc ggccatgcgg 12960 gccgctcgaa ggctggccgc gggtattgtc actgtgcccc ccaggtccag gcgacgagcg 13020 gccgccgcag cagccgcggc cattagtgct atgactcagg gtcgcagggg caacgtgtat 13080 tgggtgcgcg actcggttag cggcctgcgc gtgcccgtgc gcacccgccc cccgcgcaac 13140 tagattgcaa gaaaaaacta cttagactcg tactgttgta tgtatccagc ggcggcggcg 13200 cgcaacgaag ctatgtccaa gcgcaaaatc aaagaagaga tgctccaggt catcgcgccg 13260 gagatctatg gccccccgaa gaaggaagag caggattaca agccccgaaa gctaaagcgg 13320 gtcaaaaaga aaaagaaaga tgatgatgat gaacttgacg acgaggtgga actgctgcac 13380 gctaccgcgc ccaggcgacg ggtacagtgg aaaggtcgac gcgtaaaacg tgttttgcga 13440 cccggcacca ccgtagtctt tacgcccggt gagcgctcca cccgcaccta caagcgcgtg 13500 tatgatgagg tgtacggcga cgaggacctg cttgagcagg ccaacgagcg cctcggggag 13560 tttgcctacg gaaagcggca taaggacatg ctggcgttgc cgctggacga gggcaaccca 13620 acacctagcc taaagcccgt aacactgcag caggtgctgc ccgcgcttgc accgtccgaa 13680 gaaaagcgcg gcctaaagcg cgagtctggt gacttggcac ccaccgtgca gctgatggta 13740 cccaagcgcc agcgactgga agatgtcttg gaaaaaatga ccgtggaacc tgggctggag 13800 cccgaggtcc gcgtgcggcc aatcaagcag gtggcgccgg gactgggcgt gcagaccgtg 13860 gacgttcaga tacccactac cagtagcacc agtattgcca ccgccacaga gggcatggag 13920 acacaaacgt ccccggttgc ctcagcggtg gcggatgccg cggtgcaggc ggtcgctgcg 13980 gccgcgtcca agacctctac ggaggtgcaa acggacccgt ggatgtttcg cgtttcagcc 14040 ccccggcgcc cgcgcggttc gaggaagtac ggcgccgcca gcgcgctact gcccgaatat 14100 gccctacatc cttccattgc gcctaccccc ggctatcgtg gctacaccta ccgccccaga 14160 agacgagcaa ctacccgacg ccgaaccacc actggaaccc gccgccgccg tcgccgtcgc 14220 cagcccgtgc tggccccgat ttccgtgcgc agggtggctc gcgaaggagg caggaccctg 14280 gtgctgccaa cagcgcgcta ccaccccagc atcgtttaaa agccggtctt tgtggttctt 14340 gcagatatgg ccctcacctg ccgcctccgt ttcccggtgc cgggattccg aggaagaatg 14400 caccgtagga ggggcatggc cggccacggc ctgacgggcg gcatgcgtcg tgcgcaccac 14460 cggcggcggc gcgcgtcgca ccgtcgcatg cgcggcggta tcctgcccct ccttattcca 14520 ctgatcgccg cggcgattgg cgccgtgccc ggaattgcat ccgtggcctt gcaggcgcag 14580 agacactgat taaaaacaag ttgcatgtgg aaaaatcaaa ataaaaagtc tggactctca 14640 cgctcgcttg gtcctgtaac tattttgtag aatggaagac atcaactttg cgtctctggc 14700 cccgcgacac ggctcgcgcc cgttcatggg aaactggcaa gatatcggca ccagcaatat 14760 gagcggtggc gccttcagct ggggctcgct gtggagcggc attaaaaatt tcggttccac 14820 cgttaagaac tatggcagca aggcctggaa cagcagcaca ggccagatgc tgagggataa 14880 gttgaaagag caaaatttcc aacaaaaggt ggtagatggc ctggcctctg gcattagcgg 14940 ggtggtggac ctggccaacc aggcagtgca aaataagatt aacagtaagc ttgatccccg 15000 ccctcccgta gaggagcctc caccggccgt ggagacagtg tctccagagg ggcgtggcga 15060 aaagcgtccg cgccccgaca gggaagaaac tctggtgacg caaatagacg agcctccctc 15120 gtacgaggag gcactaaagc aaggcctgcc caccacccgt cccatcgcgc ccatggctac 15180 cggagtgctg ggccagcaca cacccgtaac gctggacctg cctccccccg ccgacaccca 15240 gcagaaacct gtgctgccag gcccgaccgc cgttgttgta acccgtccta gccgcgcgtc 15300 cctgcgccgc gccgccagcg gtccgcgatc gttgcggccc gtagccagtg gcaactggca 15360 aagcacactg aacagcatcg tgggtctggg ggtgcaatcc ctgaagcgcc gacgatgctt 15420 ctgaatagct aacgtgtcgt atgtgtgtca tgtatgcgtc catgtcgccg ccagaggagc 15480 tgctgagccg ccgcgcgccc gctttccaag atggctaccc cttcgatgat gccgcagtgg 15540 tcttacatgc acatctcggg ccaggacgcc tcggagtacc tgagccccgg gctggtgcag 15600 tttgcccgcg ccaccgagac gtacttcagc ctgaataaca agtttagaaa ccccacggtg 15660 gcgcctacgc acgacgtgac cacagaccgg tcccagcgtt tgacgctgcg gttcatccct 15720 gtggaccgtg aggatactgc gtactcgtac aaggcgcggt tcaccctagc tgtgggtgat 15780 aaccgtgtgc tggacatggc ttccacgtac tttgacatcc gcggcgtgct ggacaggggc 15840 cctactttta agccctactc tggcactgcc tacaacgccc tggctcccaa gggtgcccca 15900 aatccttgcg aatgggatga agctgctact gctcttgaaa taaacctaga agaagaggac 15960 gatgacaacg aagacgaagt agacgagcaa gctgagcagc aaaaaactca cgtatttggg 16020 caggcgcctt attctggtat aaatattaca aaggagggta ttcaaatagg tgtcgaaggt 16080 caaacaccta aatatgccga taaaacattt caacctgaac ctcaaatagg agaatctcag 16140 tggtacgaaa ctgaaattaa tcatgcagct gggagagtcc ttaaaaagac taccccaatg 16200 aaaccatgtt acggttcata tgcaaaaccc acaaatgaaa atggagggca aggcattctt 16260 gtaaagcaac aaaatggaaa gctagaaagt caagtggaaa tgcaattttt ctcaactact 16320 gaggcgaccg caggcaatgg tgataacttg actcctaaag tggtattgta cagtgaagat 16380 gtagatatag aaaccccaga cactcatatt tcttacatgc ccactattaa ggaaggtaac 16440 tcacgagaac taatgggcca acaatctatg cccaacaggc ctaattacat tgcttttagg 16500 gacaatttta ttggtctaat gtattacaac agcacgggta atatgggtgt tctggcgggc 16560 caagcatcgc agttgaatgc tgttgtagat ttgcaagaca gaaacacaga gctttcatac 16620 cagcttttgc ttgattccat tggtgataga accaggtact tttctatgtg gaatcaggct 16680 gttgacagct atgatccaga tgttagaatt attgaaaatc atggaactga agatgaactt 16740 ccaaattact gctttccact gggaggtgtg attaatacag agactcttac caaggtaaaa 16800 cctaaaacag gtcaggaaaa tggatgggaa aaagatgcta cagaattttc agataaaaat 16860 gaaataagag ttggaaataa ttttgccatg gaaatcaatc taaatgccaa cctgtggaga 16920 aatttcctgt actccaacat agcgctgtat ttgcccgaca agctaaagta cagtccttcc 16980 aacgtaaaaa tttctgataa cccaaacacc tacgactaca tgaacaagcg agtggtggct 17040 cccgggttag tggactgcta cattaacctt ggagcacgct ggtcccttga ctatatggac 17100 aacgtcaacc catttaacca ccaccgcaat gctggcctgc gctaccgctc aatgttgctg 17160 ggcaatggtc gctatgtgcc cttccacatc caggtgcctc agaagttctt tgccattaaa 17220 aacctccttc tcctgccggg ctcatacacc tacgagtgga acttcaggaa ggatgttaac 17280 atggttctgc agagctccct aggaaatgac ctaagggttg acggagccag cattaagttt 17340 gatagcattt gcctttacgc caccttcttc cccatggccc acaacaccgc ctccacgctt 17400 gaggccatgc ttagaaacga caccaacgac cagtccttta acgactatct ctccgccgcc 17460 aacatgctct accctatacc cgccaacgct accaacgtgc ccatatccat cccctcccgc 17520 aactgggcgg ctttccgcgg ctgggccttc acgcgcctta agactaagga aaccccatca 17580 ctgggctcgg gctacgaccc ttattacacc tactctggct ctatacccta cctagatgga 17640 accttttacc tcaaccacac ctttaagaag gtggccatta cctttgactc ttctgtcagc 17700 tggcctggca atgaccgcct gcttaccccc aacgagtttg aaattaagcg ctcagttgac 17760 ggggagggtt acaacgttgc ccagtgtaac atgaccaaag actggttcct ggtacaaatg 17820 ctagctaact acaacattgg ctaccagggc ttctatatcc cagagagcta caaggaccgc 17880 atgtactcct tctttagaaa cttccagccc atgagccgtc aggtggtgga tgatactaaa 17940 tacaaggact accaacaggt gggcatccta caccaacaca acaactctgg atttgttggc 18000 taccttgccc ccaccatgcg cgaaggacag gcctaccctg ctaacttccc ctatccgctt 18060 ataggcaaga ccgcagttga cagcattacc cagaaaaagt ttctttgcga tcgcaccctt 18120 tggcgcatcc cattctccag taactttatg tccatgggcg cactcacaga cctgggccaa 18180 aaccttctct acgccaactc cgcccacgcg ctagacatga cttttgaggt ggatcccatg 18240 gacgagccca cccttcttta tgttttgttt gaagtctttg acgtggtccg tgtgcaccgg 18300 ccgcaccgcg gcgtcatcga aaccgtgtac ctgcgcacgc ccttctcggc cggcaacgcc 18360 acaacataaa gaagcaagca acatcaacaa cagctgccgc catgggctcc agtgagcagg 18420 aactgaaagc cattgtcaaa gatcttggtt gtgggccata ttttttgggc acctatgaca 18480 agcgctttcc aggctttgtt tctccacaca agctcgcctg cgccatagtc aatacggccg 18540 gtcgcgagac tgggggcgta cactggatgg cctttgcctg gaacccgcac tcaaaaacat 18600 gctacctctt tgagcccttt ggcttttctg accagcgact caagcaggtt taccagtttg 18660 agtacgagtc actcctgcgc cgtagcgcca ttgcttcttc ccccgaccgc tgtataacgc 18720 tggaaaagtc cacccaaagc gtacaggggc ccaactcggc cgcctgtgga ctattctgct 18780 gcatgtttct ccacgccttt gccaactggc cccaaactcc catggatcac aaccccacca 18840 tgaaccttat taccggggta cccaactcca tgctcaacag tccccaggta cagcccaccc 18900 tgcgtcgcaa ccaggaacag ctctacagct tcctggagcg ccactcgccc tacttccgca 18960 gccacagtgc gcagattagg agcgccactt ctttttgtca cttgaaaaac atgtaaaaat 19020 aatgtactag agacactttc aataaaggca aatgctttta tttgtacact ctcgggtgat 19080 tatttacccc cacccttgcc gtctgcgccg tttaaaaatc aaaggggttc tgccgcgcat 19140 cgctatgcgc cactggcagg gacacgttgc gatactggtg tttagtgctc cacttaaact 19200 caggcacaac catccgcggc agctcggtga agttttcact ccacaggctg cgcaccatca 19260 ccaacgcgtt tagcaggtcg ggcgccgata tcttgaagtc gcagttgggg cctccgccct 19320 gcgcgcgcga gttgcgatac acagggttgc agcactggaa cactatcagc gccgggtggt 19380 gcacgctggc cagcacgctc ttgtcggaga tcagatccgc gtccaggtcc tccgcgttgc 19440 tcagggcgaa cggagtcaac tttggtagct gccttcccaa aaagggcgcg tgcccaggct 19500 ttgagttgca ctcgcaccgt agtggcatca aaaggtgacc gtgcccggtc tgggcgttag 19560 gatacagcgc ctgcataaaa gccttgatct gcttaaaagc cacctgagcc tttgcgcctt 19620 cagagaagaa catgccgcaa gacttgccgg aaaactgatt ggccggacag gccgcgtcgt 19680 gcacgcagca ccttgcgtcg gtgttggaga tctgcaccac atttcggccc caccggttct 19740 tcacgatctt ggccttgcta gactgctcct tcagcgcgcg ctgcccgttt tcgctcgtca 19800 catccatttc aatcacgtgc tccttattta tcataatgct tccgtgtaga cacttaagct 19860 cgccttcgat ctcagcgcag cggtgcagcc acaacgcgca gcccgtgggc tcgtgatgct 19920 tgtaggtcac ctctgcaaac gactgcaggt acgcctgcag gaatcgcccc atcatcgtca 19980 caaaggtctt gttgctggtg aaggtcagct gcaacccgcg gtgctcctcg ttcagccagg 20040 tcttgcatac ggccgccaga gcttccactt ggtcaggcag tagtttgaag ttcgccttta 20100 gatcgttatc cacgtggtac ttgtccatca gcgcgcgcgc agcctccatg cccttctccc 20160 acgcagacac gatcggcaca ctcagcgggt tcatcaccgt aatttcactt tccgcttcgc 20220 tgggctcttc ctcttcctct tgcgtccgca taccacgcgc cactgggtcg tcttcattca 20280 gccgccgcac tgtgcgctta cctcctttgc catgcttgat tagcaccggt gggttgctga 20340 aacccaccat ttgtagcgcc acatcttctc tttcttcctc gctgtccacg attacctctg 20400 gtgatggcgg gcgctcgggc ttgggagaag ggcgcttctt tttcttcttg ggcgcaatgg 20460 ccaaatccgc cgccgaggtc gatggccgcg ggctgggtgt gcgcggcacc agcgcgtctt 20520 gtgatgagtc ttcctcgtcc tcggactcga tacgccgcct catccgcttt tttgggggcg 20580 cccggggagg cggcggcgac ggggacgggg acgacacgtc ctccatggtt gggggacgtc 20640 gcgccgcacc gcgtccgcgc tcgggggtgg tttcgcgctg ctcctcttcc cgactggcca 20700 tttccttctc ctataggcag aaaaagatca tggagtcagt cgagaagaag gacagcctaa 20760 ccgccccctc tgagttcgcc accaccgcct ccaccgatgc cgccaacgcg cctaccacct 20820 tccccgtcga ggcacccccg cttgaggagg aggaagtgat tatcgagcag gacccaggtt 20880 ttgtaagcga agacgacgag gaccgctcag taccaacaga ggataaaaag caagaccagg 20940 acaacgcaga ggcaaacgag gaacaagtcg ggcgggggga cgaaaggcat ggcgactacc 21000 tagatgtggg agacgacgtg ctgttgaagc atctgcagcg ccagtgcgcc attatctgcg 21060 acgcgttgca agagcgcagc gatgtgcccc tcgccatagc ggatgtcagc cttgcctacg 21120 aacgccacct attctcaccg cgcgtacccc ccaaacgcca agaaaacggc acatgcgagc 21180 ccaacccgcg cctcaacttc taccccgtat ttgccgtgcc agaggtgctt gccacctatc 21240 acatcttttt ccaaaactgc aagatacccc tatcctgccg tgccaaccgc agccgagcgg 21300 acaagcagct ggccttgcgg cagggcgctg tcatacctga tatcgcctcg ctcaacgaag 21360 tgccaaaaat ctttgagggt cttggacgcg acgagaagcg cgcggcaaac gctctgcaac 21420 aggaaaacag cgaaaatgaa agtcactctg gagtgttggt ggaactcgag ggtgacaacg 21480 cgcgcctagc cgtactaaaa cgcagcatcg aggtcaccca ctttgcctac ccggcactta 21540 acctaccccc caaggtcatg agcacagtca tgagtgagct gatcgtgcgc cgtgcgcagc 21600 ccctggagag ggatgcaaat ttgcaagaac aaacagagga gggcctaccc gcagttggcg 21660 acgagcagct agcgcgctgg cttcaaacgc gcgagcctgc cgacttggag gagcgacgca 21720 aactaatgat ggccgcagtg ctcgttaccg tggagcttga gtgcatgcag cggttctttg 21780 ctgacccgga gatgcagcgc aagctagagg aaacattgca ctacaccttt cgacagggct 21840 acgtacgcca ggcctgcaag atctccaacg tggagctctg caacctggtc tcctaccttg 21900 gaattttgca cgaaaaccgc cttgggcaaa acgtgcttca ttccacgctc aagggcgagg 21960 cgcgccgcga ctacgtccgc gactgcgttt acttatttct atgctacacc tggcagacgg 22020 ccatgggcgt ttggcagcag tgcttggagg agtgcaacct caaggagctg cagaaactgc 22080 taaagcaaaa cttgaaggac ctatggacgg ccttcaacga gcgctccgtg gccgcgcacc 22140 tggcggacat cattttcccc gaacgcctgc ttaaaaccct gcaacagggt ctgccagact 22200 tcaccagtca aagcatgttg cagaacttta ggaactttat cctagagcgc tcaggaatct 22260 tgcccgccac ctgctgtgca cttcctagcg actttgtgcc cattaagtac cgcgaatgcc 22320 ctccgccgct ttggggccac tgctaccttc tgcagctagc caactacctt gcctaccact 22380 ctgacataat ggaagacgtg agcggtgacg gtctactgga gtgtcactgt cgctgcaacc 22440 tatgcacccc gcaccgctcc ctggtttgca attcgcagct gcttaacgaa agtcaaatta 22500 tcggtacctt tgagctgcag ggtccctcgc ctgacgaaaa gtccgcggct ccggggttga 22560 aactcactcc ggggctgtgg acgtcggctt accttcgcaa atttgtacct gaggactacc 22620 acgcccacga gattaggttc tacgaagacc aatcccgccc gccaaatgcg gagcttaccg 22680 cctgcgtcat tacccagggc cacattcttg gccaattgca agccatcaac aaagcccgcc 22740 aagagtttct gctacgaaag ggacgggggg tttacttgga cccccagtcc ggcgaggagc 22800 tcaacccaat ccccccgccg ccgcagccct atcagcagca gccgcgggcc cttgcttccc 22860 aggatggcac ccaaaaagaa gctgcagctg ccgccgccac ccacggacga ggaggaatac 22920 tgggacagtc aggcagagga ggttttggac gaggaggagg aggacatgat ggaagactgg 22980 gagagcctag acgaggaagc ttccgaggtc gaagaggtgt cagacgaaac accgtcaccc 23040 tcggtcgcat tcccctcgcc ggcgccccag aaatcggcaa ccggttccag catggctaca 23100 acctccgctc ctcaggcgcc gccggcactg cccgttcgcc gacccaaccg tagatgggac 23160 accactggaa ccagggccgg taagtccaag cagccgccgc cgttagccca agagcaacaa 23220 cagcgccaag gctaccgctc atggcgcggg cacaagaacg ccatagttgc ttgcttgcaa 23280 gactgtgggg gcaacatctc cttcgcccgc cgctttcttc tctaccatca cggcgtggcc 23340 ttcccccgta acatcctgca ttactaccgt catctctaca gcccatactg caccggcggc 23400 agcggcagcg gcagcaacag cagcggccac acagaagcaa aggcgaccgg atagcaagac 23460 tctgacaaag cccaagaaat ccacagcggc ggcagcagca ggaggaggag cgctgcgtct 23520 ggcgcccaac gaacccgtat cgacccgcga gcttagaaac aggatttttc ccactctgta 23580 tgctatattt caacagagca ggggccaaga acaagagctg aaaataaaaa acaggtctct 23640 gcgatccctc acccgcagct gcctgtatca caaaagcgaa gatcagcttc ggcgcacgct 23700 ggaagacgcg gaggctctct tcagtaaata ctgcgcgctg actcttaagg actagtttcg 23760 cgccctttct caaatttaag cgcgaaaact acgtcatctc cagcggccac acccggcgcc 23820 agcacctgtc gtcagcgcca ttatgagcaa ggaaattccc acgccctaca tgtggagtta 23880 ccagccacaa atgggacttg cggctggagc tgcccaagac tactcaaccc gaataaacta 23940 catgagcgcg ggaccccaca tgatatcccg ggtcaacgga atccgcgccc accgaaaccg 24000 aattctcttg gaacaggcgg ctattaccac cacacctcgt aataacctta atccccgtag 24060 ttggcccgct gccctggtgt accaggaaag tcccgctccc accactgtgg tacttcccag 24120 agacgcccag gccgaagttc agatgactaa ctcaggggcg cagcttgcgg gcggctttcg 24180 tcacagggtg cggtcgcccg ggcagggtat aactcacctg acaatcagag ggcgaggtat 24240 tcagctcaac gacgagtcgg tgagctcctc gcttggtctc cgtccggacg ggacatttca 24300 gatcggcggc gccggccgtc cttcattcac gcctcgtcag gcaatcctaa ctctgcagac 24360 ctcgtcctct gagccgcgct ctggaggcat tggaactctg caatttattg aggagtttgt 24420 gccatcggtc tactttaacc ccttctcggg acctcccggc cactatccgg atcaatttat 24480 tcctaacttt gacgcggtaa aggactcggc ggacggctac gactgaatgt taagtggaga 24540 ggcagagcaa ctgcgcctga aacacctggt ccactgtcgc cgccacaagt gctttgcccg 24600 cgactccggt gagttttgct actttgaatt gcccgaggat catatcgagg gcccggcgca 24660 cggcgtccgg cttaccgccc agggagagct tgcccgtagc ctgattcggg agtttaccca 24720 gcgccccctg ctagttgagc gggacagggg accctgtgtt ctcactgtga tttgcaactg 24780 tcctaacctt ggattacatc aagatctttg ttgccatctc tgtgctgagt ataataaata 24840 cagaaattaa aatatactgg ggctcctatc gccatcctgt aaacgccacc gtcttcaccc 24900 gcccaagcaa accaaggcga accttacctg gtacttttaa catctctccc tctgtgattt 24960 acaacagttt caacccagac ggagtgagtc tacgagagaa cctctccgag ctcagctact 25020 ccatcagaaa aaacaccacc ctccttacct gccgggaacg tacgagtgcg tcaccggccg 25080 ctgcaccaca cctaccgcct gaccgtaaac cagacttttt ccggacagac ctcaataact 25140 ctgtttacca gaacaggagg tgagcttaga aaacccttag ggtattaggc caaaggcgca 25200 gctactgtgg ggtttatgaa caattcaagc aactctacgg gctattctaa ttcaggtttc 25260 tctaatcggg gttggggtta ttctctgtct tgtgattctc tttattctta tactaacgct 25320 tctctgccta aggctcgccg cctgctgtgt gcacatttgc atttattgtc agctttttaa 25380 acgctggggt cgccacccaa gatgattagg tacataatcc taggtttact cacccttgcg 25440 tcagcccacg gtaccaccca aaaggtggat tttaaggagc cagcctgtaa tgttacattc 25500 gcagctgaag ctaatgagtg caccactctt ataaaatgca ccacagaaca tgaaaagctg 25560 cttattcgcc acaaaaacaa aattggcaag tatgctgttt atgctatttg gcagccaggt 25620 gacactacag agtataatgt tacagttttc cagggtaaaa gtcataaaac ttttatgtat 25680 acttttccat tttatgaaat gtgcgacatt accatgtaca tgagcaaaca gtataagttg 25740 tggcccccac aaaattgtgt ggaaaacact ggcactttct gctgcactgc tatgctaatt 25800 acagtgctcg ctttggtctg taccctactc tatattaaat acaaaagcag acgcagcttt 25860 attgaggaaa agaaaatgcc ttaatttact aagttacaaa gctaatgtca ccactaactg 25920 ctttactcgc tgcttgcaaa acaaattcaa aaagttagca ttataattag aataggattt 25980 aaaccccccg gtcatttcct gctcaatacc attcccctga acaattgact ctatgtggga 26040 tatgctccag cgctacaacc ttgaagtcag gcttcctgga tgtcagcatc tgactttggc 26100 cagcacctgt cccgcggatt tgttccagtc caactacagc gacccaccct aacagagatg 26160 accaacacaa ccaacgcggc cgccgctacc ggacttacat ctaccacaaa tacaccccaa 26220 gtttctgcct ttgtcaataa ctgggataac ttgggcatgt ggtggttctc catagcgctt 26280 atgtttgtat gccttattat tatgtggctc atctgctgcc taaagcgcaa acgcgcccga 26340 ccacccatct atagtcccat cattgtgcta cacccaaaca atgatggaat ccatagattg 26400 gacggactga aacacatgtt cttttctctt acagtatgat taaatgagac atgattcctc 26460 gagtttttat attactgacc cttgttgcgc ttttttgtgc gtgctccaca ttggctgcgg 26520 tttctcacat cgaagtagac tgcattccag ccttcacagt ctatttgctt tacggatttg 26580 tcaccctcac gctcatctgc agcctcatca ctgtggtcat cgcctttatc cagtgcattg 26640 actgggtctg tgtgcgcttt gcatatctca gacaccatcc ccagtacagg gacaggacta 26700 tagctgagct tcttagaaat ggacggaatt attacagagc agcgcctgct agaaagacgc 26760 agggcagcgg ccgagcaaca gcgcatgaat caagagctcc aagacatggt taacttgcac 26820 cagtgcaaaa ggggtatctt ttgtctggta aagcaggcca aagtcaccta cgacagtaat 26880 accaccggac accgccttag ctacaagttg ccaaccaagc gtcagaaatt ggtggtcatg 26940 gtgggagaaa agcccattac cataactcag cactcggtag aaaccgaagg ctgcattcac 27000 tcaccttgtc aaggacctga ggatctctgc acccttatta agaccctgtg cggtctcaaa 27060 gatcttattc cctttaacta ataaaaaaaa ataataaagc atcacttact taaaatcagt 27120 tagcaaattt ctgtccagtt tattcagcag cacctccttg ccctcctccc agctctggta 27180 ttgcagcttc ctcctggctg caaactttct ccacaatcta aatggaatgt cagtttcctc 27240 ctgttcctgt ccatccgcac ccactatctt catgttgttg cagatgaagc gcgcaagacc 27300 gtctgaagat accttcaacc ccgtgtatcc atatgacacg gaaaccggtc ctccaactgt 27360 gccttttctt actcctccct ttgtatcccc caatgggttt caagagagtc cccctggggt 27420 actctctttg cgcctatccg aacctctagt tacctccaat ggcatgcttg cgctcaaaat 27480 gggcaacggc ctctctctgg acgaggccgg caaccttacc tcccaaaatg taaccactgt 27540 gagcccacct ctcaaaaaaa ccaagtcaaa cataaacctg gaaatatctg cacccctcac 27600 agttacctca gaagccctaa ctgtggctgc cgccgcacct ctaatggtcg cgggcaacac 27660 actcaccatg caatcacagg ccccgctaac cgtgcacgac tccaaactta gcattgccac 27720 ccaaggaccc ctcacagtgt cagaaggaaa gctagccctg caaacatcag gccccctcac 27780 caccaccgat agcagtaccc ttactatcac tgcctcaccc cctctaacta ctgccactgg 27840 tagcttgggc attgacttga aagagcccat ttatacacaa aatggaaaac taggactaaa 27900 gtacggggct cctttgcatg taacagacga cctaaacact ttgaccgtag caactggtcc 27960 aggtgtgact attaataata cttccttgca aactaaagtt actggagcct tgggttttga 28020 ttcacaaggc aatatgcaac ttaatgtagc aggaggacta aggattgatt ctcaaaacag 28080 acgccttata cttgatgtta gttatccgtt tgatgctcaa aaccaactaa atctaagact 28140 aggacagggc cctcttttta taaactcagc ccacaacttg gatattaact acaacaaagg 28200 cctttacttg tttacagctt caaacaattc caaaaagctt gaggttaacc taagcactgc 28260 caaggggttg atgtttgacg ctacagccat agccattaat gcaggagatg ggcttgaatt 28320 tggttcacct aatgcaccaa acacaaatcc cctcaaaaca aaaattggcc atggcctaga 28380 atttgattca aacaaggcta tggttcctaa actaggaact ggccttagtt ttgacagcac 28440 aggtgccatt acagtaggaa acaaaaataa tgataagcta actttgtgga ccacaccagc 28500 tccatctcct aactgtagac taaatgcaga gaaagatgct aaactcactt tggtcttaac 28560 aaaatgtggc agtcaaatac ttgctacagt ttcagttttg gctgttaaag gcagtttggc 28620 tccaatatct ggaacagttc aaagtgctca tcttattata agatttgacg aaaatggagt 28680 gctactaaac aattccttcc tggacccaga atattggaac tttagaaatg gagatcttac 28740 tgaaggcaca gcctatacaa acgctgttgg atttatgcct aacctatcag cttatccaaa 28800 atctcacggt aaaactgcca aaagtaacat tgtcagtcaa gtttacttaa acggagacaa 28860 aactaaacct gtaacactaa ccattacact aaacggtaca caggaaacag gagacacaac 28920 tccaagtgca tactctatgt cattttcatg ggactggtct ggccacaact acattaatga 28980 aatatttgcc acatcctctt acactttttc atacattgcc caagaataaa gaatcgtttg 29040 tgttatgttt caacgtgttt atttttcaat tgcagaaaat ttcaagtcat ttttcattca 29100 gtagtatagc cccaccacca catagcttat acagatcacc gtaccttaat caaactcaca 29160 gaaccctagt attcaacctg ccacctccct cccaacacac agagtacaca gtcctttctc 29220 cccggctggc cttaaaaagc atcatatcat gggtaacaga catattctta ggtgttatat 29280 tccacacggt ttcctgtcga gccaaacgct catcagtgat attaataaac tccccgggca 29340 gctcacttaa gttcatgtcg ctgtccagct gctgagccac aggctgctgt ccaacttgcg 29400 gttgcttaac gggcggcgaa ggagaagtcc acgcctacat gggggtagag tcataatcgt 29460 gcatcaggat agggcggtgg tgctgcagca gcgcgcgaat aaactgctgc cgccgccgct 29520 ccgtcctgca ggaatacaac atggcagtgg tctcctcagc gatgattcgc accgcccgca 29580 gcataaggcg ccttgtcctc cgggcacagc agcgcaccct gatctcactt aaatcagcac 29640 agtaactgca gcacagcacc acaatattgt tcaaaatccc acagtgcaag gcgctgtatc 29700 caaagctcat ggcggggacc acagaaccca cgtggccatc ataccacaag cgcaggtaga 29760 ttaagtggcg acccctcata aacacgctgg acataaacat tacctctttt ggcatgttgt 29820 aattcaccac ctcccggtac catataaacc tctgattaaa catggcgcca tccaccacca 29880 tcctaaacca gctggccaaa acctgcccgc cggctataca ctgcagggaa ccgggactgg 29940 aacaatgaca gtggagagcc caggactcgt aaccatggat catcatgctc gtcatgatat 30000 caatgttggc acaacacagg cacacgtgca tacacttcct caggattaca agctcctccc 30060 gcgttagaac catatcccag ggaacaaccc attcctgaat cagcgtaaat cccacactgc 30120 agggaagacc tcgcacgtaa ctcacgttgt gcattgtcaa agtgttacat tcgggcagca 30180 gcggatgatc ctccagtatg gtagcgcggg tttctgtctc aaaaggaggt agacgatccc 30240 tactgtacgg agtgcgccga gacaaccgag atcgtgttgg tcgtagtgtc atgccaaatg 30300 gaacgccgga cgtagtcata tttcctgaag caaaaccagg tgcgggcgtg acaaacagat 30360 ctgcgtctcc ggtctcgccg cttagatcgc tctgtgtagt agttgtagta tatccactct 30420 ctcaaagcat ccaggcgccc cctggcttcg ggttctatgt aaactccttc atgcgccgct 30480 gccctgataa catccaccac cgcagaataa gccacaccca gccaacctac acattcgttc 30540 tgcgagtcac acacgggagg agcgggaaga gctggaagaa ccatgttttt ttttttattc 30600 caaaagatta tccaaaacct caaaatgaag atctattaag tgaacgcgct cccctccggt 30660 ggcgtggtca aactctacag ccaaagaaca gataatggca tttgtaagat gttgcacaat 30720 ggcttccaaa aggcaaacgg ccctcacgtc caagtggacg taaaggctaa acccttcagg 30780 gtgaatctcc tctataaaca ttccagcacc ttcaaccatg cccaaataat tctcatctcg 30840 ccaccttctc aatatatctc taagcaaatc ccgaatatta agtccggcca ttgtaaaaat 30900 ctgctccaga gcgccctcca ccttcagcct caagcagcga atcatgattg caaaaattca 30960 ggttcctcac agacctgtat aagattcaaa agcggaacat taacaaaaat accgcgatcc 31020 cgtaggtccc ttcgcagggc cagctgaaca taatcgtgca ggtctgcacg gaccagcgcg 31080 gccacttccc cgccaggaac cttgacaaaa gaacccacac tgattatgac acgcatactc 31140 ggagctatgc taaccagcgt agccccgatg taagctttgt tgcatgggcg gcgatataaa 31200 atgcaaggtg ctgctcaaaa aatcaggcaa agcctcgcgc aaaaaagaaa gcacatcgta 31260 gtcatgctca tgcagataaa ggcaggtaag ctccggaacc accacagaaa aagacaccat 31320 ttttctctca aacatgtctg cgggtttctg cataaacaca aaataaaata acaaaaaaac 31380 atttaaacat tagaagcctg tcttacaaca ggaaaaacaa cccttataag cataagacgg 31440 actacggcca tgccggcgtg accgtaaaaa aactggtcac cgtgattaaa aagcaccacc 31500 gacagctcct cggtcatgtc cggagtcata atgtaagact cggtaaacac atcaggttga 31560 ttcatcggtc agtgctaaaa agcgaccgaa atagcccggg ggaatacata cccgcaggcg 31620 tagagacaac attacagccc ccataggagg tataacaaaa ttaataggag agaaaaacac 31680 ataaacacct gaaaaaccct cctgcctagg caaaatagca ccctcccgct ccagaacaac 31740 atacagcgct tcacagcggc agcctaacag tcagccttac cagtaaaaaa gaaaacctat 31800 taaaaaaaca ccactcgaca cggcaccagc tcaatcagtc acagtgtaaa aaagggccaa 31860 gtgcagagcg agtatatata ggactaaaaa atgacgtaac ggttaaagtc cacaaaaaac 31920 acccagaaaa ccgcacgcga acctacgccc agaaacgaaa gccaaaaaac ccacaacttc 31980 ctcaaatcgt cacttccgtt ttcccacgtt acgtaacttc ccattttaag aaaactacaa 32040 ttcccaacac atacaagtta ctccgcccta aaacctacgt cacccgcccc gttcccacgc 32100 cccgcgccac gtcacaaact ccaccccctc attatcatat tggcttcaat ccaaaataag 32160 gtatat 32166 (Reason for correction 1) In paragraph “0040”, "The SEQ ID: 6 shows. And "(SEQ ID NO: 9
) ”, The sequence numbers of the corresponding nucleotide sequences and amino acid sequences in the“ Sequence Listing ”are clearly indicated.
This is a correction for the purpose of correction, and after the procedure of correction of mistranslation 3, it can also be a general correction.
It is a matter. (Reason 2 for correction) In paragraph “0279”, the point of “(SEQ ID NO: 10 and SEQ ID NO: 11)”
The sequence numbers of the corresponding nucleotide sequences and amino acid sequences in the "Sequence List" are clearly indicated.
This is a correction for the purpose of correction, and after the procedure of correction of mistranslation 3, it can also be a general correction.
It is a matter. (Reason 3 for correction) Regarding the point of “Sequence Listing” in paragraph “0342”, the description attached to the application for the present patent application (hereinafter, also referred to as the original specification)
Originally, "sequence list" was not described at the corresponding position of the
The columns and amino acid sequences are shown in the "Detailed Description of the Invention" section and figures of the original specification.
Before the correction of the mistranslation, the translation of the original specification (below
, Which is also referred to as a translated text of the specification), in the paragraph "003 of the detailed description of the invention".
5 ”, paragraph“ 0036 ”, paragraph“ 0037 ”, paragraph“ 0038 ”, paragraph“ 003 ”
9 ", paragraph" 0040 ", the nucleotide sequence and
And a part of the amino acid sequence are not described, and instead (SEQ ID NO: 1), (sequence
No .: 2), (SEQ ID NO: 3), (SEQ ID NO: 4), (SEQ ID NO: 5), (SEQ ID NO: 2)
No .: 6) is provided. Paragraphs “0035” and “0” in the “Detailed Description of the Invention” part of the translated text of the description
036 ", paragraph" 0037 ", paragraph" 0038 ", paragraph" 0039 ", paragraph" 0 "
040 ", it is preferable to describe a long base sequence and amino acid sequence.
It was judged not to be valid, and the relevant part was submitted at the international stage of the patent application.
Intended to incorporate the nucleotide and amino acid sequences listed in the supposed "Sequence List"
Because it was done. However, in the present patent application, the flexi
Bull discs were not submitted in a proper manner afterwards, so
Purpose of writing "Sequence Listing" at the end of the translation of the description in which "Sequence Listing" should be written
Then, the mistranslation correction in which the "sequence list" is described in the paragraph "0342" is corrected. It should be noted that the description corresponding to the position originally described in the original description
At the corresponding position in the "Detailed Description of the Invention" in the translation of
It is possible to correct the mistranslation by describing the amino acid sequence and the amino acid sequence.
Paragraph "0035" and Paragraph "0036" in the "Detailed Description of the Invention" part of the translated text of the book
, Paragraph “0037”, paragraph “0038”, paragraph “0039”, paragraph “0040
In, it is not preferable to describe a long base sequence and amino acid sequence.
Therefore, we will not correct the mistranslation in such a form.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 35/00 C07K 14/47 4C084 35/02 16/18 4C087 C07K 14/47 19/00 4H045 16/18 C12N 1/15 19/00 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 C12Q 1/68 A 5/10 G01N 33/15 Z C12P 21/02 33/50 Z C12Q 1/68 C12P 21/08 G01N 33/15 C12N 15/00 ZNAA 33/50 5/00 A // C12P 21/08 A61K 37/02 (31) Priority claim number 09 / 499,817 (32) Priority date February 8, 2000 (2000.2.8) (33) Priority claiming country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS) , MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA , BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ , PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR , TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Wang, Chian United States, 38117 Magnolia Drive, MN, Tennessee, 132 (72) Inventor Rinaldi, August Tinus United States, 38119 Tennessee, Memphis, North Rolling Oaks Drive, 5453 (72) Inventor Menon, Rehma United States, 38119 Tennessee, Menphys, Walnut Hall, 1350, Court Number 5F Term (Reference) 2G045 AA40 BB03 BB20 CB01 CB17 CB21 DA12 DA13 DA14 DA36 DA37 FB02 FB03 4B024 AA01 AA12 BA80 CA04 CA11 CA12 DA02 DA03 EA02 HA11 HA17 4B063 QA13 QQ08 QQ43 QQ53 QR32 QR36 QR01 QR27 QR27 QR27 QR27 QR27 QR27 QR27 QR27 QR27 QR27 QR27 QR27 QR27 QR34 AA91Y AA93X AA93Y AB01 BA01 CA24 CA44 CA46 4C084 AA02 AA07 AA13 DC50 NA14 ZB261 ZB271 4C087 BC83 CA12 NA14 ZB26 ZB27 4H045 AA10 AA11 AA20 AA30 BA10 BA41 CA41 DA76 EA28 EA51 FA74

Claims (53)

[Claims] 1. A mammalian p-Hyde protein of the p-Hyde family that induces susceptibility of cancer cells to cell death and encodes SEQ ID NOs: 1, 3, 5.
, Or an isolated nucleic acid comprising a nucleic acid sequence as described in 7, including variants and variants thereof. 2. The isolated nucleic acid of claim 1, having a nucleic acid sequence that has at least 82% similarity to the nucleic acid coding sequence of SEQ ID NO: 1, 3, 5, or 7. 3. The isolated nucleic acid of claim 1, having a nucleic acid sequence having at least 85% similarity to the nucleic acid coding sequence of SEQ ID NO: 1, 3, 5, or 7. 4. The isolated nucleic acid of claim 1, having a nucleic acid having at least 95% similarity to the nucleic acid coding sequence of SEQ ID NO: 1, 3, 5, or 7. 5. The isolated nucleic acid of claim 1, wherein the nucleic acid fragment is set forth in SEQ ID NO: 7. 6. The isolated nucleic acid of claim 1, wherein the nucleic acid fragment encodes an amino acid fragment as set forth in SEQ ID NO: 8. 7. The isolated nucleic acid of claim 1, which is DNA or RNA. 8. The isolated nucleic acid according to claim 2, which is cDNA or genomic DNA. 9. The isolated nucleic acid of claim 1, which encodes an amino acid sequence having a sequence as set forth in SEQ ID NOs: 2, 4, or 6. 10. The isolated nucleic acid of claim 1, which is labeled with a detectable marker. 11. The isolated nucleic acid of claim 10, wherein the detectable marker is a radioactive marker, a colorimetric marker, a luminescent marker, a fluorescent marker, or a gold label. 12. An oligonucleotide of at least 15 nucleotides capable of specifically hybridizing with the sequence of the nucleic acid encoding human p-Hyde of claim 1. 13. The oligonucleotide according to claim 12, wherein the nucleic acid is DNA or RNA. 14. The oligonucleotide according to claim 12, which is labeled with a detectable marker. 15. The oligonucleotide according to claim 13, which is a radioactive marker, a colorimetric marker, a luminescent marker, a fluorescent marker, or a gold label. 16. A nucleic acid having a sequence complementary to the sequence of the isolated nucleic acid of claim 1. 17. An antisense molecule capable of specifically hybridizing with the isolated nucleic acid of claim 1. 18. A vector comprising the isolated nucleic acid of claim 1. 19. The vector according to claim 18, which further comprises an expression factor functionally comprising a promoter for RNA transcription or linked to a nucleic acid. 20. The vector of claim 18, wherein the promoter comprises a bacterial, yeast, insect, or mammalian promoter. 21. A plasmid, cosmid, yeast artificial chromosome (YAC), BA
21. The vector of claim 20, which is C, adenovirus, adeno-associated virus, retrovirus, P1, bacteriophage, or eukaryotic viral DNA. 22. The adenovirus vector according to claim 21, which is a replication-defective adenovirus type 5 expression vector. 23. A deletion in the E1 and E3 regions of the genome, and p-Hyd
23. An adenovirus genome comprising within its region an insertion under the control of a promoter, a nucleic acid encoding e, an allele, fragment or variant thereof.
The described adenovirus vector. 24. The promoter is the Rous sarcoma virus promoter,
The vector according to claim 23. 25. A host vector system for the production of a polypeptide, which comprises the vector of claim 18 in a suitable host. 26. The host vector system of claim 25, wherein the suitable host is a prokaryotic cell or a eukaryotic cell. 27. The host vector system of claim 26, wherein the eukaryotic cell is a yeast, insect, plant, or mammalian cell. 28. The method of claim 18 under suitable conditions which permit the production of the polypeptide.
For the production of a polypeptide, which comprises growing the host vector system of, and recovering the polypeptide so produced. 29. (a) introducing the vector of claim 18 into a suitable host cell; (b) culturing the obtained cell to produce a polypeptide; (c) in step (b) Recovering the polypeptide produced; and (d) purifying the polypeptide so recovered: a method of obtaining the polypeptide in a purified form. 30. p-Hyde containing the amino acid sequence of mammalian p-Hyde
Family polypeptides. 31. The polypeptide according to claim 30, wherein the amino acid sequence is set forth in SEQ ID NO: 2, 4, 6, or 8. 32. A fusion protein or chimera comprising the polypeptide of claim 30. 33. An antibody that specifically binds to the polypeptide of claim 30. 34. The antibody according to claim 33, which is a monoclonal antibody or a polyclonal antibody. 35. A pharmaceutical composition comprising an amount of the polypeptide of claim 30 and a pharmaceutically effective carrier or diluent. 36. A transgenic non-human mammal comprising the isolated nucleic acid of claim 1. 37. (a) Obtaining a suitable nucleic acid sample from the subject; and (b) Whether the nucleic acid sample from step (a) is or is derived from a nucleic acid encoding a mutant p-Hyde. Determining whether the subject carries a mutation in the p-Hyde gene by determining whether the subject comprises p-Hyde
A method for determining whether to carry a mutation in a gene. 38. The nucleic acid sample in step (a) contains mRNA corresponding to a transcript of DNA encoding mutant p-Hyde, and the determination in step (b) is (i) mRNA and oligonucleotide. Contacting the mRNA with the oligonucleotide of claim 12 to form a complex under conditions that permit the binding of the complex; (ii) isolating the complex thus formed; and (iii) singly. 38. The method of claim 37, comprising determining whether the mRNA is or is derived from a nucleic acid encoding a mutant p-Hyde by identifying the mRNA in the separated complex. 39. The nucleic acid sample of step (a) under conditions wherein the determination of step (b) permits (i) digestion of the nucleic acid sample and the isolated nucleic acid into separate and distinct nucleic acid pieces. And contacting the isolated nucleic acid of claim 1 with a restriction enzyme; (ii) isolating a nucleic acid fragment; and (iii) a nucleic acid fragment derived from a nucleic acid sample derived from the isolated nucleic acid. 39. The method of claim 38, comprising: determining whether the nucleic acid sample is or is derived from a nucleic acid encoding a mutant p-Hyde by comparing the nucleic acid sample to 40. A method for screening a tumor sample from a human subject for somatic modification of the p-Hyde gene in the tumor, the p-Hyde gene from the tumor sample being derived from the tumor. The first sequence selected from the group consisting of p-Hyde RNA and p-Hyde cDNA prepared from mRNA derived from the tumor sample is used as the p-Hyde gene derived from the non-tumor sample of the subject, derived from the non-tumor sample. P-Hyde RNA, and a second sequence selected from the group consisting of p-Hyde cDNA prepared from mRNA derived from the non-tumor sample, in which case the p-Hyde gene from the tumor sample, p -Hyde RNA, or pH
of the yde cDNA sequence, p-Hyde gene, p-Hy from the non-tumor sample
A method comprising using a difference from the sequence of de RNA or p-Hyde cDNA as an index of somatic modification of the p-Hyde gene in the tumor sample. 41. A method for screening a tumor sample from a human subject for the presence of somatic alterations of the p-Hyde gene in the tumor, the method for analyzing differences between polypeptides, the method comprising: P-Hyde from the tumor sample
The polypeptide is compared to a p-Hyde polypeptide from the non-tumor sample from the subject, wherein the difference of the p-Hyde polypeptide from the tumor sample with the p-Hyde polypeptide from the non-tumor sample. Is used as an indicator of the presence of somatic modification of the p-Hyde gene in the tumor sample, the comparison comprising (i)
Detecting either the full length polypeptide or the truncated polypeptide within each sample, or (ii) specifically binding to either the modified p-Hyde polypeptide epitope or the wild-type p-Hyde polypeptide epitope. Antibody is contacted with the p-Hyde polypeptide from each sample and antibody binding is detected. 42. (a) contacting p-Hyde with a chemical compound under conditions that allow the binding of p-Hyde with the chemical compound; (b) specificity of the chemical compound and p-Hyde Detecting chemical binding; and (c) identifying a chemical compound capable of inducing susceptibility to cell death by determining whether the chemical compound inhibits p-Hyde: , A method for identifying chemical compounds capable of inducing susceptibility to cell death. 43. A step of obtaining cells and an adeno having a deletion in the E1 and E3 regions of the genome and having an insertion of a nucleic acid encoding p-Hyde under the control of the Rous sarcoma virus promoter in that region. A method for inhibiting the growth of cancer cells, which comprises the step of inhibiting the growth of cancer cells by contacting the cells of interest with a replication-defective adenovirus type 5 expression vector containing a viral genome. 44. 1) Obtaining a sample of prostate cells from a subject; 2) having a deletion in the E1 and E3 regions of the genome and inserting the p-Hyde cDNA under the control of the Rous sarcoma virus promoter. Contacting the cell with a replication-defective adenovirus type 5 expression vector containing an adenovirus genome within the region;
And 3) inhibiting the growth of cancer cells by introducing the cells into a subject: including a method of inhibiting the growth of cancer cells. 45. An amount of a nucleic acid encoding a p-Hyde protein, p
-Proliferation of cancer cells in a subject, which comprises suppressing the growth of the cancer cells in the subject by introducing into the cancer cells a nucleic acid that encodes a fragment of the Hyde protein or a nucleic acid that encodes a mutant p-Hyde protein How to suppress. 46. A therapeutically effective amount of a nucleic acid encoding a p-Hyde protein, a nucleic acid encoding a fragment of a p-Hyde protein, or a mutant p-Hyd.
Cancer cells in a subject, comprising suppressing the growth of cancer cells in the subject by administering to the subject a pharmaceutical composition comprising a nucleic acid encoding an e protein and a pharmaceutically acceptable carrier or diluent. To suppress the growth of. 47. A quantity of a nucleic acid encoding a p-Hyde protein, p
-A nucleic acid encoding a fragment of Hyde protein or a nucleic acid encoding a mutant p-Hyde protein is introduced into a cancer cell to induce susceptibility to apoptosis of the cancer cell in the subject. How to induce. 48. A therapeutically effective amount of a nucleic acid encoding a p-Hyde protein, a nucleic acid encoding a fragment of a p-Hyde protein, or a mutant p-Hyd.
to apoptosis of cancer cells in a subject, comprising inducing susceptibility to apoptosis by administering to the subject a pharmaceutical composition comprising a nucleic acid encoding an e protein and a pharmaceutically acceptable carrier or diluent. How to induce susceptibility. 49. A therapeutically effective amount of a nucleic acid encoding a p-Hyde protein, a nucleic acid encoding a fragment of a p-Hyde protein, or a mutant p-Hy.
Treating a subject with cancer, including treating a subject with cancer by administering to the subject a pharmaceutical composition comprising a nucleic acid encoding a de protein and a pharmaceutically acceptable carrier or diluent. how to. 50. 1) A therapeutically effective amount of a nucleic acid encoding a p-Hyde protein, p-Hy, in combination with radiation, chemotherapy, or a UV mimetic.
cancer by administering to a subject a pharmaceutical composition comprising a nucleic acid encoding a de protein fragment or a nucleic acid encoding a mutant p-Hyde protein and 2) a pharmaceutically acceptable carrier or diluent. A method of treating a subject having cancer, comprising treating a subject having :. 51. A therapeutically effective amount of 1) having a deletion in the E1 and E3 regions of the genome and having a full-length sense p-Hyde cDNA insertion within that region under the control of the Rous sarcoma virus promoter. Treating a subject with cancer by administering to the subject a pharmaceutical composition comprising a replication-defective adenovirus type 5 expression vector comprising the adenovirus genome and 2) a suitable carrier or diluent.
A method of treating a subject having cancer. 52. The cancer is selected from the group consisting of melanoma; lymphoma; leukemia; and carcinoma of the prostate, colorectal, pancreas, breast, brain, or stomach.
The method described in 1. 53. The pharmaceutical composition is administered parenterally, around cancer, transmucosal, transdermal, intramuscular, intravenous, intradermal, subcutaneous, intraperitoneal, intracerebroventricular, or intracranial. Fifty
The method described.