JP2002517246A - Gene expression regulatory protein - Google Patents

Abstract

  (57) [Summary] The present invention provides gene expression regulatory proteins (PRGEs) and polynucleotides that identify and encode them. The present invention also provides expression vectors and host cells, antibodies, agonists and antagonists. Further, the present invention provides a method for diagnosing, treating, or preventing a disease associated with PRGE expression.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Technical Field of the Invention The present invention, gene expression regulatory proteins (proteins regulating gene expressio
n) Nucleic acid and amino acid sequences, and use of these sequences for diagnosis, treatment, and prevention of diseases related to cell proliferation and cell differentiation, including cancer and immune disorders, developmental disorders, reproductive disorders, and neurological disorders. .

BACKGROUND OF THE INVENTION Multicellular organisms, structure and function are configured with significantly different variety of cell types.
Cell differentiation is determined by the phenotypic characteristics of the genes, and various cell types develop during development and express overlapping but distinct sets of genes. Positional and temporal regulation of gene expression is crucial for the regulation of cell proliferation and differentiation, apoptosis, cell senescence, and other cellular processes involved in the development of organisms. In addition, gene expression is regulated in response to extracellular signals that mediate intercellular communication and regulate the activity of various cell types. The cells express only those genes that perform the necessary functions, when necessary, through appropriate gene regulation.

[0003] Transcription regulatory proteins are essential for the regulation of transcription factor gene expression. Some of these proteins function as transcription factors that initiate or activate, repress, or terminate gene transcription. In general, transcription factors bind sequence-specifically to promoters and enhancers and upstream regulatory regions. However, some bind to regulatory elements within or downstream of the coding region of the gene. Transcription factors bind to specific regions of DNA, either alone or in complex with other accessory factors (Reviewed in L.
ewin, B. (1990) Genes IV , Oxford University Press, New York, NY, pp. 554
-570.).

[0004] The double helix structure and repetitive sequences of DNA form morphological and chemical features that transcription factors can recognize. These features include regular repeated extension of the sequence that induces individual folding in the helix, groups of hydrogen bond donors and acceptors, and hydrophobic patches.
(hydrophobic patch), major and minor grooves. Transcription factors usually recognize specific DNA sequence motifs of about 20 nucleotides or less. Regulation of genes may require a number of close transcription factor binding motifs.

[0005] Many transcription factors contain a DNA-binding structural motif that contains either an alpha helix or a beta sheet that binds to the major groove of DNA. Structural motifs with distinct features include Helix Stern Helix and Zn finger,
Leucine Zipper and Helix Loop Helix. Proteins containing these motifs can act alone as monomers or form homodimers or heterodimers to interact with DNA.

[0006] The helix-turn-helix motif consists of two α-helices connected by a short chain of amino acids at a fixed angle. One of them couples to the main groove. An example of a helix-turn-helix motif is the homeobox motif in homeodomain proteins. These proteins are important in identifying the anterior-posterior axis during development and are conserved in all animals. The Drosophila antennapedia and ultraviolax proteins are the original homeodomain proteins (Pabo, CO and RT Sauer (1992) Ann. Rev.
Biochem. 61: 1053-1095).

[0007] The Zn finger that binds to zinc ions typically contains a tandem repeat of about 30 amino acids consisting of periodically spaced cysteine and histidine residues. Examples of this sequence type, called C2H2 and C3HC4, have been described (Lewin, supra).
. Each Zn finger protein contains an α-helix and an antiparallel β-sheet in which adjacent structures are maintained by zinc ions. Arginine before the alpha helix and the second, third, and sixth helix residues make contact with the DNA. Z
Variants of the n-finger motif include cysteine-rich motifs that are poorly defined to bind zinc ions or other metal ions. These motifs do not contain histidine residues and are usually not repeated.

[0008] The bromodomain signature is an additional conserved region of about 70 amino acids that is found in many transcriptional regulatory proteins (ExP
ASy PROSITE document PS00633; Haynes, SR et al. (1992) Nucleic Acids R
es. 20: 2603). The exact function of this domain is not clear, but is found in the DNA-binding region of the thyroid hormone receptor co-active protein. Thyroid hormone receptors are members of the steroid / thyroid receptor family that regulate the expression of many target genes by binding to thyroid hormone response elements (Tsuyoshi, M. et al. (1997)
J. Biol. Chem. 272: 29834-29841). The bromodomain signature is the G1 of the cell cycle.
It is also found in TFIID, a eukaryotic transcription initiation factor that is a protein essential for phase progression.

[0009] The leucine zipper motif is composed of an extension of leucine-rich amino acids that can form an amphipathic alpha helix. This structure is the basis for dimerization of the two leucine zipper proteins. The region adjacent to the leucine zipper is usually basic and is located at an optimal position for binding to the major groove as the protein dimerizes.

[0010] The helix-loop-helix (HLH) motif consists of a short α-helix connected to a long α-helix in a loop. This loop is flexible, allowing the two helices to fold over each other and bind to DNA. The transcription factor Myc is the prototype H
Contains the LH motif.

[0011] Many transcription factors include characteristic D including, but not limited to, those described above.
NA binding motifs are included. The characteristics of the various motifs and the novel motifs described above are shown (Faisst, S. and S. Meyer (1992) Nucl. Acids Res. 20: 3-26).
.

[0012] In the chromatin-related protein nucleus, DNA folds into chromatin, and this compact structure restricts the access of transcription factors to DNA and plays an important role in gene regulation (Lewin, supra, pp. 409-410). The precise structure of chromatin is determined and influenced by chromatin-related proteins such as histones and chromodomain proteins, which are high mobility proteins (HMGs). Histones include H1 and H2A, H2B,
There are five classes, H3 and H4, all of which are highly basic low molecular weight proteins. The nucleosome, which is a unit structure that forms the basic structure of chromatin, is a DNA of 200 base pairs to which each of H2A and H2B, H3, and H4 is bound. H1 is associated with adjacent nucleosomes. HMG proteins are low molecular weight non-histone proteins that can play a role in unwinding DNA and stabilizing single-stranded DNA. Chromodomain proteins play an important role in the formation of transcriptionally inactive and highly dense heterochromatin.

Many of the regulation of gene expression in eukaryotic cells of RNA-related proteins occurs at a post-transcriptional stage. Messenger RNA generated by primary transcription of a gene encoding a protein in the cell nucleus
(MRNA) is processed and then transported to the cytoplasm where the protein synthesizer is located. RNA-related proteins are a group of proteins that are involved in mRNA processing and splicing, editing, transport, localization, translation, stabilization, and post-translational regulation. This protein includes RNA helicases and splicing factors, nucleases, and translation regulatory proteins. In addition, the nucleolus is a highly organized fraction within the nucleus that contains, among other things, the protein machinery responsible for the transcription and processing of ribosomal RNA. The RNA binding activity of these proteins is mediated by a series of RNA binding motifs within them. These domains include the RNP and arginine-rich motifs, the RGG box, and the KH motif (Reviewed in Burd, CG and Dreyfuss, G. (1994) Science 265: 6
15 -621.). Of these motifs, the RNP motif is the most abundant and most characteristic. The RNP motif is composed of 90 to 100 amino acids forming an RNA binding domain. This domain is present one or more times in precursor mRNA and in proteins that bind to mRNA, precursor ribosomal RNA, ribosomal RNA, and small nuclear RNA. The RNP motif is composed of two short sequences (RNP-1 and RNP-2), and many other conserved amino acids, mostly hydrophobic, are scattered throughout the motif (Burd, supra, ExPASy PROSITE Literature PDOC0030).

Many of the diseases associated with gene regulation and abnormal human tumor diseases are due to inappropriate gene regulation. Malignant cell growth is
It is often due to either overexpression of the tumor promoting gene or insufficient expression of the tumor suppressor gene (Cleary, ML (1992) Cancer Surv. 15: 89-104). Chromosomal translocations can generate chimeric loci that fuse sequences encoding one gene with regulatory regions of a second unrelated gene. Such translocations are likely to cause inappropriate gene transcription. The Wilms tumor suppressor gene product, WT1, is a DNA consisting of a transcriptionally regulatable proline-glutamine-rich region and four Zn fingers.
It is a protein containing a binding domain (ExPASy PROSITE document PR00049). Deletion of the WT1 gene or point mutations that disrupt the DNA-binding activity of proteins are associated with the development of childhood nephroblastomas and viral tumors, Denys-Drash syndrome (Rauscher, FJ (
1993) FASEB J. 7: 896-903).

[0015] Some glutamine-rich proteins are associated with various neurological disorders, including spinocerebellar ataxia and bipolar affective illness, schizophrenia, autism (Margolis, RL et al.).
(1997) Human Genetics 100: 114-122). These proteins have regions containing as many as 15 or more consecutive glutamine residues, and can function as transcription factors that can be involved in the regulation of neural development and neurogenesis.

The immune system responds to infection and trauma by activating a cascade of events that regulates the progressive selection and amplification of cell defense mechanisms, recruitment. This process involves a complex program that balances gene activation and repression. However,
Over-response of the immune system as a result of inappropriate or poor regulation of gene expression can lead to major tissue and organ damage. Such injuries have been described in the literature on arthritis and allergens, heart attacks, stroke, and the immune response associated with infection (Har
rison's Principles of Internal Medicine, 13 / e, McGraw Hill, Inc. and Tet
on Data Systems Software, 1996). Specifically, a Zn finger protein called (50 kilodalton stimulated trans-acting factor) Staf50 is a transcription factor,
Induced in various cell lines by interferons I and II. Staf50 is thought to mediate the antiviral activity of interferons by down-regulating viral transcription induced by the human immunodeficiency type I virus terminal repeat promoter region in transfected cells (Tissot, C. (1995) J. Bi
ol. Chem. 270: 14891-14898).

Furthermore, the development of multicellular organisms is based on the induction and regulation of cell differentiation at appropriate stages of development. At the heart of this process is differential gene expression, which gives cells and tissues throughout the body individual independence. Dysregulation of gene expression during development can lead to developmental disorders.

The discovery of novel gene expression regulatory proteins and polynucleotides encoding them has led to the diagnosis, treatment, and prevention of diseases related to cell proliferation and cell differentiation, including cancer and immune disorders, developmental disorders, reproductive disorders, and neurological disorders. The need in the art can be addressed by providing new compositions useful for:

[0019] SUMMARY OF THE INVENTION The present invention, each of the individual "PRGE-1", "PRGE-2 '- substantially a" PRGE-31 "and referred collectively referred to as" PRGE "gene expression regulatory proteins Provide a purified polypeptide. In one embodiment of the present invention, there is provided a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-31 and fragments thereof.

The invention further provides a substantially purified variant having at least 90% amino acid identity with at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1-31 and fragments thereof. provide. The present invention also provides an isolated and substantially purified polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-31 and fragments thereof. The present invention also relates to SEQ ID
NO: An isolated and purified polynucleotide variant having at least 90% or greater polynucleotide sequence identity to a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of 1-31 and fragments thereof. I will provide a.

In addition, the present invention provides an isolation that hybridizes under stringent conditions to a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-31 and fragments thereof. To provide a purified and purified polynucleotide.
The invention also relates to an isolated and purified polynucleotide comprising a sequence complementary to a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-31 and fragments thereof. I will provide a.

[0022] The present invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 32-62 and fragments thereof. The present invention also relates to isolated and purified polynucleotide variant sequences having 90% or more polynucleotide sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 32-62 and fragments thereof. provide. Further, the present invention provides
Provided is an isolated and purified polynucleotide having a sequence complementary to a polynucleotide comprising a polynucleotide sequence selected from the group consisting of ID NOs: 32-62 and fragments thereof.

The present invention also provides a method for detecting a polynucleotide in a sample containing nucleic acids, comprising: (a) hybridizing a polynucleotide sequence with at least one sample polynucleotide to form a hybridization complex. The present invention provides a detection method comprising the step of forming and the step of (b) detecting the hybridization complex, wherein the presence of the hybridization complex is correlated with the presence of the polynucleotide in the sample. One embodiment of the invention involves amplifying the polynucleotide prior to hybridization.

The present invention further provides an expression vector comprising at least one fragment of a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-31 and fragments thereof. In another embodiment, the expression vector is contained within a host cell. The invention also provides a method of producing a polypeptide, comprising: (a) at least one of the polynucleotides under conditions suitable for expression of the polypeptide. There is provided a production method comprising a step of culturing a host cell containing an expression vector having a fragment, and (b) a step of recovering the polypeptide from a medium of the host cell.

The present invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-31 and fragments thereof, together with a suitable pharmaceutical carrier. provide.

The present invention further provides a purified antibody that binds to a polypeptide selected from the group consisting of SEQ ID NOs: 1-31 and fragments thereof. The present invention also provides purified agonists and antagonists of the polypeptide.

The present invention also has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-31 and fragments thereof in a patient in need of treatment or prevention of a disease associated with reduced expression or activity of PRGE. Provided is a method for treating or preventing a disease associated with reduced expression or activity of PRGE, comprising administering an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide together with a suitable pharmaceutical carrier.

The present invention also has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-31 and fragments thereof in a patient in need of treatment or prevention of a disease associated with increased expression or activity of PRGE. Provided are methods for treating or preventing a disease associated with increased expression or activity of PRGE, comprising administering an effective amount of an antagonist of the polypeptide.

Before describing the protein and nucleic acid sequences and methods of the present invention in describing the present invention, it is to be understood that this invention is not limited to the particular devices, materials, and methods disclosed herein, which embodiments may be modified. I want to be understood. Further, the terms used herein are used only for the purpose of describing a specific embodiment, and are limited only by the claims described below.
It should also be understood that they are not intended to limit the scope of the present invention.

In the present specification and claims, “a”, “the” and the like refer to a singular form.
Note that the plural includes the plural unless explicitly stated in the context. Thus, for example, "a host cell" includes a plurality of host cells, and the "antibody" includes a plurality of antibodies, including equivalents well known to those skilled in the art.

All technical and scientific terms used herein are, unless otherwise defined,
This has the same meaning as commonly interpreted by a general engineer in the technical field to which the present invention belongs.
Although all devices and materials and methods similar or equivalent to those described herein can be used in the practice and testing of the present invention, the preferred devices, materials and methods are described here.
All references mentioned herein are cited to describe and disclose the cell lines, protocols, reagents, and vectors described in the literature that may be used in connection with the present invention. Citing a conventional invention does not, however, be construed as impairing the novelty of the present invention.

Definitions As used herein, the term "PRGE" refers to any species, including natural, synthetic, semi-synthetic or recombinant, from mammals including cows, sheep, pigs, mice, horses, and preferably humans. The substantially purified amino acid sequence of PRGE.

As used herein, “agonist” refers to a molecule that, when bound to PRGE, increases the effect of PRGE or prolongs its duration. The agonist may include proteins, nucleic acids, carbohydrates, and any other molecules that bind to and modulate the effect of PRGE.

As used herein, “allelic variant” is another form of the gene encoding PRGE. Allelic variant sequences result from at least one mutation in the nucleic acid sequence, resulting in a variant mRNA or variant polypeptide, whose structure or function may or may not change. All natural or recombinant genes may have no allele, one or many alleles. In general, mutations that result in allelic variants are due to natural deletions, additions, or substitutions of nucleotides. Each of these mutations may occur alone or concurrently with other mutations, and may occur one or more times within a given sequence.

As used herein, the term “mutated” nucleic acid sequence encoding PRGE refers to the same polynucleotide or PRGE even when various nucleotide deletions, insertions, or substitutions occur.
A polypeptide having at least one of the functional properties of PRGE. This definition includes improper or unexpected hybridization of the polynucleotide sequence encoding PRGE to an allelic variant at a position other than the normal chromosomal locus, and
Includes polymorphisms that are easily detectable or difficult to detect using specific oligonucleotide probes of the polynucleotide encoding PRGE. The encoded protein can also be mutated and contain deletions, insertions, or substitutions of amino acid residues that produce a silent change and are functionally equivalent to PRGE. Intentional amino acid substitutions are similar to the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of residues, as long as the activity of PRGE is retained biologically or immunologically. It can be done based on For example, a negatively charged amino acid can include aspartic acid and glutamic acid, a positively charged amino acid can include lysine and arginine, and an amino acid with a near hydrophilic value and an uncharged polar head group. Is leucine, isoleucine,
It may include valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine and tyrosine.

As used herein, “amino acid” or “amino acid sequence” refers to an oligopeptide, peptide, polypeptide, or protein sequence and fragments thereof, and is a natural molecule or a synthesized molecule. PRGE fragments, "fragments" and "immunogenic fragments", "antigenic fragments" are preferably about 5 to about 15 amino acids in length, most preferably 14 amino acids in length, Retains some biological or immunological activity of PRGE. As used herein, "an amino acid sequence is that of a naturally occurring protein molecule, but the amino acid sequence and similar terms are not limited to the complete, intact amino acid sequence associated with the listed protein molecule.

As used herein, the term “amplification” refers to generating additional copies of a nucleic acid sequence. Polymerase chain reaction (PCR) generally well known in the art.
Performed using techniques (eg, Dieffenbach, CW and GS Dveksler (1995) P
CR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, NY,
pp. 1-5.).

As used herein, an “antagonist” is a molecule that, when bound to PRGE, reduces the extent of the effect of the biological or immunological activity of PRGE or shortens its duration. Antagonists are proteins, nucleic acids, carbohydrates, antibodies or other molecules that reduce the effect of PRGE.

As used herein, “antibody” refers to Fab and F (ab ′) ₂ , and fragments thereof, Fv
An intact molecule, such as a fragment, that can bind to an antigenic determinant. Antibodies that bind to a PRGE polypeptide can be prepared using intact molecules or fragments thereof that contain the small peptide of interest to immunize the antibody. The polypeptide or oligopeptide used to immunize an animal (eg, a mouse, rat, or rabbit) is
It can be derived from the translation of NA or synthesized chemically and can be coupled to a carrier protein if necessary. Commonly used carriers that are chemically coupled to the peptide include bovine serum albumin, thyroglobin, and keyhole limpet haemonian (KLH). The animal is then immunized with the conjugated peptide.

As used herein, an “antigenic determinant” is a fragment of a molecule (ie, an epitope) that contacts a particular antibody. When a protein or a fragment of a protein is used to immunize a host animal, various regions of the protein may contain antibodies that specifically bind to antigenic determinants (defined regions or three-dimensional structures on the protein). Can be induced. Antigenic determinants can compete with the intact antigen (ie, the immunogen used to elicit the immune response) to bind the antibody.

As used herein, “antisense” is any composition that includes a nucleic acid sequence that is complementary to the sense strand of a particular nucleic acid sequence. Antisense molecules can be created by any method, including synthesis and transcription. Once introduced into a cell, the complementary nucleotide combines with the native sequence created by the cell to form a duplex and inhibits transcription and translation. The expression "minus (-)" can be said to be an antisense strand, and the expression "plus (+)" can be said to be a sense strand.

As used herein, “biological activity” refers to a protein having a structural, regulatory, or biochemical function of a naturally occurring molecule. Similarly, “immunologically active” refers to natural or recombinant PRGE, synthetic PRGE, or any of these oligopeptides, which elicits a specific immune response in a suitable animal or cell to induce a specific antibody response. The ability to combine with

As used herein, “complementary” or “complementary” refers to the spontaneous binding of polynucleotides by base pair formation under acceptable salt and acceptable temperature conditions. For example, it binds to the sequence "TCA" which is complementary to the sequence "AGT".
The complementarity between two single-stranded molecules is only partially bound by some nucleic acids,
Alternatively, there may be cases where perfect complementarity exists between the single strands, resulting in perfect complementarity.
The degree of complementarity between nucleic acid strands greatly affects the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions that depend on binding between nucleic acid strands, as well as in the design or use of peptide nucleic acid (PNA) molecules.

As used herein, the term “composition containing a predetermined polynucleotide sequence” or “composition containing a predetermined amino acid sequence” refers to any composition containing a predetermined nucleotide sequence or amino acid sequence in a broad sense. It is. The composition may include a dry formulation or an aqueous solution, a sterile composition. Compositions comprising a polynucleotide sequence encoding PRGE or a fragment of PRGE can be used as a hybridization probe. This probe can be stored in a lyophilized state, and can be bound to a stabilizer such as a carbohydrate. In hybridization, the probe can be developed into an aqueous solution containing a salt (eg, NaCl) and a surfactant (eg, SDS), and other substances (eg, Denhardt's solution, dried milk, salmon sperm DNA, etc.).

As used herein, the term “consensus sequence” refers to a nucleic acid sequence rearranged to separate unnecessary bases, and is a XL-PCR ^™ (Perkinn Elmer, Norwalk, CT)
A nucleic acid sequence that has been extended in the 5 ′ and / or 3 ′ direction and rearranged using
This refers to a nucleic acid sequence constructed from two or more overlapping sequences of Insight Clones using a computer program for constructing fragments such as the GELVIEW Fragment Assembly System (GCG, Madison, WI). Some are built into consensus sequences by both extension and construction.

As used herein, “correlated with the expression of a polynucleotide” means that the detection of a nucleic acid that is the same as or related to the nucleic acid sequence encoding PRGE by Northern analysis indicates the presence of the nucleic acid encoding PRGE in the sample. Indicates that the expression is correlated with the expression of a transcript from the polynucleotide encoding PRGE.

As used herein, a “deletion” is a change in the amino acid or nucleic acid sequence that lacks one or more amino acid residues or nucleic acid residues.

[0048] As used herein, "derivative" refers to a chemical modification of a polypeptide sequence or a polynucleotide sequence. Chemical modifications of a polynucleotide sequence include, for example,
There is replacement of hydrogen by an alkyl group, an acyl group, or an amino group. A derivative polynucleotide encodes a polypeptide that retains at least one biological or immunological function of a natural molecule (unmodified molecule). A derivative polypeptide is one that has been modified by glycosylation, polyethyleneglycolation, or any similar process that retains at least one biological or immunological function of the original polypeptide.

As used herein, “similarity” refers to the degree of complementarity. This can be partial similarity and complete similarity. This “identity” can be said to be “similarity”. Partially complementary sequences that at least partially prevent the same sequence from hybridizing to the target nucleic acid are referred to as "substantially similar." The suppression of hybridization between the completely complementary sequence and the target sequence is examined using a hybridization assay (Southern or Northern blotting, solution hybridization, etc.) under reduced stringency conditions. Substantially similar sequences or hybridization probes compete under reduced stringency conditions for binding of the completely complementary sequence to the target sequence. This means that under reduced stringency conditions, the binding of the two sequences to each other must interact specifically (ie, selectively), and under reduced stringency conditions, This does not mean that specific binding is tolerated. Using a second target sequence that is not even partially complementary (eg, less than 30% similarity or identity), it can be tested for the absence of non-specific binding. In the absence of non-specific binding, substantially similar sequences or probes will not hybridize to the second non-complementary target sequence.

As used herein, “percent identity” or “% identity” refers to the percentage of sequence similarity found in a comparison of two or more amino acid sequences or nucleic acid sequences. This percentage identity is determined, for example, by the MEGALIGN program (DNASTAR, Inc., Madison Wl).
It can be determined using an electronic device. The MEGALIGN program can be implemented in a variety of methods, such as the cluster method (eg, Higgins, DG and PM Sharp (1988) Gen.
e73: 237-244.), it is possible to create an alignment between two or more sequences. A cluster algorithm measures the distance between all sets and classifies the sequence into each cluster. These clusters are aligned by sets and then divided into groups. The percentage similarity between the sequences of two amino acids, for example, the percentage similarity between sequence A and sequence B, is calculated by calculating the total number of matching residues in sequence A and sequence B from the length of sequence A to gap residues in sequence A. It is obtained by dividing the number by subtracting the number of gap residues in sequence B and multiplying by 100. Low similarity or dissimilarity gaps between two amino acid sequences are not included in the determination of percentage similarity. Percent identity between nucleic acid sequences can also be counted or calculated by another method known in the art, such as the cluster method or the Jotun Hein method (
See, for example, Hein. J. (1990) Methods Enzymol. 183: 626-645. The identity between sequences can also be determined by other methods well-known in the art, for example, by changing the conditions for hybridization.

The human artificial chromosome (HAC) is a linear microchromosome that can contain a DNA sequence of 6 Kb to 10 Mb in size and contains all elements necessary for the separation and maintenance of stable dividing chromosomes (Harrington , JJ et al. (1997) Nat Genet. 15: 345-355)
.

As used herein, a “humanized antibody” is an antibody molecule in which the amino acid sequence of the non-antigen binding region has been mutated so as to resemble a human antibody while retaining the original binding ability.

As used herein, “hybridization” refers to any process in which a single strand of a nucleic acid forms a base pair with a complementary single strand and binds.

As used herein, the term “hybridization complex” refers to a complex formed between two nucleic acid sequences by forming hydrogen bonds between complementary base pairs. Hybridization complexes may be formed in solution (eg, C ₀ t or R ₀ t analysis) or may comprise one nucleic acid sequence in solution and a solid support (eg, paper, membrane, filter, chip, pin). Or another nucleic acid sequence immobilized on a glass slide, or any suitable substrate that immobilizes the cells and their nucleic acids.

As used herein, “insertion” or “addition” refers to a change in an amino acid sequence or a nucleic acid sequence in which one or more amino acid residues or nucleotides are added to a naturally occurring molecule sequence. It is.

As used herein, the term “immune response” refers to a symptom associated with inflammatory diseases and trauma, immune disorders, infectious diseases, genetic diseases, and the like. These conditions can be characterized by the expression of various factors that affect the cell line and the systemic defense system, such as cytokines and chemokines, and other signaling molecules.

As used herein, “microarray” refers to an array of various polynucleotides arranged on a substrate.

“Elements” or “array elements” in the description of the microarray herein refer to hybridizable polynucleotides arranged on the surface of a substrate.

As used herein, “modulation” refers to a change in PRGE activity. Examples of modulation include changes in the protein activity or binding properties of PRGE, or other biological, functional or immunological properties.

As used herein, “nucleic acid” or “nucleic acid sequence” refers to nucleotides, oligonucleotides, polynucleotides, or fragments thereof. In addition, a single-stranded or double-stranded sense or antisense strand of genomic or synthetic origin
NA or RNA, peptide nucleic acid (PNA), any DNA-like substance, RNA
Also refers to similar substances. As used herein, "fragment (or fragment)" refers to a region of a unique polynucleotide sequence that specifically identifies SEQ ID NO: 32-62 (e.g., different from any other sequence in the same genome). Nucleic acid sequence. For example,
SEQ ID NO: 32-62 is useful for hybridization and amplification techniques, and is also useful for testing similarities that distinguish SEQ ID NO: 32-62 from related polynucleotide sequences. Fragments of SEQ ID NOs: 32-62 are at least 15-20 nucleotides in length. The exact length of SEQ ID NO: 32-62 corresponding to this fragment and SEQ ID N
The region of O: 32-62 can be determined routinely using one of the techniques common in the art based on the intended use of the fragment. Also, the fragment, when translated, may form a polypeptide that retains some functional characteristics, such as antigenicity, of a full-length polypeptide or structural domain characteristics, such as an ATP binding site.

As used herein, “functionally related” or “operably linked” refers to a functionally related nucleic acid sequence. When the promoter controls the transcription of the encoded polypeptide, the promoter is operably associated with or operably linked to the coding sequence. Although functionally related or operably linked nucleic acid sequences can be in close proximity and in the same reading frame, certain genetic elements, such as repressor genes, are in close proximity to the polypeptide-encoding sequence. Unbound, but remains bound to an operator sequence that regulates expression of the polypeptide.

As used herein, “oligonucleotide” refers to a nucleic acid sequence that can be used for PCR amplification, hybridization, or microarray, and has a length of at least 6 to 60 nucleotides. Preferably it is about 15 to 30 nucleotides, more preferably about 20 to 25 nucleotides. As used herein, an oligonucleotide is substantially the same as "amplimer" and "primer", "oligomer", which are defined to be the same in the art.

As used herein, “peptide nucleic acid” (PNA) refers to an antisense molecule or an antigene comprising an oligonucleotide of about 5 or more nucleotides in length bound to a peptide backbone of amino acid residues terminating in lysine. It is an agent. This terminal lysine makes the composition soluble. PNAs can preferentially bind to complementary single-stranded DNA or RNA, stop transcript elongation, and become polyethylene glycolated to extend their lifespan in cells. (For example, Nielsen, PE et al. (1993) An
ticancer Drug Des. 8: 53-63).

As used herein, “sample” is used in its broadest sense. PR
Biological samples suspected of containing the nucleic acid encoding GE or fragments thereof, or PRGE itself, include body fluids, extracts from cells and chromosomes and organelles isolated from cells, membranes, and cells. Genomic DNA, RN fixed in a solution or on a solid support
A, cDNA, tissue or tissue print, etc. may also be included.

As used herein, “specific binding” or “specifically binds” refers to an interaction between a protein or peptide and an agonist, antibody, or antagonist. This interaction is dependent on the presence of a particular structure of the protein, such as an antigenic determinant or epitope, recognized by the binding molecule. For example, if the antibody is specific for epitope "A", then in a reaction involving unbound labeled "A" and the antibody, the presence of the polypeptide comprising epitope A or the absence of unbound polypeptide The presence of the label "A" reduces the amount of label A that binds to the antibody.

As used herein, “strict conditions” are conditions under which hybridization between a polynucleotide and a polynucleotide described in the claims is possible. The exact conditions are determined by the salt concentration and the concentration of the organic solvent (eg, formamide), the temperature, and other conditions well known in the art. Specifically, stringency can be increased by lowering the salt concentration, increasing the formamide concentration, or increasing the hybridization temperature.

As used herein, the term “substantially purified” refers to a nucleic acid sequence or amino acid sequence that has been isolated or separated after being removed from its natural environment, and is a naturally-associated component. Is at least about 60% or more removed, preferably about 75% or more removed, and most preferably about 90% or more removed.

As used herein, “substitution” refers to replacing one or more amino acids or nucleotides with another amino acid or nucleotide, respectively.

As used herein, “substrate” refers to any suitable solid or membrane, including membranes, filters, chips, slides, wafers, fibers, magnetic or non-magnetic beads, gels, tubes, plates, polymers, fine particles, capillaries. It is a semi-solid support. The substrate has various surface morphologies, such as walls and grooves, pins, channels, holes, etc., to which polynucleotides and polypeptides bind.

As used herein, “transformation” refers to the process by which foreign DNA enters and changes a recipient cell. Transformation can be accomplished by various methods well known in the art.
It can occur under natural or artificial conditions and can be performed by any known method of inserting foreign nucleic acid sequences into prokaryotic or eukaryotic host cells. The method of transformation is selected according to the type of host cell to be transformed. The methods include, but are not limited to, viral infection, electroporation, lipofection, and microparticle irradiation. A "transformed" cell includes a stably transformed cell that is capable of replicating autonomously replicating the introduced DNA as a plasmid or as part of the host chromosome. Furthermore, cells that express the introduced DNA or RNA temporarily for a limited time are also included.

As used herein, a “variant” is an amino acid sequence in which one or more amino acids have been mutated. The variants include "conservative" mutations, wherein the substituted amino acid has a similar structure or similar chemical properties, such as, for example, a substitution of leucine for isoleucine. Although rare, some mutants replace glycine with tryptophan.
Also included are "non-conservative" mutations. Similar small mutations also include amino acid deletions, insertions, or both. For example, LASERGENE ^™ software well known in the art could be used to determine which amino acid residues to substitute, insert or delete without impairing biological or immunological activity.

A “variant sequence” as used in the context of a polynucleotide sequence may include a polynucleotide sequence associated with PRGE. This definition also applies, for example, to "allelic", "splice", "species", "polymorphic" variant sequences. A splice variant may be very identical to the reference molecule, but will have more or less polynucleotides due to alternate splicing of exons during mRNA processing. The corresponding polypeptide may have additional or fewer functional domains. Species variant sequences are polynucleotide sequences that vary from species to species. The resulting polypeptides have high amino acid identity with each other.
Polymorphic variant sequences are variations in the polynucleotide sequence of a particular gene between a given species. Polymorphic variant sequences can also include "single nucleotide polymorphisms" (SNPs) that differ by one base in the polynucleotide sequence. The presence of a SNP may, for example, represent a population, condition, or characteristic of a condition.

[0073] INVENTION The present invention relates to novel human gene expression regulatory proteins (PRGE), and based on the discovery of polynucleotides encoding PRGE, cancer and immune disorders, diseases associated with cell proliferation and cell differentiation comprising the failure occurs, It relates to the use of these compositions in the diagnosis, treatment and prevention of reproductive disorders, neurological disorders.

Table 1 lists the Incyte clones used to obtain the full-length nucleotide sequence encoding PRGE. Columns 1 and 2 show amino acid and nucleic acid SEQ ID NOs. Column 3 shows the clone ID of the Incyte clone from which the nucleic acid encoding each PRGE was identified, and column 4 shows the cDNA library from which these clones were isolated. Column 5 shows the Incyte clones, their corresponding cDNA libraries, and shotgun sequences. The region of the full-length nucleotide sequence of each PRGE corresponding to the clone and shotgun sequences is listed in column 5. The clone and shotgun sequences are part of the consensus nucleotide sequence for each PRGE. These clones and fragments are useful as fragments in hybridization techniques.

Each column of Table 2 shows various properties of the polypeptide of the invention. Column 1 is the amino acid sequence number (SEQ ID NO), column 2 is the number of amino acid residues of each polypeptide, column 3 is the potential phosphorylation site, column 4 is the potential glycosylation site, column 5 is the signature sequence and motif Column 6 shows the homologous sequence of each protein, and Column 7 shows the analysis method used to identify each protein by sequence homology and protein motif.

Each column of Table 3 shows the tissue specificity of the nucleotide sequence encoding PRGE and the diseases, abnormalities and symptoms associated therewith. The first column of Table 3 shows the nucleotide sequence ID number. The second column shows the taxonomy of tissues expressing PRGE and is part of the taxonomy of all tissues. The third column shows diseases, disorders and conditions associated with tissues expressing PRGE. The fourth column shows the vector used for subcloning the cDNA library.

The fragments of the nucleotide sequence encoding PRGE shown below are useful for hybridization or amplification to identify SEQ ID NOs: 56-62 and to discriminate between SEQ ID NOs: 56-62 and related polynucleotide sequences. is there. Useful fragments include a fragment from about nucleotides 1675 to 1719 of SEQ ID NO: 56 and a fragment from about nucleotides 379 to 4 of SEQ ID NO: 57.
A fragment from SEQ ID NO: 58 to approximately nucleotides 596 to 640;
A fragment of about nucleotides 219 to 263 of Q ID NO: 59, a fragment of about nucleotides 732 to 776 of SEQ ID NO: 60, and a fragment of about nucleotides 197 to 2 of SEQ ID NO: 61.
Fragments up to 44 and a fragment from about nucleotides 217 to 261 of SEQ ID NO: 62.

The present invention also includes variants of PRGE. The variant of PRGE preferably has about 80% or more sequence identity, more preferably about 90% or more sequence identity, and most preferably about 95% or more sequence identity with the amino acid sequence of PRGE. At least one of the functional characteristics or the structural characteristics.

The present invention also includes a polynucleotide encoding PRGE. In certain embodiments, the invention includes a polynucleotide sequence having a sequence selected from the group consisting of SEQ ID NOs: 32-62 encoding PRGE.

The present invention also includes mutant sequences of the polynucleotide sequence encoding PRGE. In particular, such a mutant sequence of the polynucleotide sequence preferably has a sequence identity of 80% or more, more preferably 90% or more, with the polynucleotide sequence encoding PRGE.
It has the above sequence identity, most preferably 95% or more sequence identity. Also, in certain embodiments of the invention, at least about 80% sequence identity, preferably at least about 90% sequence identity, and most preferably with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 32-62. Mutant sequences of polynucleotide sequences comprising sequences selected from the group consisting of SEQ ID NOs: 32-62 having about 95% or greater sequence identity. All of the above-described polynucleotide variant sequences encode an amino acid sequence having at least one of the functional or structural features of PRGE.

The various polynucleotide sequences encoding PRGE that may be created by the degeneracy of the genetic code include those that have minimal similarity to the polynucleotide sequences of any known, naturally occurring genes. , One skilled in the art will understand. Thus, the present invention may include all possible polynucleotide sequence variations that may be made by selection of combinations based on possible codon choices. These combinations are made based on the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring PRGE, and all variations are considered to be explicitly disclosed.

It is desirable that the nucleotide sequence encoding PRGE and its mutant sequences be capable of hybridizing to the nucleotides of the naturally occurring PRGE under suitably selected stringent conditions, but include non-naturally occurring codons. It may be advantageous to create nucleotide sequences encoding PRGE or derivatives thereof having substantially different codon usage, such as. Codons can be selected based on the frequency with which particular codons are utilized by the host to increase the rate at which expression of the peptide occurs in a particular eukaryotic or prokaryotic host. Another reason for substantially altering the nucleotide sequence encoding PRGE and its derivatives without altering the encoded amino acid sequence is that RNA with favorable properties, such as a longer half-life, than transcripts made from naturally occurring sequences.
To make a transcript.

The present invention also includes the production of DNA sequences encoding PRGE and its derivatives, or fragments thereof, entirely by synthetic chemistry. After construction, the synthetic sequence can be inserted into any of a variety of available expression vectors and cell lines using reagents well known in the art. In addition, synthetic chemistry can be used to introduce mutations in the sequence encoding PRGE or any fragment thereof.

The invention further includes polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences under various stringency conditions. For more information,
Polynucleotide sequences capable of hybridizing to the described SEQ ID NOs: 32-62 or fragments thereof are included. (E.g., Wahl, GM and SL Berger (1987) Methods Enzy
mol. 152: 399-407; and Kimmel.AR (1987) Methods Enzymol. 152: 507-511.
See). For example, the exact salt concentration is usually less than about 750 mM sodium chloride and less than about 75 mM trisodium citrate, preferably less than about 500 mM sodium chloride and less than about 50 mM trisodium citrate, most preferably. Is less than about 250 mM sodium chloride and less than about 25 mM trisodium citrate. For example, the absence of an organic solvent such as formamide results in less stringent hybridization, while the use of about 35% or more formamide, more preferably 50% or more formamide, results in more stringent hybridization. Strict temperature conditions are usually about 30 ° C. or higher, and more preferably about 37 ° C.
° C or higher, most preferably about 42 ° C or higher. Other parameters that can be changed include hybridization time, sodium dodecyl sulfate (SDS)
And the presence or absence of carrier DNA, and are well known to those skilled in the art. By combining various necessary conditions, the degree of strictness can be changed. In a preferred embodiment, the hybridization is performed at a temperature of
Perform with 50 mM sodium chloride and 75 mM trisodium citrate, 1% SDS. In a more preferred embodiment, at a temperature of 37 ° C., 500 mM sodium chloride and 50 mM trisodium citrate, 1% SDS, 35% formamide, 10%
Performed with 0 μg / ml denatured salmon sperm DNA (ssDNA). In the most preferred embodiment, at a temperature of 42 ° C., 250 mM sodium chloride and 25 mM trisodium citrate, 1% SDS, 50% formamide, 200 μg / ml ssDNA
Do with. Useful alterations of these conditions will be apparent to those skilled in the art.

The washing process after hybridization also has varying degrees of stringency. The exact conditions for washing are determined by salt concentration and temperature. As described above, the stringency of washing can be increased by lowering the salt concentration or raising the temperature. For example, the stringent conditions for the salt concentration during the washing step are preferably less than about 30 mM sodium chloride and less than about 3 mM trisodium citrate, more preferably about 15 mM.
Less than mM mM sodium chloride and less than about 1.5 mM trisodium citrate.
Strict conditions for the temperature of the washing process are usually about 25 ° C. or higher, preferably about 42 ° C.
° C or higher, more preferably about 68 ° C or higher. In a preferred embodiment, the washing step is performed at a temperature of 25 ° C. with 30 mM sodium chloride and 3 mM trisodium citrate, 0.1% SDS. In a more preferred embodiment, the washing process is carried out at a temperature of 42 ° C. with 15 mM sodium chloride and 1.5 mM trisodium citrate, 0 mM
. Performed with 1% SDS. In the most preferred embodiment, the washing process is performed at a temperature of 68 ° C. with 15 mM sodium chloride and 1.5 mM trisodium citrate, 0.1% S
Performed in DS. Further modifications of these conditions will be apparent to those skilled in the art.

[0086] Any of the embodiments of the present invention can be performed using DNA sequencing methods well known in the art. This method includes, for example, Sequenase (Klenow fragment of DNA polymerase I (US Biochemical, Cleveland OH)), Taq polymerase (Pe
rkin Elmer), thermostable T7 polymerase (Amersham, Chicago IL), or ELON
Enzymes such as the combination of a calibrated exonuclease and a polymerase, as found in the GASE amplification system (GIBCO / BRL, Gaithersburg, MD), are used. This process is based on the HYDRA microdispenser (Robbins Scientific, Sunnyvale CA)
ilton Micro Lab2200 (Hamilton, Reno, NV), Peltier Thermal Cycler (PTC20
0; MJ Reserch, Watertown MA) and automation using equipment such as ABI Catalyst and 373 and 377 DNA sequencers (Perkin Elmer). Next, ABI 37
3 or 377 DNA sequencing system (Perkin-Elmer) or MEGABACE 1000
Sequencing is performed using one of the DNA sequencing systems (Molecular Dynamics. Sunnyvale CA). The resulting sequence is analyzed using various algorithms well known in the art (eg, Ausubei, FM (1997) Short Protocols
in Molecular Biology , John Wiley & Sons, New York NY, unit 7.7; Meyers,
RA (1995) Molecular Biology and Biotechnology , Wiley VCH, New York NY,
pp. 856-853).

Using the partial nucleotide sequence, the nucleic acid sequence encoding PRGE can be extended using a variety of PCR-based methods well known in the art to cleave upstream sequences such as promoters and regulatory elements. To detect. For example, one method utilizing restriction site PCR involves amplifying an unknown sequence from genomic DNA in a cloning vector using common primers and nested primers. (For example, Sarkar, G. (19
93) PCR Methods Applic 2: 318-322). Another method using the inverse PCR method uses primers that amplify an unknown sequence from a circularized template that has been extended in a wide range of directions. This template is derived from a restriction fragment containing the known genomic locus and surrounding sequences. (Triglia, T. et al. (1988) Nucleic Acids Res 16: 8186). Capture P
A third method using the CR method involves PCR amplification of DNA fragments adjacent to known sequences of human and yeast artificial chromosomal DNA. (Eg, Lagerstrom, M. et al. (1991)
PCR Methods Applic 1: 111-119). In this method, it is possible to insert the recombinant double-stranded sequence into a region of unknown sequence before performing PCR using digestion and ligation with multiple restriction enzymes. It is also possible to obtain unknown sequences using other methods well known in the art. (E.g. Parker, JD et al. (1991) N
ucleic Acids Res. 19: 3055-3060). Furthermore, the use of PCR, nested primers, and PromoterFinder ^™ library enables walking in genomic DNA (Clontech, Palo Alto CA). This method eliminates the need to screen libraries and is useful for finding intron / exon junctions. For all PCR-based methods, the primers are commercially available OLIGO 4.06 Primer Analysis s
Using oftware (National Biosciences, Plymouth MN) or another suitable program, the length is 22-30 nucleotides, the GC content is 50% or more, and about 6%.
It is designed to anneal to the mold at a temperature between 8C and 72C.

When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include large cDNAs. Furthermore, if the oligo d (T) library cannot produce full-length cDNA, a randomly primed library, often containing a sequence having the 5 'region of the gene, is useful. Genomic libraries may be useful for extension of sequence into 5 'non-transcribed regulatory regions.

Sequencing or PC using a commercially available capillary electrophoresis system
Analysis or confirmation of the size of the nucleotide sequence of the R product is possible. Specifically, for capillary sequencing, a flowable polymer for electrophoretic separation, a laser-activated fluorescent dye specific for four different nucleotides, and a CCD camera for detecting the emitted wavelength It is possible to use Output / light intensity is converted to electrical signal using appropriate software (e.g. Perkin Elmer Co. Genotyper ^{T M} and Sequence Navigator ^TM) of the process and electronic data display from loading of samples to computer analysis computer-controllable It is. Capillary electrophoresis is particularly suitable for sequencing small pieces of DNA that may be present in small amounts in a particular sample.

In another embodiment of the present invention, a polynucleotide sequence encoding PRGE or a fragment thereof is used in a recombinant DNA molecule to express PRGE, a fragment thereof or a functional equivalent in a suitable host cell. Is possible. Due to the inherent duplication of the genetic code, additional DNA sequences encoding substantially the same or a functionally equivalent amino acid sequence can be generated, and these sequences can be used for PRGE cloning and expression.

The nucleotide sequences of the present invention can be recombined using methods generally known in the art to alter the sequence encoding PRGE for various purposes. This purpose includes, but is not limited to, cloning, processing and / or regulating expression of the gene product. Recombination of nucleotide sequences is possible using mixing of DNA with random fragments or PCR reassembly of gene fragments and synthetic oligonucleotides. For example, oligonucleotide-mediated directed mutagenesis can be used to introduce mutations that create new restriction sites, alter glycosylation patterns, alter codon preferences, generate splice variants, and the like.

According to another embodiment, the sequence encoding PRGE can be synthesized, in whole or in part, using chemical methods well known in the art (eg, Caruthers. MH et al. (1980).
) Nuc Acids Res Symp Ser 7: 215-223; and Horn, T. et al. (1980) Nucl. Acids Re.
s See Symp. Ser. 225-232). Alternatively, PRGE itself or a fragment thereof can be synthesized using chemical methods. For example, peptide synthesis can be performed using various solid-phase techniques (eg, Roberge, JY et al. (1995) Science 269: 202-).
204). In addition, automation of synthesis can be performed, for example, using an ABI 431A peptide synthesizer (PerPerformance).
kin Elmer). Furthermore, the amino acid sequence of PRGE, or any portion thereof, may be altered by alterations during direct synthesis and / or in combination with sequences from other proteins or any portion thereof, using chemical methods. It is possible to make a polypeptide.

This peptide was purified by high performance liquid chromatography for separation (eg, Chiez, RM).
And FZ Regnier (1990) Methods Enzymol. 182: 392-421). The composition of the synthesized peptide can be confirmed by amino acid analysis or sequencing (for example, Creighton. T. (1983) Protei
ns, Structures and Molecular Properties , WH Freeman and Co., New York,
NY).

To express a biologically active PRGE, the nucleotide sequence encoding PRGE or a derivative thereof is inserted into a suitable expression vector. This expression vector contains the necessary elements for the regulation of transcription and translation of the coding sequence inserted in a suitable host. These elements include regulatory sequences such as enhancers, constitutive and expression-inducible promoters, 5 'and 3' untranslated regions in the vector and polynucleotide sequences encoding PRGE. Such elements vary in their length and specificities. With certain initiation signals, it is possible to achieve more efficient translation of the sequence encoding PRGE. Such signals include the ATG start codon and nearby sequences such as the Kozak sequence. If the sequence encoding PRGE, its initiation codon and upstream regulatory sequences are inserted into a suitable expression vector, no additional transcriptional or translational regulatory signals will be required. However,
If only the coding sequence or its fragment is inserted, the in-frame AT
Exogenous translational control signals, including the G initiation codon, must be included in the expression vector. Exogenous translational elements and initiation codons can be obtained from a variety of sources, both natural and synthetic. Increasing the efficiency of expression can be achieved by including enhancers suitable for the particular host cell line used. (For example, Scharf, D. et al. (19
94) Results Probl. Cell Differ. 20: 125-162.).

Using methods well known to those skilled in the art, it is possible to create an expression vector containing a sequence encoding PRGE and suitable transcriptional and translational regulatory elements. These methods include in
It includes in vitro recombinant DNA technology, synthetic technology, and in vivo genetic recombination technology. (For example, Sambrook, J. et al. (1989) Molecular Cloning. A Laboratom Manua
l , Cold Spring Harbor Press, Plainview, NY, Chapters 4 and 8, and 16-17; and Ausubel, FM et al. (1995, and periodicsupplements) Current Protocols i
rt Molecular Biology , John Wiley & Sons, New York, NY.ch. Chapters 9 and 13, 1
See Chapter 6).

[0096] A variety of expression vector / host systems can be used to retain and express the sequence encoding PRGE. These include, but are not limited to, microorganisms such as bacteria transformed with a recombinant bacteriophage, plasmid, or cosmid DNA expression vector, yeast transformed with a yeast expression vector, and viral expression vectors. (E.g., baculovirus) -infected insect cell lines and plants transformed with a viral expression vector (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or a bacterial expression vector (e.g., Ti or pBR322 plasmid). Cell lines and animal cell lines are included. The present invention is not limited by the host cell used.

[0097] In bacterial systems, a number of cloning and expression vectors are available depending upon the use for which the PRGE-encoding polynucleotide sequence is to be used. For example, routine cloning, subcloning,
For growth, a multifunctional E. coli vector such as Bluescript ™ (Stratagene) or pSportl ™ plasmid (GIBCO BRL) can be used. Ligation of the sequence encoding HLOR into multiple cloning sites of the vector disrupts the lacZ gene, allowing a colorimetric screening method to identify transformed bacteria containing the recombinant molecule. Furthermore, using these vectors, it is possible to transcribe cloned sequences in vitro , dideoxin screening, rescue single stranded by helper phage, and create nested deletions. (For example, Van Hee
ke. G. and SM Schuster (1989) J. Biol. Chem. 264: 5503-5509.). For example, when a large amount of PRGE is required for producing an antibody, a vector that induces PRGE expression at a high level can be used. For example, vectors containing a T5 or T7 bacteriophage promoter that strongly induces expression can be used.

[0098] Yeast expression systems can be used for PRGE expression. In the yeast Saccharomyces cerevisiae, various types of vectors containing constitutive or inducible promoters such as α-factor, alcohol oxidase, and PGH can be used. In addition, such vectors induce either the secretion or retention of the expressed protein in cells, incorporating foreign sequences into the host genome for stable growth. (For example, Aus above
ubel .; and Grant et al. (1987) Methods Enzymol. 153: 51-794: Scorer. CA
Et al. (1994) Bio / Technology 12: 181-184.) Plant systems can also be used for PRGE expression. Transcription of the PRGE-encoding sequence may be performed, for example, by using a viral promoter such as the 35S and 19S promoters derived from CaMV alone or an omega leader sequence derived from TMV (Takamatsu, N. et al. (1987) EMBO J 6: 307-311). Is promoted in combination with. These products are directly DN
It can be introduced into plant cells by A-transformation or transfection via a pathogen. (Eg, Hobbs, S. or Murry, LE in McGraw Hill Year
book of Science and Technology (1992) McGraw Hill NY, pp.191-196)
.

In mammalian cells, a variety of virus-based expression systems may be utilized. When an adenovirus is used as an expression vector, a sequence encoding PRGE may be ligated to an adenovirus transcript / translation complex consisting of the late promoter and tripartite leader sequence. Insertion of the viral genome into a non-essential E1 or E3 region makes it possible to obtain a live virus expressing PRGE in infected host cells (Logan, J.
And Shenk, T. (1984) Proc. Natl. Acad. Sci. 81: 3655-3659). In addition, transcription enhancers such as the Rous sarcoma virus (RSV) enhancer can be used to increase expression in mammalian host cells. To express proteins at high levels, vectors based on SV40 or EBV can be used.

[0100] Human artificial chromosomes (HACs) can be used to supply fragments of DNA larger than those expressed and contained in a plasmid. About 6kb-10Mb for treatment
HACs were prepared using conventional methods (ribosomes, polycation amino polymers,
Or vesicles). (For example, Harrington. JJ et al. (1997) Nat Gene
t.15: 345-355).

For long-term production of mammalian-based recombinant proteins, stable expression of PRGE in cell lines is desirable. For example, an expression vector can be used to transform a PRGE-encoding sequence into a cell line. Such expression vectors contain replication and / or endogenous expression elements of viral origin and a selectable marker gene on the same or another vector. After the introduction of the vector, the cells are allowed to grow for about 1-2 days in an enriched medium before being transferred to a selective medium. The purpose of the selectable marker confers resistance to the selective mediator, and its presence allows the growth and recovery of cells that successfully express the introduced sequence. Resistant clones of stably transformed cells can be propagated using tissue culture techniques appropriate for the cell type.

[0102] Any number of selection systems can be used to recover transformed cell lines. The selection systems include, but are not limited to, include herpes simplex virus thymidine kinase gene and adenine phosphoribosyltransferase genes, respectively tk ^- or aprt ^- used in the cell. (For example, Wigler, M. et al. (19
77) Cell 11: 223-232; and Lowy. I. et al. (1980) Cell 22: 817-823). Also, resistance to antimetabolites, antibiotics or herbicides can be used as a basis for selection. For example, dhfr confers resistance to methotrexate, neo confers resistance to aminoglycosid neomycin and G-418, als or pat confers resistance to chlorsulfuron, phosphinotricin acetyltransferase (eg, , Wigl
er, M. et al. (1980) Proc. Natl. Acad. Sci. 77: 3567-3570; Colbere-Garapin,
F. et al. (1981) J. Mol. Biol. 150: 1-14; and Murry, supra). In addition, genes available for selection, such as trpB and hi, which alter what the cell needs for metabolism
sD is described in the literature (Hartman, SC and RC Mulligan (1988) Proc. Na
tl. Acad. Sci. 85: 8047-51). Anitocyanin, green fluorescent protein (
GFP) (Clontech. Palo Alto. CA), β-glucuronidase and its substrate GUS
Luciferase and its substrate luciferin and the like.
Green fluorescent protein (GFP) (Clontech, Palo Alto, CA) can also be used. These markers can be used to not only identify transformants, but also quantify transient or stable protein expression resulting from a particular vector system (eg, Rhodes, CA et al. (1995) Methods). Mol. Biol. 55: 121-791).

Even if the presence / absence of expression of a marker gene indicates the presence of the gene of interest, it may be necessary to confirm the presence and expression of that gene. For example, if the sequence encoding PRGE is inserted into a marker gene sequence, transformed cells containing the sequence encoding PRGE can be identified by a lack of marker gene function.
Alternatively, the marker gene can be aligned with the sequence encoding PRGE under the control of one promoter. Expression of the marker gene in response to induction or selection usually also indicates expression of the tandem gene.

In general, host cells that contain a nucleic acid sequence encoding PRGE and that express PRGE can be identified using a variety of methods well known to those of skill in the art. These methods include:
Protein biology or immunological, including DNA-DNA or DNA-RNA hybridization, PCR, membrane-based, solution-based, or chip-based techniques for the detection and / or quantification of nucleic acids or proteins. Assays, including but not limited to.

PR using either specific polyclonal or monoclonal antibodies
Immunological methods for detecting and measuring the expression of GE are well known in the art. Such techniques include immunosorbent assays (ELISA), radioimmunoassays (RIAs), and fluorescence activated cell sorters (FACS) linked to enzymes. Two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on PRGE
Is preferred, but competitive binding assays can also be used. These and other assays are well known in the art. (For example, Hampton. R
. (1990) Serological Methods, a Laboratony Manual. APS Press. St Paul.
. MN, Section IV; Coligan. JE et al. (1997 and periodic supplements) Curr.
ent Protocols in Immunology, Greene Pub. Associates and Wiley-Interscien
ce, New York. NY: and Maddox. DE et al. (1983) J. Exp. Med. 158: 1211-1216.
).

A variety of labeling and conjugation methods are well known to those skilled in the art and can be used in various nucleic acid and amino acid assays. Methods for generating labeled hybridization or PCR probes for detecting sequences related to the polynucleotide encoding PRGE include oligo-labeling, nick translation, end-labeling, or labeled nucleotides. The PCR amplification used is included. Alternatively, the sequence encoding PRGE, or any fragment thereof, can be cloned into a vector for generating an mRNA probe. Such vectors, which are well known in the art and are commercially available, can be prepared by adding a suitable RNA polymerase such as T7, T3, or SP6 and labeled nucleotides in
It can be used for the synthesis of RNA probes in vitro . These methods are described, for example, in Pharmacia & Upjohn (Kalamazoo, MI) and Promega (Madison WI), US Bi
It can be performed using various kits commercially available from ochemical Corp (Cleveland OH). Suitable reporter molecules or labels that can be used for easy detection include substrates, cofactors, inhibitors, magnetic particles, and radionuclides, enzymes, fluorescent agents, chemiluminescent agents, chromogenic agents, and the like.

[0107] Host cells transformed with a nucleotide sequence encoding PRGE are cultured under conditions suitable for the appearance and recovery of the protein from cell culture. Whether a protein produced in a transformed cell is secreted or remains in the cell depends on the sequence used and / or the vector. One of skill in the art will appreciate that expression vectors containing a polynucleotide encoding PRGE can be designed to include a signal sequence that directs secretion of PRGE through prokaryotic and eukaryotic cell membranes. Another construct can be used to join a sequence encoding PRGE to a nucleotide sequence encoding a polypeptide region that facilitates purification of a soluble protein. Areas that facilitate such purification include metal chelating peptides such as the histidine tryptophan module that allow for purification on immobilized metals, protein A regions that allow for purification with immobilized immunoglobulins, and FLAGS extensions / Includes, but is not limited to, regions used in affinity purification systems (Immunex Corp., Seattle WA). Between the purified region and the PRGE encoding sequence
Purification can be facilitated with those containing a cleavable linker sequence, such as those specific for factor XA or enterokinase (Invitrogen, San Diego, CA). One such expression vector expresses a fusion protein containing PRGE and a nucleic acid encoding the six histidine residues preceding the thioredoxin or enterokinase cleavage site. Histidine residues facilitate purification on immobilized metal ion affinity chromatography (IMAC) (eg, Porath, J et al. (1992); Protein E
xp. Purif. 3: 263-281). Enterokinase cleavage sites provide a means to purify PRGE from fusion proteins (e.g., Kroll, DJ et al. (1993); DNA Cell B
iol. 12: 441-453).

In addition, a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expression of the protein in the desired fashion. Such modifications of the polypeptide include acetylation, carboxylation, glycosylation, phosphorylation, lipidation (li
pidation), and acylation. Post-translational processing that cleaves the "prepro" form of the protein can be used to identify the target protein, folding and / or activity. Different host cells (eg, CHO, HeLa, MDCK, MEK293, WI38) with specific cellular devices and characteristic mechanisms for post-translational activity are available from the American Type Culture Collection (ATC).
C; Bethesda, MD) and selected to ensure correct modification and processing of the foreign protein.

In another embodiment of the present invention, a natural or altered or recombinant nucleic acid sequence encoding PRGE is ligated to a heterologous sequence which results in translation of the fusion protein of any of the host systems described above. For example, a chimeric PR containing a heterologous moiety that can be recognized by a commercially available antibody
GE proteins can facilitate the screening of peptide libraries for inhibitors of PRGE activity. Further, the heterologous protein portion and the heterologous peptide portion are
Commercially available affinity substrates can be used to facilitate purification of the fusion protein. Such moieties include glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CB
P), 6-His, FLAG, c-mc, hemagglutinin (HA),
It is not limited to these. GST and MBP, Trx, CBP, 6-H
Is allows purification of homologous fusion proteins with immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal chelating resins, respectively. FLAG, c-mc, and hemagglutinin (HA) allow immunoaffinity purification of the fusion protein using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. Alternatively, if the fusion protein is genetically engineered so that the fusion protein contains a proteolytic cleavage site between the sequence encoding PRGE and the heterologous protein sequence, PRGE may be cleaved from the heterologous portion after purification. The method of expression and purification of the fusion protein is Au
subel. FM et al. (1995 and periodic supplements) Current Protocols
Ne Molecular Biology , John Wiley & Sons. Ne, v York. NY. ch 10. In another embodiment of the present invention, PR-labeled in vitro using TNT ^™ rabbit reticulocyte lysate or wheat germ extract system (Promega. Madison. WI).
GE synthesis is possible. These systems link transcription and translation of sequences encoding proteins operably linked to the T7 or T3, SP6 promoter. Transcription occurs in the presence of a radiolabeled amino acid precursor, preferably 35S methionine.

Fragments of PRGE can be made by direct peptide synthesis using solid phase technology as well as recombinant products (see, eg, Creighton, pp. 55-60, supra). Protein synthesis can be performed manually or automatically. Automatic synthesis, for example, Applied Bi
It can be performed using osystem 431A peptide synthesizer (Perkin Elmer). The various fragments of PRGE are synthesized separately and then ligated to produce the full-length molecule.

Therapeutic, for example, chemical and structural similarities in sequence and motif exist between PRGE and gene expression regulatory proteins. In addition, PRGE expression is closely linked to cancer and other cell proliferative conditions, differentiated cells, inflammatory and immune responses, and is found in reproductive and nervous system tissues. Thus, PRGE is thought to play a role in diseases associated with cell proliferation and differentiation, including cancer and immune disorders, developmental disorders, reproductive disorders, and neurological disorders. In the treatment of the above diseases associated with increased PRGE expression or activity, PR
It is desirable to reduce the expression or activity of GE. In treating the above-mentioned diseases associated with decreased expression or activity of PRGE, it is desirable to increase expression or activity of PRGE.

Thus, in one embodiment, it is possible to administer PRGE or a fragment or derivative thereof to such a patient for the treatment or prevention of a disease associated with reduced expression or activity of PRGE. Examples of such diseases include reproductive disorders, including abnormal production of prolactin, fallopian tube disease and ovulation, endometriosis, abnormal estrus,
Menstrual cycle abnormalities, polycystic ovary syndrome, ovarian hyperstimulation syndrome, endometrial and ovarian cancer, uterine fibroids, autoimmune abnormalities, infertility including ectopic pregnancy, teratogenesis, and breast cancer, fibrocystic mastosis, Milkletia and sperm dysplasia, physiological sperm abnormalities, testicular cancer, prostate cancer, benign prostatic hyperplasia, prostatitis, Peyronie's disease, impotence, male and gynecomastia, and Also included are disorders, including epilepsy, ischemic cerebrovascular disease, stroke, brain tumors, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis And other motor neuron diseases, progressive neuromuscular atrophy, retinitis pigmentosa, hereditary ataxia, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, dura matter Inferior pyometra, epidural abscess, Purulent intracranial thrombophlebitis, myelitis and radiculitis, and viral central nervous system disease, kuru and Creutzfeldt - Jakob disease, Gerstmann Syndrome, prion diseases including Gerstmann-Straussler-Scheinker syndrome (prion dise
ase) and fatal familial insomnia, neurotrophic and metabolic diseases, neurofibromatosis, tuberous sclerosis, cerebellar retinal hemangioblastomatosis, cerebral trigeminal neurovascular syndrome, central nervous system Mental retardation and other developmental disorders, cerebral palsy, neuroskeletal disorder, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, dermatomyositis and polymyositis Metabolic and endocrine, toxic myopathy, myasthenia gravis, periodic quadriplegia,
Mood and anxiety psychiatric disorders, schizophrenic psychiatric disorders and restlessness, amnesia, catatonia,
Includes diabetic neuropathy, extrapyramidal terminal deficiency syndrome, dystonia, delusional psychosis, postherpetic neuralgia, Tourette's disease, and further includes abnormal cell proliferation, including actinic keratosis and Arteriosclerosis, atherosclerosis, bursitis,
Cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and adenocarcinoma and leukemia, lymphoma, melanoma, bone marrow Tumors, sarcomas, and teratocarcinomas, specifically, adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglion, digestive tract, heart, kidney, liver, lung, muscle, ovary, pancreas, adrenal gland Includes thyroid, penis, prostate, salivary gland, skin, spleen, testis, thymus, uterus cancer, etc., and further includes immune abnormalities, including acquired immunodeficiency syndrome (A
IDS) and adrenal insufficiency, adult respiratory distress syndrome, allergy, ankylosing spondylitis,
Amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia,
Autoimmune thyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes, emphysema, accidental lymphopenia, erythroblastosis, erythema nodosum, atrophic gastritis , Glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, bone Osteoporosis, pancreatitis, polymyositis, psoriasis,
Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenia, ulcerative colitis, Werner syndrome, cancer complications, hemodialysis, extracorporeal circulation And viral and bacterial infections, fungal infections, parasitic infections, protozoal infections, helminth infections, trauma, Bruton's associated agammaglobulinemia, acquired immunoglobulinemia (CVI),
Di-George syndrome (thymic dysplasia), thymic dysplasia, Iga alone deficiency, severe combined immunodeficiency (SCID), immunodeficiency with thrombocytopenia and eczema (Wiscott-Aldrich syndrome), Chediak-Higashi syndrome, chronic Includes granulomatosis, hereditary angioedema, Cushing's disease-related immunodeficiency, and also includes developmental disorders,
Among them are tubular acidosis, anemia, Cushing's syndrome, achondroplasia dwarfism, Duchenne muscular dystrophy, Becker pseudohypertrophic muscular dystrophy,
Gonadal agenesis, WAGR syndrome (Wilms tumor, aniridia, genitourinary disorders, mental retardation), Miss-Magenis- syndrome, dysplastic spinal cord, hereditary mucosal epithelial dysplasia, hereditary keratosis, Hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, chorea (Syndenham's chorea)
And seizures such as cerebral palsy, spinal cord fission, encephalopathy, cranial spina bifida, congenital glaucoma, cataracts, sensory hearing loss, and systems or any tissue such as brain and adrenal glands, kidney, skeletal and reproductive systems And diseases associated with cell growth and differentiation, including embryos and organs, embryogenesis, and morphogenesis, but are not limited to those listed here.

In another embodiment, PRGE or a fragment or derivative thereof is expressed for the treatment or prevention of a disease associated with reduced expression or activity of PRGE, including but not limited to the diseases listed above. The resulting vector can be administered to a patient.

In yet another example, substantially purified PRGE is used to treat or prevent a disease associated with reduced PRGE expression or activity, including but not limited to the diseases listed above. It is also possible to administer the containing pharmaceutical composition to a patient together with a suitable pharmaceutical carrier.

In yet another embodiment, an agonist that modulates the activity of PRGE for the treatment or prevention of a disease associated with reduced expression or activity of PRGE, including but not limited to the diseases listed above, may be used. It can also be administered to patients.

Further, in another embodiment, the PRGEs include, but are not limited to, the diseases listed above.
It is possible to administer an antagonist that regulates the activity of PRGE for the treatment or prevention of a disease associated with increased expression or activity of. In one embodiment, an antibody that specifically binds PRGE can be used directly as an antagonist or indirectly as a target or delivery mechanism for delivering an agent to cells or tissues that express PRGE.

In another embodiment, the complement of a polynucleotide encoding PRGE for the treatment or prevention of a disease associated with increased expression or activity of PRGE, including but not limited to the diseases listed above. Can be administered to a patient.

In another embodiment, any of the proteins, antagonists, antibodies, agonists, complementary sequences, vectors of the invention may be administered in combination with another suitable therapeutic agent. One skilled in the art can select a suitable therapeutic agent to use in combination therapy according to conventional pharmaceutical principles. Combinations with therapeutic agents can have synergistic effects in the treatment or prevention of the various diseases listed above. Using this method, it is possible to achieve a medicinal effect with a small amount of each drug, and to reduce the possibility of a wide range of side effects.

[0119] Antagonists of PRGE can be prepared using methods common in the art. Specifically, it is possible to produce antibodies using purified PRGE, or to screen a therapeutic drug library to identify those that specifically bind to PRGE. PRGE
Can also be produced using methods common in the art. Such antibodies include polyclonal, monoclonal, chimeric, single chain,
Fab fragments and fragments produced by Fab expression libraries are included. However, it is not limited to these. For treatment, neutralizing antibodies (
That is, those which inhibit the formation of a dimer) are particularly preferred.

For the production of antibodies, various hosts, including goats, rabbits, rats, mice, humans, and others, can be injected with PRGE or any fragment, or oligopeptide thereof, having immunogenic properties. Can be immunized with. Depending on the host species, various adjuvants can be used to enhance the immune response. Such adjuvants include Freund's adjuvant, mineral gel adjuvants such as aluminum hydroxide, lysolecithin, pluronic polyols, polyanions, peptides, oily emulsions, surfactants such as keyhole limpet hemocyanin, and dinitrophenol. However, the present invention is not limited to these. Among adjuvants used for humans, BCG (bacilli Calmette-Guerin) and Corynebacterium par
vum is particularly preferred.

The oligopeptide, peptide or fragment used to elicit antibodies to PRGE preferably has an amino acid sequence of about 5 or more amino acids, more preferably about 10 or more amino acids. Desirably, these oligopeptides or peptides, or fragments thereof, are identical to a portion of the amino acid sequence of the native protein, and also include the entire amino acid sequence of small, naturally occurring molecules.
Short stretches of PRGE amino acids can be fused to sequences of another protein, such as KLH (keyhole limpet hemocyanin), to produce antibodies against the chimeric molecule.

[0122] Monoclonal antibodies to PRGE can be produced by continuous cell lines in culture using any technique that produces antibody molecules. These techniques include, but are not limited to, hybridoma technology, human B cell hybridoma technology, and EBV-hybridoma technology (eg, Kohler
(1975) Nature 256: 495-497; Kozbor, D. et al. (1985) .J. Immunol. M
ethods 81.:31-42; Cote, RJ et al. (1983) Proc. Natl. Acad. Sci. 80: 2026-2.
030; Cole, SP et al. (1984) Mol. Cell Biol. 62: 109-120).

In addition, techniques such as splicing a mouse antibody gene to a human antibody gene developed for the production of a “chimeric antibody” are used to obtain molecules with suitable antigen specificity and biological activity. (For example, Morrison, SL et al. (1984) Proc.
Natl. Acad. Sci. 81: 6851-6855; Neuberger, MS et al. (1984) Nature 312: 604.
-608; Takeda, S. et al. (1985) Nature 314: 452,454). Alternatively, applying the described techniques for the production of single chain antibodies using methods well known in the art,
Generate PRGE-specific single-chain antibodies. Antibodies with related specificities but different idiotypic compositions can also be generated by chain mixing from random combinations of immunoglobulin libraries (eg, Burton DR (1991) Proc. Natl. A
cad. Sci. 88: 11120-3).

Antibodies can be raised in vivo by inducing production in lymphocyte populations, or by screening immunoglobulin libraries or as indicated in the literature.
It can also be made by screening a panel of highly specific binding reagents (eg, Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:
3833-3837; Winter, G. et al. (1991) Nature 349: 293-299).

Antibodies containing specific binding sites for PRGE can also be generated. For example,
Such fragments include the F (ab ') generated by pepsin digestion of the antibody molecule.
2 fragments and Fab fragments generated by reducing the disulfide bridges of the F (ab ') 2 fragment, including but not limited to. Alternatively, F
By creating an ab expression library, the desired specificity and monoclonal F
It enables rapid and easy identification of ab fragments (eg, Huse, WD et al. (1989)
Science 254: 1275-1281).

Screening is performed using various immunoassays to identify antibodies with the desired specificity. Numerous protocols for competitive binding, using either polyclonal or monoclonal antibodies with sequestered specificity, or immunoradioactivity, are well known in the art. Usually such immunoassays include PRGE
And measurement of complex regulation between the antibody and its specific antibody. A two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering PRGE epitopes is preferred, but a competitive binding assay can also be used (Pound, supra).

Using various methods such as Scatchard analysis with radioimmunoassay technology, P
Evaluate the affinity of the RGE antibody. Affinity is represented by the binding constant Ka, which is the molar concentration of the PRGE antibody complex divided by the molar concentration of free antibody and free antigen under equilibrium conditions. The measured value Ka of a polyclonal antibody reagent with heterogeneous affinities for multiple PRGE epitopes represents the average affinity or avidity of the PRGE antibody. specific
The Ka of a monoclonal antibody reagent monospecific for the PRGE epitope represents a true measure of affinity. The high-affinity antibody reagent having a Ka value of 10 ⁹ to 10 ¹² L / mol was obtained by using PRGE
It is preferably used for immunoassays in which the antibody conjugate must withstand severe manipulations. A low affinity antibody reagent with a Ka value of 10 ⁶ to 10 ⁷ L / mol is preferably used for immunopurification and similar treatments where PRGE must eventually dissociate from the antibody in an activated state. (Catty, D. (1988) Antibodies, Volume I
: A Practical Approach . IRL Press, Washington, DC; Liddell, JE and Cr
yer, A. (1991) A Practical Guide to Monocional Antibodies , John Wiley &
Sons, New York NY).

The titer and avidity of the polyclonal antibody reagents are further evaluated to determine the quality and suitability of such reagents for certain downstream applications. For example, the use of a polyclonal antibody reagent containing at least 1-2 mg / ml of specific antibody, preferably 5-10 mg / ml of specific antibody, is suitable for processes where the PRGE antibody complex must be precipitated. . Guidance on antibody specificity and titer, avidity, antibody quality and usage in various applications is generally available. (For example, Cat
ty, supra, and Coligan et al., supra).

In another embodiment of the invention, a polynucleotide encoding PRGE, or any fragment or its complement, can be used for therapeutic purposes. In certain embodiments, if the complementary sequence of the polynucleotide encoding PRGE is suitable for blocking transcription of mRNA, it can be used. In particular, cells can be transformed with sequences complementary to the polynucleotide encoding PRGE. Thus, complementary molecules or fragments can be used to modulate the activity of PRGE, or to regulate gene function. Such techniques are well known in the art, and sense or antisense oligonucleotides or large fragments can be designed from the control region of the sequence encoding PRGE or from various locations along the coding region.

[0130] Nucleotide sequences can also be delivered to target organs, tissues or cell populations using expression vectors derived from retroviruses, adenoviruses, herpes or vaccinia, or various bacterial plasmids. Vectors expressing a nucleic acid sequence complementary to PRGE can be made using methods well known to those of skill in the art (see, for example, Sambrook et al., Supra, and Ausubel et al., Supra). .

The gene encoding PRGE can be stopped by transforming a cell or tissue with an expression vector that expresses a polynucleotide encoding PRGE or a fragment thereof at high levels. Such constructs can be used to introduce sense or antisense sequences that cannot be translated into cells. Such vectors, even when not incorporated into DNA, continue to transcribe mRNA molecules until their function is impaired by endogenous nucleases. Non-replicating vectors also maintain transient expression for over a month, and may last longer if suitable replication elements are part of the vector system.

As described above, gene expression is determined by the sequence complementary to the regulatory 5 ′ or regulatory region of the gene encoding PRGE or an antisense molecule (DNA or RNA, P
NA) can be adjusted by designing. As is the case when oligonucleotides derived from the transcription initiation site, i.e. between -10 and +10 from the start site, are preferred, they can be blocked using "triple helix" and base-pairing techniques.
Triple helix structures are beneficial because they prevent the double helix from spreading sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent advances in therapy using triplex DNA have been described in the literature (eg, Gee, JE et al. (1994) In: H
uber, BE and BI Carr, Molecular and Immunologic Approaches , Futura
Publishing Co., Mt. Kisco, NY). Complementary sequences or antisense molecules can also be designed to prevent translation of the mRNA by preventing the transcript from binding to the ribosome.

[0133] Ribozymes, which are enzymatic RNA molecules, can be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by nucleotide strand breaks. For example, it includes a recombinant hammerhead ribozyme molecule that specifically and effectively catalyzes nucleotide chain cleavage of a sequence encoding PRGE.

A specific ribozyme cleavage site in any potential RNA target has the sequence G
It is initially identified by scanning target molecules for ribozyme cleavage sites, including UA, GUU, and GUC. Once identified, a short RN of 15-20 ribonucleotides corresponding to the region of the target gene containing the cleavage site
The A sequence can be evaluated for secondary structural features that render the oligonucleotide dysfunctional. The suitability of a candidate target can also be assessed by using a ribonuclease protection assay to test the ease of hybridization with a complementary oligonucleotide.

[0135] Complementary ribonucleic acid molecules and ribozymes of the invention can be made for synthesis of nucleic acid molecules using methods well known in the art. These methods include chemically synthesizing oligonucleotides such as solid phase phosphoramidite compounds. Alternatively, the RNA molecule may be a DNA encoding PRGE in vitro and in vivo.
Such DNA sequences, which can be generated by transcription of the sequence, can be incorporated into a variety of vectors using a suitable RNA polymerase promoter, such as T7 or SP6. Alternatively, those cDNA constructs that synthesize complementary RNA constitutively or inducibly can be introduced into a cell line, cell, or tissue.

[0136] Modification of an RNA molecule can increase intracellular stability and increase half-life. Possible modifications include the addition of flanking sequences at the 5 'and / or 3' end of the molecule, or the use of phosphorothioate or 2'O methyl rather than a phosphodiesterase linkage in the backbone of the molecule. Are not limited to these. This concept inherent in the production of PNAs includes acetyl-, methyl-, thio-, and similar modified forms of adenine, cytidine, guanine, thymine, and uridine that are not readily recognized by endogenous endonucleases, as well as Inosine, queosine, and wybutosine
Inclusion of non-conventional bases such as, for example, can be extended throughout these molecules.

A number of methods for introducing vectors into cells or tissues are available, including in vivo , in vitr,
o and equally suitable for ex vivo use. In the case of ex vivo treatment, the vector is introduced into hepatocytes collected from a patient and cloned and propagated to return the same patient by autologous transplantation. Transfection, ribosome injection or delivery by polycation amino polymer can be performed using methods well known in the art (eg, Goldman, CK et al. (1997) Nature Biotechnology 15: 462).
-66 :).

Any of the methods of treatment described above are applicable to all subjects in need of treatment, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably humans.

Another embodiment of the present invention relates to the administration of a medicament or sterile composition with a pharmaceutically acceptable carrier for all of the above-mentioned therapeutic effects. Such pharmaceutical compositions comprise PRGE, antibodies to PRGE, mimetics, agonists, antagonists or inhibitors of PRGE, and the like. The composition can be administered alone or in combination with one or more other agents, such as stabilizers, to a sterile, biocompatible pharmaceutical carrier.
Such pharmaceutical carriers include, but are not limited to, saline, buffered saline, glucose, water, and the like. The composition can be administered alone or with another drug, such as a drug or hormone.

The pharmaceutical compositions used in the present invention can also be administered using any number of routes. This route includes oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, ectopic, sublingual Or rectum, but is not limited thereto.

In addition to the active ingredient, these pharmaceutical compositions also include suitable pharmaceutically acceptable excipients and excipients, which include excipients and adjuvants, that facilitate the conversion of the active compound into pharmaceutically acceptable agents. Carrier can be included. For detailed formulation and administration techniques, see the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., E
aston, PA).

Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art at dosages suitable for oral administration. Such carriers enable the pharmaceutical composition to be consumed by the patient in tablets, pills, dragees, capsules,
It is formulated as a liquid, gel, syrup, mud, suspension.

Pharmaceutical preparations for oral use are prepared by mixing the active compound with a solid pharmaceutical additive, treating the resulting granule mixture (after grinding if desired) into tablets or dragee cores. cores). Suitable excipients include sugars including lactose, sucrose, mannitol, or sorbitol, starch from corn, wheat, rice, potato, or other plants, cellulose such as methylcellulose, hydroxypropylmethylcellulose, or sodium carboxymethylcellulose; Carbohydrate or protein excipients such as gums, including gum arabic and tragacanth, and proteins such as gelatin and collagen.
If desired, disintegrating or solubilizing agents may be added, such as, for example, cross-linked polyvinyl pyrrolidone, tangerine, alginic acid, or a salt thereof, sodium alginate.

Dragee cores are used with suitable coatings, such as concentrated sugar solvents. Such concentrated sugar solvents can include gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solvents, and suitable organic or mixed solvents. Dyestuffs or pigments are added to the tablets or dragees for product identification or to indicate the amount of active compound, ie, dosage.

Pharmaceutical preparations for oral use include push-fit capsules made of gelatin, as well as encapsulated capsules made of gelatin with a coating, such as glycerol or sorbitol. Push-fit capsules contain the active ingredient in admixture with excipients and binders such as lactose or starch, lubricants such as talc or magnesium stearate, and stabilizers if desired. In soft capsules, the active compound is treated with or without a stabilizer.
It can be dissolved or suspended in a suitable solution such as a fatty oil, a solution, or a polyethylene glycol solution.

Pharmaceutical agents suitable for parenteral administration are formulated in aqueous solutions, with physiologically compatible buffers such as Hanks's solution, Ringer's solution, physiological saline buffer being preferred. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be formulated as suitable oily injection suspensions. Suitable hydrophilic solutions or vehicles include fatty oils such as sesame oil, and synthetic fatty acids such as ethyl oleate, triglycerides or ribosomes. Non-lipid polycationic amino polymers are used for delivery purposes. Optionally, the suspension may contain suitable stabilizers or agents that increase the solubility of the compounds to allow for concentrated solutions.

For topical or nasal administration, suitable penetrants penetrate the particular barrier will be used in the formulation. Such penetrants are well-known to those skilled in the art.

The pharmaceutical compositions of the present invention can be prepared using methods known in the art, for example, conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, encapsulating, or lyophilizing processes. Can be manufactured.

The pharmaceutical compositions are formulated as salts, and can be formed with many acids, including but not limited to hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, and the like. Salts are more soluble in aqueous or other protic solvents than the corresponding free base systems. Another preferred form of the drug comprises some or all of 1-50 mM histidine, 0.1% -2% sucrose, and 2-7% mannitol, with a pH range of 4-4.
. A lyophilized powder which is 5 to 5.5 and binds to a buffer before use can be used.

After the pharmaceutical compositions have been formulated, they can be packaged in a suitable box and labeled as medication for the indicated condition. For administration of PRGE, such labels will include quantity, frequency, and mode of administration.

Suitable pharmaceutical compositions for use in the present invention include compositions that contain an effective amount of the active ingredient to achieve its intended purpose. One skilled in the art can determine a dose that is sufficiently effective for his or her ability.

For any composition, a therapeutically effective dose can be estimated initially either in tumor cell assays, for example on tumor cells, or in animal models. Usually, a mouse, rabbit, dog, pig, or the like is used as an animal model. Animal models can also be used to determine suitable enrichment ranges and routes of administration. Based on such treatment, beneficial dosages and routes for administration in humans can be determined.

A medically effective dose is related to the amount of active ingredient that ameliorates the symptoms or condition, eg, PRGE or a fragment thereof, an antibody to PRGE, an agonist or antagonist of PRGE, an inhibitor, and the like. Medication efficacy and toxicity are calculated, for example, by calculating the ED ₅₀ (50% of the population has a medicinal effect on taking) or the LD ₅₀ (lethal is 50% of the population on taking) statistics. And the like can be determined by standard pharmaceutical techniques in cell culture or animal experiments. Dosage ratio between therapeutic and toxic effects is the therapeutic index and it can be expressed as the ratio LD ₅₀ / ED _50. Pharmaceutical compositions that exhibit high therapeutic indices are desirable. The data obtained from the cell culture assays and animal studies is used in formulating a range of dosage for human application. Such compositions dosage included are toxic not including little or no, it is desirable that a range of circulating concentrations that include the ED _50. Dosage will vary within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

The exact dosage will be determined by the practitioner, in light of factors related to the patient that requires treatment. Dosage and administration are adjusted to provide effective levels of the active ingredient or to maintain the desired effect. Factors that may be considered as dose components include disease severity, general health of the patient, age, weight, and gender of the patient, time and frequency of administration, concomitant medications, response sensitivities, and Response is included. Long-acting pharmaceutical compositions can be administered once every three or four days, once a week, once every two weeks, depending on the half-life and clearance rate of the particular formulation.

Normal dosage amounts will vary depending on the route of administration, but will range from about 0.1 to 100,000 μg.
Up to about 1 gram. Guidance on specific dosages and modes of delivery is provided in the literature and is generally available to practitioners in the art. One of skill in the art will utilize formulations of nucleotides that are different from the protein or inhibitor. Similarly, delivery of a polynucleotide or polypeptide
It will be specific for a particular cell, state, location, etc.

Diagnosis In another embodiment, an antibody that specifically binds to PRGE is used to diagnose a disease characterized by expression of PRGE, or to monitor a patient being treated with a PRGE or an agonist or antagonist or inhibitor of PRGE. Used in assays to perform Antibodies useful for diagnosis are formulated in the same manner as described for therapy. Diagnostic assays for PRGE include methods that use antibodies and labels to detect PRGE from human body fluids or from cells or tissues. These antibodies can be used with or without modification and can be labeled by covalent or non-covalent attachment of a reporter molecule. Various reporter molecules known in the art may be used, some of which are described above.

A variety of protocols for measuring PRGE, including ELISA, RIA, and FACS, are well known in the art and provide a basis for diagnosing abnormal or abnormal levels of expression of PRGE. Normal or standard PRGE expression values can be determined by combining an antibody against PRGE with body fluids or cells collected from a normal mammal, preferably a human subject, under conditions suitable for complex formation. decide. The amount of standard complex formation can be quantified in a variety of ways, but can be determined by photometry.
c) is preferred. A sample from the subject's PRGE expression level, control and disease, biopsy tissue is compared to a standard value. The deviation between the standard value and the subject is a parameter for diagnosing the disease.

According to another embodiment, a polynucleotide encoding PRGE can be used for diagnosis. Polynucleotides used include oligonucleotide sequences,
Includes complementary RNA and DNA molecules, and PNA. The polynucleotide is used to detect and quantify the expression of a gene in a biopsy tissue that expresses PRGE that can be correlated with disease. This diagnostic assay is used to determine the absence, presence, and overexpression of PRGE and to monitor the control of PRGE levels during treatment.

In one embodiment, the nucleic acid sequence encoding PRGE can be identified by hybridization with a PCR probe capable of detecting a polynucleotide sequence comprising the gene sequence encoding PRGE or a closely related molecule. is there. For example, 5 '
The specificity of the probe, whether it is created from a highly specific region that is a regulatory region or, for example, a conserved motif and a less specific region, and the stringency of hybridization or amplification (maximum, high, intermediate Or lower) if the probe is PRGE
Or only the natural sequence encoding the allele or related sequence will be identified.

Probes are also used for the detection of related sequences and preferably contain at least 50% nucleotides from any sequence encoding PRGE. The target hybridization probe of the present invention can be DNA or RNA; SEQ ID NO: 32
-62 or a genomic sequence containing the PRGE gene promoter, enhancer, and intron.

[0161] Methods for preparing hybridization probes specific for PRGE-encoding DNA include using polynucleotide sequences encoding PRGE and PRGE derivatives with mRN
There is a method of cloning into a vector for producing the A probe. Such vectors are commercially available and well known to those skilled in the art, and are used to synthesize RNA probes in vitro by adding a suitable RNA polymerase and a suitable labeled nucleotide. The hybridization probe may be a radionuclide such as 32P or 35S, or avidin / biotin.
It can be labeled with a population of various reporters, such as an enzyme label such as alkaline phosphatase, linked to the probe by a binding system.

The polynucleotide sequence encoding PRGE can be used to diagnose a disease associated with PRGE expression. Examples of such disorders include reproductive disorders, including abnormal production of prolactin, and fallopian tube and ovulation disorders, endometriosis, estrus, menstrual cycle disorders, polycystic ovary syndrome. Infertility including ovarian hyperstimulation syndrome, endometrial and ovarian cancer, uterine fibroids, autoimmune abnormalities, ectopic pregnancy, teratogenesis, breast cancer, fibrocystic mastosis, mastolacia, and spermatogenesis Physiologic sperm abnormalities, testicular cancer, prostate cancer, benign prostatic hyperplasia, prostatitis, Peyronie's disease, impotence, male and gynecomastia, and also include neurological disorders. May include epilepsy, ischemic cerebrovascular disease, stroke, brain tumors, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron diseases, progression Nervous muscular atrophy Retinitis pigmentosa, hereditary ataxia, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess,
Subdural pyometra, epidural abscess, purulent intracranial thrombophlebitis, myelitis and radiculitis,
Viral central nervous system diseases and prion diseases including prague-Kreut and Creutzfeldt-Jakob disease, Gerstmann syndrome, Gerstmann-Straussler-Scheinker syndrome (pr
ion disease), fatal familial insomnia, neurotrophic and metabolic diseases, neurofibromatosis, tuberous sclerosis, cerebellar retinal hemangioblastomatos
is), trigeminal neurovascular syndrome, central nervous system mental retardation and other developmental disorders, cerebral palsy, neuroskeletal disorder, autonomic nervous system disorder, cranial nerve disorder, spinal cord disease, muscular dystrophy and other nerves With myopathy, dermatomyositis and polymyositis,
Hereditary metabolic and endocrine, toxic myopathy, myasthenia gravis, periodic limb paralysis, mood and anxiety psychiatric disorders, schizophrenic psychiatric disorders, restlessness, amnesia,
Includes catatonia, diabetic neuropathy, extrapyramidal terminal deficiency syndrome, dystonia, paranoid psychosis, postherpetic neuralgia, Tourette's disease, and further includes abnormal cell proliferation, including sunlight angle Keratosis and arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary Thrombocythemia, and adenocarcinoma and leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, specifically, adrenal gland, bladder,
Bone, bone marrow, brain, breast, cervix, gallbladder, ganglion, digestive tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary gland, skin, spleen, testis, thymus,
Includes uterine cancer, and further includes immune disorders, including acquired immunodeficiency syndrome (AIDS) and adrenal insufficiency, adult respiratory distress syndrome, allergy, ankylosing spondylitis, amyloidosis, anemia, Asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes, emphysema, incidental lymphocytes Hypocytosis, erythroblastosis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis,
Myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, Systemic sclerosis, thrombocytopenia, ulcerative colitis, Werner syndrome, cancer complications, hemodialysis, extracorporeal circulation and viral and bacterial infections, fungal infections, parasitic infections, protozoal infections, helminths Infection, trauma, Bruton-associated agammaglobulinemia, acquired immunoglobulinemia (C
VI), Di-George syndrome (thymic dysplasia), thymic dysplasia, Iga alone deficiency, severe combined immunodeficiency (SCID), immunodeficiency with thrombocytopenia and eczema (Wiscott-Aldrich syndrome), Chediak-East Syndrome, chronic granulomatosis,
Includes hereditary angioedema, Cushing's disease-associated immunodeficiency, and also developmental disorders, including tubular acidosis, anemia, Cushing's syndrome, chondrodysplasia dwarfism, Duchenne muscle Dystrophy, Becker pseudohypertrophic muscular dystrophy, gonadal dysplasia, WAGR syndrome (Wilms tumor, aniridia, urogenital abnormalities,
Mental retardation) miss-magenis syndrome, dysplastic spinal cord,
Hereditary neuropathies such as hereditary mucosal epithelial dysplasia, hereditary keratoderma, Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, chorea (Syndenham's
chorea) and seizures such as cerebral palsy, spinal cord fission, encephalopathy, cranial spondyloarhesis, congenital glaucoma, cataract, sensorineural hearing loss, and systems such as brain and adrenal, kidney, skeletal and reproductive systems or any And any disease associated with cell proliferation and differentiation, embryogenesis and morphogenesis, including but not limited to those listed herein.

The polynucleotide sequence encoding PRGE can be obtained by Southern blotting, Northern blotting, or other membrane-based techniques, PCR, dipsticks, or the like.
ck), a pin, an ELISA assay, and a microarray used for detecting the expression of a mutant PRGE using a body fluid or tissue collected from a patient. Such qualitative or quantitative methods are well known in the art.

In certain aspects, the nucleotide sequence encoding PRGE is associated with a disease,
It will be particularly useful in assays to detect the above-mentioned diseases. The nucleotide sequence encoding PRGE could be labeled by standard methods and added to a body fluid or tissue sample taken from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared to a standard value. If the amount of signal in the patient's sample varies significantly compared to the control sample, the level of mutation in the nucleotide sequence encoding PRGE in the sample will reveal the presence of the associated disease. Such assays can be used to monitor animal studies, clinical trials, or treatment of individual patients.
It is possible to estimate the effect of a particular treatment.

A summary of normal or standard expression is established to provide a basis for the diagnosis of diseases associated with PRGE expression. This establishment is achieved by linking a PRGE-encoding sequence or a fragment thereof to a body fluid or cells extracted from a normal subject, either animal or human, under conditions suitable for hybridization or amplification. obtain. Standard hybridization can be quantified by comparing values obtained from normal subjects to values from experiments in which known amounts of substantially purified polynucleotides are used. Standard values obtained from normal samples can be compared to values obtained from subjects exhibiting symptoms of the disease. The presence of the disease is determined using the deviation between the standard value and the subject's value.

Once the presence of the disease has been determined and the treatment protocol initiated, the hybridization assay can be repeated on a regular basis to estimate whether the level of expression in the subject has begun to approach the value shown in a normal patient. It is possible. The results of the repeated assays can be used to see the effect of the treatment over a period of days to months.

In cancer, abnormal amounts of transcripts in living tissue from an individual indicate a predisposition to the development of the disease and may provide a way to detect the disease before actual clinical symptoms occur. is there. This type of more definitive diagnosis allows medical professionals to use preventative or aggressive treatments early to prevent the development or progression of cancer.

Further diagnostic uses of oligonucleotides designed from sequences encoding PRGE may include the use of PCR. Such oligomers can be produced chemically, enzymatically, or in vitro . Oligomers, preferably including fragments of a polynucleotide encoding PRGE, or fragments of a polynucleotide complementary to a polynucleotide encoding PRGE, are used to identify a particular gene or condition under optimal conditions. You. Oligomers can also be used for detection and / or quantification of closely related DNA or RNA sequences under somewhat less stringent conditions.

Methods that can be used to quantify PRGE expression include radiolabeling or biotin labeling of nucleotides, coamplification of regulatory nucleic acids, and standard curves with results added. (For example, Melby, PC, etc. (1993) J. Imm
unol. Methods, 159: 235-44; see Duplaa, C. et al. (1993) Anal. Biochem. 229-236). The rate of quantification of large numbers of samples was accelerated by using an ELISA-type assay in which the oligomer of interest was included in various diluents and quickly quantified by spectrophotometry or non-color response.

In another example, oligonucleotides or longer fragments from any of the polynucleotide sequences described herein are used as targets in a microarray. Microarrays are used to simultaneously monitor the expression levels of a very large number of genes and identify gene mutations, mutations and polymorphisms. This information is useful for determining gene function, interpreting the genetic basis of disease, diagnosing disease, and monitoring and developing the activity of therapeutic agents.

[0171] Microarrays are prepared, used and analyzed by methods well known in the art. (E.g., Brennan, TM et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Pro
c. Natl. Acad. Sci. 93: 10614-10619; Baldeschweiler et al. (1995) PCT Application No.W
O95 / 251116; Shalon, D. et al. (1995) PCT Application No.WO95 / 35505; Heller, RA et al. (1
Natl.Acad.Sci. 94: 2150-2155; and Heller, MJ et al. (1997) U.S. Pat.No. 5,605,662) Another embodiment of the invention also employs a nucleic acid sequence encoding PRGE. Thus, it is possible to generate hybridization probes useful for mapping naturally occurring genomic sequences. This array maps to:
Specific chromosomes, specific regions of chromosomes or artificially generated chromosomes, such as human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), bacterial P1 products or single chromosomal cDNA libraries. (Eg Price, CM
(1993) Blood Rev. 7: 127-134, and Trask, BJ (1991) Trends Genet. 7:14.
In situ fluorescence hybridization (FISH) will correlate with other physical chromosome mapping techniques and genetic map data (eg, by Heinz-Ulrich, et al. (1995) in Meyers, supra). , pp. 965-968.). Examples of genetic map data can be found in various scientific journals or on the Online Mendelian Inheritance in Man (OMIM) site. The correlation between the location of the gene encoding PRGE on the physical chromosomal map and a particular disease, or a predisposition to a particular disease, can help determine the region of DNA associated with such a disease. The nucleotide sequences of the present invention may also be used to detect differences in gene sequences between normal, carrier, and infected individuals.

The genetic map can also be extended using physical mapping techniques, such as in situ hybridization of chromosomal preparations and binding analysis using defined chromosomal markers. By placing the gene on the chromosome of another mammal, such as a mouse,
Even if the number or arm of a particular human chromosome is not known, relevant markers are often revealed. New arrays are created by physical mapping
It can also be assigned to chromosome arms. This is valuable information for researchers studying genetic diseases using positional cloning or other gene discovery techniques. The location of the disease or syndrome is, for example, 11q22-
Once roughly determined by gene binding to a particular gene region, such as 23, any sequence mapping for that region represents a relevant or regulatory gene for further investigation (eg, by Gatti, RA et al. (1988) Nature 336: 577-580
See). In addition, using the nucleotide sequence of the present invention of interest, normal, holder,
That is, a difference in chromosome position due to translocation or inversion between infected persons may be detected.

In another embodiment of the present invention, PRGE, its catalytic or immunogenic fragments or oligopeptides thereof, can be used to screen libraries of compounds in any of a variety of drug screening techniques. Fragments used for such screening are free in solution, fixed to a solid support, retained on the surface of cells, or present intracellularly. Formation due to binding of the complex between PRGE and the agent to be tested may be measured.

Another method used for drug screening is used to increase the screening capacity of compounds having suitable binding affinity for a protein of interest (eg, Geysen, et al. (1984) PCT Application No. See WO84 / 03564). In this method, a significant number of different small test compounds are synthesized on plastic pins or other substrates. The test compound is washed after reacting with PRGE or a fragment thereof. Next, the bound PRGE is detected by methods well known in the art. Purified PRGE can also be coated directly on plates used in the drug screening techniques described above. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.

In another example, a competitive drug screening assay can be used in which neutralizing antibodies capable of binding to PRGE specifically compete with the test compound for binding to PRGE.
In this method, the antibody detects the presence of any peptide that shares one or more antigenic determinants with PRGE.

In another embodiment, the nucleotide sequence encoding PRGE is used in developing molecular biology techniques, including but not limited to nucleotides such as, but not limited to, the currently known triplet code and specific base pairing interactions. New techniques that depend on the properties of the sequence can be provided.

Those skilled in the art will be able to utilize the present invention with the foregoing description without further explanation. Accordingly, the specific examples described below are for illustration purposes and do not limit the invention.

All patent applications, patents, publications, and US Provisional Application No. 6 mentioned above and below.
No. 0/089, 029, No. 60/094, 575, No. 60/104, 624 are incorporated herein by reference.

[0179]

【Example】

1 Preparation of cDNA Library RNA was purchased from Clontech, or was isolated from the tissues listed in Table 4. One tissue was homogenized and dissolved in a guanidinium isothiocyanate solution. On the other hand, homogenize another tissue and dissolve in phenol,
Alternatively, it was dissolved in a mixture of a suitable denaturant, such as a single phase solution of TRIZOL (Life Technologies), guanidinium isothiocyanate and phenol. The lysate was centrifuged on cesium chloride or extracted with chloroform. RNA was precipitated from this lysate with either isopropanol or sodium acetate and ethanol, or otherwise.

Extraction and precipitation of RNA with phenol were repeated as necessary to increase the purity of the RNA. Optionally, the RNA is treated with a DNase. For most libraries, poly (A +) RNA was isolated using oligo d (T) -linked paramagnetic particles (Promega) or OLIGOTEX latex particles (QIAGEN. Valencia CA) and OLIGOTEX mRNA purification kit (QIAGEN). Alternatively, the POLY (A) PURE mRNA purification kit (Arabion,
It was isolated directly from tissue lysates using another RNA isolation kit (Austin TX).

In some cases, RNA was provided to Stratagene and the corresponding cDN was
A library was prepared. Otherwise, the UNIZAP vector system (Stratag
ene) or the SUPERSCRIPT plasmid system (Life Technologies) to synthesize cDNA by recommended or similar methods known in the art. (See, for example, Ausubel, 1997, supra, units 5.1-6.6). Reverse transcription was initiated using oligo d (T) or random primers. After binding the synthetic oligonucleotide adapter to the double-stranded cDNA, the cDNA was digested with one or more suitable restriction enzymes. For most libraries, SEPHACRYL S
1000 or SEPHAROSE CL2B, SEPHAROSE CL4B column chromatography (Amersh
am Pharmacia Biotech), the size of the cDNA (300-1000 bp) was selected by agarose gel electrophoresis. PBLUESCRIPT plasmid (Stratagene) or pSPORT
1 Plasmid (Life Technologies), plNCY (Incyte Pharmaceuticals, Palo Alto
The cDNA was ligated to the compatible restriction enzyme sites of the polylinker of a suitable plasmid such as CA). This recombinant plasmid was transformed with Stratagene's XL1-Blue, XL1-BIueMRF
, SOLR, or Life Technologies DH5α or DH 10B, ElectroMAX DH 10B
Were transformed into competent E. coli cells containing

2 Isolation of cDNA Clones Plasmids were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or cell lysis. Magic or WIZARD Minipreps
DNA purification system (Promega) and AGTC Miniprep purification kit (Edge Biosystems, G
aithersburg MD), QIAGEN's QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QI
Plasmids were purified using at least one of the AWELL 8 Ultra Plasmid purification system, REAL Prep 96 plasmid kit. After precipitation, they were resuspended in 0.1 ml of distilled water and stored at 4 ° C. with or without lyophilization.

[0183] Alternatively, plasmid DNA was amplified from host cell lysates by a high-throughput direct binding PCR method. (Rao, VB (1994) Anal. Blochem. 216: 1-14). The lysis and thermal cycling process of the host cells was performed in a single reaction mixture. Samples are processed and transferred to a 384-well plate for storage and amplified plasmid DNA
Was measured fluorometrically using a PICOGREEN dye (Molecular Probes, Eugene OR) and a Fluoroskan II fluorescence scanner (Labsystems Oy, Helsinki, Finland).

3 Preparation of cDNA for Sequencing and Analytical Sequencing ABI CATALYST 800 (Perkin-E
lmer) or HYDRA microdispenser (Robbins Scientific), MICROLAB 220
The 0 system (Hamilton) was used with PTC-200 thermal cyclers (MJ Research). For cDNA sequencing, ABI PRISM 373 or 377 sequencing systems (Perkin-Elmer) and standard ABI protocols, base pairing software, kits were used. Alternatively, cDNA sequencing can be performed using MEGABACE 100
0 A DNA sequencing system (Molecular Dynamics) was used. In a further alternative, ABI PRISM BIGDYE Terminator cycling is used for cDNA amplification and sequencing.
An e sequencing ready reaction kit (Perkin-Elmer) was used. In yet another alternative, solutions and dyes from Amersham Pharmacia Biotech were used for cDNA sequencing. The reading frame of the EST is based on the standard method (reviewed in Ausubel, 1997, sup.
ra, unit 7.7). Some of the cDNA sequences were selected and extended as described in 5 of this example.

Construction and analysis of cDNA and polynucleotide sequences obtained from extension and shotgun sequencing were performed in combination with software utilizing algorithms known to those skilled in the art. Table 5 is a summary of the program name of the software used, a description of the program, references, and threshold parameters. The first column of Table 5 is the tools, programs, and algorithms used, the second column is a brief description of them, and the third column is a cited reference which is incorporated herein by reference. The fourth column is the score, probability value, and other parameters used to evaluate the degree of coincidence between the two sequences (the higher the probability value, the higher the homology). MACD for sequence analysis
NASIS PRO software (Hitachi Software Engineering, S. San Francisco CA)
And LASERGENE software (DNASTAR).

Confirmation of the polynucleotide sequence can be accomplished by using BLAST and dynamic programming, programs and algorithms based on dinucleotide nearest neighbor analysis to remove vector and linker, poly A sequences and mask ambiguous base pairs. I went in. next,
Using programs based on BLAST and FASTA, BLIMPS, these sequences can be used to select sequences from public databases such as the primates and rodents of GenBank, mammals, vertebrates, eukaryotes, and the BLOCKS database. The sequence was queried for annotation. These sequences were assembled into full-length polynucleotide sequences using programs based on Phred and Phrap, Consed, and BLAST and FASTA, BLIMPS
Screened for open reading frames with a program based on. Translate the full length polynucleotide sequence to derive the corresponding full length amino acid sequence, GenBank database (above) and SwissProt, BLOCKS, PRINTS, PFAM, Pr
These full-length sequences were analyzed by querying a database such as osite or a protein family database based on the Hidden Markov Model (HMM) such as PFAM. HMM analyzes the consensus primary structure of a gene family using probability.

The programs described above for the construction and analysis of full-length polynucleotide and amino acid sequences can also be used to identify fragments of the polynucleotide sequence from SEQ ID NOs: 32-62. Fragments of about 20 to 4000 nucleotides are useful for hybridization and amplification and have been described in the above invention.

4 Northern Analysis Northern analysis is an experimental technique used to detect the presence of a gene transcript, in which labeled nucleotides on a membrane to which RNA from a particular cell type or tissue is bound. With sequence hybridization (see, eg, Sambrook, supra).
, Chapter 7; and Ausubel. FM et al., Supra, Chapters 4 and 16).

Using a similar computer technology used for BLAST, GenBank or LIFESEQ
Search for identical or related molecules in a nucleotide database such as a database (Insight). This analysis is much faster than many membrane-based hybridizations. In addition, the sensitivity of the computer search can be changed to determine whether any particular match is classified as an exact match or a homologous match. The criteria for the search is the product score defined as (% sequence identity x% maximum BLAST score) / 100. The product score takes into account both the similarity between the two sequences and the length of the sequence match. For example, for a product score of 40, the match is 1-2
It is accurate within% error, and at 70 the match will be accurate. Similar molecules are typically identified by selecting a molecule that exhibits a product score in the range of 15 to 40, although lower scores may identify related molecules.

The results of the Northern analysis are reported as the percentage distribution of the library in which transcripts encoding PRGE occurred. The analysis also includes the classification of the cDNA library by organ / tissue and disease. Organ / tissue categories include cardiovascular, dermal, developmental, endocrine, gastrointestinal, hematopoietic / immune, musculoskeletal, nervous, reproductive, urine. Disease categories include cancer, inflammation / trauma, fetus, nerve, pooled. For each category, the number of libraries expressing the sequence of interest was counted and divided by the number of libraries in the entire range. Table 3 shows the ratio (percent) specifically expressed in each tissue and the ratio expressed in each disease.

5 Extension of the Polynucleotide Encoding PRGE The full-length nucleic acid sequence of SEQ ID NOs: 56-62 was prepared using oligonucleotide primers designed from suitable fragments of the full-length molecule. The fragment was extended and made. One primer was synthesized to initiate a 5 'extension of the known fragment and the other primer was synthesized to initiate a 3' extension of the known fragment. The starting primer was OLIGO 4.06 software (National Biosciences)
Alternatively, using any other suitable program, the primer may be used to anneal to the target sequence at a temperature of about 68-72 ° C. with a GC content of about 50 to about 50 nucleotides in length from about 22 to about 30 nucleotides. Designed. Nucleotides were extended to avoid hairpin structure and primer-primer dimer.

This sequence was extended using a selected human cDNA library. If more than one extension is needed or desired, additional or nested primer sets are designed.

Amplification was performed with high fidelity by PCR using methods known in the art. PCR is PT
The test was performed in a 96-well block plate using a C-200 thermal cycler (MJ Research, Inc.). The reaction mixture was a template DNA and 200 nmol of each primer, a buffer containing Mg ^2- and (NH ₄ ) ₂ SO ₄ and β-mercaptoethanol, Taq DNA polymerase (A
mersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene). Amplification was performed on the primer set, PCI A and PCI B, with the following parameters. Step 1 94 ° C. for 3 minutes Step 2 94 ° C. for 15 seconds Step 3 60 ° C. for 1 minute Step 4 68 ° C. for 2 minutes Step 5 Repeat Steps 2, 3 and 4 20 times Step 6 68 ° C. for 5 minutes Step 74 Alternatively, amplification was performed on the primer set, T7 and SK +, with the following parameters. Step 1 94 ° C. for 3 minutes Step 2 94 ° C. for 15 seconds Step 3 57 ° C. for 1 minute Step 4 68 ° C. for 2 minutes Step 5 Repeat Steps 2, 3 and 4 20 times Step 6 68 ° C. for 5 minutes Step 74 Store at ° C.

The DNA concentration in each well was determined by adding 100 μl of PICOGREEN quantitative reagent (0.25% (v / v) PICOGREEN; Molecular) dissolved in 1 × TE and 0.5 μl of undiluted PCR product.
Probes, Eugene OR) to opaque fluorometer plates (Coming Costar, Acton MA)
To each well, so that the DNA can bind to the reagent. The plate is scanned with Fluoroskan II (Labsystems Oy, Helsinki, Finland) and the fluorescence of the sample is measured to quantify the concentration of DNA. 5-10 μl of reaction mixture
Are analyzed by electrophoresis on a 1% agarose minigel to determine which reactions have successfully extended the sequence.

The extended nucleotides were desalted and concentrated before transferring to 384-well plates and using CviJI choleravirus endonuclease (Molecular Biology Research, Madi).
son WI) and sonicated or sheared before religating to the pUC18 vector (Amersham Pharmacia Biotech). For shotgun sequencing,
The digested nucleotides were separated on a low (0.6-0.8%) agarose gel to cut the fragments, and the agar was digested with Agar ACE (Promega). T4 ligase (New Eng
A clone extended using land Biolabs, Beverly MA) was transformed into a pUC 18 vector (Amersh
am Pharmacia Biotech) and treated with Pfu DNA polymerase (Stratagene) for extension of the restriction site to transfect competent E. coli cells. The transfected cells are selected and transferred to a medium containing antibiotics.
The cells were cultured overnight at 37 ° C. in a 384-well plate of a 2 × carbenicillin culture solution.

Cells were lysed and Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu
DNA was amplified by PCR using a DNA polymerase (Stratagene) according to the following procedure. Step 1 94 ° C. for 3 minutes Step 2 94 ° C. for 15 seconds Step 3 60 ° C. for 1 minute Step 4 72 ° C. for 2 minutes Step 5 Repeat Steps 2, 3 and 4 29 times Step 6 72 ° C. for 5 minutes Step 74 Store at ° C. DNA was quantified with PICOGREEN reagent (Molecular Probes) as described above. DN
Samples with poor A recovery were amplified again under the conditions described above. The sample was diluted with 20% dimethylsulphoxide (1: 2, v / v) and DYENAMIC D
IRECT kit (Amersham Pharmacia Biotech) or ABI PRISM BIGDYE Terminator
Sequence was performed using a cycle sequencing ready reaction kit (Perkin-Elmer).

Similarly, using the nucleotide sequence of SEQ ID NOs: 56-62, oligonucleotides designed for the above procedure and for this extension, 5 'regulatory sequences were obtained in a suitable gene library.

The nucleic acid sequence of SEQ ID NOs: 32-55 was used to design oligonucleotide primers for extending a partial nucleotide sequence to full length. In each nucleic acid sequence, one primer is synthesized to initiate extension of the antisense polynucleotide and the other primer is synthesized to initiate extension of the sense nucleotide. These primers are used to facilitate "outward" extension of a known sequence, creating an amplicon containing a new, unknown nucleotide sequence of the region of interest. The starting primer is about 22 to about 30 nucleotides in length and has a GC content of about 50% or more using OLIGO 4.06 (National Biosciences, Plymouth, Minn.) Or other suitable program. And designed to anneal to the target sequence at a temperature of about 68 to about 72 ° C. Avoid stretches of nucleotides that result in hairpin structure and primer-primer dimerization.

This sequence is extended using a selected human cDNA library (Gibco / BRL). If more than one step extension is necessary or desired, another set of primers is designed to further extend the known region.

Processing is performed according to the instructions in the instructions for the XL-PCR kit (Perkin Elmer), and high fidelity amplification is performed by thoroughly mixing the enzyme and the reaction mixture. When starting amplification with 40 pmol of each primer and all other components of the kit at the recommended concentration, Peltier Thermal Cycler (PTC200; MJ Res.
erch, Watertown MA) using the following parameters: Step 1 1 minute at 94 ° C (initial denaturation) Step 2 1 minute at 65 ° C Step 3 6 minutes at 68 ° C Step 4 15 seconds at 94 ° C Step 565 Step 6 Repeat the steps 4 to 6 for another 15 cycles Step 8 94 ° C for 15 seconds Step 9 65 ° C for 1 minute Step 10 68 ° C for 7 minutes 15 seconds Step 11 Step 8 to Step 12 is repeated for 12 cycles.

Aliquots of 5-10 μl of the reaction mixture to low concentrations (about 0.6-0.8%)
Analysis by electrophoresis on an agarose minigel determines which reactions have successfully extended the sequence. The band believed to contain the largest product was selected, excised from the gel, purified using QIAQUICK ^™ (QIAGEN Inc.), cut off the terminal extension using Klenow enzyme, and religated and cloned. Create blunt ends that will be easier.

After ethanol precipitation, the product was redissolved in 13 μl ligation buffer and 1 μl
Of T4-DNA ligase (15 units) and 1 μl of T4 polynucleotide kinase are added and the mixture is incubated for 2-3 hours at room temperature or overnight at 16 ° C. Competent E. coli cells (in 40 μl of appropriate culture)
l ligation mixture and transform with 80 μl SOC culture (eg Semb
roo, supra, see Appendix A, p. 2). After incubation at 37 ° C. for 1 hour, all transformed mixtures were washed with carbenicillin (2 × carb).
Luria Bertani (LB) agar (eg, Sembrook, supra, Appendix A, p.
Plate on top). At a later date, several colonies are randomly selected from each plate and cultured in 150 μl of liquid LB / 2xCarb medium in each well of an appropriate commercially available sterile 96-well microtiter plate. Further at a later date, each 5 μl of the overnight culture was transferred into a non-sterile 96-well plate,
After dilution 1:10 with water, transfer 5 μl of each sample to the PCR array.

For PCR amplification, 18 μl of the enriched PCR reaction mixture (3.3 ×) containing 4 units of rTth DNA polymerase, vector primers, and one or both of the gene-specific primers used in the extension reaction are added to each well. The amplification was performed under the following conditions: Step 1 at 94 ° C. for 60 seconds Step 2 at 94 ° C. for 20 seconds Step 3 at 55 ° C. for 30 seconds Step 4 at 72 ° C. for 90 seconds Step 5 Steps 2 to 4 were repeated 29 more cycles Step 6 For 180 seconds at step 74 ° C. (hold as it is).

An aliquot of the PCR reaction is run on an agarose gel with a molecular weight marker. The size of the PCR product is compared to the original partial cDNA, the appropriate clone is selected, ligated into the plasmid and sequenced.

Similarly, the procedure described above utilizes the nucleotide sequence of SEQ ID NOs: 32-55, and the oligonucleotide designed for this extension and a suitable gene library are used to 5 '
The regulatory sequence was obtained.

6 Labeling and Use of Individual Hybridization Probes cDNA, mRNA, or genomic DNA is screened using hybridization probes derived from SEQ ID NOs: 32-62. Particular mention is made of the labeling of oligonucleotides of about 20 base pairs, but the larger cDNA
The same procedure is basically used for fragments. Oligonucleotide, OL
Designed using state-of-the-art software such as IGO 4.06 software (National Bioscience), 50 pmol of each oligomer and 250 μCi of [γ- ³² P]
Labeling is performed by using adenosine triphosphate (Amersham, Chicago, IL) and T4 polynucleotide kinase (DuPont NEN, Boston MA) in combination.
The labeled oligonucleotide is substantially purified using a Sephadex ^™ G-25 ultra-fine exclusion dextran bead column (Pharmacia & Upjohn, Kalamazoo, MI). An aliquot containing probes labeled per minute 10 ⁷ counts, following endonuclease, AseI, Bgl II, Eco RI , Pst I, Xba1 or Pvu II (DuPont NEN)
Used in a typical membrane-based hybridization analysis of human genomic DNA cut with one of the following.

DNA from each cut is fractionated on a 0.7% agarose gel and transferred to a nylon membrane (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is performed at 40 ° C. for 16 hours. To remove non-specific signals, the blots are washed sequentially at room temperature up to 0.1x sodium citrate and 0.5% sodium dodecyl sulfate under increasingly stringent conditions.
After exposing the XOMAT AR ^™ film (Eastman Kodak, Rochester, NY) to the blots for transfer to film for several hours, the hybridization patterns are visually compared.

7 Using microarray chemical bonding methods and inkjet devices, it is possible to synthesize array elements on the surface of a substrate (see, eg, Baldeschweiler, supra). Using an array similar to dot or slot blots, the elements are exposed to heat, UV,
It is arranged and bonded to the surface of the substrate using a mechanical or chemical bonding method. Typical arrays can be made manually or using available methods and machines, and can include any suitable number of elements. After hybridization, unhybridized probe is removed and the level and pattern of fluorescence is determined using a scanner. The scanned images can be analyzed to determine the degree of complementarity and the relative amount / expression level of each probe that hybridizes to the element on the microarray.

[0209] Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof, can comprise elements of a microarray. Fragments suitable for hybridization can be selected using software known in the art, such as LASERGENE ^™ . Full-length cDNA corresponding to one of the nucleic acid sequences of the invention
EST, or fragments thereof, or cDNA arbitrarily selected from a cDNA library related to the present invention, are aligned on a suitable substrate such as a glass slide.
The cDNA is fixed to a slide using, for example, UV cross-linking, subjected to heat treatment and chemical treatment, and finally dried (for example, Schena,
(1995) Science 270: 467-470; and Shalon, D. et al. (1996) Genome Res.
6: 639-645). A fluorescent probe is provided and used to hybridize to an element on the substrate. This substrate is analyzed by the method described above.

8. Complementary Polynucleotides The sequence encoding the PRGE, or a sequence complementary to any portion thereof, is used to reduce or inhibit expression of the naturally occurring PRGE. Although the use of oligonucleotides containing about 15 to about 30 base pairs is described, essentially the same method can be used for fragments of smaller or larger sequences. Appropriate oligonucleotides are designed using Oligo 4.06 software and the coding sequence of PRGE. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5 'sequence and used to prevent the promoter from binding to the coding sequence. To inhibit translation, complementary oligonucleotides are designed to inhibit the ribosome from binding to the transcript encoding PRGE.

9. Expression of PRGE Expression and purification of PRGE can be performed using a bacterial or viral based expression system. For PRGE expression in bacteria, the cDNA is subcloned into a suitable vector containing an inducible promoter that increases antibiotic resistance and the level of cDNA transcription. Such promoters include, but are not limited to, the T5 or T7 bacteriophage promoter associated with the lac operator regulatory element and the trp-lac (tac) hybrid promoter. The recombinant vector is transformed into a suitable bacterial host, such as BL21 (DE3). When bacteria with antibiotic resistance are induced with isopropyl β-D thiogalactopyranoside (IPTG), HL
Express OR. Expression of PRGE in eukaryotic cells is achieved by infecting insect or mammalian cell lines with Autographica californica nuclear pleiotropic virus (AcMNPV), commonly known as baculovirus. The non-essential polyhedrin gene of the baculovirus is replaced with the cDNA encoding PRGE by either homologous recombination or bacterial-mediated gene transfer with transfer plasmid-mediated. Viral infectivity is maintained and high levels of cDN
A is transferred. Recombinant baculoviruses are often Spodoptera fru
giperda (Sf9) is used to infect insect cells, but may also be used to infect human hepatocytes. In the latter case, further genetic modification of the baculovirus is required. (For example, Engelhard. EK et al. (1994) Proc. Natl. Acad. Sci. USA
91: 3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7: 1937-1945.).

[0212] In most expression systems, PRGE is, for example, glutathione S-transferase (GST).
Or a fusion protein synthesized with a peptide epitope tag such as FLAG or 6-His, so that affinity-based purification of the recombinant fusion protein from unpurified cell lysate can be performed quickly and in one go. The 26 kilodalton enzyme GST from Schistosoma japonicum allows purification of the fusion protein with immobilized glutathione while maintaining protein activity and antigenicity. (Pharmacia,
Piscataway, NJ). After purification, the GST moiety can be proteolytically cleaved from the HLOR at a specific manipulation site. FLAG, an eight amino acid peptide, allows immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His, in which six histidine residues are continuously extended, enables purification with a metal chelating resin (QIAGEN). Methods for protein expression and purification are described in Ausubel (1995, supra, ch 10, 16). The following assays can be performed directly using HLOR purified by these methods.

10 Demonstration of PRGE activity The activity of PRGE is measured by its ability to stimulate reporter gene transcription (L
iu, HY et al. (1997); EMBO J. 16: 5289-5298.). This assay uses LexAop-LacZ, a highly characterized reporter gene construct that consists of a LexA DNA transcription regulatory element (LexA _op ) fused to a sequence encoding the E. coli LacZ enzyme. Methods for producing and expressing a fusion gene, introducing it into cells, and measuring LacZ enzyme activity are well known to those skilled in the art. The sequence encoding PRGE is cloned into a plasmid that directs the synthesis of a fusion protein LexA-PRGE, consisting of the DNA binding domain from LexA transcription factor and PRGE. The resulting plasmid encoding the LexA-PRGE fusion protein is introduced into yeast cells along with a plasmid containing the LexAop-LacZ reporter gene. The degree of activity of the LacZ enzyme associated with cells transfected with LexA-PRGE associated with regulatory cells is proportional to the amount of transcript stimulated by PRGE.

11 Functional Assays The function of PRGE is to increase physiologically elevated levels in mammalian cell culture systems.
Evaluate by expression of the sequence encoding PRGE. The cDNA is subcloned into a mammalian expression vector containing a strong promoter that expresses the cDNA at high levels. Such vectors include pCMV SPORT ^™ (Life Technologies. Gaither
sburg. MD) and pCR ^™ 3.1 (Invitrogen, Carlsbad, CA), both containing the cytomegalovirus promoter. 5-10 μg of the recombinant vector is preferably transiently transfected into human cell lines, preferably of endothelial or hematopoietic origin, by liposome preparation or electroporation. In addition, 1-2 μg of plasmid containing the sequence encoding the labeled protein is co-transfected. Expression of the labeled protein allows one to distinguish between transfected and non-transfected cells. Further, the expression of the cDNA from the recombinant vector can be accurately predicted by the expression of the labeled protein. Such labeled proteins include green fluorescent protein (GFP) (Clontech,
Palo Alto, CA), and CD64 or CD64-GFP fusion proteins. Using automatic flow cytometry (FCM), which utilizes laser optics-based technology,
Transfected cells expressing CD64-GFP are identified and characterized such as apoptotic status. In addition, the FCM detects and measures the incorporation of a fluorescent molecule that diagnoses the phenomenon of preceding or simultaneous cell death. These phenomena include changes in nuclear DNA content as measured by staining DNA with propidium iodide, down-regulation of DNA synthesis as measured by reduced uptake of bromodeoxyuridine,
Includes changes in cell surface and intracellular protein expression as measured by reactivity with specific antibodies, and changes in plasma membrane composition as measured by binding of fluorescent complex annexin V protein to the cell surface It is. Flow cytometry is described in Ormerod, M.
G. (1994) Flow Cytometry (Oxford, New York, NY.).

The effect of PRGE on gene expression was determined by the sequence encoding PRGE and CD64 or CD64-G
Highly purified cell populations transfected with either of the FPs can be evaluated. CD64 or CD64-GFP is expressed on the transformed cell surface and binds to a conserved region of human immunoglobulin G (IgG). Transformed and non-transformed cells can be separated using magnetic beads coated with either human IgG or an antibody against CD64. (See DYNAL. Lake Success. NY). mRN
A can be purified from cells by methods known in the art. Expression of mRNA encoding PRGE and other genes of interest can be analyzed by Northern analysis or microarray technology.

12 Production of antibodies specific for PRGE Polyacrylamide gel electrophoresis (PAGE) (eg, Harrington, MG (1990)
PRGE, substantially purified using Methods Enzymol. 182: 488-495) or other purification techniques, is used to immunize rabbits and generate antibodies according to standard protocols.

[0217] Alternatively, the PRGE amino acid sequence may be stored in LASERGENE software (DNASTAR).
Is used to determine a region having high immunogenicity, and the corresponding oligopeptide is synthesized and used to produce an antibody by a method well known to those skilled in the art. The selection of suitable epitopes, such as those near or adjacent to the C-terminus of the hydrophilic region, is well known in the art (see, eg, chapter 11 by Ausubel et al., Supra).

Usually, oligopeptides about 15 residues in length are synthesized by fmoc chemistry using Applied Biosystems peptide synthesizer Model 431A, using N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). KLH (Sigma, St. Louis, MO) by the reaction that has been used to enhance immunogenicity
(See, for example, Ausubel et al., Supra). Rabbits are immunized with the oligopeptide-KLH complex in Freund's complete adjuvant. To test the anti-peptide activity of the obtained antiserum, for example, the peptide is bound to plastic, blocked with 1% BSA, reacted with rabbit antiserum, washed, and further treated with radioactive iodine-labeled goat antiserum. React with rabbit IgG.

13 Purification of Spontaneous PRGE Using Specific Antibodies Spontaneous or recombinant PRGE is substantially purified by immunoaffinity chromatography using antibodies specific for PRGE. An immunoaffinity column is constructed by covalently linking a PRGE antibody to an activated chromatography resin such as CNBr-activated SEPHAROSE (Pharmacia & Upjohn). After binding, the resin is blocked and washed according to the manufacturer's instructions.

The culture containing PRGE is passed through an immunoaffinity column, and the column is washed under conditions that allow preferential adsorption of PRGE (eg, with a buffer of high ionic strength in the presence of a detergent). The column is eluted under conditions that disrupt the binding of the antibody to the PRGE (eg, with a buffer at pH 2-3 or high concentrations of chaotropic ions such as urea or thiocyanate ions) to recover the PRGE. .

14 Identification of molecules that interact with PRGE PRGE or a biologically active fragment thereof is labeled with ¹²⁵ I Bolton Hunter reagent (see, eg, Bolton et al. (1973) Biochem. J. 133: 529). . Candidate molecules, pre-arranged in a multiwell plate, are incubated with labeled PRGE, washed, and all wells with labeled PRGE complexes are assayed. Using data obtained at various PRGE concentrations, numerical values are calculated for the binding, affinity, and number of PRGEs with candidate molecules.

Those skilled in the art will be able to make various modifications of the described method and system without departing from the scope and spirit of the invention. Although the invention has been described with reference to certain preferred embodiments, it is to be understood that the scope of the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be covered by the following claims. .

[0223] BRIEF DESCRIPTION Table 1 Table were used to create a sequence of full-length encoding PRGE, ID number (SEQ ID NO) of the polypeptide sequence and nucleotide sequence, clone ID number, cD
The NA library and the cDNA fragment are shown.

Table 2 shows the characteristics of each polypeptide sequence, including latent motifs and homologous sequences, and PR
The method and algorithm used to identify GE are shown.

Table 3 shows the tissue-specific expression pattern of each nucleic acid sequence determined by Northern analysis, the diseases, abnormalities, and symptoms associated with these tissues, and the vectors into which each DNA was cloned.

Table 4 shows the tissues used to generate a cDNA library from which in-site cDNA clones encoding PRGE were isolated.

Table 5 shows the programs used for PRGE analysis, their descriptions, references, and parameter thresholds.

[Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

[Table 6]

[Table 7]

[Table 8]

[Table 9]

[Table 10]

[Table 11]

[Table 12]

[Table 13]

[Table 14]

[Table 15]

[Table 16]

[Table 17]

[Table 18]

[Table 19]

[Table 20]

[Table 21]

[Table 22]

[Table 23]

[Table 24]

[Table 25]

[Table 26]

[Table 27]

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 15/00 A61P 35/00 25/00 37/02 35/00 43/00 105 37/02 C07K 14 / 47 43/00 105 16/18 C07K 14/47 C12N 15/00 ZNA 16/18 A61K 37/02 (31) Priority claim number 60 / 104,624 (32) Priority date October 14, 1998 (1998) 10.14) (33) Priority 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, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA , CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL , TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Tongue, Wy Tom San Jose Runwick Court 95118, California, USA 4230 (72) Inventor Hillman, Jennifer El California 94040 Mountain View Anna Avenue 366 (72) Inventor Bandman, Olga 94043 Mountain Avenue Anna Avenue, California 940 (72) Inventor Bandlay, Neil Sea 94040 Mountain View, California・ # 30 ・ Dale Avenue 1240 (72) Inventor Gegler, Carl Jay 94025, California, United States of America ・ Menlo Park Oakland Ave. 1048 (72) Inventor Gorgon, Gina A (72) Inventor Bogun, Mariah Earl 14244 San Leandro Santiago Road, California 94577 United States of America Patterson, Chandra California, United States 94025, Menlo Park, # 1 Sherwood Way 490 (72) inventor Ryu, Deyungu-Aina M California, USA 95136 San Jose Park Belmont Place 55

Claims (20)

[Claims] 1. A substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-31 (SEQ ID NOs: 1-31) and fragments thereof. 2. A substantially purified variant having an amino acid sequence identity of 90% or more with the amino acid sequence of claim 1. 3. An isolated and purified polynucleotide encoding the polypeptide of claim 1. 4. An isolated and purified polynucleotide variant having 90% or more polynucleotide sequence identity to the polynucleotide of claim 3. 5. An isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide of claim 3. 6. An isolated and purified polynucleotide having a sequence complementary to the polynucleotide of claim 3. 7. A method for detecting a polynucleotide, comprising: (a) hybridizing the polynucleotide of claim 6 with at least one nucleic acid of a sample to form a hybridization complex; (b) Detecting the hybridization complex, wherein the presence of the hybridization complex is correlated with the presence of the polynucleotide in the sample. . 8. The method according to claim 7, further comprising amplifying the polynucleotide before the hybridization. 9. An isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 32-62 and fragments thereof. 10. An isolated and purified polynucleotide variant having 90% or more polynucleotide sequence identity to the polynucleotide of claim 9. 11. An isolated and purified polynucleotide having a sequence complementary to the polynucleotide of claim 9. 12. An expression vector comprising a fragment of at least one polynucleotide of claim 3. 13. A host cell comprising the expression vector according to claim 12. 14. A method for producing a polypeptide, comprising: (a) culturing the host cell of claim 13 under conditions suitable for expression of the polypeptide; and (b) medium for the host cell. And recovering the polypeptide from the protein. 15. A pharmaceutical composition comprising the polypeptide of claim 1 together with a suitable pharmaceutical carrier. 16. A purified antibody that specifically binds to the polypeptide of claim 1. 17. A purified agonist of the polypeptide of claim 1. 18. A purified antagonist of the polypeptide of claim 1. 19. The expression or activity of PRGE, comprising the step of administering an effective amount of the pharmaceutical composition of claim 15 to a patient in need of treatment or prevention of a disease associated with reduced expression or activity of PRGE. A method for treating or preventing a disease associated with a decline. 20. An agent for increasing the expression or activity of PRGE, comprising administering an effective amount of the antagonist of claim 18 to a patient in need of treatment or prevention of a disease associated with the expression or activity of PRGE. A method of treating or preventing a related disorder.